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How to cite this thesis

Surname, Initial(s). (2012). Title of the thesis or dissertation (Doctoral Thesis / Master’s Dissertation). Johannesburg: University of Johannesburg. Available from: http://hdl.handle.net/102000/0002 (Accessed: 22 August 2017).

A synopsis of medicinally important indigenous species of the genus Scabiosa (Caprifoliaceae), an evaluation of their biological activity and synergistic properties of Scabiosa columbaria By Ndinne Wendy Mugwena (201400888)

Dissertation submitted in fulfilment of the requirements for the degree of Magister Scientiae (MSc) In Botany In the Faculty of Science at the University of Johannesburg South Africa

Supervisor: Prof. A.N. Moteetee (UJ) Co-supervisor: Prof S. Van Vuuren (WITS)

August 2020

DECLARATION

I declare that this dissertation hereby submitted to the University of Johannesburg for the degree MAGISTER SCIENTIAE (Botany), is my own work and has not been previously

submitted by me for a degree at another institution.

Ndinne W. Mugwena (August 2020)

DEDICATION

I dedicate this dissertation to my parents, Livhuwani & Aaron Mugwena as well as my grandmother Naume Nethanani “Momi”

Ndi livhuwa thikhedzo ye vha mpha yone, na u kondelela havho u bva mathomoni u swika zwino. Ndi livhuwa zwihulwane u pfesesa ndila ye nda di nangela yone isingo doweleyaho. “Momi” thabelo dzavho ndi dzone dzi ntikaho misi yothe.

My Omnipresent God of Grace, I thank You!

Proverbs 16:3 “Ask the LORD to bless your plans, and you will be successful in carrying them out.”

ACKNOWLEDGMENT

I would like to express my gratitude to the following persons and institutions:

. Prof Annah Moteetee, my supervisor, for the opportunity to learn under your supervision and guidance. I appreciate your patience, support and perseverance to complete this dissertation with me amid a pandemic. . Prof Sandy Van Vuuren for co-supervising my study, together with your laboratory technician Phumzile Moerane for assisting with antimicrobial laboratory experience at University of Witwatersrand. . Mr Thinus Fourie, for always being willing to assist with all my laboratory needs. . Johnathan and the Random Harvest Nursery stuff, for helping me grow some of the plants that I needed. . Dube Sifelani & Dance Mabu from the Department of Chemistry for assisting with antacid activity practical section . The University of Johannesburg and Department of Botany and Plant Biotechnology for the support. . National Research Foundation (NRF) for their financial support during my studies. . To my siblings, Nkhangweleni, Tshifhiwa, Ndivhuwo and Nanne, you have all been amazing cheerlearders.

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ABSTRACT

The genus Scabiosa L., so called because of its traditional use for the treatment of scabies, belongs to the family Caprifoliaceae. This genus is relatively large comprising about 80 species distributed mainly in the Mediterranean, with only nine species occurring naturally in southern Africa. In southern Africa the genus was last revised by Harvey and Sonder (1865), however, the purpose of the current study was to provide a synopsis of the medicinally important species of the genus. In addition, the study aimed to record the ethnomedicinal uses of these species and identify the plants with which Scabiosa columbaria L. is used in combination. The third aim of the study was to evaluate S. columbaria and the plant combinations for antibacterial and anti-inflammatory activities and then assess interactive effects. The fourth aim was to evaluate S. columbaria for its antacid and acid neutralising potential and lastly, to investigate all the plants studied here for their toxicity levels. Herbarium specimens were examined to study the morphological characteristics and the geographical distribution patterns of the medicinal species. For the antibacterial activity, the minimum inhibitory concentration assay was used. The pathogens tested were Neisseria gonorrhoeae (sexual transmitted infections), Bacillus cereus, Enterococcus faecalis and Escherichia coli (gastrointestinal pathogens) and skin pathogens (Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis). The anti-inflammatory activities were investigated through inhibition of the cyclooxygenase (COX) isoenzymes COX-1 and COX-2, as well as 15-lipoxygenase enzyme (15-LOX). Back titration of sodium hydroxide and Fordtran’s model were used to assess the antacid activities and neutralization potential of the selected plants. Lastly the brine shrimp lethality assay was used to test the toxicity of S. columbaria and plants used in combination. The results indicate that leaf morphology is of diagnostic importance in distinguishing the closely related taxa with differences observed in pubescence density, leaf shape, size and margins. Nine species of Scabiosa occur in southern African and four of these are used for medicinal purposes, namely: Scabiosa columbaria, S. transvaalensis S. Moore, S. incisa Mill., and S. albanensis R.A. Dyer. Scabiosa columbaria is sometimes mixed with other species, for example Afroaster hispida (Thunb.) J.C. Manning & Goldblatt., Cussonia paniculata subsp sinuata (Reyneke & Kok)

De Winter., Dicoma anomala Sond., Helichrysum caespititium (DC.) Sond. Ex Harv., Searsia divaricata (Eckl. & Zeyh) Moffett., and Zantedeschia albomaculata (Hook). Baill. where it is used as a remedy for a wide variety of ailments ranging from heartburn to infections associated with the gastrointestinal system, respiratory tract, eyes, and the skin, as well as inflammatory diseases. Only S. columbaria has hitherto been assessed for its antibacterial activity against pathogens responsible for sexually transmitted and respiratory tract infections. Interestingly, although S. columbaria and plants used in combination with it, have been documented frequently in the literature for medicinal uses, their antibacterial screening showed no noteworthy activity against all the tested pathogens. Scabiosa columbaria was found to be non-toxic. However, extracts from two plants, i.e. C. paniculata subsp sinuata (organic and aqueous) and D. anomala (organic) used in combination with S. columbaria were found to be toxic. It was also noted that their presence in combination with S. columbaria increased the mortality rate of the brine shrimp. When the concentration was decreased, a drastic decrease in the mortality rate was observed. For anti-inflammatory activity, S. columbaria exhibited selective inhibition of COX-1, which is associated with negative side effects and showed moderate activity against 15-LOX. The combination of S. columbaria with C. paniculata subsp sinuata and S. divaricata had noteworthy anti-inflammatory activity, being able to inhibit both LOX and COX enzymes at low concentrations. For antacid activity, the immediate neutralization of artificial gastric juice was higher for the combinations when compared to S. columbaria used individually. When comparing the results of S. columbaria and the positive control (Rennie®) in the back titration protocol using both NaOH and artificial gastric juice, the results showed that the plant was a weak antacid when used individually and in combination with other plants. Although most of the results were not in support of the use of Scabiosa species in ethnomedicine, the species should not be neglected completely. Future studies should focus on understanding the behaviour of ethnomedicinally important Scabiosa sp when tested in complex environments.

TABLE OF CONTENTS

DECLARATION ...... i DEDICATION ...... ii ACKNOWLEDGMENT ...... iii ABSTRACT ...... iv TABLE OF CONTENTS ...... vi LIST OF FIGURES ...... viii LIST OF TABLES ...... ix LIST OF EQUATIONS ...... x LIST OF ABBREVIATIONS & ACRONYM ...... xi LIST OF SYMBOLS & UNITS ...... xiii CHAPTER 1 LITERATURE REVIEW ...... 1 1.1 Brief taxonomic overview ...... 1 1.1.1 Family Caprifoliaceae ...... 1 1.1.2 Subfamily Dipsacoideae ...... 2 1.1.3 The genus Scabiosa ...... 2 1.2 Ethnobotanical uses of subfamily Dipsacoideae ...... 3 1.3 Antimicrobial activity of the subfamily Dipsacoideae...... 4 1.4 Anti-inflammatory properties of the subfamily Dipsacoideae ...... 6 1.5 Medicinal plants as antacids for heartburn ...... 8 1.6 Synergistic effects of medicinal plants ...... 11 1.7 Toxicity of medicinal plants ...... 12 1.8 Aims and objectives of the study ...... 13 CHAPTER 2 SYNOPSIS OF MEDICINALLY IMPORTANT SPECIES OF SCABIOSA ...... 15 2.1 Introduction ...... 15 2.2 Materials and methods ...... 15 2.3 Results ...... 16 2.3.1 Vegetative morphology ...... 16 2.3.2 Reproductive morphology ...... 16 2.3.3 The synopsis of medicinally important southern African Scabiosa species ...... 18 CHAPTER 3 ETHNOBOTANY ...... 34 3.1 Introduction ...... 34 3.2 Materials and methods ...... 34

3.3 Results and discussion ...... Error! Bookmark not defined. 3.4 Chapter summary ...... 43 CHAPTER 4 BIOLOGICAL ACTIVITY...... 44 4.1 Introduction ...... 44 4.1.1 Antimicrobial activity ...... 44 4.1.2 Anti-inflammatory activity ...... 45 4.1.3 Antacid activity...... 45 4.1.4 Toxicity ...... 46 4.2 Material and methods ...... 46 4.2.1 Collection of plant material ...... 46 4.2.2 Biological activity ...... 47 4.2.2.1 Antibacterial activity of Scabiosa sp ...... 47 4.2.2.2 Anti-inflammatory activity of S. columbaria and plants used in combination ....50 4.2.2.3 Antacid and acid neutralising activity of S. columbaria and plants used in combination ...... 52 4.2.2.4 Toxicity of the selected medicinal plants...... 54 4.3 Results and discussion ...... 57 4.3.1 Antibacterial activity ...... 57 4.3.2 Anti-inflammatory activity ...... 62 4.3.3 Antacid activity...... 66 4.3.4 Toxicity: Brine shrimp assay ...... 70 4.4 Chapter summary ...... 73 CHAPTER 5 CONCLUSION ...... 75 5.1 Summary ...... 75 5.1.1 The synopsis ...... 75 5.1.2 Ethnobotany ...... 75 5.1.3 Biological activity ...... 76 5.2 Limitations ...... 76 5.3 Recommendations for future work ...... 76 5.4 General conclusion ...... 77 CHAPTER 6 REFERENCE LIST...... 78

vii

LIST OF FIGURES

Figure 1.1: Scabiosa sp showing the young plants and a blossomed pincushion flower from matured plants…………………………………………………………………………………………………... 3 Figure 2.1: Line drawing of Scabiosa flower ...... 17 Figure 2.2: Line drawing of Scabiosa albanensis plant structure with leaves occurring densely throughout the stem...... 19 Figure 2.3: Map showing the known geographic distribution pattern of Scabiosa albanensis. ...21 Figure 2.4: Line drawing of Scabiosa columbaria plant structure showing heterophyllous leaves forming a rosette at the base...... 22 Figure 2.5: Map showing the known geographic distribution of Scabiosa columbaria...... 27 Figure 2.6: Line drawing of Scabiosa incisa plants structure with bi–pinnate and lyrate leaves occurring basally...... 28 Figure 2.7: Map showing the known geographic distribution of Scabiosa incisa ...... 30 Figure 2.8: Line drawing of Scabiosa transvaalensis showing sessile leaves distributed evenly on stem...... 31 Figure 2.9: Map showing the known geographic distribution pattern of Scabiosa transvaalensis...... 33 Figure 4.1: The two possible colour changes expected after reaching an end point using NaOH for back titration ...... 53 Figure 4.2: Adult brine shrimp when viewed at 40X magnification under a light microscope ...... 56 Figure 4.3: Effect on the pH of extracts at temperatures ranging from 10°C to 37°C...... 66 Figure 4.4: Dosage response of the toxic plant extracts...... 73

viii

LIST OF TABLES

Table 3.1: The ethnomedicinal uses of Scabiosa species & plants used in conjunction with Scabiosa columbaria...... 35 Table 3.2: Literature review of the plants used in synergism with Scabiosa columbaria, their common name, medicinal uses and plant part used ...... 38 Table 4.1: Minimum inhibitory concentration (mg/ml) of the organic (DCM: Methanol) extracts of selected plant...... 57 Table 4.2: MIC and (ΣFIC) values in brackets, with interpretation of extracts tested in combination with Scabiosa columbaria extracts. The ΣFIC could not be calculated for plants that did not have a definite MIC value (for example when MIC value is ≥ 8.00)...... 60 Table 4.3: Anti-inflammatory activity of S. columbaria and plants used in combination with the

species. IC50 was reported with enzyme inhibitory effects determined in the concentration of 12.5, 25, 50, and 100 μg/ml. The data is expressed as the mean of three replicates...... 62 Table 4.4: Antacid and acid neutralization activity of S. columbaria as well as plants used in combination with the species using back titration. The data was presented as mean± standard deviation of triplicates...... 67 Table 4.5: Average mortality rate of the aqueous and organic extracts of all the plants selected...... 70

LIST OF EQUATIONS

Equation 4.1 ( )× 47 ( ) …………………………………………………………………… 𝑋𝑋 𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤ℎ𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 1000 32 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 Equation 4.2 = + ……………………………………………………………………… 48

Ʃ 𝐹𝐹𝐹𝐹𝐹𝐹 𝐹𝐹𝐹𝐹𝐹𝐹1 𝐹𝐹𝐹𝐹𝐹𝐹2

Equation 4.3 % Inhibition = × 100 ……………………………………………………. 51 𝐼𝐼𝐼𝐼−𝑖𝑖𝑖𝑖ℎ𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖 � 𝐼𝐼𝐼𝐼 � Equation 4.4 = ( ) ( ) = ( × 𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑜𝑜𝑜𝑜 𝐻𝐻𝐻𝐻𝐻𝐻 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 − 52 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑜𝑜𝑜𝑜 𝑁𝑁 )𝑁𝑁𝑁𝑁(𝑁𝑁 𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟 𝑓𝑓𝑓𝑓𝑓𝑓 𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏×𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 )𝑀𝑀𝑀𝑀𝑀𝑀 ………………………………….𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀 𝑜𝑜𝑜𝑜 𝐻𝐻𝐻𝐻𝐻𝐻 𝑉𝑉 𝑉𝑉𝑀𝑀𝑛𝑛𝑚𝑚𝑀𝑀𝐻𝐻𝐻𝐻𝐻𝐻 − 𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑛𝑛𝑎𝑎𝑛𝑛𝑀𝑀 𝑜𝑜𝑜𝑜 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 Equation 4.5 0.063096 ( . / ) × ( ) …………………………………. 53

𝑚𝑚 𝑚𝑚𝑜𝑜𝑙𝑙 𝑚𝑚𝑚𝑚 𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑚𝑚𝑚𝑚

Equation 4.6 ( ) × 53 ( ) ………………………………………………………………….. 𝑋𝑋 𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤ℎ𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 1000 2 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 ( ) ( ) × Equation 4.7 % = 55 ( ) ……… 𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷 𝑠𝑠ℎ𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟 𝑎𝑎𝑎𝑎 48 ℎ𝑟𝑟𝑟𝑟 𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 −𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑 𝑠𝑠ℎ𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 0 100 𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀 𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷 𝑠𝑠ℎ𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎

LIST OF ABBREVIATIONS & ACRONYM

APG III Angiosperm Phylogeny Group III

Bc Bacillus cereus

BHI Brain heart infusion

BSLA Brine shrimp lethality assay

CO2 Carbon dioxide

Combination 1 S. columbaria + A. hispida

Combination 2 S. columbaria + H. caespititium + Z. albomaculata + D. anomala

Combination 3 S. columbaria + C. paniculata + S. divaricata

Combination 4 S. columbaria + E. ornithogaloides + G. perpensa

Combination 5 S. columbaria + C. paniculata + D. anomala + S. divaricata

COX Cyclooxygenase

CPdA C. paniculata aqueous dilution

CPdO C. paniculata organic dilution

DAdO D. anomala organic dilution

DCM Dichloromethane

DMSO Dimethyl sulfoxide

Ec Escherichia coli

Ef Enterococcus faecalis

FIC Fractional Inhibitory Concentration

H+ Hydrogen ion

H2 Histamine 2

HCl Hydrochloric acid

IA Initial activity

iNOS Inducible Nitric Oxide synthase

INT p-iodonitrotetrazolium violet

JRAU University of Johannesburg herbarium

KHP Potassium acid phthalate

KOH Potassium hydroxide

LOX Lipoxygenase

MIC Minimum Inhibitory Concentration

NaCl Sodium Chloride

NaOH Sodium Hydroxide

NDGA Nordihydroguaiaretic acid

Ng Neisseria gonorrhoeae

NSAID Non-Steroidal Anti-inflammatory Drugs

NU Herbarium Natal University

Pa Pseudomonas aeruginosa

PRE National Herbarium Pretoria

Sa Staphylococcus aureus

Se Staphylococcus epidermidis

Sp Species

TBS Tryptone Soya broth

Var Variety

LIST OF SYMBOLS & UNITS

% Percentage > Greater than ≥ Greater than or equal to < Less than ≤ Less than or equal to °C Degree Celsius µl Microliter Hrs Hours m Meter mmol Millimoles mg Milligram mg/kg p.o. Milligram of medication per kilogram of the body weight mg/ml Milligrams per millilitre min Minutes ml Millilitre mM Millimolar nm Nanometre V Volts v Volume μg/ml Micrograms per millilitres

xiii

CHAPTER 1

LITERATURE REVIEW

1.1 Brief taxonomic overview 1.1.1 Family Caprifoliaceae The genus Scabiosa was previously placed in the family Dipsacaceae Juss., however, Dipsacaceae, also known as the teasel family, is now no longer recognised as a separate family (APG III, 2009). It traditionally comprises about 290–320 species of sub-shrubs or perennial to annual herbs occurring throughout the Mediterranean basin, bordering western Eurasia, Africa, and Asia. Scabiosa is now placed in the honeysuckle family Caprifoliaceae as a subfamily Dipsacoideae. According to Goldblatt and Manning (2019), the family Caprifoliaceae as currently circumscribed comprises of 39 genera and 900 species of shrubs, trees, herbs or woody climbers. They occur mainly in temperate areas in the Northern Hemisphere and in mountainous tropical regions of the Southern Hemisphere. This family is characterised by petiolate leaves which sometimes have a sheathing base, and are simple or compound, with or without stipules, opposite or whorled, and with toothed or entire margins (Simpson, 2011). Inflorescences are simple to compound 1–3 flowered cymes or heads of actinomorphic or zygomorphic flowers subtended by a pair of bracts or bracteoles. Sepals may be fused or absent, while the petals are fused and often bilabiate with either two upper lobes and three lower lobes or one upper lobe and four lower lobes. Four to five stamens may occur in a single flower with the filaments attached to petals. The ovary is inferior to rarely semi-inferior, with 2–8 fused carpels and 2–8 locules. The fruit may be dry or fleshy, i.e. achenes and cypsela. All these characteristics are the reason for the transfer of Dipsacaceae to the Caprifoliaceae (Byng, 2014). Caprifoliaceae s.l. is divided into seven clades, namely: Caprifolioideae, Diervilloideae, Dipsacoideae, Linnaeoideae, Morinoideae, Valerianoideae and Zabelia. The subfamily level remains controversial and unresolved (Wang, 2020).

1.1.2 Subfamily Dipsacoideae According to Naghiloo and Claßen-Bockhoff -Bockhoff (2017), there are 14 genera in the subfamily Dipsacoideae. It is divided into three tribes: (1) Knautieae (Knautia L.), (2) Dipsaceae (Dipsacus L. and Cephalaria Schrad. Ex Roem. and Schult), and (3) Scabioseae (Pycnocomon Hoffm, Succisa Hall, Succisella G. Beck., Scabiosa L., Tremastelma Raf. and Pterocephalus Adans.) (William, 1984). Dipsacoideae is unique in its capitate inflorescences, cup-shaped, or divided calyx of usually four to numerous bristles (Carlson et al., 2009). In some species, the corona may be reduced or absent, and a pappus-like structure is formed by the increasing number of calyx bristles. Species of Dipsacoideae have opposite leaves, sympetalous corollas, and inferior ovaries. Dipsacoideae fruits have a single seed with calyx segments shaped with bristles and tightly enclosed by four fused bracts that form an epicalyx and acentric dispersal structures which together with the adjacent calyx form the diaspore (Mayer and Ehrendorfer, 1997). The epicalyx arises close below the calyx and develops into a tubular organ that surrounds the ovary. It functions in ovary protection, germination, and seed dispersal (Carlson et al., 2009).

1.1.3 The genus Scabiosa Scabiosa, so called because of its traditional use for the treatment of scabies, is now firmly placed in the family Caprifoliaceae (Quattrocchi, 2012; Manning, 2014). It is often confused with the closely related Cephalaria due to its pincushion-shaped flowers. Cephalaria species differ from Scabiosa species in being taller and more robust, having stems that are more erect, ridges that are more prominent, and broader leaves (Armitage, 2008). Scabiosa is a relatively large genus comprising about 80 species distributed mainly in the Mediterranean, with about 43 occurring in Europe and the rest in Africa and Asia. In southern Africa, the genus is represented by nine species (Germishuizen and Meyer, 2003).

Figure 1.1: Scabiosa sp showing the young plants and a blossomed pincushion flower from a matured plant.

1.2 Ethnobotanical uses of subfamily Dipsacoideae People have relied on the use of natural products to maintain their health as well as their livestock for ages. Medicinal plants still play a major role in the lives of some people in South Africa. According to Zhang et al. (2013), the World Health Organisation reported that about 60% of the world population and 80% of the population in developing countries use traditional medicine that mainly consists of extracts from medicinal plants. This is because these plants are affordable and easily accessible. Traditional medication may be prescribed by traditional healers or used through self-medication (Moteetee and Van Wyk, 2011; Seleteng Kose et al., 2015). It has been estimated that about 3 000 species are used in southern Africa for medicinal purposes and fourteen of these species have been commercialized (Van Wyk, 2011). The information of medicinally useful plants is verbally passed from one generation to the other, therefore, it is necessary to document this information for its preservation and conservation before it is completely eroded (Tshikalange, 2016). In this regard, much progress has been made in southern Africa in the past few years, in an attempt to record most of this information. For example, in South Africa alone, numerous studies have been undertaken to document ethnomedicinal uses of plants by different ethnic groups in different provinces. For example, Bapedi (Semenya et al., 2012; Mogale et al., 2019), VhaVenda (Masevhe et al., 2015; Magwede et al., 2019), amaXhosa (Afolayan et al., 2014; Bhat, 2014) amaZulu (De Wet et al., 2010; Corrigan et al., 2011; Mhlongo and Van Wyk 2019) and many others.

Scabiosa species are used in the food industry, cosmetics and mostly for medicinal purposes. Ethnomedicinally, they are used for several ailments including reproductive conditions (dysmenorrhoea), respiratory ailments (tuberculosis), and digestive problems (colic) (Watt and Breyer-Brandwijk, 1962; Besbes Hlila et al., 2013). Examples of members of the subfamily Dipsacoideae used medicinally include Cephalaria gigantea (Ledeb.) Bobrov. used for its anti-inflammatory activities and as a sedative (Mbhele et al., 2015), Dipsacus asperoides L. known to be used by the Chinese for preventing uterine bleeding, tendon and bone strengthening and maintain kidney and liver health (Dai et al., 2015). The Icelandic Succisa pratensis Moench. whole plant is used for the treatment of coughs, fever, internal inflammation, sore throat, and for external use on itchy skin, rashes, and bruises (Robertsdottir, 2016). The species is also used as a diuretic and for the treatment of ulcers, bronchitis, influenza, and asthma, as well as externally for ringworms and scabies (Besbes et al., 2012). Dipsacus fullonum L. (Teasel) has been used by the Chinese for the treatment of joint and tendon injuries, muscle pains, inflammation, and chronic arthritis. Recently, western herbalists started using it for Lyme disease and fibromyalgia (Bruton-Seal and Seal, 2009). Dipsacoideae species are used because they are known to contain phytochemicals such as alkaloids, flavonoids, iridoid and lignan glycosides, as well as triterpenoids which are generally considered to have medicinal properties (Kayce and Kirmizigul, 2010). Some species are ornamental plants because of their large flowers i.e. Cephalaria gigantea (Ledeb.) Bobrov., Knautia macedonica Gris., Lomelosia caucasica (M. Bieb.) Greuter. & Burdet. and Lomelosia graminifolia (L.) Greuter. and Burdet. (Kadereit and Bittrich, 2016).

1.3 Antimicrobial activity of the subfamily Dipsacoideae Researchers have gained interest in studies that focus on the antimicrobial activity of natural products due to the rise in their use in therapeutics. There are many factors that determine the antimicrobial activity of a plant. These include geographical location, the plant part being tested, the type of extraction, and the microorganism and protocol used to do the test (Lawal et al., 2014). Plants are known to contain secondary metabolites (phytochemicals) such as coumarins, tannins, terpenoids and quinones. The phytochemicals function in the plant’s self-defence against predation from microorganisms, insects and herbivores, and have other functions within plants. In

4 addition, these phytochemicals have been proven to have antimicrobial properties (Wintola and Afolayan, 2015). The plant extracts containing isolated compounds are responsible for the antimicrobial activity of the plants. Knowing the antimicrobial activity of plants can help in the discovery of drugs.

