THE PHYTOCHEMISTRY AND BIOLOGICAL ACTIVITIES OF ATHRIXIA PHYLICOIDES KUMESHNIE PADAYACHEE 9803723R A Research Report submitted to the Faculty of Health Sciences, University of Witwatersrand, in fulfillment of the requirements for the degree of Master of Science in Medicine. February 2011 I DECLARATION I, Kumeshnie Padayachee declare that this research report is my own work. It is being submitted for the degree of Master of Science in Medicine at the University of Witwatersrand, Johannesburg. It has not been submitted before for any degree or examination at this or any other University. Signature?????????.. ????day of???????2011 II DEDICATION I dedicate this research report to my husband, Prinevin my son, Praveen and my parents, Kassey and Savie For always loving and supporting me and for all the sacrifices you have made for me. III ACKNOWLEDGEMENTS Firstly I would like to thank my supervisors, Prof. Sandy van Vuuren and Dr Robyn van Zyl and my co-supervisors, Prof. Alvaro Viljoen and Prof. Jana Olivier for their guidance and full support throughout the research period. A special thanks to Prof. K. H?sn? C Ba?er, Dr Bet?l Demirci and Dr Temel ?zek at the Department of Pharmacognosy, Faculty of Pharmacy, Anadolu University, Eski?ehir, Turkey. I appreciate their assistance in the GC-MS analysis of the essential oils. Thank you to Prof. F.R. van Heerden (University of KwaZulu-Natal) who assisted me to structurally characterize the isolated compounds. A sincere thank you to my family for always loving and supporting me. I appreciate all the sacrifices you have made for me. To all those who have contributed to the completion of this research report and whose names do not appear here, I would like to express my sincere gratitude. IV ABSTRACT Herbal medicines are an important part of the African culture and tradition and about 80% of Africa?s population relies on traditional remedies for their primary health care needs (WHO, 2008). Athrixia phylicoides is widely used as a traditional remedy, but despite its substantial use, literature on its chemical composition and biological activities is limited. In this study, the chemical composition of the essential oil was determined using gas chromatography combined with mass spectroscopy (GC/MS) and analysis resulted in the identification of 182 compounds. The major compounds identified in selected samples were ?-pinene, ?-pinene, caryophyllene oxide, ?-caryophyllene, myrcene and spathulenol. Based on the traditional uses of A. phylicoides, various in vitro biological activities were investigated. The extensive use of A. phylicoides for the treatment of boils, sores, bad acne, infected wounds and cuts, prompted the antimicrobial (bacterial and fungal) study using the minimum inhibitory concentration (MIC) assay. The methanol extract and essential oil were more selective for the Gram- positive bacteria than the Gram-negative bacteria. The extract exhibited stronger activity against all micro-organisms tested compared to the essential oil, with the highest activity against S. aureus, B. cereus and B. subtilis (MIC = 1 mg/ml). The bioactive compound, (4-hydroxyphenyl) propyl coumaroate was isolated from the extract and proved to be most active against S. aureus (MIC = 19.5 ?g/ml). Using the DPPH? assay, a comparative anti- oxidant study was performed. The anti-oxidant activity of the aqueous extract of A. phylicoides (IC50 = 14.01 ? 2.68 ?g/ml) was greater than rooibos and Ceylon (black) tea (IC50 > 25.00 ?g/ml); comparable to green rooibos and honeybush tea (IC50 = 18.01 ? 4.06 ?g/ml; 18.02 ? 4.27 ?g/ml, respectively), but less active than green tea (IC50 = 9.64 ? 0.96 ?g/ml). The antimalarial activity was determined using the [3H] hypoxanthine incorporation method. The essential oil exhibited greater antimalarial activity against the chloroquine-resistant Plasmodium falciparum (FCR-3) strain (IC50 = 1.006 ? 0.06 ?g/ml) than the methanol extract. The 5-lipoxygenase assay was used to assess the anti-inflammatory activity of the methanol extract and essential oil. Only the essential oil displayed anti-inflammatory activity (IC50 = 25.68 ?g/ml). Low safety indices were reported for both the methanol extract (SI = 2.28) and essential oil (SI = 1.77). The in vitro biological activities may validate the use of A. phylicoides in traditional medicine. V TABLE OF CONTENTS Page DECLARATION I DEDICATION II ACKNOWLEDGEMENTS III ABSTRACT IV TABLE OF CONTENTS V LIST OF FIGURES IX LIST OF TABLES X LIST OF ABBREVIATIONS AND SYMBOLS XI CHAPTER 1: GENERAL INTRODUCTION 1 1.1 Medicinal plants and their place in our society 1 1.2 Athrixia phylicoides 3 1.2.1 Name derivation and common names 5 1.2.2 Description 5 1.2.3 Distribution 7 1.2.4 Traditional uses 7 1.2.5 A review of previous studies on A. phylicoides 7 1.3 Objectives of the study 9 CHAPTER 2: PLANT COLLECTION AND PREPARATION OF SAMPLES 10 2.1 Collection of plant material 10 2.2 Preparation of samples 11 2.2.1 Isolation of essential oils 11 2.2.2 Preparation of the solvent extracts 11 2.2.3 Preparation of the aqueous tea infusions 12 VI CHAPTER 3: COMPOSITION OF ESSENTIAL OILS OF ATHRIXIA PHYLICOIDES 13 3.1 Introduction 13 3.2 Materials and methods 14 3.2.1 Principle of the method 14 3.2.2 Gas chromatography 14 3.2.3 Gas chromatography combined with mass spectrometry 14 3.3 Results and discussion 15 3.3.1 Essential oil yield 15 3.3.2 Essential oil composition 16 3.4 Conclusions 26 CHAPTER 4: ANTIMICROBIAL ACTIVITY AND ISOLATION OF BIOACTIVE COMPOUND 27 4.1 Introduction 27 4.2 Materials and methods 28 4.2.1 Minimum inhibitory concentration assay 28 4.2.1.1 Principle of the method 28 4.2.1.2 Protocol 28 4.2.2 Isolation of bioactive compound 30 4.2.2.1 Column chromatography 30 4.2.2.2 Thin layer chromatography and bio-autographic assay 30 4.2.2.3 Bioactivity-guided fractionation and isolation 32 4.2.2.4 Identification, elucidation and minimum inhibitory concentration of the isolated compound 32 4.2.2.4.1 Nuclear magnetic resonance 32 4.2.2.4.2 Antimicrobial activity of the isolated compound 34 VII 4.3 Results and discussion 34 4.3.1 Antimicrobial activity: Extract, essential oil and bioactive compound 34 4.3.2 Bioactivity-guided fractionation and isolation 37 4.3.3 Elucidation and identification of the isolated compound 37 4.4 Conclusions 38 CHAPTER 5: ANTI-OXIDANT ACTIVITY 39 5.1 Introduction 39 5.2 Materials and methods 40 5.2.1 Principle of the method 41 5.2.2 Protocol 41 5.3 Results and discussion 42 5.4 Conclusions 46 CHAPTER 6: ANTIMALARIAL ACTIVITY 47 6.1 Introduction 47 6.2 Materials and methods 48 6.2.1 Parasite suspension 48 6.2.2 Tritrated hypoxanthine incorporation assay 49 6.2.3 Principle of the method 49 6.2.4 Protocol 49 6.3 Results and discussion 50 6.4 Conclusions 52 CHAPTER 7: ANTI-INFLAMMATORY ACTIVITY 53 7.1 Introduction 53 7.2 Materials and methods 55 7.2.1 Principle of the method 55 7.2.2 Protocol 55 VIII 7.3 Results and discussion 56 7.4 Conclusions 57 CHAPTER 8: CYTOTOXICITY 58 8.1 Introduction 58 8.2 Materials and methods 59 8.2.1 Microculture tetrazolium cellular viability assay 59 8.2.2 Principle of the method 59 8.2.3 Protocol 59 8.3 Results and discussion 61 8.4 Conclusions 63 CHAPTER 9: GENERAL CONCLUSIONS AND RECOMMENDATIONS 64 9.1 Summary 64 9.2 Recommendations for future work 65 REFERENCES 67 APPENDIX A: NMR data for the (4-hydroxyphenyl)propyl coumaroate 77 IX LIST OF FIGURES Figure Page 1.1A Speaking to a local woman collecting A. phylicoides in Haenertsberg 4 1.1B/C Athrixia phylicoides during the non-flowering seasons 4 1.1D/E Athrixia phylicoides during the flowering season 4 1.2 Distribution of A. phylicoides in South Africa (SANBI) 7 2.1 Hydrodistillation of plant material using the Clevenger-type apparatus 11 3.1 Percentage essential oil yield for samples of A. phylicoides 15 3.2 Chemical structures for major compounds identified in the essential oil of A. phylicoides 23 3.3 Major compound identified in A. phylicoides 25 4.1 Microtitre plate for Salmonella typhimurium 29 4.2 Preparing a silica column for column chromatography used to separate fractions 31 4.3 Bio-autographic assay of A. phylicoides against the test pathogen S. aureus 32 4.4 Bioactivity-guided fractionation procedure 33 4.5 Structure of (4-hydroxyphenyl)propyl coumaroate isolated from A. phylicoides 37 5.1 Commercial packs of the teas tested for anti-oxidant activity 41 5.2 In vitro anti-oxidant activity of aqueous tea extracts 43 6.1 The life cycle of the malaria parasite 48 7.1 Inflammatory cascade, where PG is prostaglandins, LT is leukotrienes, TXA2- thromboxane and HPETE is hydroperoxyeicosatetraenoic acid 54 A.1 The NMR carbon spectra (1H) for (4-hydroxyphenyl)propyl coumaroate 78 A.2. The NMR carbon spectra (13C) for (4-hydroxyphenyl)propyl coumaroate 79 X LIST OF TABLES Table Page 1.1 Examples of active compounds isolated from plants 2 1.2 Traditional uses of A. phylicoides as a medicine by various ethnic groups 6 2.1 Plant collection, voucher and sample reference number and percentage oil yield 10 2.2 Botanical name, common name and percentage aqueous extract yield 12 3.1 Essential oil composition of seven samples of A. phylicoides 16 4.1 Antimicrobial activity of A. phylicoides methanol extract, essential oil and bioactive compound 35 5.1 Botanical names and common names of samples tested in DPPH? assay 41 6.1 In vitro antimalarial activity of A. phylicoides 50 8.1 In vitro cytotoxic properties of A. phylicoides 61 9.1 Summary of the phytochemistry and in vitro biological activities of the essential oils and extracts of A. phylicoides 66 A.1 NMR data (in CDCl3 + CD3OD) of the compound isolated from A. phylicoides 77 XI LIST OF ABBREVIATIONS AND SYMBOLS % Percent ?C: Degrees celsius CAT: Catalase CFU: Colony forming unit CO2: Carbon dioxide COSY: Correlation spectroscopy COX: Cyclooxygenase DEPT: Distortionless enhanced polarization transfert DPPH: 2,2-diphenyl-1-picrylhydrazl DMSO: Dimethyl sulfoxide DNA: Deoxyribonucleic acid EC50: Extract concentration 50% EDTA: Ethylenediaminetetraacetic acid EIMS: Electron ionization mass spectrometry eV: Electron volt FID: Flame ionization detection FCS: Foetal calf serum GC: Gas chromatography GC/MS: Gas chromatography coupled to mass spectrometry GPX: Gluthathione peroxidase Gram -ve: Gram-negative Gram +ve: Gram-positive H2O2: Hydrogen peroxide HEPES: [N-(2-hydroxethyl)piperazine-N?-(2-ethanesulfonic acid)] HP: Hewlett Packard HPETE: Hydroperoxyeicosatetraenoic HPLC: High pressure liquid chromatography IC50: Inhibitory concentration (Concentration that reduced the effect by 50%) INT: ?-Iodonitrotetrazolium XII LC50: Lethal concentration 50% LT: Leukotrienes m: Meter mg: Milligram MHZ: Megahertz ml: Millilitre mm: Millimeter mM: Millimolar MIC: Minimum inhibitory concentration min: Minutes MS: Mass spectroscopy MTT: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide n: Number of repetitions N2: Nitrogen NaHCO3: Sodium hydrogen carbonate ND: Not determined NIST: National Institute of Standards and Technology NDGA: Nordihydroguaiaretic NMR: Nuclear magnetic resonance No.: Number NSAIDs: Non-steroidal anti-inflammatory drugs O2?: Superoxide radical OH?: Hydroxyl radicals PG: Prostaglandin pH: Potential hydrogen ppm: Parts per million rpm: Revolutions per minute RPMI 1640: Roswell Park Memorial Institute 1640 RRI: Relative retention index SANBI: South African National Botanical Institute SI: Safety index XIII s.d.: Standard deviation SOD: Superoxide dismutase t: Trace TEAC: Trolox? equivalent anti-oxidant capacity TLC: Thin layer chromatography TXA2: Thromboxane ?l: Microlitre ?m: Micrometer ?M: Micromolar UV: Ultraviolet UV-VIS: Ultraviolet-visible WHO: World Health Organisation v/v: Volume per volume w/v: Weight per volume w/w: Weight per weight XTT: Sodium 3?-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate 1 Chapter 1 ? General introduction 1.1 Medicinal plants and their place in our society ?Medicinal plants are something of the future, not the past? (van Wyk et al., 2002). Herbal medicines are an important part of the culture and tradition of African people (Fennell et al., 2004). The World Health Organisation (WHO) estimates that in some African countries, up to 80% of the population relies on traditional materia medica (medicinal plants) for their primary health care needs (WHO, 2008). Africa is a continent endowed with an enormous wealth of plant resources. Southern Africa has well over 30 000 species of higher plants. In South Africa, approximately 3 000 species have been used for several centuries as medicine for the prevention and treatment of diseases (van Wyk et al., 2002). Some renowned African medicinal plants include Acacia senegal (gum Arabic), Agathosma betulina (buchu), Aloe ferox (Cape aloes), Artemisia afra (African wormwood), Aspalathus linearis (rooibos tea), Boswellia sacra (frankincense), Catha edulis (khat), Commiphora myrrha (myrrh), Harpagophytum procumbens (devil?s claw), Hibiscus sabdariffa (hibiscus or roselle), Hypoxis hemerocallidea (African potato) and Prunus africana (African cherry or red stinkwood) (van Wyk and Wink, 2004). It is believed that the profound knowledge of medicinal plants was developed through trial and error over several centuries and the most important remedies were passed on verbally from one generation to the next. The founders and users of the medicinal plants may not have understood the scientific rationale behind the efficacy of these plants, but knew from personal experience that they were highly effective (van Wyk and Wink, 2004). History reveals that for centuries plants have been the main source of medicine for most cultures. The long history of herbal medicine and the number of allopathic drugs derived from plant material (Table 1.1) is an indicator that medicinal plants are effective and can stimulate the discovery of new compounds for the treatment of new and old diseases. 2 Table 1.1: Examples of active compounds isolated from plants. Plant Active compound Use/Action Literature source Aloe ferox Aloe emodin Chrysophanol Aloin Purgative Kambizi et al., 2004 Artemisia annua Artemisinin Antimalarial Clark, 1996 Atropa belladonna Atropine Anticholinergic De Smet, 1997; Rates, 2001 Camellia sinesis Theophylline Caffeine Bronchodilator CNS stimulant De Smet, 1997 Cassia spp. Sennosides A&B Laxative De Smet, 1997 Catharanthus roseus Vinblastine & Vincristine Antitumor De Smet, 1997; Rates, 2001 Chondodendron tomentosum Tubocurarine Skeletal muscle relaxant De Smet, 1997 Cinchona ledgeriana Quinine Quinidine Antimalarial Antiarrhythmic De Smet, 1997; Rates, 2001 Colchicum autumnale Colchicine Anti-gout De Smet, 1997 Digitalis purpurea Digoxin Cardiotonic De Smet, 1997; Rates, 2001 Ephedra sinica Ephedrine Sympathomimetic De Smet, 1997 Gaultheria procumbens Methyl salicylate Rubefacient De Smet, 1997 Helichrysum cymosum Helihumulone Antibacterial and antifungal (yeasts) van Vuuren et al., 2006 Hyoscyamus niger Hyocyamine Anticholinergic De Smet, 1997 Larrea divaricata Nordihydroguaiaretic acid Anti-oxidant De Smet, 1997 Mucuna deeringiana Levodopa Anti-parkinsonism De Smet, 1997 Papaver somniferum Codeine Morphine Noscapine Analgesic/antitussive Analgesic Antitussive De Smet, 1997; Rates, 2001 Physostigma venenosum Physostigmine Cholinesterase inhibitor De Smet, 1997 Pilocarpus jaborandi Pilocarpine Parasympathomimetic De Smet, 1997 Podophyllum peltatum Podophyllotoxin Topical treatment De Smet, 1997 Rauwolfia serpentine Reserpine Antihypertensive De Smet, 1997 Salix capensis Catechol 2-Hydroxybenzyl alcohol Antibacterial Masika et al., 2005 Salvia chamelaeagnea Carnosol Antibacterial Kamatou et al., 2007 Terminalia sericea Anolignan Antibacterial Eldeen et al., 2006 3 The integration of medicinal plants into the mainstream healthcare system in South Africa presents its own challenges with the following to overcome (WHO, 2008 adapted): ? National policy and regulation: The prescription and use of medicinal plants is currently not regulated. The result is that there is always the danger of misadministration, especially of toxic plants. ? Patient safety and use: It is believed that because herbal medicines are natural, they are safe. Herbal medicines can cause harmful adverse reactions, if the medicine is of a poor quality, taken incorrectly or in conjunction with modern medicines. ? Scientific evidence: Evidence from in vivo studies to evaluate the safety and efficacy of traditional medicines is limited. ? Sustainability: Herbal medicines are collected from wild plant populations and cultivated from medicinal plants. Poorly managed collections and cultivations could lead to the extinction of endangered plant species and destruction of natural resources. ? Knowledge: The use of African traditional medicine is not well documented and this is becoming increasing urgent due to the rapid loss of the natural habitat of some of the plants. Since only one-third of all diseases can be treated efficiently with currently available innovative drugs (Muller et al., 2000), the need for research and development of medicinal plants is on the increase. Phytochemical and pharmacological studies of medicinal plants are essential to provide us with sound scientific data on their safety and efficacy and possibly provide mankind with a sound solution to the current problem of ineffective drugs. 