Different in vitro screening methods (bioassays) can be used to test the antimicrobial activity of plants. These methods fall into three categories, namely bioautographic, diffusion (qualitative assay) and dilution (quantitative assay) (Valgas et al., 2007). Examples of these bioassays are disk diffusion, well diffusion, broth or agar dilution, flow cytofluorometric and bioluminescence. The susceptibility of microorganisms tested can be measured on a quantitative criterion such as the minimum inhibitory concentration (MIC), where the MIC value is the minimum concentration of the plant extract that would inhibit visible growth of the organism. The antimicrobial activity is categorised into sensitive, intermediate and resistant (Brennan-Krohn, 2017) when examining conventional antimicrobials. The results of the microbial growth can be read visually by colour changes using dye reagents such as tetrazolium salts or by spectrophotometer using absorbance reading (Kumar & Egbuna, 2019). The broth or agar dilution method is more suitable in testing the MIC because of the ability to estimate the concentration of the tested antimicrobial agent in the medium (Balouiri et al., 2015). In the current study, the broth micro-dilution technique was used to determine the MIC values. The technique does have limitations such as a longer preparation time and contamination is not easily recognisable when compared to the agar dilution method. The advantages of the broth micro-dilution method are its accuracy to within ±1 dilution and no special equipment or reagents are needed to conduct the experiments (Kumar and Egbuna, 2019).

Mbhele et al. (2015), investigated the antimicrobial activity of Cephalaria gigantean (subfamily Dipsacoideae) using the MIC assay. The pathogens tested were four Gram- positive and eight Gram-negative bacterial strains as well as four fungal strains. The acetone, methanol and ethanol leaf extracts of the plant showed low antimicrobial activity (with MIC values ranging between 1.60 mg/mL and 3.10 mg/mL) when compared to distilled water (3.10 mg/mL–6.20 mg/mL) and hydro-ethanol (3.10 mg/mL–12.50 mg/mL). The roots exhibited even lower activity (MIC 6.20 mg/mL–12.50 mg/mL), with only the

ethanol extracts showing antifungal activity with an MIC value of 3.10 mg/mL for all the fungi tested.

1.4 Anti-inflammatory properties of the subfamily Dipsacoideae Plants that have anti-inflammatory properties have been of interest in the search for new effective agents with little or no side effects since commercial drugs have been proven to be toxic when used over long periods (Oguntibeju, 2018). The denaturation of proteins is the main cause of inflammation, and this can be brought about by factors such as the application of external stress, light, pressure, heat and chemical agents such as organic solvents, acids, alkalies, as well as concentrated inorganic salts (Reshma et al., 2014; Sangeetha and Vidhya, 2016). Inflammation can also be caused by various factors such as microbial infections, surgery, high blood pressure, oestrogen therapy, tobacco usage, excessive sugar diet and over processed food (Appleton and Jacobs, 2005). Inflammation occurs when the body releases tissue hormones called histamines and kinins in response to tissue injury (Calixto et al., 2004). Other chemicals that are released in the body which are responsible for inflammation are cytokines, eosinophils, prostaglandins, leukotrienes, free radicals, serotonin and insulin (Martini et al., 2015).

There are many methods used to test the anti-inflammatory activity of plant extracts for example, the in vivo assay where oedema is induced on different body parts. This can be achieved by using different reagents such as arachidonic acid (Romay et al., 1998) and phorbol myristate acetate (Griswold, 1998) on mice ears or carrageenan, formalin, histamine and serotonin on mice paws (Dimo et al., 2006). There are also in vitro assays to test anti-inflammatory activity for example the inhibition of enzymes such as cyclooxygenase, lipoxygenase, proteinase, inducible nitric oxide synthase (iNOS) and membrane stabilization (Gunathilake et al., 2018).

Lipoxygenase (LOX) is an enzyme that has four isoforms, 5-, 8-, 12- and 15-lipoxygenase. The 5- and 15-lipoxygenase are commonly found in human beings where they cause undesirable physiological conditions. They are also important for inflammatory response (Abdelall et al., 2016; Singh and Rao, 2019). Cyclooxygenase (COX) is an enzyme known for catalysing the formation of eicosanoids, the lipid mediators of inflammation (Cherian, 2013). There are two well-known isoforms of cyclooxygenase enzymes (COXs). The

constitutive COX-1 produces prostaglandins that are important for maintaining homeostatic functioning of the human body such as, promoting blood clotting, maintain renal functioning and protect stomach lining (Limongelli, 2010; Atta-ur-Rahman, 2015). The inducible isoform COX-2 produces undesirable prostaglandins that mediate pain and maintain inflammatory processes (Simon, 1999; Limongelli, 2010). The third isoform, COX-3, is still under investigation. Most anti-inflammatory drugs (aspirin, ibuprofen, indomethacin and diclofenac) work by unselectively inhibiting both COX-1 and COX-2 enzymes resulting in undesirable side effects in the gastrointestinal system (Vane and Botting, 2003; Argoff et al., 2009). Some anti-inflammatory drugs (rofecoxib and valdecoxib, currently withdrawn from the market) have been developed that selectively inhibit COX-2, but these drugs also have undesirable effects causing disorders associated with myocardial infarctions (Abdelall et al., 2016). According to Weissmann et al. (1987), a good non-steroidal anti-inflammatory drug will inhibit the synthesis and release of prostaglandins, inhibit activation of neutrophils as well as calcium movement. Current studies are focused on finding a drug that will be able to have a dual inhibition against COX-2 and LOX activity (Abdelall et al., 2016). Of all the numerous secondary metabolites produced by plants, flavonoids are known to have anti-inflammatory properties through the inhibition of iNOS, COX-2 and the 5-LOX metabolic pathway of arachidonic acid (González-Gallego et al., 2007; Soheila et al., 2019). The inhibition of lipoxygenase is a sufficient model for testing the anti-inflammatory properties of plants because the lipoxygenase pathway in plants is equivalent to the arachidonic acid cascades in animals which is the central regulator of inflammatory response (Leelaprakash and Mohan Dass, 2011).

Numerous ways have been documented to treat inflammation. These include eating nutritionally rich foods, commercial drugs i.e. corticosteroids such as, cortisone, hydrocortisone, prednisone, triamcinolone and methylprednisolone (Romich, 2012) and nonsteroidal anti-inflammatory drugs (NSAID’s) for example, naproxen, ibuprofen, diclofenac, ketorolac and celebrex. However, some of these NSAID’s have side effects such as dizziness, nausea and diarrhoea. Furthermore, long term use of these drugs may result in gastric erosion and bleeding, ulceration and hepatic failure (Sinatra et al., 2010). Corticosteroids have also been reported to have side effects such as an increase in the

individual’s appetite resulting in weight gain, high blood pressure, diabetes and muscle weakness (Parvizi, 2010). A different approach to treat inflammation is by the utilization of herbal plants. Some plants known to have high anti-inflammatory activities are Aloe ferox Mill. (Mwale and Masika, 2010), Malva parviflora L., Pachycarpus rigidus E. Mey., Solanum nigrum L. (Shale et al., 1999), Peltophorum africanum Sond. and Zanthoxylum capense (Thunb.) Harv. (Adebayo et al., 2015).

Some Caprifoliaceae species have been reported to be used for the relief of ailments that result from inflammation for example, the flower buds of Lonicera japonica are used by the Chinese in Japan, Korea and Taiwan for the treatment of osteoarthritis/rheumatoid arthritis, enteritis and skin inflammations (Lim, 2013). Sambucus javanica Reinw. ex. Blume is used in Indonesia to treat rheumatism (Wiart, 2007). Shen et al., (2017), investigated the anti-inflammatory properties of the total glycosides of Pterocephalus hookeri (C.B.Clarke) Hock. (Caprifoliaceae). This was achieved through oral administration of the plant extracts and the control drug (25 mg/kg indomethacin, an NSAID) on male Sprague–Dawley rats as well as female and male Kunming mice induced for an oedema using xylene, carrageenan-induced paw oedema, agar-induced granuloma formation and adjuvant-induced arthritis. The results showed that the xylene- induced oedema treated with total glycosides had insignificant inhibition compared to indomethacin. On the other hand, the carrageenan-induced paw oedema as well as the agar-induced granuloma both treated with total glycoside had inhibition comparable to that of indomethacin. It was concluded that the total glycosides from P. hookeri has anti- inflammatory, anti-arthritic, and analgesic properties.

1.5 Medicinal plants as antacids for heartburn Heartburn occurs when the stomach acid (hydrochloric acid) is not enough to digest food, causing the partially digested food to start fermenting in the stomach, resulting in harmful gas being produced that push stomach acid into the oesophagus (Hargreaves, 2007). When stomach acid touches the delicate oesophagus lining, the burning sensation/pain is felt (Raymond and Beaver, 2014). Unlike the stomach, the oesophagus does not have protection against hydrochloric acid that is why it becomes irritated. Heartburn gets its name from the fact that the oesophagus passes right next to the heart and so the burning

sensation feels like it is occurring on the heart (Bassett, 2007; Burns & Shah, 2007). Causes of heartburn include certain foods, overeating, not chewing enough, eating just before bedtime, posture and stress (Hargreaves, 2007). Food and drinks that trigger the secretion of gastric juices in the stomach include dairy products, caffeine (coffee/chocolate), oily food, food containing gluten and citric juices (Usman and Davidson, 2013). Ingestion of the drugs such as aspirin, nonsteroidal anti-inflammatory drugs, bisphosphonate, metformin, antibiotics, digitalis, potassium and iron supplements, and theophylline derivatives also causes heartburn. People who are pregnant are more susceptible to experiencing heartburn after a meal or even in their sleep because of delayed gastric emptying, increased intra-abdominal pressure and an increased oestrogen level facilitating oesophageal reflux (Halpern, 2004; Seller and Symons, 2011).

Symptoms of heartburn include sore throat, dry cough, difficulty in swallowing, sour taste in the mouth, vomiting, and nausea. Usually one would take antacid medication such as Gaviscon®, Mi-acid® and Mintox®. Antacids are remedies that act by neutralising the stomach acid [hydrochloric acid, (HCl)] through the reduction of hydrogen ion (H+) concentration and inhibiting the activity of pepsin (Singh and Terrell, 2019). Pepsin is a potent acidic enzyme which breaks down proteins in the stomach. The activity of pepsin is optimum at pH 2 and low at pH above 5. Antacids that contain aluminium hydroxide and magnesium hydroxide increase gastric pH to be greater than four, leading to the inhibition of pepsin activity together with the HCl (Bayless and Diehl, 2005). Antacids are composed of various salts of aluminium, calcium and magnesium that function as the active ingredients. These ingredients work through different mechanisms with the common goal of neutralizing the gastric acid (Washington, 1991). Supplements may also be taken to reduce heartburn. These include L-glutamine, ascorbic acid, and vitamin E (Best-Boss and Edelberg, 2009). Although these antacids relieve the burning sensation, if used for long periods of time, they may result in side effects such as, constipation, diarrhoea, increased risk of intestinal infections, and tumours of the stomach (Halpern, 2004). As an alternative from antacids, people have resorted to medicinal plants as they are believed to have fewer side effects.

The in vitro methods that can be used to assess the antacid and neutralising properties of plant extracts are: assessing the inhibition of H+ K+ ATP’ase (enzyme which secrets acid in stomach), modified model of Vatier’s artificial stomach, back titration of Fordtran’s model method and the neutralising effects on artificial gastric acid (Thabrew and Arawwawala, 2016). In the back titration method, a known excess of acid (HCl) is reacted with an antacid then the solution is back titrated to assess the amount of acid consumed by titrating the unreacted acid with a standardized base (NaOH) (Washington, 1991). Gastric juices in the human stomach are made up of many components including water, HCl, digestive enzymes, electrolytes and intrinsic factors (Dev, 2002). For back titration of Fordtran’s model, artificial gastric juice is made up of salt, pepsin enzyme, HCl and water (Wu, 2010). To be considered a good antacid, the remedy should be in liquid form, constantly neutralising the artificial gastric juice to a pH of 3 for a period of three hrs (Rutter, 2020). The in vitro methods are valuable in preliminary screening for testing plant material for the presence of antacid properties that need to be present to remedy heartburn (Thabrew and Arawwawala, 2016).

Different plants have been reported to be used traditionally to relieve heartburn. Such plants include Elephantorrhiza elephantina (Burch.) Skeels., Pentanisia prunelloides (Klotzsch ex Eckl. & Zeyh.) Walp. (Mpofu et al., 2014), Gazania krebsiana Less., Trifolium burchellianum Ser. (Quattrocchi, 2016) and Xysmalobium undulatum (L.) W.T. Aiton f., (Seleteng Kose et al., 2015). Some of the medicinal plants used for this purpose have been scientifically proven to have antacid and/or acid neutralising properties. For example, Sandhya et al. (2015), evaluated the in vitro antacid activity of methanol extracts of Tephrosia calophylla Bedd. and Tephrosia maxima (L.) Pers. roots using different methods. The standards (synthetic drugs) used were sodium bicarbonate (A1) and aluminium hydroxide mixed with magnesium hydroxide (A2). The results of the neutralization effects on artificial gastric acid showed that T. maxima and A1 had the best neutralization effect, while the neutralization effects of A2 were statistically non- significant. When testing the duration of consistent neutralization effect on artificial gastric acid using the modified Vatier's artificial stomach model, T. maxima was the most potent with 254.33 min. Aluminium hydroxide mixed with magnesium hydroxide had the second- best activity with neutralization of 206.33 min. For the in vitro titration method of Fordtran’s

10 model to determine the neutralization capacity, T. maxima had the most significant gastric acid neutralizing effect of 94.17 mL, consuming 6.0124 hydrogen ions while the standards had less neutralization effects. It was concluded that both T. maxima and T. calophylla can be considered as efficient substitutes for synthetic antacids.

1.6 Synergistic effects of medicinal plants The synergistic effects of ethnomedicinally important plants has been proven to be successful due to their activity on multiple targets, reduction of side effects, reduced bacterial resistance and the improvement of bioavailability (Yuan et al., 2017). According to Kent (1998), synergy, additivity and antagonistic effects may occur as a result of a combination of two or more agents. Synergism would be when the combination of agents results in an effect much greater than that of individual agents. Additive effects occur when the combination of agents result in a slightly improved effect to that of individual agents, while antagonism is the undesirable outcome where the combination of the agents result in less effects compared to the effects of individual agents. Non-interactive effects demonstrate no improved efficacy upon combination (Roell et al., 2007). The degree of synergism is calculated using sum of fraction inhibitory inhibition (ΣFIC). According to van Vuuren and Viljoen (2011), FIC is expressed as “the interaction of two agents where the concentration of each test agent in combination is expressed as a fraction of the concentration that would produce the same effect when used independently”.

In Van Vuuren et al. (2015), the antimicrobial activity of 23 plants used for treatment of diarrhoea in northern Maputaland, KwaZulu-Natal were studied. The study also focused on the synergistic properties of four plant combinations against seven diarrhoea-related pathogens using the minimum inhibitory concentration assay. The herbal combinations of organic extracts of Acanthospermum glabratum (DC.) Wild. and Krauseola mosambicina (Moss) Pax & K. Hoffm., A. glabratum and Psidium guajava L., as well as Brachylaena transvaalensis Hutch. ex E. Phillips & Schweick. with Sclerocarya birrea (A. Rich.) Hochst. were evaluated using ΣFIC. These combinations exhibited synergistic interactions against four of the seven pathogens studied with average sum of fraction inhibitory inhibition (ΣFIC) value of 0.30, 0.46 and 0.88 respectively against all pathogens.

The combination of B. transvaalensis and P. guajava had synergistic interaction against five pathogens with average ΣFIC value of 0.39. Of all the combinations, A. glabratum with K. mosambicina exhibited the most noteworthy interaction against Staphylococcus aureus where the mean MIC value for individual organic plant extract were 0.04 mg/mL and the mean ΣFIC value was 0.01. While most of the organic extracts plant combinations displayed favourable results of synergism, all the aqueous extracts combinations had a non-interactive effect (Van Vuuren et al., 2015)

1.7 Toxicity of medicinal plants Plant toxicity studies focus on determining the toxicity of biological activity of extracts or active compounds isolated from plants (McGaw, 2014). Herbs have been utilised around the world by different cultures due to the wide variety of secondary metabolites they constitute with potential therapeutic properties. Regardless of this worldwide usage, there are still concerns with respect to the usage of herbal products and the potential threat that they may have on human health. The mode of action of secondary metabolites from most of these herbs in the human body, lack scientific evidence. Metabolites that are produced as a form of defence from adverse conditions for example, against insects, may also end up toxic to human beings (Subramanian et al., 2018).

Secondary metabolites known to be of concern to human health are: capsaicin (active compound in peri-peri, used commercially in pain relief creams and supplements) (Abdel- Salam, 2014), phytoestrogens (mainly found in soy and supplements) (Patisaul and Jefferson, 2010) and safrole (low concentrations found in camphor, black pepper, cinnamon) (Maga and Tu, 1994). The toxicity of a plant depends on the dosage, the plant part used, the growth stage of the plant and susceptibility of the consumer (Tamokou and Kuete, 2014). Toxicity of plants may also be caused by contaminants (toxic minerals or heavy metals). Children with underdeveloped immune and digestive systems and older people with an aged immune system are most susceptible to plant toxicity (Ghorani-Azam et al., 2018). It has become necessary to do tests to evaluate the toxicity levels of these plants to ensure safety when consumed, more specifically in crude form for medicinal practices. There are many different types of tests used to assess toxicity levels of medicinal plants. These include acute toxicity, allergenicity, biokinetics, carcinogenicity,

genotoxicity, mutagenicity, repeat-dose toxicity and reproductive/developmental toxicity (Worth, 2019). The brine shrimp lethality assay (BSLA) is an acute systemic toxicity assay that uses larvae (nauplii) of Artemia spp (sea monkeys) to measure its relative toxicity response to a brief exposure to the test substance (Libralato et al., 2016). It can be used to study the toxicity of any substance that can be dissolved or dispersed into water. These include plant extracts, agricultural chemicals, food additives and pharmaceuticals (Lieberman, 1999). Acute systemic toxicity exposure routes (ways in which people can encounter the toxic substance) are dermal (skin), oral and through inhalation (Seidle et al., 2011). Artemia salina is the most widely used Artemia species. This brine shrimp is used in 90% of these types of toxicity assays; for example, to test plant extracts, cyanobacteria, heavy metals and metal ions toxicity (Hamidi et al., 2014). The BSLA requires killing the shrimp and calculating percentage mortality. An advantage of using BSLA is that it is a low-cost assay that can produce relevant results after 24 hrs with no special skills needed for equipment setup and reading assay results. BSLA has its limitation in that the results do not provide information about the mechanism of toxicity action. Nevertheless, BSLA was chosen as the method of determining the toxicity of the plant extracts in the present study.

1.8 Aims and objectives of the study The aim of this study is to provide a synopsis of the medicinally useful species of the genus Scabiosa, to compile a review of their ethnomedicinal uses and assess their biological activities to validate their traditional use.

The specific objectives are to:

(i) Record the morphological characteristics of four medicinally important species of Scabiosa and plot their distribution ranges in southern Africa,

(ii) Screen Scabiosa columbaria against specific pathogens based on its medicinal traditional uses,

(iii) Evaluate Scabiosa columbaria for its anti-inflammatory activity, antacid activity and neutralization capacity as well as its toxicity,

(iv) Assess the synergistic properties of Scabiosa columbaria antibacterial, anti- inflammatory, antacid activity and toxicity when used in combination with other plants.

CHAPTER 2

SYNOPSIS OF MEDICINALLY IMPORTANT SPECIES OF SCABIOSA

2.1 Introduction The genus Scabiosa was erected by Linnaeus (1753), to accommodate the 18 species known to him at the time. The species originated from different continents across the globe; such as, Africa (South Africa), Asia (India, Syria), Europe (France, Germany, Greece, Portugal, Romania, Spain, Switzerland) and North America (Cuba). Due to its relatively large size and wide distribution, the genus has not been revised in its entirety. Taxonomic revisions have rather been at regional or country level, for example Spain and Balearic Islands (Devesa, 1989) and Egypt (El bous and Gazer, 2016). In southern Africa, the genus was last revised by Harvey and Sonder (1865), however, the main focus of the current study is to provide a synopsis of the medicinally important species of the genus. According to the latest checklist (Germishuizen and Meyer, 2003) and Carlson et al. (2012), the genus is represented in the region by nine indigenous species namely, S. africana L., S. albanensis R.A. Dyer, S. angustiloba (Sond.) Hutch., S. buekiana Eckl. & Zeyh, S. columbaria L., S. drakensbergensis B.L. Burtt, S. incisa Mill., S. transvaalensis S. Moore., and S. tysonii L. Bolus. Of the nine species, four have been reported to be medicinally important. These are, S. albanensis, S. incisa, S. columbaria and S. transvaalensis.

2.2 Materials and methods Herbarium specimens were loaned from the National Herbarium (PRE) and University of KwaZulu-Natal Herbarium (NU) and these were examined to identify vegetative and reproductive morphological characteristics that can be used to distinguish between the medicinally important species of the genus. Living material was also studied during field trips. The information about geographical distributions was obtained from the herbarium specimens. The localities (longitude and latitude) were recorded according to the quarter– degree grid reference system of Leistner and Morris (1976). The maps were plotted using CorelDRAW X7. The type specimens were studied online on the JSTOR database.

2.3 Results 2.3.1 Vegetative morphology Scabiosa plants are erect perennials and annuals of herbs or sometimes shrubs. Stems are branched and have ridges that are less obvious. Each southern African Scabiosa species has a unique leaf type. The differences can be seen in the pubescence, shape, size, margins and other features. Leaves of Scabiosa may be heterophyllous, usually clumped at the base, always exstipulate, greyish green, oppositely arranged, mostly hairy. The blade is usually pinnatipartite or pinnatisect and rarely has entire margins.

2.3.2 Reproductive morphology Scabiosa species are characterised by flowers shaped like a pincushion (from which their common name is derived). The inflorescence is a capitulum occurring singularly at the tip of a thin peduncle. Individual flowers cluster together to form the flower head. When the flowers blossom, the outer row forms a radiant frilly edge of the longer petals than the inner flowers. They have a pilose epicalyx with 5 long awns and involucral bracts in one or two rows of varying lengths. The corolla is usually dimorphic, conspicuously veined, tubular, with soft hairs on outer surface and blue, purple, pink or white coloured. The tubular corolla is bilabiate with the upper lip usually shorter and bilobed while the lower lip is trilobed, obovate or obovate–oblong, and raylike. The pistil has an inferior ovary with one locule which develops into an achene fruit, elongated style and capitate stigma. The flower has four equally exserted epipetalous stamens and pappus that are bright red, hairy and project above the florets only when the flower heads are still young (Figure 2.1). The plants flower almost throughout the year.

Figure 2.1: Line drawing of Scabiosa flower with A– Seed pod which forms at the end of flowering season. B– Single flower showing anthers hidden within the fused petals with pistil protruding and vice–verse for E (dimorphism). C– Immature flower head composed of flower buds with pappus sticking out between them, flower head also ringed by hairy sepals. D– Capitulum inflorescence composed of large frilly petals on the outer edge and central disk of smaller petaled flowers/flower buds of which either stamens or pistils would be protruding. E– Single flower showing stamens exerted and inferior pistil within the tubular corolla. Scale bar A–F = 1 mm.

2.3.3 The synopsis of medicinally important southern African Scabiosa species

2.3.3.1 Scabiosa albanensis R.A. Dyer, in Bull. Misc. Inform. Kew 1934(6): 267 (1934); Type: South Africa. Eastern Cape Province, 3326 (Grahamstown): Beggars Bush near Grahamstown (-BC), (14 May 1919) Britten 1965 (GRA–image!, lectotype, here designated; K–image!, PRE–image!, isolectotypes). [Note: The specimen in K is chosen as lectotype because it is likely that Dyer was looking at it when he described this species since he was based in Kew at the time]. [Syntypes: South Africa. Eastern Cape Province, 3326 (Grahamstown): Assegai Bush (-AD), Drege s.n. (specimen not seen); Featherstone Kloof (-BC), MacOwan 204 (specimen not seen). = S. columbaria L. var. dissecta Sond., in Harv. & Sond., Fl. Cap. 3: 43 (1864) pro parte. Type as above.