1.2 Athrixia phylicoides Athrixia phylicoides DC. is a well-known, multi-purpose, versatile plant and according to the literature, it is used as a tea and a medicine (van Wyk and Gericke, 2000). A recent survey revealed that A. phylicoides is still frequently utilised as a tea and a medicine by both rural and urban dwellers in South Africa (Figure 1.1A) and will be purchased if commercially available (Rampedi and Olivier, 2005). But despite the substantial demand for A. phylicoides, little scientific data on its chemical composition, toxicity and pharmacological activity has been published. In contrast to the lack of scientific data available for A. phylicoides, extensive 4 A B C D E Figure 1.1: A: Speaking to a local woman collecting A. phylicoides in Haenertsberg. B & C: Athrixia phylicoides during the non-flowering seasons. D & E: Athrixia phylicoides during the flowering season. [Source: Myself (A&C), A.M. Viljoen (B&E) and G. Mbambezeli (D)] 5 research on the chemical composition, pharmacological activity and toxicity has been performed on another well-known and frequently consumed tea, Rooibos tea (Rabe et al, 1994; von Gadow et al., 1997; Manzocco et al., 1998; McGaw et al., 2007; Almajano et al., 2008; Marnewick et al., 2009). Guided by the medicinal uses (Table 1.2) and the substantial demand for A. phylicoides, this study encompasses a phytochemical and pharmacological investigation of the indigenous plant. The biological activities investigated were based on the traditional uses of A. phylicoides. Its use as a substitute for tea prompted the investigation as an anti-oxidant agent and the comparative anti-oxidant activity with other commercially available teas. Athrixia phylicoides is also used to treat boils, sores, bad acne, infected wounds and cuts, and therefore its antimicrobial activity was studied. The anti-inflammatory activity of the plant was investigated because of its use to treat sore feet and sore throats. According to the WHO, malaria has been responsible for nearly one million deaths in 2008 as a result of drug-resistant strains of Plasmodium falciparum (WHO, 2010). These statistics prompted the investigation of A. phylicoides as an antimalarial agent. A toxicity study was conducted due to the substantial use and consumption of A. phylicoides, and the lack of safety data for the volatile oils. 1.2.1 Name derivation and common names The genus name Athrixia is derived from the Greek word thrix meaning hair describing the appearance of the leaves. Athrixia phylicoides is commonly known as Bushman?s tea, Bush tea, Zulu tea (English), Boesmanstee, Bostee (Afrikaans) and Icholocholo, Itshelo, Umthsanelo (Zulu) (Mbambezeli, 2005). 1.2.2 Description Athrixia belongs to the Asteraceae family which is the largest family of flowering plants comprising of about 1 100 genera and 20 000 species. Nine species of the genus Athrixia occur in southern Africa, A. phylicoides and A. elata (wildtee) being the most common. Athrixia phylicoides is an erect shrub with small aromatic leaves (Figure 1.1B/C). The upper surface of the leaf is dark green. The lower surface of the leaf is covered by a mass of fine woolly hair giving the shrub a silvery appearance. Athrixia phylicoides bears small purple flowers in late autumn (van Wyk and Gericke, 2000) (Figure 1.1D/E). 6 Table 1.2: Traditional uses of A. phylicoides as a medicine by various ethnic groups. Medicinal use/ailments Ethnic group Part of plant used Literature source All communities Not specified Plowes and Drummond, 1976 Vhavenda Branches and leaves Mabogo, 1990 Tea Zulu, Xhosa and Vhavenda Leaves Hutchings et al., 1996 Sore throats Sotho and Xhosa Leaves chewed Roberts, 1990; Hutchings et al., 1996 Decoction of leaves for bathing sore feet. Watt and Breyer- Brandwijk, 1962; Hutchings et al., 1996 Sore feet Sotho A strong tea used as a soothing wash Roberts, 1990 All communities Not specified Plowes and Drummond, 1976 Zulu Root decoctions Watt and Breyer- Brandwijk, 1962; Roberts, 1990; Hutchings et al., 1996 Cough remedy Sotho and Xhosa Leaves chewed Roberts, 1990; Hutchings et al., 1996 Anti-helmintics Vhavenda Extracts from soaked leaves and roots Mabogo, 1990; Hutchings et al., 1996 Aphrodisiac Vhavenda Root infusions Hutchings et al., 1996; van Wyk and Gericke, 2000 Boils, bad acne, infected wounds and cuts All communities Leaves Plowes and Drummond, 1976; Roberts, 1990 ?Blood purifier? for sores and boils Zulu Plant infusion (no part specified) Hutchings et al., 1996 Purgative Zulu Root decoctions Watt and Breyer- Brandwijk, 1962; Roberts, 1990; Hutchings et al., 1996 7 1.2.3 Distribution Athrixia phylicoides grows in the eastern hilly and mountainous parts of South Africa, from the Eastern Cape in the south to the Soutpanberg in the Limpopo Province in the north (Figure 1.2). Figure 1.2: Distribution of A. phylicoides in South Africa (SANBI). 1.2.4 Traditional uses Athrixia phylicoides is a multi-purpose plant with a variety of uses such as a tea, a medicine and for the making of brooms (van Wyk and Gericke, 2000). The attractive plant with its small purple flowers and silvery leaves is very useful for decorative purposes, alone in a vase or as foliage in flower arrangements (Olivier and De Jager, 2005). Athrixia phylicoides is also used to make baskets and is burnt as incense at ceremonies such as weddings and funerals (Olivier and De Jager, 2005). The medicinal use of A. phylicoides by various ethnic groups in South Africa is summarised in Table 1.2. 1.2.5 A review of previous studies on A. phylicoides Most of the literature refers to the extensive traditional use of A. phylicoides (van Wyk and Gericke, 2000; Olivier and De Jager, 2005; Rampedi and Olivier, 2005) and thus, it is remarkable that only limited research has been conducted on this indigenous plant. This 8 implies that very little information on the chemical composition, toxicity and pharmacological activity of A. phylicoides is available. Phytochemical studies conducted on A. phylicoides revealed the presence of chemical compounds known to possess pharmacological activity, such as flavonoids (5-hydroxy- 6,7,8,3?,4?,5?-hexamethoxyflavon-3-ol; 3-O-demethyldigicitrin; 5,6,7,8,3?,4?-hexamethoxy- flavone and quercertin) and polyphenols (Mudau et al., 2006; Mashimbye et al., 2006; Mavundza et al., 2010). Further analysis revealed the absence of caffeine (CNS stimulant) stimulant) and pyrrolizidine alkaloids (plant toxins associated with disease in humans and animals) in A. phylicoides (McGaw et al., 2007). A study on the nutrient content of A. phylicoides revealed the presence of approximately 13% non-structural carbohydrates, 8% proteins, 2.5% fatty acids, minerals (calcium, magnesium, phosphorus, potassium, sodium, iron, manganese, zinc, copper, aluminium, sulphur and fluoride) and traces of tannins and vitamins B1, B2, C and E (Olivier and Rampedi, 2008). The effect of fermentation temperature and time on the chemical composition of A. phylicoides was investigated by Hlahla et al. (2010). The study revealed that neither fermentation temperature nor time had any significant influence on the anti-oxidant content, however, increasing fermentation temperature increased polyphenols and decreased tannin content. In addition increasing fermentation time increased both polyphenols and tannin content. Few reports have been undertaken on the anti-oxidant and antimicrobial activity of A. phylicoides. McGaw et al. (2007) reported that the ethanol extracts of A. phylicoides exhibited anti-oxidant activity comparable to that found in rooibos when using Trolox?equivalent anti-oxidant capacity (TEAC) assay. It has also been reported that the ethanol extract of A. phylicoides displayed anti-oxidant activity in the 2,2-diphenyl-1- picrylhydrazyl (DPPH?) free radical assay (Mavundza et al., 2007, 2010). Mavundza et al. (2007) reported that the ethanol extract displayed antimicrobial activity against five micro- organisms tested. 9 Some toxicity studies have been conducted on A. phylicoides (McGaw et al., 2007; Chellan et al., 2008; Mavundza et al., 2010). Athrixia phylicoides traditional preparations (decoctions and infusions), aqueous and ethanol extracts were tested for toxic effects to brine shrimp larvae and the Vero kidney cell line. The traditional preparations and aqueous extract had little effect in both assays, but the ethanol extract was toxic. In another study, the ethanol extract showed low toxicity on Vero kidney cells (Mavundza et al., 2010). Chellan et al. (2008) investigated the cytotoxic effect of the aqueous extract of A. phylicoides on Wistar rats for three months, but the extract did not cause any morbidity or mortality. Some reviews have been undertaken on A. phylicoides. In a review conducted by Joubert et al. (2008), it was noted that due to its substantial use, A. phylicoides is at high risk of over- exploitation. The review also confirmed that very few studies have been conducted on A. phylicoides, and to date it is the only indigenous South African tea that has been subjected to a sub-chronic toxicity study. In another review by Gulumian and Savolainen (2008), the results of the toxicity study conducted by Chellan et al. (2008) on A. phylicoides was discussed. 