= S. pallida E. Mey., in Drége, Zwei Pfl. Docum.: 218 (07 August 1843). Type: South Africa. Western Cape Province, 3318 (Cape Town): Leeuwenberg [the lion mountain] (- CD), no date, Drége a (HAL-image!, lectotype here designated). [Note: Meyer listed 11 specimens of S. pallida collected by Drége and of these, this is the only one available].

= S. pallida var. α E. Mey, in Drége, Zwei Pfl. Docum.: 218 (1843). Type: South Africa, Western Cape Province, 3318 (Cape Town): Paarlberg (-DB), no date, Drége var. a (HAL- image!, lectotype here designated).

= S. pallida var. β E. Mey, in Drége, Zwei Pfl. Docum.: 218 (1843). Type: South Africa, Western Cape Province, 3326 (Grahamstown): Near Assagaybosch and Botram (-AD), no date, Drége var. b (HAL-image!, lectotype here designated).

Slender semi–erected plant, sparsely or freely branched, herbaceous on the upper part, woody stem base, 80–340 mm long. Petiolate leaves, 20–30 mm, occur densely on the stem. Lower leaves long, bipinnately lobed, upper leaves pinnately or bipinnately lobed, sparsely pubescent (Figure 2.2). Peduncle 150–200 mm long, sparse hairs along the stalk, dense hairs below the capitulum. Peduncle bears flower head, subglobose, involucral bracts in single row, linear to linear–lanceolate in a single series. Corolla pale blue to white colour, fade to mauve on dry. Creamy stigma, mauve anthers, pale blue filaments.

Phenology: From summer to late autumn, December to May.

Etymology: The specific epithet is derived from distribution pattern of the species as it occurs in the Albany District in the Eastern Cape Province.

Figure 2.2: Line drawing of S. albanensis plant with leaves occurring densely throughout the stem. Scale bar 7 mm.

Distribution and Habitat: Scabiosa albanensis has a restricted distribution, where it is found only in the Eastern Cape Province of South Africa, (Figure 2.3) growing in a grassveld at altitudes of 20– 2310 m.a.s.l.

Conservation status: Least concern (Victor, 2005)

Specimens examined

EASTERN CAPE – 3027 (Lady Grey): Road from Barkley East to Farfield farm (–DC), 07 November 1995, J.E. Victor 1490 (PRE). 3226 (Queenstown): Katberg Pass (–BC), 1524 m, 17 January 1980, P. Goldblatt 5458 (PRE); Hogsback fire above Kettlespork Falls (–DB), 09 December 1977 B.L. Burtt & O.M. Hilliard 10983 (NU). 3227 (Stutterheim): Komgha (–DB), 1891, H.G. Flanagan 602 (PRE). 3228 (East London): Morgan’s Bay (–CD), 28 January 1979, O.M. Hillard & B.L. Burtt 12439 (NU). 3325 (Port Elizabeth): Uitenhage, Van Staden’s Pass (–CC), 1928, J.C. Letty 102 (PRE). 3326 (Grahamstown): Between Kleinemonde and Fish river on Sherwood (–AC), 01 May 2000, P.B. Philipson 5237 (PRE); Kwandwe Private Game Reserve (–AD), 02 November 1977, B.L. Burtt & O.M. Hilliard 10800 (NU); 7 miles from Grahamstown on Mr. Jolly’s farm (–BC), 20 December 1930, R.A. Dyer 3304 (PRE); Potters pass (–BB), 19 September 1984, A.R. Palmer 1148 (PRE). 3327 (Peddie): Bulura River mouth (–BB), 3 April 1950, J.P.H. Acocks 15778 (PRE); Between Hamburger and Port Alfred (–CA), 19 October 1986, P.B. Phillipson 1513 (PRE).

Figure 2.3: Map showing the known geographic distribution pattern of S. albanensis.

2.3.3.2 Scabiosa columbaria L., Sp. Pl. 1:99 (1753). Type: Sweden. Gotland, LINN– 120.17, lectotype designated by Napper Fl. Trop. E. Afr., Dipsacaceae 7 (1968); = S. austroafricana Heine., in Mitt. Bot. Staatssamml. München. 1 (9–10): 445 (1954). Type: South Africa. Western Cape Province, 3318 (Cape Town): “Inter fruticeta altitude. 2. mont. tabul. et leonis. Octbr.’ [Among thickets between Table Mountain and Lion’s Head] (-CD), Ecklon 725, (M-image!, holotype). – HT M IT K.

= S. ochroleuca Thunb., Prodr. Fl. Cap. 1:29 (1794), non L.: 101 (1753), nom illegit.

= S. anthemifolia Eckl. & Zeyh., Enum. Pl. Afr. Austral.3:371 (1837). Type: South Africa. Eastern Cape Province, 3325 (Uitenhage): Van Stadensriviersberge (-CC), Date not specified, C.F. Ecklon & C.L.P. Zeyher 2337

Hairy perennial plant up to 1 m high, growing from a woody rootstock. Leaves lacy grey– green, hairy on both sides, elongated petioles, usually basal forming a rosette and markedly dimorphic. Lower leaves, 100–175 mm, oblanceolate to lyrate–pinnatified, margin varying from smooth, toothed to incised. Elongated stem containing upper small pinnatisect leaves, 20–80 mm, often deeply cut to the midrib into slender lobes, entire margins (Figure 2.4). Peduncle up to 200 mm, bearing puberulent corolla, 20–30 mm wide, white to mauve pink and blue. Involucral bracts lanceolate, acute, varying length of shorter and longer than marginal flowers. Receptacle bracts linear–lanceolate, acute, long and adaxial hairy.

Phenology: From early spring to late summer, between August and February.

Figure 2.4: Line drawing of S. columbaria plant showing heterophyllous leaves forming a rosette at the base. Scale bar 7 mm

Diagnostic characteristics: Heterophyllous leaves form a rosette at the base of stem.

Distribution and habitat: Scabiosa columbaria is distributed in Namibia, eSwatini, Lesotho, and in all South African provinces (Figure 2.5). It mostly occurs in grassland, rocky slopes and bushveld habitats at altitudes up to 2500 m.a.s.l.

Conservation status: Least concern (William et al., 2008)

Specimens examined

LIMPOPO– 2228 (Maasstroom): Blauwbergsvlei (–DB), 13 January 1955, R.A. Dyer & L.E. Codd 9162 (PRE). 2229 (Soutpansberg): Near Lake Fundudzi (–CD), 29 October 1948, R.A. Dyer & L.E. Codd 4498 (PRE). 2230 (Musina): Soutpansberg (–CC), December 1930, A.A. Obermeyer 575 (PRE); Tshaulu Mutanzhela (–DC), 10 April 1980, A.E. Van Wyk 3845 (PRE). 2329 (Polokwane): Polokwane game reserve (–CD), 08 January 1979, Brendenkamp & Van Vuuren 69 (PRE). 2329 (Makhado): 500 m from Cloud End Hotel (–BB), 06 November 1985, B.J. Pienaar 690 (PRE). 2330 (Wolkberg): Wolkberg Wilderness Area, Wolkberg farm (–CC), 23 October 1985, S. Venter 11,117, (PRE). 2427 (Thabazimbi): Kransberg, south east from Groothoek (–DA), 11 December 1979, R.H. Westfall 767 (PRE).

NORTH WEST– 2526 (Zeerust): Swartruggens (–DA), 07 November 1981, E. Van Hoepen 1747 (PRE); Marico District, Koster station (–DD), 12 November 1907, J.B. Davy 7176 (PRE). 2527 (Rustenberg): Pilanesberg (–AC), 04 December 1984, Selaledi & Sekhaolelo 60 (PRE); Magaliesberg Castle Gorge (–DA), 10 December 1978, G. Matthews 2964 (PRE); Bokfontein (–DB), December 1908, T.J. Jenkins TRV 6920 (PRE); West of Hartbeespoort dam (–DB), 26 November 1984, Ekokonsult 30–60 (PRE). 2625 (Mahikeng): Biesiesvlei, side to Sannieshof (–BD), 21 January 1987, E. Retief 1798 (PRE); Farm Boschkop (–CB), 22 January 1987, E. Retief 1832 (PRE). 2626 (Delareyville): Hendriksrust (–AA), 30 January 1968, J.W. Morris 1156 (PRE); Klerksdorp (–CD), 07 November 1974, W.J. Hanekom 2445 (PRE).

GAUTENG – 2528 (Pretoria): Faerie Glen (–CB), 27 November 1971, SAGP– SAAB/12/41 (PRE). 2627 (Krugersdoorp): Ruimsig, Equestrian Road (–BB), 03 November 1998, S.L. Turner 345 (PRE); Hertzenbergfontein (–CA), 19 April 1975, S.

Allcock sn (NU). 2628 (Johannesburg): Kelland (–BB), October 1976, L.C.C. Liebenberg 8436 (PRE); Suikerbosrand (–CA), 09 October 1971, G.J. Bredenkamp 113 (PRE).

MPUMALANGA – 2430 (Pilgrim’s Rest): West of Blyde River Canyon (–DB), 20 November 1993, P.M. Burgoyne 1981 (PRE). 2530 (Mashishing): Vertroosting Nature Reserve (–BB), 02 February 2013, J.E. Burrows & S.M. Burrows 13349 (NU). 2531 (Barberton): Songimvelo Game Reserve (–CB), 08 December 1992, M. Jordan 2468 (PRE). 2729 (Volksrust): ±15.4 km WSW of Volksrust (–BC), 02 December 2015, S.P. Bester 12965 (PRE).

FREE STATE – 2726 (Odendaalsrus): Rustfontein Makwassie (–AC), 11 December 1968, S.E. Morris 54 (PRE). 2727 (Kroonstad): Vredefort Dome (–CA), 16 November 2011, D.M. Komape, S.J. Siebert & L.L Mabe KMS 175 (PRE). 2826 (Brandfort): Brandfort (–CD), 02 March 1971, JA VD Berg 3886 (PRE). 2828 (Bethlehem): (–CB), November 1917, Dr Van Hoepen PRE 59897 (PRE). 3026 (Aliwal North): Cliftonvale farm (–CA), 27 November 1983, H.H. Burrows 2177 (PRE); Burghersdorp (–CD), November 1908, T.W. Pocock 95 (TRV 10808) (PRE).

KWAZULU–NATAL – 2632 (uMkhanyakude): eNkovukeni (DD), 30 November 2002, D.G.A Styles 1282 (NU). 2732 (Ubombo): Sodwana Bay (–DA), 19 February 1996, P.M. Burgonye & N. Snow 4621 (PRE); Natal Parks Board Camp (–DD), 27 November 1967, E.J. Moll & R.J. Strey 3920 (PRE). 2828 (Bethlehem): Royal Natal Nation Park (–DB), February 1964, W.R. Trousel 24 (NU); Royal Natal Nation Park (–DD), 24 January 2014, C. Brochmann CB52 (2014) (NU). 2831 (Nkandla): Hluhluwe Game Reserve (–BB), 21 November 1983, A.J. Phelan 754 (NU). 2832 (Mtubatuba): Dukuduku State Forest (– AD), 09 December 1989, S. Hobson 379 (PRE). 2929 (Underberg) : Upper Loteni Valley (–AD), 05 February 1985 O.M. Hillard & B.L. Burtt 18114 (NU); Between Sani Pass and Polela Valley (–BC), 03 December 1983, O.M. Hillard & B.L. Burtt 17060 (NU); Mlambonja Valley (–CA), 06 January 1982, O.M. Hillard & B.L. Burtt 14987 (NU); Bamboo Mountain(– CB), 08 November 1997, O.M. Hillard & B.L. Burtt 10076 (NU); Garden Castle State Forest (–CD), May 1983 M. Grice sn (NU). 2930 (Pietermaritzburg): Howick, Lions River district (–AC), 29 September 1964, E.J. Moll 1047 (NU); Richmond side of Umkomaas Valley (–CD), 22 December 1965, B.L. Burtt 3403 (NU); Camperdown district,

Manderston (–DA), May 1949, S.V. Runcie 23 (NU); Botha’s Hill (–DC), 08 October 1991, C.J. Potgieter 01 (NU); Pinetown, Sarnia Beacon Hill (–DD), 13 January 1971, C.J. Ward 7542 (NU). 2931 (Stanger): On the North Coast (BA), 27 October 2000, C. Potgieter 476 (NU). 3029 (Kokstad): Ntsikeni Nature Reserve (–CB), 09 February 2019, D.P. Philips 118 (NU). 3030 (Umzinto): Vernon Crookes Nature Reserve (–BC), 14 October 2006, P. Wragg 1742 (NU). 3130 (Port Edward): mouth of Mzamba River (–AA), 25 October 1975, C.H. Stirton 5620 (PRE).

NORTHERN CAPE – 3017 (Hondeklipbaai): Kamieskroon (–BB), 09 August 1972, J.V. Van Der Westhuizen 297 (PRE).

WESTERN CAPE – 3118 (Vanrhynsdorp): Outskirts of Klawer Vanrhynsdorp (–DC), 8 September 1083, D. Snijman 750 (PRE). 3219 (Wuppertal): Cedarberg Wilderness Area (–AA), 16 January 1977, R.A. Haynes 1251 (PRE). 3222 (Beaufort West): Karoo National park Mountain View Farm (–AB), 02 January 1985, D.A.M.B Shearing 825 (PRE); Karoo National park (–BC), 09 December 2005. E. Retief ER/KSAN86 (PRE). 3318 (Cape Town): Mountain slope above Clifton near Camps Bay High School (–CD), 02 November 1982, P. Goldblatt 6646 (NU). 3320 (Montagu): Warmwaterberg (–DD), 16 January 1989, P. Burger 51 (PRE); Konstabel (–AD), 15 November 1978, C. Boucher 4106 (PRE). 3321 (Ladismith): Swartberg (–AD), 03 February 1992, E.G.H. Oliver 10004a (PRE); Kruisrivier (–BD), 30 December 1969, H.C. Taylor 7561 (PRE). 3322 (Oudtshoorn): Swartberg Mountains (–AC), 28 June 1974, R.O Moffett 266 (PRE); Grootrivier (–CB), 28 November 1972, W.J. Hanekom, 1774 (PRE). 3323 (Willowmore): North of the foot Outeniquas near Joubertina (–DD), 15 January 1947, E. Esterhuysen 13593 (PRE). 3418 (Simonstown): Nordhoek Sand Dunes (–AB), 03 October 1980, O.M. Hillard & B.L. Burtt 13094 (NU). 3419 (Caledon): Strandskloof (–CB), 24 August 1946, L.M. Leighton 1919 (PRE); Riviersonderend Mountains (–CD), February 1984, E.G.H. Oliver 8456 (PRE). 3422 (Mossel bay): Hartenbos on road to Mossel bay, Robinson (– AA), 50 m, 13 January 1955, L. Hugo 105 (PRE).

EASTERN CAPE – 3027 (Lady Grey): Witteberg Joubert Pass (–BC), 18 January 1979, O.M. Hillard & B.L. Burtt 1202 (NU); Rhodes District (–DB), 12 December 1999, M.S. Mothogoane, 236 (PRE). 3028 (Matatiele): Ongeluksnek Nature Reserve (–AD), 21

February 1999, A.T.D. Abbott 7497 (PRE). 3126 (Queenstown): Small kloof on road to Dordrecht (–DD), 13 January 1997, C.L. Bredenkamp 1206 (PRE); Near Birds River (– BD), 13 January 1997, P.N. Sebothoma 52 (PRE). 3129 (Port St Johns): Lusikisiki (– BD), 20 October 1984, C. Shackleton 61 (PRE); Ntsubane (–BC), 30 August 1969, R.G. Strey 9011 (PRE); Pondoland (–BD), 21 January 1937, A.O.D. Moss 13602 (PRE). 3224 (Graaft Reinet): Hills above Valley of Desoletion (–AB), 28 December 1977, O.M. Hillard & B.L. Burtt 10731 (NU). 3225 (Somerset East): Somerset East District (–CB), 06 December 1977, O.M. Hillard & B.L. Burtt 10890 (NU). 3226 (Fort Beaufort): Kartberg pass (–BC), 10 December 1977, O.M. Hillard & B.L. Burtt 10970 (NU); Mpofu Nature Reserve (–DA), 27 February 2006, L.P. Steenkamp 243 (PRE); Amathole mountains (– DB), 12 December 1984, O.M. Hillard & B.L. Burtt 8818 (NU). 3227 ( Stutterheim): South east of King Williams Town (–CD), 15 December 1977, O.M. Hillard & B.L. Burtt 11021 (NU); On road west of Komgha (DA), 15 December 1977 O.M. Hillard & B.L. Burtt 11097 (NU); Near Bridle Rift Dam (–DC), 19 December 1980, O.M. Hillard & B.L. Burtt 13180 (NU). 3228 (Butterworth): Kentani (–CB), A. Pegler 410 (PRE); Hill above Kwaelegha River (–CC), 02 October 1981, O.M. Hillard & B.L. Burtt 14812 (NU). 3326 (Grahamstown): About 5 km from Grahamstown in a grass veld (–BC), 7 December 1935, R.A. Dyer 3300 (PRE). 3424 (Humansdorp): Humefield Hills (–AA), April–August 1930, Rhode A2521 (PRE).

NAMIBIA – 1917 (Tsumeb): Otavi district, north of Kombat on farm Gauss (–DA), 02 March 1995, G. Germishuizen 7371 (PRE). 2417 (Mariental): Hardap, Farm Auros (– AD), 11 March 1973, W. Giess 12572 (PRE).

ZIMBABWE – 1830 (Dodhill): Cleveland Dam north gate (–CB), 06 September 1984, S. Winter 24 (NU).

ESWATINI – 2631 (Mbabane): Malolotja Nature Reserve 20 km from Pigg’s peak (–AA), 03 March 1993, C.L. Bredenkamp 576 (PRE); Malolotja Nature Reserve, near fence on west facing slope of picnic site (–AA), 29 December 1988, K.P. Braun 491 (PRE); Hhohho, Malandzela Area (–AB), 25 January 1994, S.R. Hobson 2078 (PRE); Usutu Forest (–CA), December 1947, J.M. Watson 9 (PRE). 2632 (Bela Vista): Mlawula farm (–AC), 05 March 1977, J. Culverwell 0621 (PRE).

LESOTHO – 2927 (Maseru): Mountain road near Lithabaneng (–BD), March 1978, M. Schmitz 8263 (PRE). 2928 (Marakabei): Malibamatso River (–AD), K.P. Braun 2247 (PRE); Near Pelaneng River (–BA), 12 December 1995, K.P. Braun 2218 (PRE).

Figure 2.5: Map showing the known geographic distribution of S. columbaria.

2.3.3.3 Scabiosa incisa Mill. Gardeners Dictionary, Edition 8. London n.18 (1768). Type: South Africa, precise locality unknown, Anon s.n. (BM–image!, lectotype here designated). [Note: When Miller described this species, he did not mention the type locality or designate a type specimen. However, the specimen in BM is chosen as a lectotype as it originated from his herbarium and is the only one available. It is also clear that he was referring to Linnaeus’ S. africana var. γ]. = S. africana L. var. γ, Sp. Pl. 2nd ed. 1: 145 (1762). Type as above.

Herbaceous perennial herb, rosette–like with spreading form up to 50 mm wide, 800 mm high. Stem procumbent and pubescent. Compound petiolate leaves, grey–green, pubescent on both sides, mostly basal, not markedly dimorphic, 70–130 mm long, lyrate– pinnatisect to bi–pinnatisect arrangement (Figure 2.6). Leaflets oppositely, 10–15 mm long, appear to be deeply divided with entire margins, obtuse to acute tips and round base. Inflorescence of hairy flowers heads, 20–50 mm wide on flower stalks about 315 mm long. Flowers are sweet scented, white to lilac hued.

Phenology: Flowers in spring, between September and November.

Etymology: Named after the structure of leaves with great incision

Figure 2.6: Line drawing of S. incisa plants structure with bi–pinnate and lyrate leaves occurring basally. Scale bar 5 mm

Diagnostic characteristics: Scabiosa incisa is often confused with S. albanensis due to their similar leaf structure (bi–pinnately compound). Scabiosa incisa can be distinguished by its spreading growth form, as well as lyrate–pinnate leaves as opposed to S. albanensis which has more erect habit and lacks lyrate–pinnatisect leaves.

Distribution and habitat: Scabiosa incisa occurs only in the Western Cape and Eastern Cape Provinces, South Africa (Figure 2.7) at altitudes ranging from 152 –1450 m.a.s.l. Occur naturally on coastal sand and limestone region habitat.

Conservation status: Least concern (Foden and Potter, 2005)

Specimens examined

WESTERN CAPE – 3318 (Cape Town): Malmesberg (–CB), 16 September 1980, B.L. Burtt & O.M. Hilliard 13021 (NU). 3419 (Caledon): 15 km South east of Hermanus (–AC), 29 September 1976, L. Hugo 574 (PRE); Gansbaai (–CB), 19 November 1944, F.M. Leighton 811 (PRE). 3420 (Bredasdorp): De Hoop–Potberg Nature Reserve (–BC), 07 September 1978, C.J. Burgers 1082 (PRE).

EASTERN CAPE – 3226 (Fort Beaufort): Amathole mountains (–DB), 03 December 1982, H.P. Furness & P.B. Phillipson 56 (PRE); 5.5 km away from Grahamstown (–DC), October 1973, T.H. Arnold 624 (PRE). 3228 (Butterworth): Dwesa–Cwebe Nature Reserve (–BD), 22 November 1978, H.P. Linder 1852 (PRE). 3326 (Grahamstown): Grahamstown (–BC), 29 April 1995, J.E. Victor 1353 (PRE); Port Alfred, Bathurst (–DB), 17 November 1964, M.J. Wells 2915 (PRE); Alexandria (–CB), 26 December 1955, E.E.A. Archibald 6109 (PRE). 3227 (Stutterheim): King Williams Rd (–CB), 14 December 1977, B.L. Burtt & O.M. Hilliard 11064 (NU). 3327 (Peddie): East London, Kidds Beach (–BA), 07 December 1977, B.L. Burtt & O.M. Hilliard 11127 (NU). 3424 (Humansdorp): Kruisfontein (–BA), 26 November 1921, E.P. Phillips 3333 (PRE).

Figure 2.7: Map showing the known geographic distribution of S. incisa

2.3.3.4 Scabiosa transvaalensis, S. Moore., in J. Bot. 56:6 (1918). Type: South Africa. Mpumalanga Province, 2430 (Pilgrim’s Rest): Pilgrim’s Rest (-DB); (28 December 1914) F.A. Rogers 14361 (K–image!, lectotype, designated here). [Syntypes: South Africa. Mpumalanga Province, 2430 (Pilgrim’s Rest): Pilgrim’s Rest (-DB), (December 1915) F.A. Rogers 14999 (BM–image!, K-image!, S-image!); 2629 (Spitzkop): Spitzkop (-BD), Wilms 619 (specimen not seen)].

Herbaceous plant, stem visibly ridged, up to 2 m tall. Leaves evenly scattered along the stem, light green, opposite, simple, without stipules, sessile, oblong–lanceolate to linear– lanceolate. Basal leaves with toothed margins, 55–130 mm long, forming pseudo–whorls

of four, upper leaves pinnatisect, 40–100 mm, entire margins (Figure 2.8). Flower stalk 150–280 mm long. Corolla 11 mm wide, creamy white coloured.

Phenology: From summer to early autumn, December to March.

Etymology: The species is named for its distribution pattern, occurring in the Transvaal (now Gauteng, Limpopo and Mpumalanga Provinces).

Figure 2.8: Line drawing of S. transvaalensis showing sessile leaves distributed evenly on stem. Scale bar 7 mm.

Diagnostic characteristics: Stem ridges are more prominent as compared to other species, leaves occur throughout the whole stem and form pseudo–whorls of four.

Distribution and habitat: Scabiosa transvaalensis occurs only in the Limpopo and Mpumalanga Provinces in South Africa (Figure 2.9). Occurs on scarp of forest or grassy slopes near forest margins at altitude as high as 1076 m.a.s.l. 31

Conservation status: Vulnerable D2 (Area of occupancy is <20km2 or the number of locations ≤5). This is a result of timber plantation in the past and presently, the species is competing with invasive species (von Staden, 2016).

Specimens examined

LIMPOPO – 2328 (Lephalale): Lephalale Riverbank (–CD), 31 March 2004, N. Swelankomo 102 (PRE). 2427 (Thabazimbi): Thabazimbi (–BD), 16 May 1978, G. Germishuizen 840 (PRE). 2428 (Modimolle): Waterberg Massif (–BC),15 March 1919, E.E. Galpin M175 (PRE).

MPUMALANGA – 2430 (Pilgrim’s Rest): Blyde Nature Reserve (–DB), 07 March 2000, S. Krynauw 2019 (PRE). 2530 (Mashishing): Tweefontein (–BB), February 1932, V. Wager B183 (PRE).

Figure 2.9: Map showing the known geographic distribution pattern of S. transvaalensis.

CHAPTER 3

ETHNOBOTANY

3.1 Introduction Although the genus Scabiosa is relatively large, only a few studies have documented the ethnomedicinal uses of its members within its distribution range while in southern Africa, only S. columbaria L. has been studied (Seleteng Kose, 2017; Maroyi, 2019). In Peru, Scabiosa atropurpurea L. is used to regulate menstruation, while in Tunisia it is used as a diuretic (Bussmann and Glenn, 2010; Wannes and Marzouk, 2016). In Spain, the same plant is taken orally as a hypoglycemic by Iberian Peninsula people while in Catalonia it is used for measles and boils (Hlila et al., 2015; Pinto et al., 2018). In Mongolia, the flowers of Scabiosa comosa Fisch. ex Roem. & Schult. are used in combination with six other plants to make Gurigumu-7, an ethnic medicine used to treat liver disease (Xu et al., 2016). In mainland southeast Asia, Scabiosa japonica Miq. leaves are used to treat stomach disorders, nausea and to strengthen teeth (Perry and Metzger, 1980). In Tunisia Scabiosa pratensis Moench. is used for treatment of respiratory ailments such as asthma, bronchitis and influenza. It is also helpful for skin problems, herpes, ringworms, skin rashes and for ulcers (Besbes et al., 2012). In Morocco, Scabiosa stellata L. is used to treat cracked heels (Rahmouni, 2018). Scabiosa contributes to traditional medicinal use because of its richness in secondary metabolites i.e. flavonoids, glycosides, saponins and pentacyclic triterpenoids (Pinto et al., 2018). The aim of this chapter is to provide information on the ethnomedicinal uses of Scabiosa species as well as the plants with which it is used in combination in southern Africa.

3.2 Materials and methods A literature survey on the traditional uses of indigenous African Scabiosa species, as well as plants used in combination with S. columbaria was carried out. The information was sourced from various databases (Science Direct, Springer Link, Google Scholar, Biodiversity Heritage Library and National Center of Biotechnology Information) including published and unpublished thesis and dissertations, E-books, paperback books and journals.

3.3 Results and discussion

Table 3.1: The ethnomedicinal uses of Scabiosa species & plants used in conjunction with Scabiosa columbaria.

Plants Medicinal uses Reference Scabiosa albanensis Treatment of tuberculosis Lawal et al. (2014) Wound healing, skin rashes, venereal Seleteng Kose et al. (2015); sores, prevents uterine disorders and Kılınç et al. (2020) liver diseases, sore eyes, acute respiratory infections, tuberculosis, diphtheria, heart problems, Scabiosa columbaria dysmenorrhea, abdominal pains, heartburn, colic, intestinal problems, female sterility, cleanses womb, difficult childbirth, high blood pressure, reduction of transmission of HIV from mother to child Scabiosa incisa Lotion for wounds, dusting powder Van der Walt (2003) Watt & Breyer-Brandwijk Scabiosa transvaalensis Lotion for sore eyes (1962) Combinations Watt & Breyer-Brandwijk S. columbaria + Haplocarpha scaposa + Massonia jasminiflora Ophthalmic application (1962) Moteetee & Van Wyk S columbaria + Asclepias humilis + Gunnera perpensa Regulate menstrual cycles (2011)

Watt & Breyer-Brandwijk S. columbaria roots + Dicoma anomala + Helichrysum caespititium, + Venereal sores (1962); Moteetee & Van Zantedeschia albomaculata Wyk (2011) Moteetee & Seleteng Kose S. columbaria + Afroaster hispida roots Skin rashes (2017) S. columbaria + Cussonia paniculata + Dicoma anomala + Searsia divaricata OR Moteetee & Van Wyk Dysmenorrhoea S. columbaria + Searsia divaricata + Cussonia paniculata (2011) S. columbaria + Cussonia paniculata + Searsia divaricata OR Watt & Breyer-Brandwijk S. columbaria + Cussonia paniculata + Searsia divaricata + Searsia zeyheri OR (1962); Moteetee & Van Treatment of colic Scabiosa columbaria roots + Eriospermum ornithogaloides bulb + Gunnera Wyk (2011); Quattrocchi perpensa roots + Mentha sp (2012) S. columbaria roots + Cussonia paniculata + Dicoma anomala + Searsia Watt & Breyer-Brandwijk divaricata OR Heartburn (1962); Quattrocchi (2012) S. columbaria roots + Searsia divaricata+ Cussonia paniculata S columbaria + Eulophia ovalis + Gunnera perpensa Moteetee & Van Wyk S columbaria + Eulophia ovalis + Eriospermum ornithogaloides (2011); Womb purifier to enhance fertility Scabiosa columbaria roots + Eulophia ovalis + Eriospermum ornithogaloides bulb Quattrocchi (2012) + Gunnera perpensa roots + Mentha sp Stricture of urethra, causes release of Watt & Breyer-Brandwijk S columbaria + Gazania jurineifolia pus (1962)

The medicinal uses of Scabiosa species are presented in Table 3.1. According to the latest checklist of southern African plants (Germishuizen and Meyer, 2003), there are nine species of Scabiosa occurring in the region. It was noted that four of these have medicinal uses, these are: S. albanensis, S. columbaria, S. incisa and S. transvaalensis. When compared to the other three species, Scabiosa columbaria has numerous documented uses which is not surprising since it has a much wider distribution range while the other three species have restricted distributions and occur only in South Africa. In the southern African region, S. columbaria occurs in Lesotho, eSwatini and South Africa where it is used by different ethnic groups, but mostly by the Basotho (Sesotho speaking people of Lesotho). For example, Basotho use the charred roots mixed with kerosene as an ointment for venereal sores (Moffett, 2010). The Basotho also use the plant for the treatment of colic, period pains, for stricture of urethra resulting in pus coming out, and to promote sterility (Moteetee and Van Wyk, 2011; Maroyi, 2019). The Tswana people (Setwana speaking people of South Africa) chew the roots for relief from heartburn, while the amaXhosa people (isiXhosa speaking people of South Africa) apply a lotion prepared from the roots to sore eyes and use the fragranced powder of the dried plant as dusting powder for infants (Van Wyk et al., 2009; Moffett, 2016). The distribution of Scabiosa columbaria extends beyond southern Africa, it occurs in North Africa, Europe, northern Asia (Siberia), and western Asia, where it is also used for medicinal purposes as well. In Italy, it is used for chilblains while in Turkey it is utilised as a diuretic and for the relief of constipation (Maroyi 2019). The other three species only have a single medicinal use each. The roots of S. transvaalensis are used by the Tswana people as a lotion for sore eyes (Watt & Breyer-Brandwijk, 1962). The leaves and roots of S. albanensis are used to treat tuberculosis by the people of Nkonkobe Municipality in the Eastern Cape Province. For this purpose, it is reported that the plant is extracted for five days in alcohol and taken three times a day for a month (Lawal et al., 2014). Scabiosa incisa is used in the coast of South Africa as a lotion for wound and a dusting powder. Although Scabiosa species treat various infections, most of the medicinal uses of southern African Scabiosa species are yet to be scientifically validated.

Interestingly, in southern Africa the Basotho people (Lesotho and Free State Province of South Africa) are the only group which uses S. columbaria in conjunction with other plants,

where it is used in herb-herb combinations (Seleteng Kose et al., 2015; Mugomeri et al., 2016; Mabaleha et al., 2019). Herb-herb combinations are done by combining two or more herbs with the desired result being an interaction of greater therapeutic benefits (Che et al., 2013). Scabiosa columbaria is used in conjunction with several well-known southern African medicinal species such as Cussonia paniculata, Dicoma anomala and Gunnera perpensa. For example, it is mixed with D. anomala, Helichrysum caespititium and Zantedeschia albomaculata for the treatment of venereal sores. Such herbal combinations sometimes consist of up to five plants, for example a mixture of Eulophia ovalis, Eriospermum ornithogaloides, Gunnera perpensa, Mentha spp. and S. columbaria is used for cleansing the womb in order to improve fertility (Moteetee and Van Wyk, 2011; Quattrocchi, 2012).

Table 3.2: Literature review of the plants used in synergism with Scabiosa columbaria, their common name, medicinal uses and plant part used.

Plant part Species name & (family) Common name/s Medicinal uses References used Afroaster hispida (Thunb.) Uhloshana Tonic for pregnant women, treats female sterility, sores, Shale et al. (1999); Van Wyk et al. J.C. Manning & Goldblatt Unozixhekana syphilis, urinary infections, headaches, earaches, fever, Roots (2009); Mabona & Van Vuuren (Asteraceae) Phoa stomach aches, snake bite and coughs (2013) Cussonia paniculata Eckl. & Colic remedy, treat kidney and bladder disorders, Mountain cabbage Zeyh subsp. sinuata heartburn, mental illness, malaria, dysmenorrhea, Roots Tetyana et al. (2001); Seleteng tree (Reyneke & Kok) DeWinter. breast cancer, early nervous and mental disease, works Leaves Kose et al. (2015) Mots’ets’e (Araliaceae) as an analgesic and promotes wound healing Remedy for dysentery, treat heartburn, fever, cold, coughs, toothache, decoction used for intestinal worm Roots Dicoma anomala Sond. Fever bush Stomach infestations, diarrhoea, gall sickness, treat abdominal Balogun and Ashafa (2017); Satyajit Stem (Asteraceae) bush Hloenya pains, colic, ulcers, dysmenorrhea, uterine disorders, et al. (2017) Leaves labour pains, syphilis, gonorrhoea, malaria, skin sores, breast cancer, purgative and as a toothache remedy Eriospermum ornithogaloides River pumpkin, Baker. Iphuzi-Lomlambo Treat earache, anaemia and cleanse uterus Bulb Moffett (2010) (Eriospermaceae) Isibaha Plant part Species name & (family) Common name/s Medicinal uses References used Eulophia ovalis subsp ovalis Iphamba Painful limbs & infetlity Chinsamy et al., (2011) Lindl Gazania jurineifolia DC. Dandelion No individual uses reported Roots Watt & Breyer-Brandwijk (1962) (Asteraceae) Sweswe

Mpipete Mixed with S. columbaria for stricture of urethra, causes release of pus Rhizome: treats impotence, female infertility, induce labour, treat endometritis, abdominal pain, bladder problem, bleeding stomach, gastrointestinal parasites, River pumpkin kidney problems, period pain, urinary infections, urinary Gunnera perpensa L. Rhizome Wild rhubarb stones, urine bleeding earache, heart disease, Maroyi (2016) (Gunneraceae) Leaves shamboḓavhadzimu hypertension, swelling, scabies, cold, rheumatic fever, and gonorrhoea Leaves: treats boils, cancer, for wound healing, stomachache, constipation, ulcers and headache Common Used for chest colds, cancer, wound healing, treats Haplocarpha scaposa Harv. haplocarpha Roots dysmenorrhea, amenorrhoea, infertility, STIs and paste Shale et al. (1999) (Asteraceae) False gerbera Leaves applied on sore ears Dwaba Helichrysum caespititium Speelwonderboom Roots Increase virility in males, treat gonorrhoea, relief head Lourens et al. (2008); Maroyi (DC.) Sond. Ex Harv. Boriba Stem and chest colds and treats nausea (2019); Mabaleha et al. (2019) (Asteraceae) Phate ea ngaka Leaves Khongoana- Massonia jasminiflora Burch. tšingoana Ophthalmic application, treat sterility in women, powder ex Bulb Watt and Breyer-Brandwijk (1962) Lematla placed in incisions (Hyacinthaceae)

Plant part Species name & (family) Common name/s Medicinal uses References used Mentha longifolia (L.) L. Wild mint Treats dysentery, used for deworming, a diuretic, Roots Mikaili et al. (2013) (Lamiaceae) Inxina constipation, gall stones, indigestion, toothaches, Leaves

Isambalabwaba asthma, cough, headache, kidney and bladder stones, Flowers jaundice and wound healing Rusty-leaved Coughs, cold, remedy for bloating/constipation when Searsia divaricata (Eckl. & Moteetee and Van Wyk (2011); currant, mixed with Aloe striatula, decoction used for back pains, Zeyh) Moffett. Roots Quattrocchi (2012); Lennox et al. Roesblaartaaibos kidney and bladder problems, treats dysmenorrhea and (Anacardiaceae) (2015) Koditsane colic when mixed with S. columbaria and cure diabetes. Searsia zeyheri (Sond.) Blue currant No individual use recorded Moffett Roots Watt and Breyer-Brandwijk (1962 Bloutaaibos Colic (mixed with S. columbaria) (Anacardiaceae) Zantedeschia albomaculata Arum lily Treatment of sore throat, kidney and bladder infections, (Hook). Baill. Mohalalitoe mouth ulcers, cysts in uterus (rhizomes mixed with Rhizomes Seleteng Kose et al. (2015) (Araceae) Eucomis autumnalis)

The medicinal uses of the plants used in combination with S. columbaria are shown in Table 3.2. The family Asteraceae is commonly used in conjunction with S. columbaria, with four species, followed by two plant species from Anacardiaceae. Although there is knowledge of these plant combinations, it is necessary to note that the ratios of how these plants are taken is unknown. In some cases, the medicinal uses of a species are similar when used individually to when used in combination with S. columbaria. For example, C. paniculata is used for the treatment of colic and heartburn, and it is used for the same purposes in a herbal concoction with S. columbaria and Searsia divaricata. These species may be combined due to their ability to enhance medicinal effect or because they produce different pharmaceutical effects that are necessary to alleviate symptoms associated with these ailments. Surprisingly, some species namely, Searsia zeyheri and Gazania jurineifolia have no other recorded medicinal uses except when used in conjunction with S. columbaria. However, in southern Africa, the use of herbal combinations is not unique to the Basotho people. It has also been reported for amaZulu where Adenia gummifera (Harv) Harms var. gummifera roots are mixed with leaves of Erianthemum dregei (Eckl. & Zeyh.) Tiegh. and Sarcophyte sanguinea Sparrm. stems, or Ranunculus multifidus Forssk. are mixed with Hypoxis hemerocallidea C.A. Mey. & Avé-Lall. and Senecio serratuloides DC. to treat STI related infections (De Wet et al., 2010). VhaVenda people combine Dodonaea angustifolia L.f. with Dovyalis zeyheri (Sond.) Warb. or Hippocratea longipetiolata Oliv. with Olinia rochetiana A. Juss. to treat candidal infections (Masevhe et al., 2015). In Zimbabwe Elephantorrhiza goetzei (Harms.) Harms. is mixed with Piliostigma thonningii (Schumach.) Milne-Redh. for the treatment of bilharzia or Eucalyptus camaldulensis Dehnh. mixed with Psidium guajava L. and Citrus limon (L.) Osbeck. as a remedy for coughs, flu and fever (Maroyi, 2013), while in Tanzania Carissa edulis (Forssk.) Vahl. is mixed with Ximenia caffra Sond. for the treatment of pneumonia and X. caffra mixed with Harrisonia abyssinica Oliv. for treatment of measles (Otieno et al., 2008).

Generally, the plant parts that are mostly used are roots, however, this is regarded as an unsustainable method of collection as it threatens the survival of the plant. This is because the plant species may have a limited abundance and slow growth. Other harvesting

practices that leave plants vulnerable are using the whole-plant or bark. There are about 15 000 species of flowering plants that are threatened with extinction due to overharvesting and habitat destruction, therefore finding alternative plant species, cultivation and the harvesting of non-destructive plant parts can help sustain medicinally important species (Chen et al., 2016).

3.4 Chapter summary • Four Scabiosa species, viz, S. albanensis, S. columbaria, S. incisa and S. transvaalensis found in southern Africa were documented to be used medicinally for different ailment. • Only S. columbaria was documented to be used as an antacid and for ailments associated with inflammation. • Scabiosa columbaria was also the only species documented to be used in combination with other plants species

CHAPTER 4

BIOLOGICAL ACTIVITY

4.1 Introduction 4.1.1 Antimicrobial activity Four species of Scabiosa are used medicinally against different ailments in southern Africa, but these species have been largely neglected in studies validating traditional uses. Some examples of research work done on antibacterial activity of other Scabiosa sp was by Hafsa et al. (2009), where Scabiosa arenaria Forsk. exhibited low antibacterial activity against Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. All extracts exhibited MIC values of 2.5 mg/ml. In Bussmann et al. (2010), the ethanol extracts of Scabiosa atropurpurea showed low activity against S. aureus with an MIC value of 32 mg/ml while the aqueous extracts showed no activity against the same pathogen. The ethanol and methanol extracts exhibited no activity against E. coli. In Alipoor Birgani et al. (2019), Scabiosa Olivieri Coult. was tested against E. coli and S. aureus and exhibited no activity against both pathogens with MIC values of 9.37 mg/ml and 4.68 mg/ml respectively. Mouffouk et al. (2018), showed Scabiosa stellata to have antibacterial activity with zone of inhibition ranging from 9 to 20 mm against Gram- positive (S. aureus, Enterococcus sp, Streptococcus D) and Gram negative (E. coli, Cinetobacter baumannii, Proteus mirabilis, Enterobacter sakazaki) bacteria. In this study, the antibacterial properties of Scabiosa columbaria were investigated based on the traditional uses, whereby the use relates to possible antimicrobial activity. Due to their restricted distribution ranges, it was not possible to locate the other medicinal species of Scabiosa (S. albanensis and S. transvaalensis) despite several attempts, therefore only S. columbaria and S. incisa were evaluated for antibacterial activity. The synergistic properties of S. columbaria when used in combination with other plant species i.e. Afroaster hispida, Cussonia paniculata, Dicoma anomala, Helichrysum caespititium, Searsia divaricata and Zantedeschia albomaculata for the treatment of skin, gastrointestinal and sexually transmitted infections were also evaluated.

4.1.2 Anti-inflammatory activity Scabiosa columbaria has been reported to treat ailments associated with inflammation such as dysmenorrhea and stricture of the urethra. As far as we know, no research has been done to study the anti-inflammatory properties of southern African Scabiosa species and combinations of S. columbaria with other plant species. In a study by Mouffouk et al. (2018), Scabiosa stellata L. was tested for anti-inflammatory effects using the carrageenan induced paw oedema assay, and for its anti-arthritic potential using the inhibition of albumin denaturation method using Diclofenac is a positive control. The anti- inflammatory effects of both ethyl acetate extract and Diclofenac were observed at the first hour, and the high effects were from ethyl acetate extracts with inhibition of 72.73% while the maximum of Diclofenac was only observed at three hrs with inhibition of 61.59%. For the anti-arthritic activity assay, inhibition was found to be dose-dependent and the crude ethyl acetate extract showed significant inhibition of 78.86% while the n-butanol extracts had 3.21% inhibition. The study concluded that the ethyl acetate extracts had high anti-arthritic and anti-inflammatory activities and S. stellata extract could be used therapeutically in pharmaceutical formulations. This study evaluates the anti- inflammatory activity of S. columbaria and plants (Dicoma anomala, Cussonia paniculata subsp. sinuata, Eriospermum ornithogaloides, Gunnera perpensa, S. columbaria and Searsia divaricata) used in combination with it using COX and LOX inhibition.

4.1.3 Antacid activity Many plant species have been recorded to be used as remedies for heartburn but there has not been much scientific evidence to validate their use. Some common herbs used for treatment of heartburn are chamomile, ginger, liquorice and calcium rich food that helps strengthen the lower oesophageal sphincter and prevent reflux of acid (Hargreaves, 2007). Apart from two species, Lonicera japonica Thunb. and Scabiosa columbaria, the family Caprifoliaceae is not commonly known for treatment of heartburn. In an in vivo study by Ku et al. (2009), the extracts of L. japonica were tested for their ability to decrease mucosal damage in rats. The mucosal damage was a result of induced reflux esophagitis (i.e. heartburn and acid reflux). It was found that the extracts of L. japonica were effective in attenuating the severity of reflux esophagitis. Scabiosa columbaria was recorded in Watt & Breyer-Brandwijk (1962) and Quattrocchi (2012) as a remedy for

heartburn. It is not only used independently, but also used in combination with other plant species such as Cussonia paniculata, Dicoma anomala and Searsia divaricata. There has been no research conducted to validate the antacid efficacy of S. columbaria and combinations. This study was carried out to determine the antacid capacity of these plants and verify the claims using in vitro methods.

4.1.4 Toxicity Most medicinally important Scabiosa species have not been evaluated for their toxicity except for Scabiosa stellata L. (starflower pincushion), used medicinally for the treatment of hypoglycaemia, respiratory diseases, to regulate menstruation and as a diuretic. The crude plant extracts of S. stellata were prepared in DMSO and the toxicity was evaluated using the BSLA at concentrations 10, 20, 40 and 80 µg/mL by Mouffouk et al., (2018). The various extracts (ethyl acetate, n-butanol and petroleum ether) showed varying levels of toxicity at different concentrations. N-butanol extract displayed the highest mortality rate of 57.2 % at 80 µg/ml, while the ethyl acetate and petroleum ether extract had mortality rates of 28.5% and 42.8% respectively. The study concluded that S. stellata should be used with caution. None of the southern African ethnomedicinal Scabiosa species have been accessed for their toxicity. In the present study, S. columbaria will be assessed for its toxicity when used independently and also when combined with other plant species with which it is used medicinally.

4.2 Material and methods 4.2.1 Collection of plant material

Collection permits were applied for and upon acceptance, fieldtrips were taken for plant collection according to the legislations of the Biodiversity and Bioprospecting act. Plant material was identified with the assistance of taxonomist Professor A.N. Moteetee. The selection of the plant parts collected (and tested for the different biological activity tests) was based on the traditional use as documented in the literature as well as the accessibility/availability of the plants. In cases where plants could not be found in the field, the plants were bought from Random Harvest Nursery located in Muldersdrift, South Africa. Voucher specimens were prepared and stored at University of Johannesburg Herbarium (JRAU). The plant material was washed, dried in a greenhouse, ground with

an electric grinder (MRC/ Mellerware) into fine powder. The ground samples were then stored in airtight plastic containers to prevent absorption of moisture and used when needed for all the biological activities tests undertaken.

4.2.2 Biological activity 4.2.2.1 Antibacterial activity of Scabiosa sp • Plant sample extraction

Dry ground and weighed plant material (sample) was placed in a conical flask. For the organic extracts, dichloromethane (DCM) (Sigma-Aldrich): methanol (Sigma-Aldrich) in a 1:1 ratio was used. The solvent, twice the quantity of the plant material was added. The conical flask top was covered by a foil and placed in a shaker incubator at 37°C for 24 hrs. The supernatant was transferred into another container using autoclaved cotton wool by withdrawing the supernatant from the cotton wool. The conical flask was topped again with solvent equal to quantity of pellet and incubated at 37°C for 24 hrs. The two supernatant solutions were mixed and evaporated to dryness for three days in a fume hood. The percentage yield was determined. The completely dry mass was then dissolved in 100% acetone (Van Vuuren et al., 2015).

For the aqueous extracts, sterile water was added to the plant material at equal ratios in the conical flasks. The conical flask tops were covered with foil and incubated for 24 hrs at 30°C. The filtered supernatant was transferred into weighed vials and frozen for 24 hrs at -80°C. The vials with plant samples were freeze dried in a lyophiliser (United Scientific). Once lyophilised, the vials were weighed again, and the yield was calculated (Van Vuuren et al., 2015).

• Culture preparation

Bacterial test organisms were selected based on conditions that the plants are reported to treat. The bacterial cultures were grown in Tryptone Soya broth (TSB) (Oxoid, Ltd) (except for Neisseria gonorrhoeae grown in Brain Heart Infusion (Oxoid Ltd)) for 24 hrs at 37°C and were modified to optimise the growth requirements of the micro-organism. The chosen pathogens were: Bacillus cereus ATCC 13778, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 8739 for gastrointestinal diseases (ethnobotanically

47 referred to as colic), Neisseria gonorrhoeae ATCC 19424 for sexual transmitted infections (ethnobotanically referred to as venereal sores), and Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC12223 for skin problems (ethnobotanically referred to as rashes). All the pathogens where originally obtained from Davies Diagnostics (South Africa) and kept viable within the Department of Pharmacy and Pharmacology, University of Witwatersrand in Johannesburg.