1.3 Objectives of the study The objectives of the study were to: ? Investigate the chemical composition of the essential oil using gas chromatography (GC) and gas chromatography combined with mass spectrometry (GC/MS). ? Evaluate the antimicrobial activity of the essential oil and methanol extract on selected micro-organisms using the minimum inhibitory concentration method. ? Isolate and identify the compound(s) responsible for the antimicrobial activity. ? Investigate the anti-oxidant activity of the aqueous extract of A. phylicoides and to compare its activity to other commercially available teas. ? Investigate the effect of the methanol extract and essential oil on Plasmodium falciparum, the protozoan responsible for malaria. ? Investigate the anti-inflammatory activity of the methanol extract and essential oil of A. phylicoides. ? Determine the toxic properties of the methanol extract and essential oil. 10 Chapter 2 ? Plant collection and preparation of samples 2.1 Collection of plant material A total of seven samples of A. phylicoides were collected from two natural populations over a period of four months from February 2004 to May 2004. The sample populations were Haenertsberg in Limpopo and Dundee in KwaZulu-Natal. Pooled samples (a number of many individual plants) and two individual plants were collected (Table 2.1). Professors J. Olivier and A.M. Viljoen assisted with the collection of plants. The taxonomic identification of all samples was confirmed by botanists at the South African National Botanical Institute (SANBI) in Pretoria. Voucher specimens have been maintained at the Department of Pharmacy and Pharmacology at the University of Witwatersrand. Pooled samples for both the essential oils and extracts have been used in the antimicrobial, anti-oxidant, antimalarial, anti-inflammatory and toxicity studies. Table 2.1: Plant collection, voucher and sample reference numbers, and percentage oil yield. Plant Population Month of collection Type of sample Voucher no. Sample reference no. Yield (%) Haenertsberg February Pooled sample AV914 1A 0.22 Haenertsberg February Individual AV916 1B 0.17 Haenertsberg February Individual AV917 1C 0.18 Haenertsberg March Pooled sample AV936 2A 0.10 Dundee March Pooled sample AV958 2B 0.17 Haenertsberg April Pooled sample AV977 3A 0.47 Haenertsberg May Pooled sample AV999 4A 0.31 11 2.2 Preparation of samples 2.2.1 Isolation of essential oils The fresh aerial parts of the plant were allowed to dry for 24 hours at room temperature. Conventional hydrodistillation was then carried out using a Clevenger-type apparatus for three hours, to selectively isolate the essential oils from the aerial parts of the plant (Figure 2.1). The pure essential oils were collected and the essential oil yield (Table 2.1) was recorded prior to storing the essential oils in amber vials at 4 ?C. Figure 2.1: Hydrodistillation of plant material using the Clevenger-type apparatus. 2.2.2 Preparation of the solvent extracts Fresh aerial parts were dried at room temperature for seven days and pulverized into a fine powder, using a mill (Diaf R/S, sieve no. 2). The pulverized aerial parts (5 g) were macerated for 24 hours with methanol (50 ml) at room temperature, than filtered through Whatman? No.1 filter paper (pore size: 20-25 ?m). The extraction solvent was evaporated at 35 ?C using a rotavapor (B?chi Rotavapor R-114). The extracts were then concentrated to dryness in the fume-hood. The concentrated extract was stored at 4 ?C prior to analysis. A yield of 0.04% was recorded for extract of sample 1A. It is important to note that various solvent systems (acetone, methanol, dichloromethane, chloroform and ethanol) were assayed before methanol 12 was finally selected as the extracting solvent because this solvent extracted compounds over a range of polarities. 2.2.3 Preparation of aqueous tea infusions Aqueous tea extracts were prepared by pouring boiling distilled water (250 ml) onto dry leaves (1.7 g), followed by stirring with a magnetic stirrer. The leaves were brewed for 15 min. The aqueous extracts were strained and filtered through Pasteur pipettes plugged with cotton wool. Aliquots of the filtered extracts were freeze-dried and the yield of the extracts was recorded (Table 2.2). Table 2.2: Botanical name, common name and percentage aqueous extract yield. Botanical name Common name Yield (%) Aspalathus linearis Green rooibos tea 1.06 Aspalathus linearis (fermented) Rooibos tea 1.48 Athrixia phylicoides Bush tea (Zulu tea) 0.59 Camellia sinesis Green tea 0.94 Camellia sinesis (fermented) Black tea (Ceylon) 0.88 Cyclopia intermedia Honeybush tea 1.93 13 Chapter 3 ? Composition of essential oils of Athrixia phylicoides 3.1 Introduction Essential oils are volatile, natural, complex compounds formed by aromatic plants as secondary metabolites. Initially essential oils were thought to be waste products or storage products; however, in recent times we have begun to uncover the biological and ecological roles of essential oils. Research has shown that essential oils can act as pollination agents, dispersal agents, defense compounds against insects and animals and as antimicrobial agents (Hili, 2001). Essential oils have been used for centuries in medicine. It is believed that Arnald de Villanova was the first doctor to use essential oils therapeutically in the 13th century. By the 17th century essential oils were widely used by pharmacists as topical preparations. During the 19th and 20th century much research was conducted on the nature of essential oils and aromatic compounds. Physical and chemical procedures were established to analyze aromatics (Hili, 2001). Today essential oils are used in perfumes, make-up, sanitary products, dentistry and agriculture, as food preservers and additives and as natural remedies (Bakkali et al., 2008). The use of essential oils as natural remedies has triggered a renewed interest in essential oil research. In recent years many researchers have dedicated time to establish the relationship between essential oils and their biological activities (Hammer et al., 1999; Hernandez et al., 2007; van Vuuren et al., 2007). Even though the literature describes A. phylicoides as a well-known, multi-purpose, versatile plant, utilised as a tea and a medicine (van Wyk and Gericke, 2000), to date no data on the essential oil composition of this plant has been published. This study is the first report on the essential oil composition of A. phylicoides. The aim of this aspect of the study was to determine the chemical composition of the essential oil of A. phylicoides. 14 3.2 Materials and methods Essential oils were distilled from the aerial parts of the plant using hydrodistillation as explained in Section 2.2.1 and were analysed using gas chromatography (GC) and gas chromatography combined with mass spectrometry (GC/MS) in collaboration with Professor Ba?er at the Department of Pharmacognosy, Faculty of Pharmacy, Anadolu University, Eskisehir, Turkey. 3.2.1 Principle of the method Gas chromatography is a separating technique comparing retention times of the chromatogram peaks with those of reference compounds run under identical conditions to identify the peaks. Mass spectrometry records a fragmentation pattern of a substance and provides information about the molecular structures. Since the mass spectrum of a substance is unique to that substance, the spectrum can be compared to previously recorded spectra to identify the substance (Gillespie et al., 1994). 3.2.2 Gas chromatography Analytical gas chromatography was performed using the Hewlett Packard 6890 system. An HP-Innowax FSC column (60 m x 0.25 mm, 0.25 ?m film thickness) was used with nitrogen gas as the carrier gas (1 ml/min). The oven temperature was kept at 60 ?C for 10 min and programmed to increase to 220 ?C at a rate of 4 ?C/min, then kept constant at 220 ?C for 10 min and then programmed to increase to 240 ?C at a rate of 1 ?C/min. The split ratio was adjusted to 50:1. The injector temperature was set at 250 ?C. The percentage amounts were obtained from electronic integration measurements using flame ionization detection (FID) set at 250 ?C. Alkanes were used as reference points in the calculation of relative retention indices (RRI). 3.2.3 Gas chromatography combined with mass spectrometry Analysis of the essential oils by GC/MS was performed using a Hewlett Packard (HP) 1800A GCD system operating under the following conditions: column: HP-Innowax (60 m x 0.25 mm, 0.25 ?m film thickness); temperatures: injection port 250 ?C, column 60 ?C for 10 min, 4 ?C/min to 220 ?C, 220 ?C for 10 min, 1 ?C/min to 240 ?C (total = 80 minutes). Percentage 15 compounds were calculated from total ion chromatograms, by the computer. To identify the essential oil components, the mass spectra and retention indices of the components were compared to the Ba?er, Adams and Wiley Libraries of essential oil constituents. 