Tryptone Soya broth was prepared by adding 40 g TSB in 1 L of purified water. It was brought to boil to dissolve the powders completely and then sterilised by autoclaving at 121ºC for 30 min and left to cool down. Brain heart infusion was prepared by adding 37 g to 1 L of distilled water and sterilised as previously described.

• Sample preparation

Samples were prepared with a starting concentration of 32 mg/ml. The extracts were weighed to amount between 0.08-0.09. The amount of solvent to be added was determined by using Equation 4.1 below.

( ) × 1000

32 ( ) 𝑋𝑋 𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤ℎ𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 Equation 4.1

Solvent [sterile water for aqueous extracts and acetone (AEC- Amersham) for organic extracts] was added to the plant extracts and vortexed. For the negative control, acetone (32 mg/m/L) was used and the positive control Ciprofloxacin at a starting concentration of 0.01 mg/ml was used. The culture control was the broth stored overnight at 37ºC with organism inoculated into the broth to determine viability of the culture.

• MIC assay

The microtiter plates were prepared aseptically. An amount of 100 µl of TSB broth was added into all the 96 wells of the microtiter plate. An amount of 100 µl of prepared extract sample at a starting concentration of 32 mg/ml was added in row A1-A12. Each extract

48 sample was duplicated. A serial dilution of 100 µl was done for the extracts and 100 µl was discarded from the final row H.

A 0.5 McFarland standard was prepared by adding approximately 1 ml of 24 hr culture into 10 ml sterile broth and vortexed. A 1:100 dilution of culture was prepared by adding 1% of McFarland standard into the chosen volume for example, adding 1 ml of McFarland standard in 100 ml broth. The 1:1 dilution culture (100 µl) was then added in all the wells. The plate was sealed with sterile adhesive sealers (AEC Amersham) to prevent evaporation during incubation. The culture was streaked on an agar plate to assess contamination. The left over 1:1 dilution cultures, streaked plates and microtiter plates were incubated at 37ºC for 24 hrs (N. gonorrhoeae for 48 hrs in a carbon dioxide incubator (AEC- Amersham) at five percent carbon dioxide and temperature 37°C).

A serial microdilution (MIC) assay was used to quantify the MIC values of the plant extracts using p-iodonitrotetrazolium violet (INT) (Sigma-Aldrich) as an indicator of growth (Eloff, 1998; NCCLS, 2003). The p-iodonitrotetrazolium violet was prepared with 0.08 g INT powder and 200 ml sterile water. It was left to incubate at 37ºC for an hr until the powder dissolved, then kept at 4°C.

After 24 hrs of incubation, 40 µl of INT was added into each well of the microtiter plates. The plates were left to rest until the formazan (red/pink) developed. Reading was done only after the controls had developed the formazan. The time left for the colour to develop differs for each bacterium, ranging from 4-6 hrs. The presence of a pink colour indicated pathogen growth and lack of pink-red colour indicated inhibition. The negative control confirmed that the solvents had little to no antibacterial effects while the positive control confirmed the susceptibility of the bacteria.

For analysis of combination studies, the Fractional Inhibitory Concentration (FIC) index was calculated as shown in Equation 4.2 below.

= + , where

1 2 Ʃ𝐹𝐹𝐹𝐹𝐹𝐹= 𝐹𝐹𝐹𝐹𝐹𝐹 ( 𝐹𝐹𝐹𝐹𝐹𝐹 ) = ( ) and 𝑀𝑀 𝑀𝑀𝑀𝑀 𝑃𝑃𝑃𝑃𝑃𝑃𝑐𝑐𝑒𝑒 𝐴𝐴 𝑖𝑖𝑖𝑖 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑤𝑤𝑤𝑤𝑡𝑡ℎ 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝐵𝐵 𝑀𝑀 𝑀𝑀𝑀𝑀 𝑃𝑃𝑃𝑃𝑃𝑃𝑐𝑐𝑒𝑒 𝐵𝐵 𝑖𝑖𝑖𝑖 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑤𝑤𝑤𝑤𝑤𝑤ℎ 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝐴𝐴 𝐹𝐹𝐹𝐹𝐹𝐹1 𝑀𝑀𝑀𝑀𝑀𝑀 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝐴𝐴 𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖 𝐹𝐹𝐹𝐹𝐹𝐹2 𝑀𝑀𝑀𝑀𝑀𝑀 𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝐵𝐵 𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖 Equation 4.2

4.2.2.2 Anti-inflammatory activity of S. columbaria and plants used in combination • Plant extraction

Reverse osmosis water and methanol were used as extraction solvents. About 2-7 g of ground plant material was extracted in 30-40 ml of cold solvent and sonicated in an ultrasonic water bath (Rayos) for 1 hr. The extracts were left on a shaker to saturate overnight and then filtered under a fume hood using Whatman No. 1 filter paper. The resultant aqueous extracts were freeze dried (United Scientific) while the organic extracts were left to evaporate and fully dry in a fume hood. A stock concentration of 200 μg/ml was made from plant extract and methanol. The methanolic extracts were tested at concentrations 100, 50, 25 and 12.5 μg/ml (Chinsamy et al., 2014).

• Assays perfomed according to the corresponding kits.

Lipoxygenase inhibitor screening assay was used where the first step was to prepare reagent and enzymes using the following steps: Three millimeters of assay buffer concentrate was diluted in 27 ml of HPLC-grade water to make final assay buffer. The final 1X assay buffer (0.1 M Tris-HCl, pH 7.4) was used for the dilution of samples and the 15-LOX standard prior to assaying. Chromogen was made through addition of equal volumes (500 µl) of developing reagents 1 and 2 in a test tube and vortexed. An amount of 100 µl was added to each of the wells. Soybean 15-LOX was used as the positive control by diluting 10 µl of the enzyme in 990 µl of 1X assay buffer in a vial and stored in ice. In a vial, 25 µl of substrate and 25 µl of KOH were added and vortexed to make arachidonic acid (substrate). This was followed by dilution using 950 µl of HPLC-grade water to achieve a working concentration of 1 mM. To make Nordihydroguaiaretic acid (NDGA) i.e. the positive control inhibitor, the vial contents supplied in the kit (550 nm non- selective lipoxygenase inhibitor NDGA) were resuspended in 500 µl of 1X assay buffer to make a 1.1 mM stock.

The second step was to perform the assay using the following steps: For the blank wells, 100 µl of assay buffer was added to two wells. An amount of 90 µl 15-LOX and 10 µl of assay buffer were added into two wells as the positive control wells. For 100% initial

50 activity (IA) wells, 90 µl of lipoxygenase enzyme and 10 µl of methanol were added to two wells. The inhibitor wells had 90 µl of lipoxygenase enzyme and 10 µl of the plant extracts. The plate was incubated for 5 min at room temperature. The reaction was initiated by adding 100 µl of arachidonic substrate to all the wells. The plate was then placed on a shaker for 10 min. An amount of 100 µl Chromogen was added in each well to stop enzyme catalysis and to develop the reaction. The plate was covered with a plate cover and placed in a shaker again for 5 min. The cover was removed, and the absorbance was read at 490-500 nm using a plate reader.

The second enzyme assessed was cyclooxygenase inhibition using COX colorimetric inhibitor screening assay kit (Cayman Chemicals, No. 701050) where the first step was to prepare reagent and enzymes using the following steps: Three millimeters of assay buffer concentrate was diluted with 27 ml of HPLC-grade water. The final 1X assay buffer (0.1 M Tris-HCl, pH 7.4) was used for the dilution of Hemin and COX enzymes prior to assaying. An amount of 88 µl of Hemin was diluted with 1.912 ml of diluted assay buffer. Colorimetric COX assay COX-1 (ovine) and COX-2 (human) were made through dilution of 120 µl of enzyme in 360 µl of diluted assay buffer and stored in ice. An amount of 100 µl of substrate and 100 µl of KOH were added into a vial and vortexed to make Arachidonic acid (substrate). The solution was diluted using 1.8 ml of HPLC-grade water to get a final concentration of 1.1 mM.

To perform the cyclooxygenase inhibition assay, three wells were allocated as background wells that had 160 µl of assay buffer and 10 µl of hemin added to them. For the 100% IA and inhibitors wells, 150 µl of assay buffer, 10 µl of Hemin and 10 µl of enzyme (COX-1/ COX-2) were added into three wells. An amount of 10 µl of plant extract was added to the inhibitor wells and 10 µl of methanol was added to 100% IA wells and the background wells. The plate was shaken for a few seconds and incubated for 5 min at 25°C. An amount of 20 µl of colorimetric substrate solution was added quickly into all the wells, followed by 20 µl of arachidonic acid. The plate was shaken again for a few seconds and incubated for 2 min at 25°C. The absorbance was read at 590 nm.

• Analysis of COX and LOX inhibition.

The average absorbance was determined for the blank, 100% initial activity (IA) and inhibitor wells. The average absorbance of the blank well was subtracted from the average absorbance of 100% IA and inhibitor wells. The percentage inhibition for each inhibitor was determined using Equation 4.3 below.

% Inhibition = [ ] × 100 𝐼𝐼𝐼𝐼 − 𝑖𝑖𝑖𝑖ℎ𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖 Equation 4.3 𝐼𝐼𝐼𝐼

® The software GraphPad8 Prism (GraphPad Software, Inc) was used to calculate the IC50

values of the samples. The IC50 value represents the concentration of a test substance necessary to have an inhibition effect of 50% on the enzyme tested.

4.2.2.3 Antacid and acid neutralising activity of S. columbaria and plants used in combination • Standardization of sodium hydroxide (NaOH)

Sodium hydroxide was standardized using potassium hydrogen phthalate (KHP) (Associated Chemical Enterprises) as a primary standard according to the method used by Houshia et al. (2012), with slight modifications. An amount of 4.6 g of KHPh was dissolved in 100 ml distilled water. The KHP required 22.95 ml of 0.994 M NaOH (Rochelle Chemicals) solution to reach the endpoint. At the endpoint, the number of moles of NaOH equalled the number of moles of KHP used. This standardized NaOH was used for titrating (in a 50 ml burette) the herbal plants (C. paniculata subsp. sinuata, D. anomala, S. columbaria and S. divaricata).

• Sample preparation and titration

One gram of the ground plant sample was transferred to a 250 ml Erlenmeyer flask. An amount of 25 ml of 0.521 M HCl (Rochelle Chemicals) was added to the flask and gently swirled. The flask was heated gently to boil for 5 min to remove any interference from

carbonic acid resulting from the carbon dioxide (CO2) dissolved in water. The CO2 was driven off by heating the contents of the flask to boil, the flask was removed from the heat and allowed to cool until it was comfortable to hold. Three drops of Phenolphthalein

52 indicator were added into the solution. Phenolphthalein indicator shows colour change in pH 8.0-10.0, colourless in acidic conditions and pink (Figure 4.1) in basic conditions. An amount of 75 ml of distilled water was added to the flask and swirled. Then the exact antacid capacity was calculated using the back titration technique (Houshia et al., 2012). The moles of acid neutralized were calculated as shown in Equation 4.4 below.

= ( ) ( ) 𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛 = ( × ) ( × ) 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑜𝑜𝑜𝑜 𝐻𝐻𝐻𝐻𝐻𝐻 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 − 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑜𝑜𝑜𝑜 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟 𝑓𝑓𝑓𝑓𝑓𝑓 𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 𝐻𝐻𝐻𝐻𝐻𝐻 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀 𝑜𝑜𝑜𝑜 𝐻𝐻𝐻𝐻𝐻𝐻 𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉 − 𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑎𝑎𝑛𝑛𝑀𝑀 𝑜𝑜𝑜𝑜 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉Equation𝑒𝑒 4.4

Figure 4.1: The colour change expected after reaching an end point using NaOH for back titration.

• Fordtran’s model with modifications.

This model was conducted according to Sandhya et al. (2012), with some modifications. The extracts were prepared by taking one gram ground plant sample and dissolve in 90 ml distilled water then the pH of the plant extracts and the control solutions were measured at different temperatures ranging from 10°C to 37°C.

To prepare artificial gastric juice, an amount of two grams of Sodium Chloride (NaCl) (Associated Chemical Enterprises) and 3.2 mg of pepsin (Roche) were dissolved in 500 ml distilled water. About 7 ml of hydrochloric acid (Associated Chemical Enterprises) and

adequate water were added to make a 1000 ml solution. The pH of the solution was adjusted with hydrochloric acid to 1.2.

To evaluate the neutralization effects on artificial gastric juice, a test solution of 90 ml of the plant extracts were added to 100 ml artificial gastric juices at pH 1.2. The pH values were determined to examine the neutralizing effect.

• In vitro titration method of Fordtran’s model for determination of the neutralization capacity

An amount of 90 ml of sample warmed to 37°C and aeration given at 136 air bubbles per minute to imitate the peristaltic movements in the stomach. The samples were titrated with the artificial gastric juice to obtain an end point of pH 3. The consumed volume (V) of artificial gastric juice was recorded. The total consumed hydrogen ion (mmol) was measured as shown in Equation 4.5 below.

0.063096 ( . / ) × ( ).

𝑚𝑚 𝑚𝑚𝑚𝑚𝑚𝑚 𝑚𝑚𝑚𝑚 𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑚𝑚𝑚𝑚 Equation 4.5

The Scabiosa species chosen for the antacid test was S. columbaria as well as plant species used in combination for the relief of heartburn.

4.2.2.4 Toxicity of the selected medicinal plants

This method for extraction of plants and the assay to test toxicity of the plants were done according to Bussmann et al. (2011), and Hübsch (2014). For the organic extracts, 2% dimethyl sulfoxide (DMSO) was used as solvent and for the aqueous extracts, sterile water was used as the solvent. The test samples were prepared to a concentration of 2 mg/ml. The amount of solvent to be added was determined by Equation 4.6 shown below.

( ) × 1000

2 ( ) 𝑋𝑋 𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤ℎ𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 Equation 4.6

Samples tested in combination were in a 1:1 ratio. The samples were combined in a sample bottle and vortexed before they were added into the wells during the assay.

The artificial saltwater was prepared by dissolving 16 g of Tropic Marine® sea salt in 500 ml of distilled water in an inverted, bottomless, transparent plastic bottle. The rubber pipe with metal filter was placed inside the water so that the pipe reached the bottom of the container to promote water aeration and constant motion that always kept the Artemia franciscana, brine shrimp eggs (Ocean Nutrition) well dispersed. Then 0.5 g of dried, brine shrimp eggs were added into the prepared sea water. The eggs were exposed to constant light using lamps (230 V) for 24 hrs and aeration by use of a rotary pump (Kiho). After 24 hrs, the artificial saltwater containing the brine shrimp was transferred to a shallow rectangular container and placed at an angle. The container was exposed to a concentrated light source for 30 min in order to attract the brine shrimp towards the light. This allowed the withdrawal of high quantities of brine shrimp when needed.

An amount of 400 μl saltwater containing brine shrimp was withdrawn and added to each well of a 48-well microtiter plate. For each sample, 400 µl was added to the wells with the brine shrimp in triplicate. For the negative control, 400 µl, (prepared in triplicate) of prepared saltwater was added in wells as a toxin-free control to mimic the natural environment of the brine shrimp, thereby supporting their survival and growth. For the positive control, 400 µl of 1.6 mg/ml potassium dichromate (Saarchem) solution was added to the wells (triplicate) to ensure the mortality of all brine shrimp present in the well. The plates were immediately observed under the light microscope (Olympus) (magnification of 40x) after the brine shrimp was added to the plates (Figure 4.2). A count of the amount of dead brine shrimp before the addition of the sample was done and recorded as T0.

Figure 4.2: Adult brine shrimp when viewed at 40X magnification under a light microscope. The sample extracts were added and the number of dead shrimp after the addition was recorded as dead shrimp for T1 (excluded when doing the total mortality calculation). The plates, with their lids on were left in the laboratory at room temperature (24°C) with the lights on. A count of the number of dead brine shrimp at 24 and 48 hrs of exposure to test samples was done after the count, a lethal dose of 50 μl of 100% v/v glacial acetic acid (Saarchem) was added to each well. A final count of the total dead brine shrimp was conducted, and the percentage mortality was calculated as shown in Equation 4.7 below.

% 48 ( ) ( 0) × 100 = 𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀 ( ) 𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷 𝑠𝑠ℎ𝑟𝑟𝑖𝑖𝑖𝑖𝑖𝑖 𝑎𝑎𝑎𝑎 ℎ𝑟𝑟𝑟𝑟 𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 − 𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑 𝑠𝑠ℎ𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷 𝑠𝑠ℎ𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 Equation 4.7

Samples that induced a percentage mortality greater than 50% were tested for dose response toxicity at concentrations of 2, 1, 0.5, 0.25, 0.125 and 0.063 mg/ml. Each of the dilutions were tested in triplicate, in two independent experiments, including the positive and negative controls.

4.3 Results and discussion The antibacterial activity of Scabiosa incisa and Scabiosa columbaria were assessed as well as the synergistic properties of S. columbaria used in combination with other species. Furthermore, S. columbaria is the only species used for the treatment of heartburn and ailments associated with inflammation, therefore it was the only one assessed for antacid and acid neutralising activities as well as for anti-inflammatory activity.

4.3.1 Antibacterial activity For this study, the MIC results were interpreted in the following manner: low antimicrobial activity >1 mg/ml; moderate antimicrobial activity between ≥0.16-1 mg/ml (Kuete and Efferth, 2010); noteworthy antimicrobial activity ≤0.16 mg/ml (Van Vuuren and Holl, 2017).

Table 4.1: Minimum inhibitory concentration (mg/ml) of the organic (DCM: Methanol) extracts of selected plant.

MIC values (mg/ml) Voucher Plant part Bc Ef Sa Se Ng Ec Pa Plant species number used (ATCC (ATCC (ATCC (ATCC (ATCC (ATCC (ATCC 13778) 29212) 25923) 25923) 19424) 8739) 27853) Cussonia NWM paniculata 001 Leaves 1.00 2.00 2.00 4.00 2.00 2.00 1.00 subsp. sinuata Dicoma KSK Roots 0.50 2.00 2.00 2.00 2.00 1.00 ≥ 8.00 anomala 0002

Helichrysum KSKP Roots ≥ 8.00 2.00 2.00 ≥ 8.00 ≥ 8.00 4.00 2.00 caespititium 0029 Scabiosa Leaves 1.00 1.00 2.00 2.00 2.00 2.00 1.00 columbaria KSK Scabiosa 0035 Roots 2.00 2.00 4.00 2.00 2.00 2.00 1.00 columbaria

Scabiosa incisa NWM Leaves 4.00 2.00 2.00 ND ND 005 Scabiosa incisa Roots 2.00 2.00 2.00

Searsia NWM Roots 4.00 2.00 ≥ 8.00 1.00 ≥ 8.00 2.00 ≥ 8.00 divaricata 004

MIC values (mg/ml) Voucher Plant part Bc Ef Sa Se Ng Ec Pa Plant species number used (ATCC (ATCC (ATCC (ATCC (ATCC (ATCC (ATCC 13778) 29212) 25923) 25923) 19424) 8739) 27853) Zantedeschia KSK Roots 2.00 4.00 2.00 2.00 4.00 2.00 4.00 albomaculata 0011 Ciprofloxacin Positive control 0.08 0.63 0.04 0.31 0.63 1.25 0.04 (ug/ml) Acetone + Negative control ≥ 8.00 water (ug/ml) Culture control TSB ≥ 8.00 Pathogens: Bc, Bacillus cereus, Ef, Enterococcus faecalis, Ec, Escherichia coli, Ng, Neisseria gonorrhoeae, Pa, Pseudomonas aeruginosa, Sa, Staphylococcus aureus, Se, Staphylococcus epidermidis. Bold value indicates moderate activity; shaded areas are Scabiosa species, ND- not determined due to insufficient material.

Of the seven plant species studied, four plants had previously been assessed for antimicrobial activity. In this study, Scabiosa columbaria and the species with which it is used in combination, were tested individually for antibacterial activity against selected bacteria based on their medicinal uses and the results are shown in Table 4.1. All the plants studied had no activity against all pathogens tested except D. anomala which had moderate activity against B. cereus with MIC value of 0.5 mg/ml.

Scabiosa columbaria had weak antibacterial activity despite being recorded numerous times in literature for its traditional medicinal uses. The results of this study are comparable to those of Van Vuuren and Naidoo (2010), where the organic extracts (DCM: Methanol) of the roots and leaves of S. columbaria were evaluated against N. gonorrhoeae and other causative agents linked to sexually transmitted infections, resulting in MIC values >2.00 mg/ml, i.e. no antimicrobial activity against all pathogens tested. It is interesting to note that the antimicrobial activity of S. columbaria does extend beyond the current study against skin, gastrointestinal and STI pathogens. In Seleteng Kose (2017), the plant was screened for its antimicrobial activity against pathogens associated with respiratory tract infections (Citrobacter frendii, Enterobacter hormaendis, Klebsiella pneumoniae, Moraxella catarrhalis and S. aureus) and still showed no activity

against all the pathogens tested. These studies do not support the use of this plant against those ailments. In a previous study (Benli et al., 2008), S. columbaria subsp. paphlagonica (Bornm.) Matthews leaf extracts were investigated for their antimicrobial activity against ten bacterial and four yeast strains using the disk-diffusion agar method. The extracts had no inhibition i.e. no antimicrobial activity against the pathogens tested.

Scabiosa incisa was reported to be used as a dusting powder and wound lotion. In this study, S. incisa was tested exclusively against pathogens responsible for skin problems i.e. S. aureus, S. epidermidis and P. aeruginosa. It exhibited weak antibacterial activity against all these pathogens with MIC values 2-4 mg/ml. These results did not support the ethnomedicinal use of this plant in the treatment of wounds.

In the current study, Dicoma anomala showed moderate antibacterial activity against B. cereus with MIC value of 0.50 mg/ml supporting its use as a gastrointestinal remedy i.e. colic. However, both the aqueous and organic leaf extracts had no activity against the rest of the pathogens tested. Results of this study are similar to those of Tafadzwa (2013), where D. anomala showed antibacterial activity against S. aureus, S. aureus group A and P. aeruginosa. These results do somewhat support the use of D. anomala for gastrointestinal infections.

Cussonia paniculata subsp sinuata leaves showed very weak to no activity against all pathogens tested in this study. A similar interaction was noted in De Villiers et al. (2010), where the methanolic extracts of the leaves of the same plant exhibited no inhibition against pathogens S. aureus, E. faecalis, E. coli and P. aeruginosa

In the present study, Helichrysum caespititium showed no activity against all the pathogens tested. Comparable to the present results, Mathekga (2001) showed that H. caespititium extracts did not possess antimicrobial activity against B. cereus, S. aureus, E. coli and P. aeruginosa. These results indicate that the plant is not effective against the pathogens tested although contrary to this study, H. caespititium exhibited noteworthy activity with MIC value 0.06 mg/ml against N. gonorrhoeae (Seleteng Kose, 2017).

The activity of Zantedeschia albomaculata organic extracts against the pathogens ranged between 2.00-4.00 mg/ml. Searsia divaricata organic extracts had antibacterial activity of

MIC 1.00-8.00 mg/ml against the pathogens tested. Both these plants are being tested for their antibacterial activity for the first time in this study and they were found to be ineffective against all the pathogens tested. Whether the negative result obtained from the Z. albomaculata extract invalidates the traditional use of this plant for venereal sores, is inconclusive since it was tested against only one STI pathogen, however, it was also ineffective against pathogens associated with skin infections.

Most of the plants tested in this study have shown low or no antibacterial activity against the pathogens tested, although they had shown good activity in previous studies. The antimicrobial activity of the plants could have been influenced by environmental and phenological factors. According to Inácio et al. (2016), plants that experience higher level of stress due to seasonal changes exhibit higher concentrations of secondary metabolites responsible for antimicrobial activity i.e. in spring activity is expected to be non-significant as compared to autumn/winter where activity is at its peak. De Zoysa et al. (2019), mentions that a plant may not exhibit the presence of antimicrobial activity during the tests, but this does not mean that the plant does not have the bioactive compounds. The negative results can also be attributed to the presence of inadequate quantities of the active bioactive constituents in the plant extracts. This can also be caused by the difference in type of extraction solvents used because different solvents extract phytochemicals at different concentrations therefore affecting the antimicrobial activity capacity of the plant (Thouri et al., 2017).

Table 4.2: MIC and (ΣFIC) values in brackets, with interpretation of extracts tested in combination with Scabiosa columbaria extracts. The ΣFIC could not be calculated for plants that did not have a definite MIC value (e.g. when MIC value is ≥ 8.00).

MIC values mg/ml and (ΣFIC values) Plant Plant species part Bc Ef Sa Se Ng Ec Pa used (ATCC (ATCC (ATCC (ATCC (ATCC (ATCC (ATCC 13778) 29212) 25923) 25923) 19424) 8739) 27853) Combination 1 S. columbaria + Roots N/A 4.00 N/A 2.00 4.00 N/A 4.00 A. hispida

Combination 2 S. columbaria + H. caespititium + Roots 2.00 2.00 ≥ 8.00 2.00 2.00 2.00 2.00 Z. albomaculata (0.88=A) (0.88=A) (1.13=NI) + D. anomala Combination 3 S. columbaria + C. paniculata Roots 2.00 2.00 2.00 1.00 ≥ 8.00 2.00 2.00 subsp sinuata + (1.16=NI) (0.99=A) (0.57=A) (0.99=A) S. divaricata Pathogens: Bc, Bacillus cereus, Ef, Enterococcus faecalis, Ec, Escherichia coli, Ng, Neisseria gonorrhoeae, Pa, Pseudomonas aeruginosa, Sa, Staphylococcus aureus, Se, Staphylococcus epidermidis. N/A- insufficient plant material, test not conducted

• Combinations

Afroaster hispida is used in combination with S. columbaria for rashes (combination 1). Due to lack of sufficient plant material, A. hispida was only tested for selected combinations (i.e. against S. epidermidis and P. aeruginosa, pathogens responsible for skin problems), however, in previous studies, extracts from the plant showed good activity against some pathogens while inactive against others. For example, using the disk diffusion method Shale et al. (1999), showed that the organic extracts (hexane and methanol) of A. hispida roots displayed high activity against S. epidermidis (0.80 mm), medium inhibition against S. aureus (0.58 mm) and P. aeruginosa (0.33 mm) and no inhibition against E. coli (0.00 mm). In Seleteng Kose (2017), A. hispida was tested against N. gonorrhoeae using the MIC assay and showed no antibacterial activity.

Table 4.2 shows the ΣFIC values of the plants used in combination with S. columbaria. The interpretation of the results for the ΣFIC are based on Van Vuuren and Viljoen (2011), where interactions were classified as synergistic (≤0.50), additive (>0.50 - 1.00), indifferent (>1.0 - ≤4.00) and antagonistic (>4.00). In this study, as shown in Table 4.2, none of the plant combinations had any synergistic interactions. Combinations 2 (S. columbaria + H. caespititium + Z. albomaculata + D. anomala) and 3 (S. columbaria + C. paniculata subsp sinuata + S. divaricata) showed additive effect against Enterococcus faecalis with ΣFIC values 0.88 and 0.99 respectively. All the combinations that had

61 additive effect may be used in traditional medicine due to their similarity in medicinal use with the hope of increased healing properties. Combinations 2 and 3 also had indifferent effects of 1.13 against E. coli and 1.16 against B. cereus. These ΣFIC values mean that the antibacterial effect of the combined extracts is more or less equal to the antibacterial activity of the individual plants. All the combinations of S. columbaria with other plants had previously not been studied for the antibacterial activity.

4.3.2 Anti-inflammatory activity Table 4.3: Anti-inflammatory activity of S. columbaria and plants used in combination with the species. IC50 was reported with enzyme inhibitory effects determined in the concentration of 12.5, 25, 50, and 100 μg/ml. The data is expressed as the mean of three replicates.

Voucher Aqueous extracts Methanol extracts Species name number 15-LOX COX-1 COX-2 15-LOX COX-1 COX-2 D. anomala KSK 002 1.19 1.38 22.86 1.18 1.04 70.63 C. paniculata subsp. sinuata NWM 001 1.10 49.04 1.05 48.71 35.80 1.08 E. ornithogaloides NWM 002 1.15 12.48 10.14 1.16 24.17 1.08 G. perpensa NWM 003 1.14 8.50 189.02 7.33 190.47 1.12 S. columbaria KSK 0035 23.43 1.03 182.41 24.25 1.03 194.37 S. divaricata NWM 004 12.74 1.04 48.29 13.26 25.04 11.58 Combination 3 (S. columbaria + C. paniculata 15.63 1.16 14.14 71.87 12.90 25.64 subsp sinuata + S. divaricata) Combination 4 (S. columbaria + E. ND 73.86 1.03 ND 1.02 26.30 ornithogaloides + G. perpensa) Combination 5 (S. columbaria + C. paniculata 1.07 1.04 194.79 192.21 1.05 43.10 subsp sinuata + D. anomala + S. divaricata) Positive control COX = aspirin 0.500 97.703 1.297 0.500 97.703 1.297 LOX = NDGA In bold- noteworthy active plant extracts when compared to the positive control NDGA- Nordihydroguaiaretic acid

ND = IC50 not determined

Table 4.3 shows the anti-inflammatory activity of the plants studied. The activities of the

plant’s extract were evaluated by comparing their IC50 values with the positive control. As

previously mentioned, the IC50 value represents the concentration of a test substance necessary to have an inhibition effect of 50% on the enzyme tested. Not all the plant extracts inhibited the enzymes tested, but their overall activity was somewhat noteworthy. This supports the traditional use of these plants to alleviate symptoms associated with inflammation resulting from the complex process of inflammation (such as, stricture of uterus and period pains). Aspirin was used as the positive control for cyclooxygenase inhibition and it was noted to have selective inhibition of COX-2, similar to a study by Hamsin et al., (2014). Some extracts that were noted to exhibit the same activity were extracts of C. paniculata subsp sinuata, aqueous extracts of combination 4 (S. columbaria + E. ornithogaloides + G. perpensa) as well as methanol extracts of G. perpensa and E. ornithogaloides. Although this is a desirable action, there has to be a moderate balance between the cyclooxygenase inhibition because highly selective inhibition against COX- 2 can cause disorders associated with myocardial infarctions (Abdelall et al., 2016).

Scabiosa columbaria had not previously been investigated for anti-inflammatory activity, neither individually nor in combination with other plants. Scabiosa columbaria exhibited weak activity against COX-2 with IC50 value of ≤194.37 μg/ml, while it showed noteworthy

activity against COX-1 with IC50 ≤1.03 μg/ml. It also had moderate anti-inflammatory activity against 15-LOX enzyme (IC50 ≤24.43 μg/ml). Plants like S. columbaria of which the extracts selectively inhibit COX-1 could possibly have negative side effects in humans. Inhibition of COX-1 on its own can lead to gastrointestinal bleeding, irritation and ulcerations. Which suggest that precautions have to be taken when this plant is taken as traditional medicine (Argoff et al., 2009).

Both aqueous and methanol extracts of Dicoma anomala had high inhibition activity

against COX-1 with an IC50 value of ≤1.38 μg/ml. The methanol extracts had low activity against COX-2 enzyme with IC50 value of 70.63 μg/ml. Comparable to this study, Shale et al. (1999), studied the anti-inflammatory activity of D. anomala using cyclooxygenase bioassay at 200 µg/ml. The leaves and roots aqueous extracts had noteworthy inhibition of ≤7%. The roots methanol extract had low inhibition of 27%. Both aqueous and methanol

extracts had noteworthy activity against 15-LOX. Dicoma anomala extracts are effective against COX-1 and 15-LOX but need higher concentration to inhibit COX-2.

Cussonia paniculata subsp sinuata had moderate activity against COX-1 with IC50 ≤49.04

μg/ml, however, the inhibition was high for COX-2 with IC50 ≤1.08 μg/ml. Cussonia

paniculata subsp sinuata inhibition of 15-LOX was prominent with an IC50 value of 48.71 μg/ml for methanol extracts and high for aqueous extracts (1.10 μg/ml). Similar results were observed in Tetyana at al. (2001), where the ethyl acetate and ethanolic extracts of the plant were screened for prostaglandin-synthesis inhibitory activity using the COX-1 assay at 0.2 µg/µg. The roots had low inhibition of ≤38% across all extracts tested while the organic extracts of leaves, bark and stem had high percentage inhibition of ≥78%. Cussonia paniculata subsp sinuata methanolic extract can be considered to be an effective anti-inflammatory agent because the results were even better than that of aspirin. It was able to inhibit both the cyclooxygenase and 15-LOX enzymes.

The anti-inflammatory activity of Eriospermum ornithogaloides is being reported for the first time in this study. The results show that extracts of this plant are noteworthy anti- inflammatory agents. Both the organic and aqueous extracts displayed noteworthy anti-

inflammatory activity as they inhibited both COX enzymes with an IC50 ≤24.48 μg/ml. Inhibition against the 15-LOX enzyme was also high for both aqueous and methanol

extracts with IC50 ≤1.15 μg/ml. The methanolic extract of E. ornithogaloides is an effective anti-inflammatory agent, with IC50 value better than that of aspirin, i.e. inhibition of both COXs and 15-LOX enzymes at low concentration.

Gunnera perpensa roots exhibited high activity against 15-LOX for both aqueous and organic extracts with IC50 ≤ 7.33 μg/ml. These results are comparable to Mzindle (2017), where extracts of G. perpensa showed high activity with a percentage inhibition of 71.5% and 66.8% for aqueous and methanol extracts respectively against 15-LOX at 1 mg/ml. In contrast, a previous study by Muleya et al. (2014), tested G. perpensa against 15-LOX

at a concentration of 25 µg/ml, and the results exhibited low inhibition with an IC50 value of 81.18 µg/ml. The different levels of activity may be attributed to the difference in concentrations tested assuming that the anti-inflammatory activity against LOX is dose- depend. For the cyclooxygenase inhibition, in this study, the organic extracts showed a

very low anti-inflammatory activity against COX-1 enzyme with an IC50 value of 190.47

μg/ml while the inhibition by aqueous extracts was high at IC50 ≤8.50 μg/ml. These results are similar to Ndhlala et al. (2011), where the aqueous extracts of the same plant exhibited high activity against COX-1 with inhibition ≥71%. The 80% ethanol and dichloromethane extracts had insignificant activity of ≤13% against COX-1. The aqueous extracts of G. perpensa had weak inhibition against the COX-2 enzyme while the

methanol extracts had high anti-inflammatory activity with IC50 1.12 μg/ml. Although it is more desirable to have agents that have a higher COX-2 inhibition than COX-1, such circumstances would still have negative side effects like renal failure, oedema or increased blood pressure that cannot be negated.

The extracts of Searsia divaricata selectively inhibit COX-1 enzyme with a concentration lower than that of aspirin, while higher concentrations of the extracts were needed to inhibit COX-2 as well as LOX enzyme. As previously stated these results are not favourable, suggesting that S. divaricata extracts are not effective anti-inflammatory agents.

For the evaluation of synergism, the methanol extracts of combinations 3 (S. columbaria + C. paniculata subsp sinuata + S. divaricata) and 4 (S. columbaria + E. ornithogaloides + G. perpensa) had a good anti-inflammatory activity, being able to inhibit both COX-1

(IC50 ≤12.90) and COX-2 (IC50 ≤26.30). The aqueous extracts of combinations 3 and 5 (S. columbaria + C. paniculata subsp sinuata + D. anomala + S. divaricata) where highly selective towards one of the cyclooxygenase enzymes, requiring a higher concentration for one or the other of which was previously shown to have negative side effects. These results do not support the use of these combinations as remedies for ailments associated with inflammation. Overall, combination 3 (S. columbaria + C. paniculata subsp sinuata + S. divaricata) exhibited noteworthy anti-inflammatory activity, being able to inhibit both LOX and COX enzymes at low concentrations.

Some plant extracts exhibited weak to no activity which does not mean that they do not have anti-inflammatory activity. Through the IC50 values given in the results, it can be seen that the doses (concentrations) used for some of the plants were not high enough to achieve the desired outcomes. The complete absence of anti-inflammatory action can

65 consequently be confirmed by utilizing higher dosages before completely ruling out the ability of the plant to inhibit the enzymes (Eldeen et al., 2005). Additionally, plant extracts studied may be active at other complex pathways in the human body that are responsible for inflammation and these were not investigated in this study. For example, inhibition of 5-LOX and nuclear factor NF-κB activity or the reduction of nitric oxide, cytokines and many more pro-inflammation pathways (Azab et al., 2016). The plants could also contain compounds that function better under in vivo conditions in which they may undergo metabolic transformation (Fawole et al., 2009).

4.3.3 Antacid activity

8,5

7,5

6,5 pH ofpH plant extracts

5,5

5 10 15 20 25 30 37 Temperature of plant extract Scabiosa columbaria Searsia divaricata Cussonia paniculata Dicoma anomala Combination 5 Combination 3 Rennie®

Figure 4.3: Effect on the pH of extracts at temperatures ranging from 10°C to 37°C.

The results depicted in Figure 4.3 indicate that the pH of the extracts was not affected by temperature. All the evaluated extracts had an acidic pH of up to ≤6.42 while the positive control (Rennie®) had high alkalinity with pH of up to 8.93. The pH of the extracts

66 remained relatively constant throughout the temperature changes, with only decimal points changes being observed. For example, S. columbaria pH ranged between 5.97- 6.14 while Rennie® pH was between 8.84-8.91. This signified that the plant extracts were good candidates for traditional use as antacids.

Table 4.4: Antacid and acid neutralization activity of S. columbaria as well as plants used in combination with the species using back titration. The data was presented as mean± standard deviation of triplicates.

Back titration of acid using Back titration of acid using Immediate standardized NaOH artificial gastric juice neutralization Volume of Volumes of Plants species Moles of H+ ions effect on NaOH gastric juice acid consumed gastric juice consumed consumed neutralized (m/mol) (ml) (ml) C. paniculata subsp. sinuata 1.52 10.40 ± 0.71 0.0027 10.63 ± 1.10 0.67 D. anomala 1.43 10.55 ± 0.92 0.0025 6.00 ± 1.65 0.38 S. columbaria 1.49 10.20 ± 0.71 0.0029 7.93 ± 1.50 0.50 0.0024 S. divaricata 1.51 10.70 ± 1.41 3.57 ± 0.81 0.23

Combination 3 (S. columbaria + C. paniculata subsp sinuata 1.59 10.65 ± 0.78 0.0024 6.50 ± 1.75 0.41 + S. divaricata) Combination 5 (S. columbaria + C. paniculata subsp sinuata 1.60 07.35 ± 2.90 0.0057 7.07 ± 0.31 0.45 + D. anomala + S. divaricata) 0.0145 ® Rennie 1.77 ND (Houshia et al., 3.92 62.13 ± 7.04 2012)

Water 1.51 ND 5.60 0.35

ND-Not determined; shaded area is Scabiosa species

Currently there are no set standard values that define a good antacid agent. Values of samples being studied are usually compared to commercial antacids. When assessing products/samples for their antacid properties using the normal (NaOH) titration procedure, carbon dioxide is produced and interferes with the process. The back titration

method used in this study helps drive off the carbon dioxide through addition of excess acid, while boiling the solution before back titration promotes accuracy of the results (Kenkel, 2002).

InTable 4.4, the results for the immediate neutralization effects shows both combinations 3 (S. columbaria + C. paniculata subsp sinuata + S. divaricata) and 5 (S. columbaria + C. paniculata subsp sinuata + D. anomala + S. divaricata) had noteworthy neutralization capacity with pH ≤1.60 and Rennie® at pH 1.77. The neutralization capacity of the combinations was better than that of all the plants when used individually. For example, S. columbaria had immediate neutralization effects of pH 1.49 and Dicoma anomala had pH 1.43.

For back titration using NaOH show that the positive control i.e. the commercial drug Rennie® has antacid capacity of 0.0145 moles. Despite the traditional use as a heartburn remedy, the plant extracts tested in this study showed little to no antacid capacity when compared to Rennie. A positive observation was that none of the plants studied had any acidic composition released into the solution, therefore they would not contribute negatively to the intensity of the heartburn (i.e. acidic content in the stomach). Compared to other extracts, combination 5 (S. columbaria + C. paniculata subsp sinuata + D. anomala + S. divaricata) showed the highest amount of antacid capacity (50% of Rennie) by neutralizing 0.005720 moles of acid. While extracts from other plants as well as combination 3 (S. columbaria + C. paniculata subsp sinuata + S. divaricata) had similar antacid capacity with ≤0.002887 moles of acid neutralized.

The neutralization capacity evaluated through Fordtran’s in vitro titration model revealed that C. paniculata subsp sinuata had better activity when compared to all the other plant species. It was able to neutralize 10.63 ml of artificial gastric juice where 0.67 m/mol of H+ ions were consumed. This was followed by S. columbaria which neutralized 7.93 ml artificial gastric juice, consuming 0.50 m/mol H+ ions. Dicoma anomala and Searsia divaricata were only able to neutralize ≤6.00 ml artificial gastric juice, consuming ≤0.38 m/mol H+ ions. All the plant extracts except S. divaricata consumed higher H+ ions than water, thus exhibiting potent neutralizing capacities. This is contrary to a study by Karamanolis et al. (2008), where water was reported to increase the stomach pH to >4

within a minute. The results also show that S. columbaria performs best when used individually than in combination with other plants because both combinations were only able to consume ≤7.07 ml of gastric juice. When compared with the commercial product Rennie®, the antacid capacity of the plant extracts was found to be weak. Rennie® was able to consume 62.13 ml artificial gastric juice with 3.92 m/mol H+ ions consumed. Rennie® tablets contain 680 mg calcium carbonate and 80 mg of magnesium carbonate (rennie.co.za). Calcium carbonate (chalk) is known to have a rapid and prolonged impact in increasing the Ph of gastric juices. However, it has been reported to cause constipation as a side effect (Washington, 1991), while magnesium carbonate is reported to have mild laxative effect. Hence these two compounds are combined in the product to counteract each other’s negative effects (Mahadik and Kuchekar, 2008). The Rennie® tablet falls under the category of non-systemic antacids, these kinds of antacids remain only in the gastrointestinal tract and cannot be absorbed into blood circulatory system because of their cationic nature (Barar, 2000). The chemical composition of the plant extracts is unknown therefore it is not possible to deduce any side effects they may possibly cause as well as the path they take in the human body to achieve their antacid properties.

Some of these plants displayed low or no antacid activity despite their continued traditional use for the treatment of heartburn. This suggests that they may have a different biological or physiological mode of action. For example, these plants could be functioning as biological blockers or alginates. Plants that function as biological blockers i.e. histamine 2 (H2) blockers, function through acid reduction that decreases acid concentration in the stomach. They competitively block the H2 receptors of the parietal cells in the stomach resulting in a reduced gastric acid secretion (Romich, 2012). Those that function as alginates, form a precipitation in the presence of gastric acid causing formation of a gel layer which prevents the acid from irritating the oesophagus (Mandel et al., 2000).

4.3.4 Toxicity: Brine shrimp assay Table 4.5: Average mortality rate of the aqueous and organic extracts of all the plants selected.

Percentage mortality Plant part Plant name Organic extracts Aqueous extracts used 24 hrs 48 hrs 24 hrs 48 hrs Afroaster hispida Roots ND ND 6.50 12.67 C. paniculata subsp sinuata Leaves 42.71 92.58 97.51 100.00 C. paniculata subsp sinuata Roots 96.14 100 71.19 87.91 Dicoma anomala Roots 57.98 83.93 0.61 0.61 Eriospermum Roots ND ND 4.30 4.77 ornithogaloides Gunnera perpensa Roots 1.49 1.49 1.56 2.47 Helichrysum caespititium Roots 22.03 40.97 0.00 2.56 Scabiosa columbaria Leaves 0.00 1.18 0.71 1.88 Scabiosa columbaria Roots 0.16 0.16 0.00 1.36 Scabiosa incisa Leaves 2.13 3.02 1.57 2.95 Scabiosa incisa Roots 0.40 0.83 ND ND Searsia divaricata Roots 0.00 10.21 0.00 3.69 Zantedeschia albomaculata Roots 1.20 3.36 0.46 2.48 Combination 1 (S. Roots ND ND 1.41 7.36 columbaria + A. hispida) Combination 2 (S. columbaria + H. caespititium Roots 1.75 27.78 9.01 37.75 + Z. albomaculata + D. anomala) Combination 3 (S. columbaria + C. paniculata Roots & 0.48 3.62 18.75 46.48 subsp sinuata + S. leaves divaricata) Negative control Salt water 0.00 0.00 1.12 1.67 Potassium Positive control 100.00 dichromate Toxic species are given in bold; ND= Not determined due to insufficient plant material; shaded areas are Scabiosa species

Of the ten plants evaluated for their toxicity levels in this study, two of the plants assesed were found to be toxic (Table 4.5). The results were interpreted according to Bussman et al. (2011), where toxicity is defined by mortality rate of >50% of brine shrimp that are in contact with extracts for up to 48 hrs. The organic and aqueous extracts (leaves and roots) of C. paniculata subsp sinuata were found to be highly toxic at 48 hrs with mortality rates of ≥92.58% and ≥87.91% respectively. The toxicity of C. paniculata had previously been assessed by Adedapo et al. (2008), whereby rats were given different doses of the plant aqueous extracts in an acute toxicity test. The overall mortality of the rats was 80%, with death of all rats that consumed extracts at 400 to 3200 mg/kg p.o., while those given a 200 mg/kg dose survived after 72 hrs of exposure.

Dicoma anomala organic extract was toxic at 83.93%, while the aqueous extract was not toxic. These results support the use of water by most traditional medicine practitioners for plant extraction when administering medication, assuming that the organic solvents extract phytochemicals at concentration which may be high, resulting in the plants being toxic. The results of this study are similar to Balogun et al. (2016), where the aqueous extracts of this plant were given orally to Wistar rats. The results showed no sign of toxicity

(i.e no mortality). It was assumed that the LD50 of D. anomala aqueous extracts was >2000 mg/kg p.o.

In this study, both organic and aqueous extracts of Gunnera perpensa were found to be non-toxic. In a study by McGaw (2005), different solvents were used (i.e hexane, dichloromethane, acetone, methanol, ethanol/water) to test the toxicity of the root extracts against brine shrimp. The results were dose dependent with 100% mortality obtained for all the extracts except hexane at 10 mg/ml while at 1 mg/ml, only acetone and methanol were toxic with a mortality rate of ≥69%.

The toxicity of Scabiosa columbaria and Scabiosa incisa is being reported here for the first time. Both S. columbaria and S. incisa extracts were non-toxic. The mortality rate of leaves was slightly higher than that of roots in both plants. These results suggest that the plant extracts can be safely used with no concern of toxicity.

The extracts of Helichrysum caespititium were found to be non-toxic with the highest mortality rate recorded for organic extracts with 40.97% at 48 hrs. Helichrysum

71 caespititium was previously studied by Mamabolo et al. (2017), where dichloromethane, methanol and aqueous extracts of the whole plant were found to be non-toxic with LC50

≥357.39 ± 2.81 μg/ml while the hexane extracts had a lower LC50 value (82.86 ± 3.36

μg/ml). These were compared to the positive control which had an LC50 value of 10.80 ± 1.63 μg/ml. Although these mortality rates are non-toxic, this plant should be used with caution.

Afroaster hispida, Eriospermum ornithogaloides, Searsia divaricata and Zantedeschia albomaculata (plants used in combination with S. columbaria) were assessed for the first time in this study and were found to be non-toxic.

For the toxicity of the combined extracts, combination 1 (S. columbaria + A. hispida) aqueous extracts and combination 2 (S. columbaria + H. caespititium + Z. albomaculata + D. anomala) organic extracts had a low percentage mortality of 7.36% and 27.78% respectively. This was a positive observation because sometimes a combination of plant extracts can cause antagonistic effects due to the interaction/accumulation of compounds (Kent, 1998). Combination 3 (S. columbaria + C. paniculata subsp sinuata + S. divaricata) aqueous extracts had moderate toxicity (46.48%) due to the presence of the highly toxic plant C. paniculata subsp sinuata. It could be assumed that the combination during ethnomedicinal use helps mitigate the toxic effects of C. paniculata subsp sinuata.

100

30 Percentage mortality rate (%) 20

0 2 1 0,5 0,25 0,125 0,0625 Plant extracts concentration mg/ml

CPdA CPdO DAdO

Figure 4.4: Dosage response of the toxic plant extracts where CpdA = C. paniculata subsp sinuata aqueous dilution; CpdO = C. paniculata subsp sinuata organic dilution and DadO = D. anomala organic dilution.