3.3 Results and discussion 3.3.1 Essential oil yield The essential oil yield among the samples studied ranged from 0.1 to 0.47% (w/w), with the highest essential oil yield recorded in April (0.47% w/w) and May (0.31% w/w) (Figure 3.1). The flowering season of A. phylicoides is late autumn (van Wyk and Gericke, 2000). A higher oil yield had been recorded during the flowering months of April and May as compared to the non-flowering months of February and March. Since the amount of essential oil was highest during the flowering months, it is possible that the plant increases the amount of oil to favour pollination (Pal?-P?ul et al., 2001). A similar trend was observed in the leaf oil yield of Agathosma ovata, with values ranging from 0.23% in early spring to 0.85% in late autumn (flowering season) (Viljoen et al., 2006). 0.22 0.17 0.18 0.1 0.17 0.47 0.31 0 0.1 0.2 0.3 0.4 0.5 0.6 1A/Feb 1B/Feb 1C/Feb 2A/Mar 2B/Mar 3A/Apr 4A/May Samples % E ss en tia l o il yi el d Figure 3.1: Percentage essential oil yield for samples of A. phylicoides. In a study performed by M?ller et al. (2006), it was reported that the glandular trichomes present on the leaf surfaces of A. phylicoides, which are believed to be responsible for 16 essential oil synthesis, bursts open during late autumn. The time at which the trichomes bursts open coincided with the highest essential oil yield reported in this study. 3.3.2 Essential oil composition The chemical compounds identified in seven samples using GC/MS analysis are reported in Table 3.1. The essential oil compositions determined by GC and GC/MS revealed 182 components representing 75.4% to 99.8% of the total oil composition. Table 3.1: Essential oil composition of seven samples of A. phylicoides. RRI Compound 1A 1B 1C 2A 2B 3A 4A 1014 Tricyclene t t t - t t t 1032 ?-Pinene 13.7 4.6 1.9 1.5 0.8 13.9 14.8 1035 ?-Thujene 0.1 t 0.1 t t 0.3 0.2 1048 2-Methyl-3-buten-2-ol t t t t t - - 1050 Decene t t t - - t t 1072 ?-Fenchene - - - - - t t 1076 Camphene 0.1 - t - t 0.1 0.1 1093 Hexanal - - - - - t t 1100 Undecane - - t t t 5.0 2.5 1118 ?-Pinene 10.2 7.0 2.5 1.1 1.0 12.6 12.9 1132 Sabinene 0.8 0.9 0.3 t 0.1 1.0 0.8 1134 Thuja-2(4),10-diene t - 0.2 t 0.1 t t 1174 Myrcene 4.1 1.5 0.2 - t 11.6 9.9 1176 ? -Phellandrene - - - t - 0.5 t 1183 ?-Mentha-1,7(8)-diene (=Pseudolimonene) - - - t t t t 1195 Dehydro-1,8-cineole t t t - t t t 1203 Limonene 1.0 0.9 1.0 1.8 0.7 4.0 3.6 1213 1,8-Cineole 0.3 0.1 1.8 2.0 2.5 2.3 2.6 1218 ?-Phellandrene t - - t - 0.5 0.2 1225 (Z)-3-Hexenal - - - - - t t 17 RRI Compound 1A 1B 1C 2A 2B 3A 4A 1244 2-Pentyl furan - - - - t t t 1246 (Z)- ? -Ocimene t t - - - 1.7 0.7 1247 6-Methyl-2-heptanone - t t t t - - 1255 ?-Terpinene - - - - t 0.2 t 1257 1-Dodecene t - t - t - - 1266 (E)-?-Ocimene t - - - - 1.0 0.2 1280 ?-Cymene 2.8 4.0 1.4 0.9 1.0 2.1 2.5 1290 Terpinolene - - - - - 0.1 t 1327 (Z)-3-Hexenyl acetate 0.2 0.2 0.1 - - - - 1348 6-Methyl-5-hepten-2- one t - 0.1 0.1 t - t 1356 1-Tridecene t - - 0.1 0.1 t t 1382 cis-Allo-ocimene - - - - - t t 1384 ?-Pinene oxide 0.2 0.2 0.3 t 0.1 - t 1391 (Z)-3-Hexenol t t 0.1 - - t t 1393 3-Octanol 0.1 t 0.1 t 0.1 t t 1400 Nonanal - 0.1 - t t - - 1402 Tetradecane - - - t - - - 1405 4,8-Dimethyl-1,3,7- nonatriene 0.3 0.3 0.4 0.1 0.1 t 0.2 1429 Perillen 0.3 0.8 0.7 t 0.1 t 0.1 1432 7-?-(H)-Silphiperfol-5- ene t t t 0.1 0.1 t t 1437 ?-Thujone t t t t t t - 1439 ?-Campholene aldehyde - - t - - - - 1443 Dimethyltetradecane t t 0.1 0.7 0.1 t t 1450 trans-Linalool oxide (Furanoid) t t 0.1 0.1 - - - 1451 ?-Thujone t 0.3 0.3 t t t t 1452 ?,?-Dimethylstryene - - - - - t t 1452 1-Octen-3-ol 0.1 t t t 0.1 0.1 0.1 18 RRI Compound 1A 1B 1C 2A 2B 3A 4A 1458 cis-1,2-Limonene epoxide - - t - t - - 1460 7?-(H)-Silphiperfol-5- ene t t - - t t t 1466 ?-Cubebene t t 0.1 0.1 0.1 0.1 0.1 1475 Menthone - t - - - - - 1477 4,8-Epoxyterpinolene - t - - - - - 1478 cis-Linalool oxide (Furanoid) t t 0.1 0.1 0.1 t t 1481 Longipinene t - - - - - - 1493 ?-Ylangene 0.1 0.1 0.1 t t 0.1 0.1 1495 Bicycloelemene - - - - - t t 1496 3-Nonanol t - - - - - - 1497 ?-Copaene 0.4 0.2 0.6 t 0.3 0.5 0.4 1499 ?-Campholene aldehyde t - - 0.2 - - - 1503 Isomenthone - t - - - - - 1507 (E,E)-2,4-Heptadienal - - - - - 0.1 t 1528 ?-Bourbonene t t t t t t t 1532 Camphor - - t t - - - 1535 ?-Boubonene 0.1 0.1 0.2 0.2 t 0.1 0.2 1544 ?-Gurjunene t t - - t t t 1549 ?-Cubebene - t - t - 0.1 - 1553 Linalool 1.3 0.5 1.7 0.1 2.4 1.1 1.0 1556 cis-Sabinene hydrate - - - - 0.1 - - 1557 1-Nonen-3-ol t 0.1 0.1 - - - t 1559 8,9-Limonene epoxide I - - - - t - - 1562 Octanol - - - t - - - 1571 trans-?-Menth-2-en-1-ol t 0.1 0.1 t t t t 1574 8,9- Limonene epoxide II - - t - t - - 1586 Pinocarvone 0.3 0.2 0.7 0.2 0.3 0.1 0.2 1589 ?-Ylangene 0.1 t 1.3 0.1 0.3 0.3 0.2 19 RRI Compound 1A 1B 1C 2A 2B 3A 4A 1592 Nopinone - t 0.6 0.1 0.1 - - 1594 trans-?-Bergamotene - 0.2 - t t 0.5 0.1 1600 ?-Elemene 0.6 2.4 0.3 1.1 0.1 0.3 t 1602 ?-Copaene - t 0.1 0.3 0.1 0.1 t 1604 6-Methyl-3,5-heptadien- 2-one - t - - - - - 1611 Terpinen-4-ol 0.2 2.7 0.2 0.2 0.1 0.4 0.5 1612 ?-Caryophyllene 8.3 5.4 0.3 0.2 2.2 11.9 10.0 1628 Aromadendrene 0.1 0.1 t 0.4 0.1 - t 1638 cis-?-Menth-2-en-1-ol - 0.1 - - - t t 1639 trans-?-Mentha-2,8- dien-1-ol - - t t 0.1 - - 1648 Myrtenal 0.6 0.7 1.7 0.3 0.8 0.1 0.3 1661 Alloaromadendrene 0.3 0.2 0.2 0.2 0.1 0.2 0.2 1668 (Z)-?-Farnesene - - - - - 0.1 0.1 1670 trans-Pinocarveol 0.7 0.9 2.4 0.4 1.4 0.1 0.4 1677 epi-Zonarene - - - - - t 0.1 1678 cis-?-Mentha-2,8-dien- 1-ol - - - t 0.1 - - 1683 trans-Verbenol - 0.1 1.4 0.3 t t 0.3 1687 ?-Humulene 0.9 0.1 - - 1.2 0.4 0.5 1688 Selina-4,11-diene (=4,11- Eudesmadiene) - - - 0.1 0.1 0.1 t 1695 (E)- ?-Farnesene - 0.1 - t t 0.1 0.1 1700 ?-Mentha-1,8-dien-4-ol (=Limonen-4-ol) - - - 0.1 t - - 1704 ?-Muurolene 0.5 0.8 0.6 1.0 0.5 0.2 1.1 1706 ?-Terpineol - t t t t - - 1708 Ledene - t - - t t 0.1 1719 Borneol t t t 0.1 0.1 t t 1720 trans-Sabinol t - - - - 0.1 t 1725 Verbenone 0.3 0.1 0.8 0.2 0.4 - t 20 RRI Compound 1A 1B 1C 2A 2B 3A 4A 1726 Germacrene D 2.7 0.1 - - t 13.5 5.5 1740 ?-Muurolene 0.7 0.4 0.7 0.7 0.8 0.4 0.7 1741 Valencene 0.1 t t t t - - 1742 ?-Selinene 0.1 - t 0.7 t 0.1 0.2 1751 Carvone - - 0.1 0.2 0.2 - - 1755 Bicyclogermacrene 0.2 0.1 t - - 2.4 0.4 1758 (E,E)-?-Franesene t - - - - 0.2 0.1 1760 1-Heptadecene t - - - - - - 1764 cis-Chrysanthenol - 0.1 0.1 - 0.3 - - 1773 ?-Cadinene 0.4 t t t t 0.9 1.0 1776 ?-Cadinene 0.6 0.6 0.3 0.6 0.1 0.3 0.6 1783 ?-Sesquiphellandrene - - - - - t t 1784 (E)-?-Bisabolene t - - - - t t 1797 ?-Methyl acetophenone - - 0.1 t 0.1 - - 1804 Myrtenol 0.4 0.6 1.6 0.4 0.9 0.1 0.3 1807 ?-Cadinene 0.1 t - - 0.1 0.1 0.1 1845 trans-Carveol 0.1 t 0.4 0.2 0.3 t 0.1 1853 cis-Calamenene 0.2 0.3 0.1 0.1 0.1 0.1 0.3 1857 Geraniol - 0.1 t - - 0.1 t 1864 ? -Cymen-8-ol 0.1 0.4 0.2 0.1 t t 0.1 1868 (E)-Geranyl acetone t t t 0.2 0.2 t 0.1 1882 cis-Carveol - - 0.1 0.1 0.2 t t 1900 epi-Cubebol 0.3 t 0.2 0.2 0.2 0.2 0.2 1937 ?-Calacorene I - 0.3 0.1 0.1 0.1 0.1 t 1945 1,5-Epoxy-salvial(4)14- ene 0.2 0.5 1.3 1.5 1.1 t 0.3 1949 (Z)-3-Hexenyl nonanoate t t 0.2 t t - t 1953 Palustrol - 0.2 0.2 t t t t 1957 Cubebol t 0.2 - 0.2 0.3 0.2 0.2 1984 ?-Calacorene 0.1 0.1 0.2 t 0.2 t - 2001 Isocaryophyllene oxide 1.4 1.3 4.0 0.7 4.0 0.1 0.7 21 RRI Compound 1A 1B 1C 2A 2B 3A 4A 2008 Caryophyllene oxide 6.2 21.2 21.4 34.2 18.5 1.0 4.1 2033 Epiglobulol - - - 0.1 0.1 - - 2037 Salvial-4(14)-en-1-one 0.2 0.4 0.1 0.2 0.2 t 0.2 2041 Pentadecanal 0.1 - - 0.2 - t t 2050 (E)-Nerolidol - - t - - t t 2057 Ledol 0.2 0.2 0.2 0.3 0.1 0.1 0.1 2071 Humulene epoxide-II 0.2 0.2 0.9 0.9 0.7 t 0.1 2080 1,10-di-epi-Cubenol 0.2 0.3 0.1 0.1 t 0.1 0.2 2088 1-epi-Cubenol 0.7 0.2 0.4 0.3 0.6 0.3 0.5 2098 Globulol 0.2 0.1 - 1.2 0.6 0.2 0.3 2104 Viridiflorol 0.2 0.1 0.1 0.4 0.3 0.1 0.1 2113 Cumin alcohol - - t 0.1 t - - 2131 Hexahydrofarnesyl acetone - t 0.3 - t t 0.1 2144 Rosifoliol 0.1 0.1 t t 0.1 t 0.1 2151 Salviadienol 0.4 0.3 0.3 0.1 0.1 t 0.2 2164 Spathulenol 4.1 8.5 2.7 9.9 12.6 1.5 3.4 2169 6-epi-Cubenol 0.1 0.4 0.1 0.2 - 0.1 0.2 2179 Nor-Copaonone - t 0.1 - - - - 2187 T-Cadinol 0.7 1.0 0.5 0.3 0.3 0.3 0.6 2209 T-Muurolol 0.6 0.9 0.5 0.4 0.3 0.3 0.6 2214 Torreyol 0.4 0.3 0.3 0.3 0.2 0.3 0.3 2219 Dimyrcene II-a - 0.1 0.1 - - 0.3 0.1 2228 Isospathulenol 0.1 t - 0.2 0.2 - - 2247 trans-?-Bergamotol 0.4 0.1 0.1 0.1 0.4 0.2 0.3 2249 Torilenol 1.0 2.2 0.8 0.2 0.3 t 0.3 2255 ?-Cadinol 1.1 2.0 1.3 1.1 0.9 0.5 1.0 2268 4,7-Dimethyl-1-tetralone - 0.1 0.3 0.1 - - - 2269 Dimyrcene II-b - 0.3 - - - 0.1 0.1 2273 Selin-11-en-4?-ol 0.1 0.1 0.1 0.7 0.1 - - 2287 4-Oxo-?-ylangene (= Mustakone) - t 0.3 0.5 0.3 - - 22 RRI Compound 1A 1B 1C 2A 2B 3A 4A 2315 Eudesma-4(5),7-dienol isomer? 1.1 0.8 0.4 0.1 0.2 0.3 0.6 2316 9-Geranyl-?-cymene t t t - t t t 2320 Caryophylla- 2(12),6(13)-dien-?-ol (=Caryophylladienol I) 0.2 0.2 0.5 0.3 0.5 0.1 0.2 2323 14-nor-Cadin-5-en-4- one* 0.3 0.2 0.6 0.5 1.0 - - 2389 Caryophylla-2(12),6- dien-5?-ol (=Caryophyllenol I) 0.1 0.1 0.1 0.4 0.3 0.1 0.1 2395 cis-Calamenenol? 0.2 0.1 0.1 t t t t 2415 Eudesma-4(5),7-dien- 1?-ol 0.9 0.9 1.0 t 0.6 0.2 0.4 2425 trans-Calamenenol? 0.4 0.3 0.5 0.8 1.3 t 0.1 2427 Farnesyl acetone - t - - 0.1 t t 2433 Caryophylla-2(12),6- dien-5?-ol (=Caryophyllenol II) 0.4 0.5 1.4 2.0 0.2 0.1 0.2 2489 Demethoxyencecaline - - t - - t t 2500 Pentacosane - - t - 0.2 - - 2552 Oplopanone - 0.5 0.6 - - - - 2554 Geranyl linalool 0.2 0.1 0.6 - 0.2 0.2 0.6 2570 14-Hydroxy-?- muurolene 0.1 0.2 0.1 t - t t 2575 ent-4?,10?-Dihydroxy- aromadendrane? 0.1 t 0.3 0.4 1.5 t 0.1 2651 ent-4?,10?-Dihydroxy- aromadendrane isomer? - - t 0.4 0.6 t - 2622 Phytol - - - - 0.2 0.1 0.1 2700 Heptacosane - - - - 0.3 - - 2816 Unknown? 0.9 1.5 2.0 1.1 3.3 0.3 1.1 23 RRI Compound 1A 1B 1C 2A 2B 3A 4A 2908 Unknown?? 2.0 1.1 1.0 3.6 2.1 0.1 0.6 2931 Hexadecanoic acid 0.2 t t t t t t Total 81.2 87.0 75.4 82.1 76.8 99.8 95.3 RRI Relative retention indices calculated against n-alkanes; t Trace (< 0.1%); % calculated from FID data; * Correct isomer not characterized; ? Tentatively identified from Wiley, Adams, NIST libraries; ? Mass spectrum of unknown constituent: EIMS, 70 eV, m/z (rel. int.): 107(100), 121(82), 81(76), 93(71), 43(64), 159(63), 55(58), 177(54), 220(44), 69(41), 189(24), 202(22). ?? Mass spectrum of unknown constituent: EIMS, 70 eV, m/z (rel. int.): 193(100), 91(92), 123(86), 107(82), 133(75), 81(69), 41(61), 55(60), 175(54), 147(52), 236(48), 69(38), 153(26), 218(17). Bold text represents major compounds. Major compounds: Major compounds (>10%) identified in the volatile oil of A. phylicoides studied were ?-pinene, ?-pinene, myrcene, germacrene D, ?-caryophyllene, spathulenol and caryophyllene oxide (Figure 3.2). ?-pinene ?-pinene myrcene germacrene D ?-caryophyllene H H 24 spathulenol caryophyllene oxide Figure 3.2: Chemical structures for major compounds identified in the essential oil of A. phylicoides. Essential oils are known to exhibit various pharmacological activities, however the constituents responsible for these activities remain largely unidentified (van Zyl et al., 2006). Some of the major essential oil constituents present in A. phylicoides are known to possess pharmacological activities. Essential oil constituents, ?-caryophyllene (10 ? IC50 ? 30 ppm); germacrene D (10 ? IC50 ? 30 ppm) and ?-pinene (31 ? IC50 ? 50 ppm) exhibited anti- inflammatory activity against the 5-lipoxygenase enzyme (Baylac and Racine, 2003). In a study conducted by van Zyl et al. (2006), it was reported that ?-pinene displayed potent antimalarial activity against the chloroquine- resistant strain of P. falciparum (FRC-3) (IC50 = 1.2 ? 0.2 ?M). The toxicity of some of the major essential oil constituents of A. phylicoides have also been investigated. Caryophyllene oxide has been reported to have cytotoxic activity (IC50 values range from 147 to 351 ?M) against human tumour cells (Sibanda et al., 2004). In another study, it has been reported that ?-pinene (IC50 = 172.2 ? 13.4 ?M) and ?-pinene (IC50 = 166.7 ? 19.6 ?M) displayed some degree of toxicity against human kidney epithelial cells (van Zyl et al., 2006). Quantitative variation in major compounds of the essential oil in the seven samples studied has been observed (Figure 3.3). No specific trend was noticed for the major compounds, however remarkable fluctuations in quantities included caryophyllene oxide (1.0% to 34.2%), ?-pinene (1.0% to 12.9%), ?-pinene (0.8% to 14.8%), spathulenol (1.5% to 12.6%), germacrene D (0% to 13.5%), ?-caryophyllene (0.2% to 11.9%) and mycrene (0% to 11.6%). The most H H OH R O 25 conspicuous variation in major compounds was noted for caryophyllene oxide, ranging from 1.0% to 34.2%. ?-Pinene was found to be the main constituent of samples 1A/Feb (13.7%), 3A/Apr (13.9%) and 4A/May (14.8%) and caryophyllene oxide was the main constituent of samples 1B/Feb (21.2%), 1C/Feb (21.4%), 2A/Mar (34.2%) and 2B/Mar (18.5%). Spathulenol (12.6%) was identified as a major compound in sample 2B/Mar only. It was also noted that the essential oil compositions of samples 3A/Apr and 4A/May were similar (Figure 3.3). The major compounds represented between 21.2% and 63.5% of the total oil composition (Table 3.1). 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 1A/Feb 1B/Feb 1C/Feb 2A/Mar 2B/Mar 3A/Apr 4A/May Samples % C om po si tio n Other Spathulenol Caryophyllene oxide Germacrene D ?-Caryophyllene Myrcene ?-Pinene ?-Pinene Figure 3.3: Major compounds identified in A. phylicoides. The results from this study revealed that variation in essential oil composition exists in the same species (A. phylicoides) collected from the same location. The composition of the essential oils can be affected by many factors such as development or reproductive stages, seasons, location, temperature, extraction method and the conditions of analysis (Putievsky et al., 1986; Sangwan et al., 2001; Kim and Lee, 2004). In a study by Simmons et al. (1987), it was reported that the variation in essential oil composition of Eucalyptus ovata and Eucalyptus camphora could be attributed to the leaf ageing effect. Kamatou et al. (2008) reported seasonal variation in essential oil composition for three South African Salvia species. The composition of the essential oil of sage has been found to change under the influence of temperature (Avato et al., 2005). The variation in essential oil composition noted in this study 26 may be as a result of various factors, such as temperature, age of the leaves, development stages and genetic factors. In a study conducted on Athrixia elata, the following constituents were found in the aerial parts: squalene, germacrene D, caryophyllene, ?-humulene and cinnamates (Bohlmann et al., 1982). Some of the constituents found in A. elata are also present in A. phylicoides in varying quantities: germacrene D ? trace to 13.5%; caryophyllene ? 0.2 to 11.9% and ?-humulene ? 0.1 to 1.2%. 3.4 Conclusions ? A higher percentage essential oil yield was recorded for A. phylicoides collected during the flowering months of April and May (0.47% and 0.31%) compared to the non- flowering months (0.1% to 0.2%). ? Seven major compounds (>10%) were identified in the samples of A. phylicoides studied: caryophyllene oxide, spathulenol, germacrene D, ?-caryophyllene, ?-pinene, ?-pinene and myrcene. ? Variation in the essential oil composition of A. phylicoides from within the same population and between populations has been noted. 27 Chapter 4 ? Antimicrobial activity and isolation of bioactive compound 4.1 Introduction The discovery of antimicrobial agents during the twentieth century substantially reduced the threat posed by infectious diseases. Antimicrobial agents combined with improvements in sanitation, housing and nutrition, and the advent of widespread immunization programmes, ensured a dramatic drop in deaths from diseases that were previously widespread, untreatable and frequently fatal. Over the years, antimicrobial agents have saved lives, eased the suffering of millions of people and contributed to increasing life expectancy (WHO, 2002). This significant advancement in medicine is now under severe threat by another recent development, the emergence and spread of micro-organisms that are resistant to antimicrobial agents (WHO, 2002). The consequences of resistant micro-organisms are severe. Infections caused by resistant micro-organisms do not respond to treatment with antimicrobial agents, for example nosocomial infections caused by methicillin resistant Staphylococcus aureus will not respond to methicillin and community acquired pneumonia caused by strains resistant to penicillin will not respond to penicillin and even sometimes cephalosporins. The result is prolonged illness, increasing the risk of death. In addition, the prolonged illness leads to longer periods of infectivity, which can increase the number of infected people moving in the community and thus expose the general population to the risk of contracting a resistant strain of the infection (WHO, 2002). With the continuous escalation of antimicrobial resistance, the acceptance of traditional medicine as an alternative form of health care by South Africans has increased. For this reason, medicinal plants have become the focus of intense study to determine whether their traditional uses can be supported by pharmacological effects or are merely based on folklore (Rabe and van Staden, 1997). A survey of the literature revealed that the leaves of A. phylicoides have been used for the treatment of boils, sores, bad acne, infected wounds and cuts (Table 1.2) (Plowes and 28 Drummond, 1976; Roberts, 1990; Hutchings et al., 1996). This information prompted an investigation into the in vitro antimicrobial activity of A. phylicoides. The aims of this study were to: ? investigate the essential oil and methanol extract of the indigenous plant A. phylicoides for potential antimicrobial activity. ? isolate and identify the compound/s exhibiting antimicrobial activity. 