For species that showed levels of toxicity (extracts of C. paniculata subsp sinuata and organic extracts of D. anomala), a dose response assessment was conducted, and the results are presented in Figure 4.4. For all the plants, the mortality rate decreases with a decrease in concentration of the plant extracts. For example, when the aqueous extracts of C. paniculata subsp sinuata were diluted from 2 mg/ml to 1 mg/ml, the toxicity had a drastic decrease from 100% to 5.94%.

4.4 Chapter summary • Scabiosa incisa, S. columbaria and the combinations were screened for their biological activities for the first time in the present study. The antibacterial results do not support the traditional medicinal use of all the evaluated plants species of Scabiosa and combinaton of S. columbaria with other plants. • Scabiosa columbaria was the only southern African species reported to remedy ailments which are caused by inflammation and therefore the only one tested for

the first time for its antiinflamatory activity in this study. It possessed selective inhibition of COX-1 and 15-LOX enzyme while it needed higher concentration to

inhibit COX-2 with IC50 value of ≤194.37 μg/ml. From the three combinations tested, combination 3 (S. columbaria + C. paniculata subsp sinuata + S. divaricata) exhibited noteworthy anti-inflammatory activity, being able to inhibit both LOX and COX enzymes at low concentrations. • All the plants tested displayed weak antacid capacity. Compared to the other extracts, combination 5 (S. columbaria + C. paniculata subsp sinuata + D. anomala + S. divaricata) displayed the highest amount of antacid capacity (50% of Rennie) by neutralizing 0.005720 moles of acid using back titration of NaOH. • None of the Scabiosa sp tested were found to be toxic. Two species used in combination with Scabiosa columbaria were found to be toxic at ≥2 mg/ml.

CHAPTER 5 CONCLUSION

5.1 Summary

The aim of the study was to compile a synopsis of medicinally important Scabiosa species found in southern Africa and asses their biological activities and validate their medicinal uses. The biological activities investigated were antibacterial, anti-inflammatory and antacid activities, as well as toxicity of the plants. The findings are summarized as follows:

5.1.1 The synopsis In southern Africa, Scabiosa species leaf morphology appeared to be of diagnostic importance in distinguishing closely related Scabiosa species of southern Africa. Scabiosa albanensis can be easily recognized by its more erect habit, lack of lyrate– pinnatisect leaves and isolated geographical distribution in Eastern Cape. Scabiosa columbaria has grey green heterophyllous leaves that form a rosette basally and occur abundantly in all provinces. Scabiosa incisa can be distinguished by its bright green leaves that are in spreading growth form, as well as lyrate–pinnate leaves. Scabiosa transvaalensis stem ridges are more prominent as compared to other species, leaves occur throughout the whole stem and form pseudo–whorls of four and is distributed in Limpopo and Mpumalanga Provinces. There seems to be no distinct variation in their reproductive morphology.

5.1.2 Ethnobotany This study identified knowledge gaps in the validation of the efficacy of medicinally important Scabiosa species in southern Africa and identified use of plants in combination with Scabiosa columbaria. Fifteen plants used in combination with S. columbaria are from various families with Asteraceae being the most common family used. The most recorded Scabiosa species used medicinally in southern Africa is S. columbaria, which is not surprising because of its widespread distribution. It is abundant and readily available for people to use.

5.1.3 Biological activity The poor antibacterial as well as antacid activities of S. columbaria when tested individually and in combination with other plants species did not support the popular use of the plant in traditional medicine. However, it exhibited anti-inflammatory activity through selective inhibition of COX-1 and 15-LOX. The selective inhibition of COX-1 is associated with some negative side effects and therefore S. columbaria should be used with caution. It was encouraging to note that neither leaves nor roots of S. columbaria were found to be toxic. Although three extracts of plants used with S. columbaria were toxic, they were found to be dose dependent and non-toxic at 1mg/ml.

5.2 Limitations • Titration error may have compromised the antacid results because sometimes with back titration it is impossible to back-titrate the antacid if it is not fully dissolved. • Not being able to locate the other Scabiosa species and some of the other plants used in combination with Scabiosa columbaria.

5.3 Recommendations for future work • More concerted efforts to try and locate the other three medicinally important Scabiosa species in the field. • A re-evaluation of conservation status will be necessary since the Red List of South African Plants listed S. albanensis and S. incisa as least concern (population high and unlikely to go extinct soon), while S. transvaalensis is listed as vulnerable D2, in a single small population with an area of occupancy of <20km2 or the number of locations it’s found in are ≤5. • Testing the antimicrobial activity of Scabiosa columbaria using other solvents against a wide range of pathogens associated with the various ailments for which the plant is used. • Test different Scabiosa spp against scabies causing pathogens. • Assess Scabiosa columbaria and the combinations using an artificial stomach model, as well as in vivo antacid test to see how the plants can perform in complex environments

• Previous literature from another study indicated that during interviews conducted with traditional medical practitioners, A. hispida and Z. albomaculata (plants used with S. columbaria) were reported to be toxic at high doses. It would be interesting in future to validate these claims using other toxicity studies such as MTT assay, because the concentration used within this study showed these plants to be non- toxic. • Most of the results in this study were negative (although novel), of which other researchers may have previously investigated but could not publish because of poor results. To avoid the scenario of repetition, studies of negative results should also be published.

5.4 General conclusion This study has highlighted that the medicinal Scabiosa species can easily be distinguished from each other. The uses of Scabiosa plants for medicinal purposes in southern Africa is common among the Basotho people. The fact that the results of the evaluation of the antibacterial and antacid activities did not support or validate the use of these plants in traditional medicine, may not necessarily mean that the plants do not work. Many factors could have contributed to these results, for example, in the screening of antibacterial activity, as mentioned earlier, seasonal variations and extraction solvent impact extraction and presence of phytochemical compounds needed. While for the assessment of antacid and anti-inflammatory activities, the plants may work through different pathways that were not explored in this study. The overall results of brine shrimp lethality assay show that water extracts are less toxic than organic extracts suggesting that water extracts compounds in concentrations that are less/not toxic. The toxicity of the extracts is dependent on the dosage concentration.

CHAPTER 6

REFERENCE LIST

Abdelall, E.K.A., Lamie, P.F. and Ali, W.A.M. 2016. Cyclooxygenase-2 and 15- Lipoxygenase inhibition, synthesis, anti-inflammatory activity and ulcer liability of new celecoxib analogues: determination of region-specific pyrazole ring formation by NOESY. Bioorganic and Medicinal Chemistry Letters. 26: 2893-2899.

Abdel-Salam, O.M.E. 2014. Capsaicin as a Therapeutic Molecule. Springer. Basel. Pg 140-141.

Adebayo, S.A., Dzoyem, J.P., Shai, L.J. and Eloff, J.N. 2015. The anti-inflammatory and antioxidant activity of 25 plant species used traditionally to treat pain in southern African. BMC Complementary and Alternative Medicine. 15: 1-10.

Adedapo, A.A., Sofidiya, M.O., Maphosa, V., Moyo, B., Masika, P.J. and Afolayan, A.J. 2008. Anti-Inflammatory and analgesic activities of the aqueous extract of Cussonia paniculata stem bark. Records of Natural Products. 2 (2): 46-53.

Afolayan, A.J., Grierson, D.S. and Mbeng, W.O. 2014. Ethnobotanical survey of medicinal plants used in the management of skin disorders among the Xhosa communities of the Amathole District, Eastern Cape, South Africa. Journal of Ethnopharmacology. 153: 220-232.

Alipoor Birgani, A., Sartipnia, N., Hamdi, S.M.M., Naghizadeh, M. and Arasteh, J. 2019. Antimicrobial activity of Scabiosa Olivieri extract and its effect on TNF-α and IL-1 expression in human peripheral blood cells (PBMCs). Journal of Fasa University of Medical Sciences. 9(4): 1749-1756

Appleton, N. and Jacobs, G.N. 2005. Stopping Inflammation: Relieving the Cause of Degenerative Diseases. Square One Publishers, Inc. Garden City Park, New York. Pg 39-53.

Argoff, C.E., Dubin, A., Pilitsis, J. and McCleane, G. 2009. Pain Management Secrets E-book. 3rd Ed. Mosby Elsevier. Philadelphia. Pg 253.

Armitage, A.M. 2008. Herbaceous Perennial Plants: A Treatise on Their Identification, Culture, and Garden Attributes. 3rd Ed. Stipes Publishing. Georgia. Pg 254.

Atta-ur-Rahman., Choudhary, M.I. and Perry, G. 2015. Recent Advances in Medicinal Chemistry. Elsevier. Amsterdam. Pg 313-314.

Azab, A., Nassar, A. and Azab, A.B. 2016. Anti-Inflammatory activity of natural products. Molecules. 21(10): 1321.

Balogun, F.O. and Ashafa, A.O.T. 2017. Aqueous root extracts of Dicoma anomala (Sond.) extenuates postprandial hyperglycaemia in vitro and its modulation on the activities of carbohydrate-metabolizing enzymes in streptozotocin-induced diabetic Wistar rats. South African Journal of Botany. 112: 102-111.

Balogun, F.O., Omotayo, A. & Ashafa, T. 2016. Acute and Subchronic Oral Toxicity Evaluation of Aqueous Root Extract of Dicoma anomala Sond. In Wistar Rats. Evidence-Based Complementary and Alternative Medicine: Hindawi Publishing Corporation. 2016(7): 1-11

Balouiri, M., Sadiki, M. and Ibnsouda, S.K. 2015. Methods for in vitro evaluating antimicrobial activity: a review. Journal of Pharmaceutical Analysis. 6: 71-79.

Barar, F.S.K. 2000. Essentials of Pharmacotherapeutics. S. Chand & Company Pvt. Ltd. New Delhi. Pg 550.

Bassett, S. 2007. Cliffs Study Solver: Anatomy and Physiology. Wiley Publishing Inc. Hoboken, New Jersey. Pg 291.

Bayless, T.M. and Diehl, A.M. 2005. Advance Therapy in Gastroenterology and Liver Disease. 5th Ed. BC Decker Inc. Hamilton. Pg 162-163.

Benli, M., Bingol, U., Geven, F., Guney, K. and Yigit, N. 2008. An investigation on the antimicrobial activity of some endemic plant species from Turkey. African Journal of Biotechnology. 7(1): 001-005.

Besbes, H.M., Omri, A., Jannet, H.B., Lamari, A., Aouni, M. and Selmi, B. 2013. Phenolic composition, antioxidant and anti-acetylcholinesterase activities of the Tunisian Scabiosa arenaria. Pharmaceutical Biology. 51(5): 525-532.

Besbes, M., Omri, A., Cheraif, I., Daami, M., Jannet, H.B., Mastouri, M., Aouni, M. and Selmi, B. 2012. Chemical composition and antimicrobial activity of essential oils from Scabiosa arenaria Forssk. growing wild in Tunisia. Chemistry and Biodiversity. 9(4): 829-839.

Best-Boss, A. and Edelberg, D. 2009. The Everything Digestive Health Book: What You Need to Know to Eat Well, Be Healthy, and Feel Great. Adams Media. Massachusetts.

Bhat, R.B. 2014. Medicinal plants and traditional practices of Xhosa people in the Transkei Region of Eastern Cape, South Africa. Indian Journal of Traditional Knowledge. 13(2): 292-298.

Brennan-Krohn, T., Truelson, K.A., Smith, K.P. and Kirby, J.E. 2017. Screening for synergistic activity of antimicrobial combinations against carbapenem-resistant Enterobacteriaceae using inkjet printer-based technology. Journal of Antimicrobial Chemotherapy. 72: 2775-278.

Bruton-Seal, J. and Seal, M. 2009. Backyard Medicine: Harvest and Make Your Own Herbal Remedies. Skyhorse. New York. Pg 457-462.

Burns, D.L. and Shah, N.L. 2007. 100 Questions & Answers About Gastroesophageal Reflux Disease (GERD): A Lahey Clinic Guide. Jones and Barlett Publishers, Inc. Sudbury, Massachusetts. Pg 4.

Bussmann, R.W. and Glenn, A. 2010. Medicinal plants used in Northern Peru for reproductive problems and female health. Journal of Ethnobiology and Ethnomedicine. 6:30.

Bussmann, R. W., Malca-Garcıa, G., Glenn, A., Sharon, D., Chait, G., Dıaz, D., Pourmand, K., Jonat, B., Somogy, S., Guardado, G., Aguirr, C., Chan, R., Meyer, K., Kuhlman, A., Townesmith, A., Effio-Carbajal, J., Frıas-Fernandez, F. and Benito, M.

2010. Minimum inhibitory concentrations of medicinal plants used in Northern Peru as antibacterial remedies. Journal of Ethnopharmacology. 132 (1): 101-108.

Bussmann, R.W., Malca, G., Glenn, A., Sharon, D., Nilsen, B., Parris, B., Dubose, D., Ruiz, D., Saleda, J., Martinez, M., Carillo, L., Walker, K., Kuhlman, A. and Townesmith, A., 2011. Toxicity of medicinal plants used in traditional medicine in Northern Peru. Journal of Ethnopharmacology. 137: 121 – 140.

Byng, J.W. 2014. The Flowering Plants Handbook: A Practical Guide to Families and Genera of the World. Plant Gateway Ltd. United Kingdom. Pg 509.

Calixto, J.B., Campos, M.M., Otuki, M.F. and Santos, A.R.S. 2004. Anti-inflammatory compounds from plant origin. Part II. Modulation of proinflammatory cytokines, chemokines and adhesion molecules. Planta Medica. 70(2): 93-103.

Carlson, S.E., Linder, H.P. and Donoghue, M.J. 2012. The historical biogeography of Scabiosa (Dipsacaceae): implications for Old World plant disjunctions. Journal of Biogeography. 39(6): 1086-1100.

Carlson, S.E., Mayer, V. and Donoghue, M.J., 2009. Phylogenetic relationships, taxonomy, and morphological evolution in Dipsacaceae (Dipsacales) Inferred by DNA sequence data. Taxon. 58(4): 1075-1091.

Che, C.T., Wang, Z.J., Chow, M.S. and Lam, C.W. 2013. Herb-herb combination for therapeutic enhancement and advancement: theory, practice and future perspectives. Molecules. 18(5): 5124-5141.

Chen, S., Yu, H., Luo, H., Wu, Q., Li, C. and Steinmetz, A. 2016. Conservation and sustainable use of medicinal plants: problems, progress and prospects. Chinese Medicine. 11(37): 1-10.

Cherian, G. 2013. Chapter 28 - Omega-3 Fatty Acids and Early Life Nutritional Programming: Lessons from The Avian Model. In: Watson, R.R. and Preedy, V.R. 2013. Bioactive Food as Dietary Interventions for Liver and Gastrointestinal Disease. Academic Press. Boston. Pg 437-448.

Chinsamy, M., Finnie, J.F. and Van Staden, J. 2011. The ethnobotany of South African medicinal orchids. South African Journal of Botany. 77: 2-9.

Chinsamy, M., Finnie, J.F. and Van Staden, J. 2014. Anti-Inflammatory, antioxidant, anti-cholinesterase activity and mutagenicity of South African Medicinal orchids. South African Journal of Botany. 91: 88-98.

Corrigan, B.M., Van Wyk, B.E., Geldenhuys, C.J. and Jardine, J.M. 2011. Ethnobotanical plant uses in the KwaNibela Peninsula, St Lucia, South Africa. South African Journal of Botany. 77: 346-359.

Dai, J., Lin, H., Nui, G., Wu, X., Wu, Y. and Zhang, H. 2015. Total alkaloids in Dipsacus asperoides and their effects on proliferation of osteosarcoma SaOS-2 cell lines and gene expression of VEGF. Biomedical Research. 26(1): 37-42.

De Villiers, B.J., Van Vuuren, S.F., Van Zyl, R.L. and Van Wyk, B.E. Antimicrobial and antimalarial activity on Cussonia species (Araliaceae). Journal of Ethnopharmacology. 129: 189-196.

De Wet, H., Nkwanyana, M.N. and Van Vuuren, S.F. 2010. Medicinal plants used for the treatment of diarrhoea in Northern Maputaland, KwaZulu-Natal Province, South Africa. Journal of Ethnopharmacology. 130: 284-289.

De Zoysa, M.H.N., Rathnayake, H., Hewawasam, R.P. and Wijayaratne, W.M.D.G.B. 2019. Determination of in vitro antimicrobial activity of five Sri Lankan medicinal plants against selected human pathogenic bacteria. International Journal of Microbiology. 2019(5): 1-8.

Dev, A.B. 2002. Yoga for Better Health. Diamond Pocket books (P) Ltd. New Delhi. Pg 13.

Devesa, J. A. 1984. Revisión del genero Scabiosa en la Peninsula Ibérica e Islas Baleares. Lagascalia. 12(2): 143-12.

Dimo, T., Fotio, A.L., Nguelefack, T.B. Asongalem, E.A. and Kamtchouing. 2006. Anti- inflammatory activity of Kalanchoe crenata Andr. Indian Journal of Pharmacology. 38(2): 115-119.

El bous, M.M. and Gazer, M. 2016. Taxonomic studies on Dipsacaceae Juss. in Egypt. Egyptian Journal of Botany and Microbiology. 2: 307-331.

Eldeen, I.M.S., Elgorashi, E.E. Van Staden, J. 2005. Antibacterial, anti-inflammatory, anti-cholinesterase and mutagenic effects of extracts obtained from some trees used in South African traditional medicine. Journal of Ethnopharmacology. 102: 457-464.

Eloff J.N. 1998. A sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extracts for bacteria. Planta Medica. 64(8): 711-713.

Fawole, O.A., Ndhlala, A.R., Amoo, S.O., Finnie, J.F. and Van Staden, J. 2009. Anti- inflammatory and phytochemical properties of twelve medicinal plants used for treating gastro-intestinal ailments in South Africa. Journal of Ethnopharmacology. 123: 237-243.

Foden, W. & Potter, L. 2005. Scabiosa incisa Mill. National Assessment: Red List of South African Plants version 2020.1. Retrieved from: Date accessed: http://redlist.sanbi.org/species.php?species=2742-13. 17/08/2020.

Germishuizen, G. and Meyer, N.L. 2003. Plants of southern Africa: an annotated checklist. Strelitzia. National Botanical Institute, Pretoria. 14: 80-83.

Germishuizen, G. and Meyer, N.L. 2003. Plants of Southern Africa: An Annotated Checklist. Strelitzia. National Botanical Institute, Pretoria. 14: 80-83.

Ghorani-Azam, A., Sepahi, S., Riahi-Zanjani, B., Alizadeh Ghamsari, A., Mohajeri, S.A., and Balali-Mood, M. 2018. Plant toxins and acute medicinal plant poisoning in children: a systematic literature review. Journal of Research in Medical Sciences. 23: 26.

Goldblatt, P. and Manning, J.C. 2019. Temperate Garden Plant Families: The Essential Guide to Identification and Classification. Timber Press, Inc. Portland. Pg 101-105.

González-Gallego, J., Sánchez-Campos, S. and Tuῆón, M.J. 2007. Anti-inflammatory properties of dietary flavonoids. Nutrición Hospitalaria. 22(3): 287-293.

Griswold, D.E., Martin, L.D., Badger, A.M., Breton, J. and Chabot-Fletcher, M. 1998. Evaluation of the cutaneous anti-inflammatory activity of azapiranes. Inflammation Research. 47(2): 56–61.

Gunathilake, K.D.P.P., Ranaweera, K.K.D.S. and Vasantha Rupasinghe, H.P. 2018. In Vitro anti-inflammatory properties of selected green leafy vegetables. Biomedicine. 6: 107.

Hafsa, M.B., Besbes, M., Mahjoub, M.A. and Mighri, Z. 2009. Antimicrobial, antioxidant activities and total phenolic content of the acetonic, ethanolic and aqueous extracts of Scabiosa arenaria (Forsk). Tunisian Journal of Medicinal Plants and Natural Products. TJMPNP 1(2009): 59-68

Halpern, G.M. 2004. Ulcer free!: nature's safe & effective remedy for ulcers. Square One Publishers Inc., Garden City Park, New York. Pg 71-82.

Hamidi, M.R., Jovanova, B. and Panovska, T.K. 2014. Toxicоlogical evaluation of the plant products using brine shrimp (Artemia salina L.) model. Macedonian Pharmaceutical Bulletin. 60(1): 9-18.

Hamsin, D.E.Z.A, Hamid, R.A., Yazan, L.S., Mat Taib, C.N. and Yeong, L.T. 2014. Ardisia crispa roots inhibit cyclooxygenase and suppress angiogenesis. BMC Complementary and Alternative Medicine. 14(1): 102.

Hargreaves, A.D. 2007. Digest Alive the Natural Cure to Heartburn. Lulu.com. Morrisville, North Carolina. Pg 34-38.

Hlila, M.B., Mosbah, H., Majouli, K., Msaada, K., Jannet, B.H., Aouni, M. and Selmi, B. 2015. α-Glucosidase inhibition by Tunisian Scabiosa arenaria Forssk. extracts. International Journal of Biological Macromolecules. 77: 383-389.

Houshia, O.J., AbuEid, M., Zaid, O., Zaid, M. and Al-daqqa, N. 2012. Assessment of the value of the antacid contents of selected Palestinian plants. American Journal of Chemistry. 2(6): 322-325.

Hübsch, Z., 2014. Antimicrobial efficacy and toxicity profiles of conventional antimicrobial agents in combination with commercially relevant southern African medicinal plants. MSc (Pharmacy). University of the Witwatersrand, Johannesburg. Retrieved from: http://wiredspace.wits.ac.za/bitstream/handle/10539/15308/Zelna%20Hubsch%20%283 00896%29%20dissertation%2008.04.14.pdf?sequence=1&isAllowed=y. Date accessed: 13/12/2020

Inácio, M.C., Paz, T.A., Bertoni, B.W. and Pereira, A.M.S. 2016. Effect of environmental and phenological factors on the antimicrobial activity of Cochlospermum regium (Schrank) Pilg. roots. Acta Scientiarum. Agronomy. 38(4): 467-473.

Kadereit, J.W. and Bittrich, V. 2016. The Families and Genera of Vascular Plants. Springer Nature. Switzerland. Pg 153-154.

Karamanolis, G., Theofanidou, I., Yiasemidou, M., Giannoulis, E., Triantafyllou, K. and Ladas, D.S. 2008. A glass of water immediately increases gastric pH in healthy subjects. Digestive Diseases and Science. 53: 3128-3132.

Kayce, P. and Kirmizigul, S. 2010. Chemical constituents of two endemic Cephalaria species. Records of Natural Products. 4(3): 141-148.

Kenkel, J. 2002. Analytical Chemistry for Technicians. CRC Press, Inc. Boca Raton, Florida. Pg 124.

Kent, C. 1998. Basics of Toxicology. John Wiley & Sons. Canada. Pg 80-82.

Kılınç, H., Masullo, M., D’Urso, G., Karayildirim, T., Alankus, O. and Piacente, S. 2020. Phytochemical investigation of Scabiosa sicula guided by a preliminary HPLC-ESIMSn profiling. Phytochemistry. 174: 112350.

Ku, S., Seo., B., Park, J., Park, G., Seo, Y., Kim, J., Lee, H. and Roh, S. 2009. Effect of Lonicerae Flos extracts on reflux esophagitis with antioxidant activity. World Journal of Gastroenterology. 15(38): 4799-4805.

Kuete, V. and Efferth, T. 2010. Cameroonian medicinal plants: pharmacology and derived natural products. Frontiers in Pharmacology. 1: 123.

Kumar, S. and Egbuna, C. 2019. Phytochemistry: An In-Silico and In-Vitro Update: Advances in Phytochemical Research. Springer Nature Singapore Pte Ltd. Singapore. Pg 291-292.

Lawal, I. O., Grierson, D. S., and Afolayan, A. J. 2014. Phytotherapeutic information on plants used for the treatment of tuberculosis in Eastern Cape Province, South Africa. Hindawi Publishing Corporation. 2014(5): 1-11.

Lawal, I.O., Grierson, D.S. and Afolayan, A.J. 2014. Phytotherapeutic information on plants used for the treatment of tuberculosis in Eastern Cape Province, South Africa. Hindawi Publishing Corporation. Evidence-Based Complementary and Alternative Medicine. Retrieved from: https://www.hindawi.com/journals/ecam/2014/735423/. Date accessed: 30/03/2020.