4.2 Materials and methods 4.2.1 Minimum inhibitory concentration assay The anitmicrobial activity of the essential oil and methanol extract of A. phylicoides was assessed using the minimum inhibitory concentration microdilution (MIC) assay (Eloff, 1998). 4.2.1.1 Principle of the method The minimum inhibitory concentration (MIC) is the lowest concentration at which the growth of the bacteria is inhibited. The ?-iodonitrotetrazolium (INT) microplate bioassay technique was used to determine the MIC (Eloff, 1998). Microbial growth is indicated by the presence of a reddish colour, which is produced when INT (colourless product) is reduced by metabolically active micro-organisms to the corresponding intensely coloured formazan. Therefore the MIC value is the concentration of the first well in which no colour change is observed (Figure 4.1). 4.2.1.2 Protocol Five Gram-positive bacteria: Staphylococcus aureus (ATCC 12600), Enterococcus faecalis (ATCC 29212), Bacillus cereus (ATCC 11778), Staphylococcus epidermidis (ATCC 2223) and Bacillus subtilis (ATCC 6051); four Gram-negative bacteria: Klebsiella pneumoniae (NCTC 9633), Escherichia coli (ATCC 8739), Yersinia entercolitica (ATCC 23715) and Salmonella typhimurium (ATCC 14028) and two yeasts: Candida albicans (ATCC 10231) and Candida tropicalis (ATCC 750) were used to evaluate the antimicrobial activity of the essential oil and methanol extract of A. phylicoides. 29 Figure 4.1: Microtitre plate for Salmonella typhimurium where column 1 is the essential oil (concentrations decreasing from 32 mg/ml in row A to 0.25 mg/ml in row H), column 2 is the extract (concentrations decreasing from 16 mg/ml in row A to 0.125 mg/ml in row H), column 3 is a positive control and column 4 is a negative control. The test solutions were prepared by diluting the essential oil and methanol extract in acetone to achieve a final concentration of 128 mg/ml and 64 mg/ml, respectively. The inoculum was prepared by mixing 1 ml of the respective bacteria and yeast grown overnight with 99 ml of fresh Tryptone Soya broth. INT was prepared by dissolving 40 mg in 100 ml sterile water. For each micro-organism, columns in the microtitre plate were reserved for positive and negative controls. Commercial antimicrobials (ciprofloxacin and amphothericin B) were used as positive controls for the bacteria and yeast, respectively. Ciprofloxacin (1 mg/ml) and amphothericin B (1 mg/ml) was diluted (1:10) with sterile water and DMSO, respectively to achieve a concentration of 0.01 mg/ml. For negative controls, wells containing acetone (100 ?l) and no plant extract were prepared for the observation of normal bacterial and fungal growth. In the microtitre plate, 100 ?l sterile water was added to each well. The test solution (100 ?l) was added to each well in row A of the plate. Serial dilutions (1:1) were made in the Red well ? bacterial growth Clear well ? inhibition of bacterial growth. MIC - 8 mg/ml and 4 mg/ml, respectively Serial dilution (1:1) ROW A ROW H 1 2 3 4 1 2 3 4 30 microtitre plates. Inoculum (100 ?l), yielding an inoculum size of 1 x 106 CFU/ml was then added to each well to achieve final concentrations of 32 mg/ml in row A to 0.25 mg/ml in row H for the essential oil and 16 mg/ml in row A to 0.125 mg/ml in row H for the extract, respectively. The plates were incubated at 37 ?C for 24 hours for bacteria and 48 hours for yeast. After incubation, 50 ?l INT (0.04% w/v) was added to each well and the plates were left at room temperature for six hours for bacteria and 24 hours for yeast before MIC?s were determined visually. All organisms were tested in duplicate (two columns each) for the essential oil and the methanol extract (Figure 4.1). The minimum inhibitory assays were repeated and the mean was calculated. 4.2.2 Isolation of the bioactive compound The methanol extract of A. phylicoides was selected to isolate the bioactive compound/s, since it displayed favourable antimicrobial activity. 4.2.2.1 Column chromatography Column chromatography was conducted to isolate the compound/s responsible for antimicrobial activity (Figure 4.2). Silica mixed with hexane and ethyl acetate was poured into the column and allowed to settle for a few minutes. The plant extract dissolved in ethyl acetate was carefully pipetted along the rim of the column. The column was eluted successively with hexane:ethyl acetate (6:4), then hexane:ethyl acetate (7:3) followed by 100% ethyl acetate and finally stripped with 100% methanol. The collection of these crude fractions was based on colour change. The fractions were collected in 250 ml conical flasks and spotted on TLC plates. TLC plates were developed in hexane:ethyl acetate (6:4). Fractions displaying similar chromatographic profiles on the TLC plates were combined to yield four main fractions (A-D). 4.2.2.2 Thin layer chromatography and bio-autographic assay A direct antimicrobial bioassay on a thin layer chromatography plate was employed using the agar-overlay method with Staphylococcus aureus (ATCC 12600) as the test organism to isolate the bioactive compound. S. aureus was chosen as the test organism because it is one 31 Figure 4.2: Preparing a silica column for column chromatography used to separate fractions. of the most difficult bacterial strains to treat with conventional antibiotics (Esposito et al., 2009). Each of the four fractions were spotted on a TLC plate (Alugram? Sil G/UV254, 0.200 mm) and developed in a hexane:ethyl acetate (6:4) solvent system. The TLC plate was then dried and sterilised under UV light (254 mm) for one hour. Tryptone Soya agar (15 ml) was poured into a petri dish and allowed to solidify. The sterilised TLC plate was placed onto the agar surface with the silica side facing upwards. An overlayed layer of Tryptone Soya agar (15 ml) containing 150 ?l of S. aureus (approximate inoculum size, 1 x 106 CFU/ml)) was placed on top of the TLC plate. The petri-dish was placed in the refrigerator (4 ?C) for one hour to allow the compounds on the TLC plate to diffuse into the agar. The plate was then removed and incubated at 37 ?C for 24 hours. At the end of the incubation period, the TLC plate was sprayed with INT (0.04% w/v) to visualise the inhibition zone. The inhibition zone was indicated by the clear zone on the red background (Figure 4.3). On evaluation of the bio- autographic assay of the four fractions, the best zone of inhibition was observed around Fraction D and was thus selected for further purification (Figure 4.3). 32 Figure 4.3: Bio-autographic assay of A. phylicoides against the test pathogen S. aureus. (Inhibition zones are shown by clear spots on the red background.) 4.2.2.3 Bioactivity-guided fractionation and isolation Column chromatography yielded four fractions of which Fraction D was the most bioactive (Figure 4.3). A second column was prepared in order to fractionate Fraction D. A total of 57 sub-fractions were collected and spotted on TLC plates. The bioactivity-guided fractionation procedure is summarized in Figure 4.4. 4.2.2.4 Identification, elucidation and minimum inhibitory concentration of the isolated compound 4.2.2.4.1 Nuclear magnetic resonance The isolated compound was structurally characterized using nuclear magnetic resonance (NMR) and MS and data compared to those found in the literature in collaboration with Professor F.R. van Heerden at the University of KwaZulu-Natal. Spectra were recorded on a Varian Inova 2000 300 MHz spectrometer. All spectra were recorded at 25 ?C in denatured chloroform and the chemical shifts were recorded in ppm referenced to tetramethylsilane. Electron ionization MS of the isolated compounds was performed by direct inlet at 70 eV on the GC-MS QP 2010 GC coupled to MS. Crude extract Fraction D ? bioactive fraction 33 Crude A. phylicoides methanol extract 13.57 g Silica gel 60 Hexane:ethyl acetate (6:4) Hexane:ethyl acetate (7:3) Ethyl acetate 100% Methanol 100% 340 fractions Fraction A Fraction B Fraction C Fraction D (13-25) (45-51) (61-87) (91-102) 70 mg 37.7 mg 48.9 mg 41.5 mg 57 fractions Sephadex LH-20 Methanol 100% Chloroform Subfractions 16-18 21.5 mg Figure 4.4: Bioactivity-guided fractionation procedure (All fractions and sub-fractions not listed had low activity and were not considered for further purification). Antimicrobial compound 34 4.2.2.4.2 Antimicrobial activity of the isolated compound The antimicrobial activity of the isolated compound of A. phylicoides was assessed using the MIC assay as described in Section 4.2.1. Three micro-organisms were chosen for the MIC assay, i.e. one Gram-positive bacterium: Staphylococcus aureus (ATCC 12600); one Gram- negative bacterium: Escherichia coli (ATCC 8739) and one yeast Candida albicans (ATCC 10231). Only three micro-organisms could be used in the MIC assay because of a limited quantity of isolated compound. Commercial antimicrobials (ciprofloxacin and amphothericin B) were used as positive controls for the bacteria and yeast, respectively. 