Leelaprakash, G. and Mohan Daas, S. 2011. In vitro anti-inflammatory activity of methanol extract of Enicostemma axillare. International Journal of Drug Development & Research. 3(3): 189-196.

Leistner, O.A. and Morris, J.W. 1976. South African Place Names. Annals of the Cape Provincial Museums. 12: 1-565.

Lennox, S., Wadley, L. and Bamford, M. 2015. Charcoal analysis from 49 000-year-old hearths at Sibudu: implications for wood uses and the Kwazulu-Natal Environment. South African Archaeological Bulletin. 70(201): 36-52.

Libralato, G., Prato, E., Migliore, L., Cicero, A.M., and Manfra, L. 2016. A review of toxicity testing protocols and endpoints with Artemia spp. Ecological indicators. 69: 35- 49.

Lieberman, M. 1999. A brine shrimp bioassay for measuring toxicity and remediation of chemicals. Journal of Chemical Education. 76 (12): 1689-1691.

Lim, T.K. 2013. Edible Medicinal and Non-Medicinal Plants: Volume 7, Flowers. Springer Science and Business Media. New York. Pg 674-675.

Limongelli, V., Bonomi, M., Marinelli, L., Gervasio, F.L., Cavalli, A., Novellino, E. and Parrinello, M. 2010. Molecular basis of Cyclooxygenase enzymes (COXs) selective inhibition. Proceedings of the National Academy of Sciences. 107(12): 5411-5416.

Linnaeus, C. 1753. Species plantarum, Volume 1. Impensis Laurentii Salvii. Stockholm. Pg 98-101.

Lourens, A.C.U., Viljoen, A.M. and van Heerden, F.R. 2008. South African Helichrysum species: a review of the traditional uses, biological activity and phytochemistry. Journal of Ethnopharmacology. 119: 630-652.

Mabaleha, M.B., Zietsman, P.C., Wilhelm, A. and Bonnet, S.L. 2019. Ethnobotanical survey of medicinal plants used to treat mental illnesses in the Berea, Leribe and Maseru Districts of Lesotho. Natural Product Communications. Retrieved from: https://journals.sagepub.com/doi/pdf/10.1177/1934578X19864215. Date accessed: 28/07/2020.

Mabona, U. and Van Vuuren, S.F. 2013. Southern African medicinal plants used to treat skin diseases. South African Journal of Botany. 87: 175-193.

Maga, J.A. and Tu, A.T. 1994. Food Additive Toxicology. Marcel Dekker. New York. Pg 371.

Magwede, K., Van Wyk, B.E. and Van Wyk, A.E. 2019. An inventory of VhaVenḓa useful plants. South African Journal of Botany. 122: 57-89.

Mahadik, K.R. and Kuchekar, B.S. 2008. Concise Inorganic Pharmaceutical Chemistry. Nirali Prakashan. Pune. Pg 2-29.

Mamabolo, M.P., Muganza, F.M., Olivier, M.T., Olaokun, O.O. and Nemutavhanani, L.D. 2017. Evaluation of antigonorrhea activity and cytotoxicity of Helichrysum caespititium (DC) Harv. whole plant extracts. Journal of Biology and Medicine. 9(6): 1-4

Mandel, K.G., Daggy, B.P., Brodie, D.A. and Jacoby H.I. 2000. Review article: alginate- raft formulations in the treatment of heartburn and acid reflux. Aliments Pharmacology Therapeutics. 14(6): 669-690.

Manning, J.C., Goldblatt, P., and Johns, A., 2014. A taxonomic review of Cephalaria (Dipsacaceae) in the Cape Floristic Region. South African Journal of Botany. 94: 195- 203.

Maroyi, A. 2013. Traditional use of medicinal plants in South-Central Zimbabwe: review and perspectives. Journal of Ethnobiology and Ethnomedicine. 9:31.

Maroyi, A. 2016. From traditional usage to pharmacological evidence: systematic review of Gunnera perpensa L. Evidence-Based Complementary and Alternative Medicine. Retrieved from: http://downloads.hindawi.com/journals/ecam/2016/1720123.pdf. Date accessed: 13/05/2020.

Maroyi, A. 2019. Helichrysum caespititium (DC.) Harv.: review of its medicinal uses, phytochemistry and biological activities. Journal of Applied Pharmaceutical Science. 9(6): 111-118.

Maroyi, A. 2019. Scabiosa columbaria: a review of its medicinal uses, phytochemistry, and biological activities. Asian Journal of Pharmaceutical and Clinical Research. 12(8): 10-14.

Martini, F.H., Nath, J.L. and Bartholomew, E.F. 2015. Fundamentals of Anatomy and Physiology. 10th Ed. Pearson Education, Inc. California. Pg 144.

Masevhe, N.A., McGaw, L.J. and Eloff, J.N. 2015. The Traditional Use of Plants to Manage Candidiasis and Related Infections in Venda, South Africa. Journal of Ethnopharmacology. 168: 364-372.

Mathekga A.D.M. 2001. Antimicrobial activity of Helichrysum species and the isolation of a new phloroglucinol from Helichrysum caespititium. PhD. (Chemistry). University of Pretoria. Retrieved from: https://repository.up.ac.za/bitstream/handle/2263/23672/Complete.pdf?sequence=15&is Allowed=y Date accessed: 19/10/2019

Mayer, V. and Ehrendorfer, F. 1997. Fruit differentiation, palynology, and systematics in the Scabiosa group of genera and Pseudoscabiosa (Dipsacaceae). Plant Systematics and Evolution. 216: 135-166.

Mbhele, N., Balogun, F.O., Kazeem, M.I. and Ashafa, T. 2015. In vitro studies on the antimicrobial, antioxidant and antidiabetic potential of Cephalaria gigantea. A Journal of the Bangladesh Pharmacological Society. 10: 214-221.

McGaw, L.J., Elgorashi, E.E. and Eloff, J.N. 2014. Cytotoxicity of African Medicinal Plants Against Normal Animal and Human Cells. In: Toxicological Survey of African Medicinal Plants. Elsevier. Pg 181-233.

McGaw, L.J., Gehring, R., Katsoulis, L. & Eloff, J.N. 2005. Is the use of Gunnera perpensa extracts in endometritis related to antibacterial activity? Onderstepoort Journal of Veterinary Research. 72:129–134

Mhlongo, L.S. and Van Wyk, B.E. 2019. Zulu medicinal ethnobotany: new records from the Amandawe area of KwaZulu-Natal, South Africa. South African Journal of Botany. 122: 266-290.

Mikaili P., Mojaverrostami S., Moloudizargari, M. and Aghajanshakeri, S. 2013. Pharmacological and therapeutic effects of Mentha longifolia L. and its main constituent, Menthol. Ancient Science of Life. 33(2): 131-138.

Moffet, R. 2010. Sesotho plant and animal names and plants used by the Basotho. AFRICAN SUN MeDIA. Bloemfontein.

Moffett, R. 2016. Basotho medicinal plants- Meriana ya dimela tsa Basotho. SUN MeDIA. Bloemfontein.

Mogale, M.M.P., Van Wyk, B.E. and Raimondo, D.C. 2019. The ethnobotany of central Sekhukhuneland, South Africa. South African Journal of Botany. 122: 90-119.

Moteetee, A. and Seleteng Kose, L. 2017. A review of medicinal plants used by the Basotho for treatment of skin disorders: their phytochemical, antimicrobial, and anti- inflammatory potential. African Journal of Traditional, Complementary & Alternative Medicines. 14(5): 121-137.

Moteetee, A.N., & Van Wyk, B.E., 2011. The medicinal ethnobotany of Lesotho: a review, Bothalia. 41(1): 209-228.

Mouffouk, C., Hambaba, L., Haba, H., Mouffouk, S. and Bensouici, C. 2018. Evaluation of cytotoxic effect, anti-cholinesterase, hemolytic and antibacterial activities of the species Scabiosa stellata L. Current Bioactive Compounds. 14: 000-000.

Mouffouk, C., Hambaba, L., Haba, H., Mouffouk, S. and Bensouici, C., Mouffouk, S., Hachemi, M. and Khadraoui, H. 2018. Acute toxicity and in vivo anti-inflammatory effects and in vivo antioxidant and anti-arthritic potential of Scabiosa stellata. Oriental Pharmacy and Experimental Medicine. Retrieved from: https://www.researchgate.net/publication/325723357_Acute_toxicity_and_in_vivo_anti- inflammatory_effects_and_in_vitro_antioxidant_and_anti- arthritic_potential_of_Scabiosa_stellata. Date accessed: 06/11/2020

Mpofu, S., Ndinteh, D.T., Van Vuuren, S.F., Olivier, D.K. and Krause, R.W.M. 2014. Interactive efficacies of Elephantorrhiza elephantina and Pentanisia prunelloides extracts and isolated compounds against gastrointestinal bacteria. South African Journal of Botany. 94: 224-230.

Mugomeri, E., Chatanga, P., Raditladi, T., Makara, M. and Tarirai, C. 2016. Ethnobotanical study and conservation status of local medicinal plants: towards a repository and monograph of herbal medicines in Lesotho. African journal of Traditional, Complementary and Alternative Medicines. 13(1): 143-156.

Muleya, E., Ahmed, A.S., Sipamla, A.M., Mtunzi, F.M. and Mutatu, W. 2014. Evaluation of anti-microbial, anti-inflammatory and anti-oxidative properties Artemisia afra, Gunnera perpensa and Eucomis autumnalis. Journal of Nutrition & Food Sciences. 4(6): 1-6.

Mwale, M. and Masika, P.J. 2010. Analgesic and anti-inflammatory activities of Aloe ferox Mill. aqueous extract. African Journal of Pharmacy and Pharmacology. 4(6): 291- 297

Mzindle, N.B. 2017. Anti-inflammatory, antioxidant and wound-healing properties of selected South African medicinal plants. Master of Applied Sciences (Biotechnology). [Unpublished]: Durban University of Technology. Retrieved from:

https://openscholar.dut.ac.za/bitstream/10321/2893/1/MZINDLE_NB_2017.pdf. Date accessed: 01/04/2020.

Naghiloo, S. and Claßen-Bockhoff, R. 2017. Developmental changes in time and space promote evolutionary diversification of flowers: a case study in Dipsacoideae. Frontiers in Plant Science. 8: 1665.

NCCLS. 2003. National Committee for Clinical Laboratory Standards. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically. 6th Ed. Wayne, Pennsylvania.

Ndhlala, A.R., Finnie, J.F. and Van Staden, J. 2011. Plant composition, pharmacological properties and mutagenic evaluation of a commercial Zulu herbal mixture: Imbiza ephuzwato. Journal of Ethnopharmacology. 133: 663-674.

Oguntibeju, O.O. 2018. Medicinal plants with anti-inflammatory activities from selected countries and regions of Africa. Journal of Inflammation Research. 11: 307-317.

Otieno, J.N., Matengo Hosea, K.M., Lyaruu, H.V. and Anselm Mahunnah, R.L. 2008. Multi-plant or single-plant extracts, which is the most effective for local healing in Tanzania? African Journal of Traditional, Complementary and Alternative Medicines. 5(2): 165-172.

Parvizi, J. 2010. High Yield Orthopaedics. Saunders Elsevier. Philadelphia, Pennsylvania. Pg 120-123.

Patisaul, H.B. and Jefferson, W. 2010. The pros and cons of phytoestrogens. Frontiers in Neuroendocrinology. 31(4): 400-419.

Perry, L.M. and Metzger, J. 1980. Medicinal Plants of East and Southeast Asia: Attributed Properties and Uses. MIT Press. Massachusetts. Pg 128.

Pinto, D.C.G.A., Rahmouni, N., Beghidja, N. and Silva, A.M.S. 2018. Scabiosa genus: a rich source of bioactive metabolites. Medicines (Basel). 5(4): e110.

Quattrocchi, U. 2012. CRC World Dictionary of Medicinal and Poisonous Plants: Common Names, Scientific Names, Eponyms, Synonyms, And Etymology (5 volume Set). CRC Press. Boca Raton. Pg 3341.

Rahmouni, N. 2018. Phytochemical and biological studies of Scabiosa stellata L. (Caprifoliaceae) & Sedum carealeum Vahl. (Crassulaceae). PhD (Chemistry). Mentoutri Brothers University Contantine 1. Retrieved from: https://bu.umc.edu.dz/theses/chimie/RAH7341.pdf. Date accessed: 03/04/2020.

Raymond, P. and Beaver, M. 2014. Acid Reflux Diet and Cookbook for Dummies. John Wiley & Sons. New Jersey. Pg 19.

Reshma, Arun, K.P. and Brindha, P. 2014. In vitro anti-inflammatory, antioxidant and nephroprotective studies on leaves of Aegle marmelos and Ocimum sanctum. Asian Journal of Pharmaceutical and Clinical Research. 7(4):121-129.

Robertsdottir, A.R. 2016. Icelandic Herbs and Their Medicinal Uses. North Atlantic Books. California. Pg 144-147.

Roell, K.R., Reif, D.M. and Motsinger-Reif, A.A. 2017. An introduction to terminology and methodology of chemical synergy—perspectives from across disciplines. Frontiers in Pharmacology. 8:158.

Romay, C., Ledón, N., and González, R. 1998. Further studies on anti-inflammatory activity of phycocyanin in some animal models of inflammation. Inflammation Research. 47(8): 334–8.

Romich, J.A. 2012. Fundamentals of Pharmacology for Veterinary Technicians. 2nd Ed. Cengage Learning. Delmar. Pg 329-330.

Rutter, P. 2020. Community Pharmacy: Symptoms, Diagnosis and Treatment. 5th Ed. Elsevier Health Science. London. Pg 181.

Sandhya, S., Venkata, R.K. and Vinod, K.R. 2015. A comparative evaluation of in vitro antacid activity of two Tephrosia species using modified artificial stomach model. Hygeia: Journal for Drugs and Medicines. 7(2): 9-17.

Sandhya, S., Venkata, R.K., Vinod, K.R. and Chaitanya, R. 2012. Assessment of in vitro antacid activity of different root extracts of Tephrosia purpurea (L) Pers by modified artificial stomach model. Asian Pacific Journal of Tropical Biomedicine. 2(3): S1487- S1492.

Sangeetha, G. and Vidhya, R. 2016. In vitro anti-inflammatory activity of different parts of Pedalium murex (L.). International journal of Herbal Medicine. 4(3): 31-36.

Satyajit, T.A.W. and Matsabisa, M.G. 2017. Dicoma Anomala Sond [Asteraceae]: a possible resource of future antimalarial agents. Research Journal of Biology. 5(4): 19- 20.

Seidle T., Prieto, P. Bulgheroni, A. 2011. Examining the regulatory value of multi-route mammalian acute systemic toxicity studies. ALTEX. 28(2): 95-102.

Seleteng Kose, L., Moteetee, A. and Van Vuuren, S. 2015. Ethnobotanical survey of medicinal plants used in the Maseru District of Lesotho. Journal of Ethnopharmacology. 170: 184-200.

Seleteng Kose, L.S, 2017. Evaluation of commonly used medicinal plants of Maseru District in Lesotho for their ethnobotanical uses, antimicrobial properties and phytochemical composition. PhD. (Botany) [unpublished]. University of Johannesburg. Retrieved from: https://ujcontent.uj.ac.za/vital/access/manager/Repository/uj:25093?site_name=Resear ch+Output&query=kose&f0=sm_type%3A%22Doctoral+%28Thesis%29%22&sort=ss_d ateNormalized+desc%2Csort_ss_title+asc&queryType=vitalDismax. Date accessed: 14/10/2019.

Seller, R.H. & Symons, A.B. 2011. Differential Diagnosis of Common Complaints. 6th Ed. Elsevier Health Science. Philadelphia. Pg 207-215.

Semenya, S., Potgieter, M.J. and Erasmus, L. 2012. Ethnobotanical survey of medicinal plants used by Bapedi healers to treat diabetes mellitus in the Limpopo Province, South Africa. Journal of Ethnopharmacology. 141(1): 440-445

Shale, T.L., Stirk, W.A. & van Staden, J. 1999. Screening of medicinal plants used in Lesotho for anti-bacterial and anti-inflammatory activity. Journal of Ethnopharmacology. 67: 347-354.

Shen, X., Zeng, Y., Li, J., Tang, C., Zhang, Y. & Meng, X. 2017. The anti-arthritic activity of total glycosides from Pterocephalus hookeri, a traditional Tibetan herbal medicine. Pharmaceutical Biology. 55(1): 560-570.

Simon, L.S. 1999. Role and regulation of Cyclooxygenase-2 during inflammation. The American Journal of Medicine. 106(5): 37S-42S.

Simpson M.G. 2011. Plant Systematics. Elsevier Academic Press. Massachusetts. Pg 331-333.

Sinatra, R.S., Jahr, J.S. and Watkins-Pitchford, J. 2010. The Essence of Analgesia and Analgesics. Cambridge University Press. New York. Pg 211-214.

Singh, N.K. and Rao, G.N. 2019. Emerging role of 12/15-Lipoxygenase (ALOX15) in human pathologies. Progress in Lipid Research. 73: 24-45.

Singh, P. and Terrell, J.M. 2019. Antacids. StatPearls Publishing. Treasure Island, Florida. Retrieved from: https://www.ncbi.nlm.nih.gov/books/NBK526049/. Date accessed: 23/02/2020.

Soheila, J.M., Crespo, J.F. and Cabanillas, B. 2019. Anti-inflammatory effects of flavonoids. Food Chemistry. 299: 125124.

Subramanian, K., Sankaramourthy, D. and Gunasekaran, M. 2018. ‘Toxicity Studies Related to Medicinal Plants’ in Natural Products and Drug Discovery, An Integrated Approach. Elsevier. Pg 491-505.

Tamokou, J.D. and Kuete, V. 2014. 'Toxic Plants Used in African Traditional Medicine' in Toxicological Survey of African Medicinal Plants. Elsevier, Pg 136-180.

Tetyana, P., Prozesky, E.A., Jager, A.K., Meyer, J.J.M. and Van Staden, J. 2002. Some medicinal properties of Cussonia and Schefflera species used in traditional medicine. South African Journal of Botany. 68: 51-54.

Thabrew, M.I. and Arawwawala, L.D.A.M. 2016. An overview of in vivo and in vitro models that can be used for evaluating anti-gastric ulcer potential of medicinal plants. Austin Biology. 1(2): 1007.

Thouri, A., Chahdoura, H., El Arem, A., Hichri, A.O., Hassin, R.B. and Achour, L. 2017. Effect of solvents extraction on phytochemical components and biological activities of Tunisian date seeds (var. Korkobbi and Arechti). BMC Complementary and Alternative Medicine. 17: 248.

Tshikalange, T.E., Mophuting, B.C., Mahore, J., Winterboer, S. and Lall, N. 2016. An ethnobotanical study of medicinal plants used in villages under Jongilanga Tribal Council, Mpumalanga, South Africa. African Journal of Traditional, Complementary Alternative Medicines. 3(6): 83-89.

Usman, M. & Davidson, J. 2013. How to eliminate heartburn and acid reflux naturally. JD-Biz Corp Publishing. Utah.

Valgas, C., de Souza, S.M., Smânia, E.F.A and Smânia Jr., A.S. 2007. Screening methods to determine antibacterial activity of natural products. Brazilian Journal of Microbiology. 38: 369-380.

Van der Walt, L. 2003. Scabiosa incisa Mill. Retrieved from: http://pza.sanbi.org/scabiosa-incisa. Date accessed: 01/02/2020.

Van Vuuren and Naidoo, D. 2010. An antimicrobial investigation of plants used traditionally in southern Africa to treat sexually transmitted infections. Journal of Ethnopharmacology. 130: 552-558.

Van Vuuren, S. and Holl, D. 2017. Antimicrobial natural product research: A review from a South African perspective for the years 2009–2016. Journal of Ethnopharmacology. 208: 236-252.

Van Vuuren, S. and Viljoen, A. 2011. Plant-Based antimicrobial studies- methods and approaches to study the interaction between natural products. Planta Medica. 77: 1168- 1182.

Van Vuuren, S.F., Nkwanyana, M.N. and De Wet, H. 2015. Antimicrobial evaluation of plants used for the treatment of diarrhoea in a rural community in Northern Maputaland, KwaZulu-Natal, South Africa. BMC Complementary & Alternative Medicine. 15:53.

Van Wyk, B-E., Van Oudtshoorn, B., Gericke, N. 2009. Medicinal Plants of South Africa. Briza Publications. Pretoria.

Van Wyk, B.E. 2011. The potential of South African plants in the development of new medicinal products. South African Journal of Botany. 77: 812-829.

Vane, J.R. and Botting, R.M. 2003. The mechanism of action of aspirin. Thrombosis Research. 110(5-6): 255-258.

Victor, J.E. 2005. Scabiosa albanensis R.A. Dyer. National Assessment: Red List of South African Plants version 2020.1. Retrieved from: http://redlist.sanbi.org/species.php?species=2742-2. Date accessed: 17/08/2020.

Von Staden, L. 2016. Scabiosa transvaalensis S. Moore. National Assessment: Red List of South African Plants version 2020.1. Retrieved from: http://redlist.sanbi.org/species.php?species=2742-16. Date accessed: 17/08/2020.

Wang, H., Liu, H., Moore, M.J., Landrein, S., Liu, B., Zhu, Z. and Wang, H. 2020. Plastid phylogenomic insights into the evolution of the Caprifoliaceae s.l. (Dipsacales). Molecular Phylogenetics and Evolution. 142: 106641.

Wannes, W.A. and Marzouk, B. 2016. Research progress of Tunisian medicinal plants used for acute diabetes. Journal of Acute Disease. 5(5): 357-363.

Washington, N. 1991. Antacids and Anti-reflux Agents. CRC Press, Inc. Boca Raton, Florida. Pg 2-12.

Watt, J.M. & Breyer-Brandwijk, M.G. 1962. The Medicinal and Poisonous Plants Of Southern and Eastern Africa. 2nd Ed. E. & S. Livingstone Ltd. South Africa. Pg 49, 229, 387, 495 & 707.

Weissmann, G., Korchak, H., Ludewig, R., Edelson, H., Haines, K., Levin, R.I., Herman, R., Rider, L., Kimmel, S. and Abramson, S. 1987. Non-steroidal anti-inflammatory

drugs: how do they work? European Journal of Rheumatology and Inflammation. 8(1): 6-17.

Wiart, C. 2007. Ethnopharmacology of Medicinal Plants: Asia and the Pacific. Springer Science & Business Media. Totowa, New Jersey. Pg 10-12.

William, F.G. 1984. Plant Biosystematics. Academic Press. Canada. Pg 307-309.

Williams, V.L., Raimondo, D., Crouch, N.R., Cunningham, A.B., Scott-Shaw, C.R., Lötter, M. and Ngwenya, A.M. 2008. Scabiosa columbaria L. National Assessment: Red List of South African Plants version 2020.1. Retrieved from: http://redlist.sanbi.org/species.php?species=2742-9. Date accessed: 17/08/2020.

Wintola, O.A. and Afolayan, A.J. 2015. The antibacterial, phytochemicals and antioxidants evaluation of the root extracts of Hydnora africana Thunb. used as antidysenteric in Eastern Cape Province, South Africa. BMC Complementary and Alternative Medicine. 15(307): 1-12.

Worth, A.P. 2019. The history of alternative test methods in toxicology: Chapter 1.2 - Types of toxicity and applications of toxicity testing. History of Toxicology and Environmental Health. 7-10.

Wu, T., Chen, I. and Chen, L. 2010. Antacid effects of Chinese herbal prescriptions assessed by a modified artificial stomach model. World Journal of Gastroenterology. 16(35): 4455-4459.

www.rennie.co.za. Retrieved from: https://www.rennie.co.za/en/product- range/peppermint/ Date accessed: 18 May 2020

Xu, H., Ma, Q., Ma, J., Wu, Z., Wang, Y. and Ma, C. 2016. Hepato-protective effects and chemical constituents of a bioactive fraction of the traditional compound Medicine- Gurigumu-7. BMC Complementary and Alternative Medicine. 16: 179.

Yuan, H., Ma, Q., Cui, H., Liu, G., Zhao, X., Li, W. & Piao, G. 2017. How can synergism of traditional medicines benefit from network pharmacology? Molecules. 22(1135): 1-19.

Zhang, F., Xie, J., Zhang, Y., Kong, L. and Li, S. 2013. What is important during the selection of traditional Chinese medicine (TCM) in a health care reimbursement or insurance system? Critical issues of assessment from the perspective of TCM practitioners. Value Health Regional Issues. 2(1): 141-146.