4.3 Results and discussion 4.3.1 Antimicrobial activity: Extract, essential oil and bioactive compound The MIC assay revealed that both the extract and essential oil were active against some of the test organisms (Table 4.1). The methanol extract displayed stronger antimicrobial activity towards all test organisms than the essential oil. The average MIC values obtained for the extract against each organism ranged from 1 to 6 mg/ml; while the average MIC values obtained for the essential oil against each organism ranged from 3 to >32 mg/ml. It has been reported that an essential oil with a MIC ?2 mg/ml, an extract with a MIC <8 mg/ml and a bioactive compound with a MIC value ranging between 64 ? 100 ?g/ml are considered to possess noteworthy antimicrobial activity (Fabry et al., 1998; Gibbons, 2004; van Vuuren, 2008). The highest sensitivities for the methanol extract were against S. aureus, B. cereus and B. subtilis (MIC value: 1 mg/ml) and the essential oil was moderately active against B. cereus (MIC value: 3 mg/ml). The MIC assay revealed that A. phylicoides bioactive compound displayed antimicrobial activity and was most active against the Gram-positive bacterium, S. aureus (MIC value: 19.5 ?g/ml). The bioactive compound also displayed some antimicrobial activity against the Gram- negative bacterium, E. coli and the yeast, C. albicans. The bioactive compound was 51 times more active compared to the methanol extract against S. aureus, 26 times more active compared to the methanol extract against C. albicans and 24 times more active compared to the methanol extract against E. coli. 35 Table 4.1: Antimicrobial activity of A. phylicoides methanol extract, essential oil and bioactive compound (n = 2). Test Organism Essential Oil (mg/ml) Extract (mg/ml) Bioactive compound (?g/ml) Control (?g/ml) Bacillus cereus (ATCC 11778) 3 1 ND 0.6 (a) Bacillus subtilis (ATCC 6051) 6 1 ND 0.3 (a) Enterococcus faecalis (ATCC 292192) > 32 4 ND 0.3 (a) Staphylococcus aureus (ATCC 12600) 24 1 19.5 0.6 (a) Staphylococcus epidermidis (ATCC 2223) 24 3 ND 0.6 (a) Escherichia coli (ATCC 8739) 12 3 125 0.15 (a) Klebsiella pneumoniae (NCTC 9633) 12 6 ND 0.3 (a) Salmonella typhimurium (ATCC 14028) > 32 6 ND 0.6 (a) Yersenia entercolitica (ATCC 23715) 12 3 ND 0.3 (a) Candida albicans (ATCC 10231) 6 2 78 0.15 (b) Candida tropicalis (ATCC 750) 4 1.5 ND 0.15 (b) (a) Ciprofloxacin (b) Amphothercin B ND ? Not determined 36 The plant extract, the bioactive compound and the essential oil displayed better activity against the Gram-positive bacteria than the Gram-negative bacteria. These results are consistent with results from previous in vitro studies with plant extracts and essential oils (Hernandez et al., 2007; Nyiligira et al., 2008; Mahboubi and Haghi, 2008; Paraskeva et al., 2008; Lalli et al., 2008). It is believed that the difference in structure, that is, the hydrophobic lipopolysaccharide in the outer membrane of Gram-negative bacteria provides an additional barrier to agents making them more resistant to the inhibitory effects of agents compared to Gram-positive bacteria (Delaquis et al., 2002; Mahboubi and Haghi, 2008). In a study conducted by Mavundza et al. (2007), the antimicrobial activity of the ethanol extract of A. phylicoides (collected from Venda in the Limpopo Province, South Africa in mid- July) was investigated. The micro-organisms tested were S. aureus (ATCC 12600), B. cereus (ATCC 11778), E. faecalis (ATCC 292192), E. coli (ATCC 11775) and Mycobacterium smegmatis (ATCC X). The extract displayed inhibitory activity against all micro-organisms tested. These results are congruent with the results obtained in this study, in which the methanol extract also displayed inhibitory activity against S. aureus, B. cereus, E. faecalis and E. coli (Table 4.1). Athrixia phylicoides plant extract had MIC values below 8 mg/ml for all organisms tested (Table 4.1) and the bioactive compound had MIC values of 19.5 ?g/ml and 78 ?g/ml for S. aureus (Gram-positive bacteria) and C. albicans (yeast) respectively. As such it can be concluded that A. phylicoides plant extract possesses some antimicrobial activity (Fabry et al., 1998; Gibbons, 2004). Furthermore the antimicrobial activities exhibited by the crude plant extract and the bioactive compound of A. phylicoides against S. aureus (MIC values: 1 mg/ml and 19.5 ?g/ml) must be emphasised. Infections caused by S. aureus are difficult to treat because often it does not respond to conventional antibiotic therapy, which can result in high morbidity and substantial costs (Esposito et al., 2009). In addition, S. aureus has been recognised as a significant cause of nosocomial infections (Esposito et al., 2009). This noteworthy antimicrobial activity of A. phylicoides extract and bioactive compound against S. aureus (Table 4.1) can therefore pave the way for further in vitro and possibly in vivo tests to yield compounds that can improve the treatment of infections caused by S. aureus. The in 37 vitro antimicrobial activity of A. phylicoides obtained from this study, justifies its use as a traditional remedy to treat boils, sores, bad acne, infected wounds and cuts (Table1.2). 4.3.2 Bioactivity-guided fractionation and isolation Two active substances were visible on the TLC plate (Figure 4.3) for Fraction D, whereas only one active substance was visible for the crude extract. However following the TLC of fractions of the second column which separated the constituents of Fraction D, only active substance was visible for both the fractions and the crude extract. It can therefore be concluded that the second compound for Fraction D was probably due to degradation during isolation or due to concentration effects. Only one active compound was present in the A. phylicoides methanol extract. 4.3.3 Elucidation and identification of the isolated compound Based on the1H-NMR, 13C-NMR, DEPT, COSY spectra and comparison with data obtained in the literature, the compound was identified as (4-hydroxyphenyl)propyl coumaroate (Figure 4.5). The chemical shift data (Table A.1) as well as the 13C and 1H spectra (Figures A.1 and A.2) for (4-hydroxyphenyl)propyl coumaroate has been included in Appendix A. (4-Hydroxyphenyl)propyl coumaroate has been previously identified in A. elata, however, no data has been published on any of the biological activities of the compound (Bohlmann and Zdero, 1977; Bohlmann et al., 1982). (4-Hydroxyphenyl)propyl coumaroate is an ester of coumaric acid. HO O O OH 1 2 3 1' 2' 3' 4' 1" 2" 3" 4" ? ? Figure 4.5: Structure of (4-hydroxyphenyl)propyl coumaroate isolated from A. phylicoides. 38 Coumarins are found in many families of plants such as Apiaceae, Asteraceae, Leguminoseae, Rosaceae, Rubiaceae, Rutaceae and Solanaceae (Weinmann, 1997). Their structural diversity can greatly influence their biological activities (Kostova, 2005). Several papers have been published on the antimicrobial activity of coumarins. The antimicrobial properties of coumarins were first recognized in 1945 when an investigation with dicoumarol was conducted and it was found to inhibit the growth of several strains of bacteria (Goth, 1945). Smyth et al. (2009) evaluated the antimicrobial activity of 43 naturally occurring and synthetic coumarins against six strains of bacteria, using the MIC microtitre assay. Approximately half of the coumarins tested were found to have varying antimicrobial activity with MICs ranging from 1.56 to >1000 ?g/ml. The antimicrobial activity of three coumarins (aesculetin, prenyletin and haplopinol) isolated from Haplopappus multifolius were investigated and it was found to inhibit the growth of three bacterial strains (Sarcina lutea, E. coli and S. aureus) with MICs ranging from 250 to 1000 ?g/ml (Chiang et al., 1982). 4.4 Conclusions ? The plant extract of A. phylicoides (MIC range: 1 to 6 mg/ml) exhibited greater antimicrobial activity than the essential oil (MIC range: 3 to >32 mg/ml). ? Greater antimicrobial activity was displayed against Gram-positive bacteria than Gram-negative bacteria. ? Bioactivity guided fractionation led to the isolation of (4-hydroxyphenyl)propyl coumaroate. ? (4-Hydroxyphenyl)propyl coumaroate was most active against the Gram-positive bacteria, S. aureus with a MIC of 19.5 ?g/ml compared to a MIC of 1 mg/ml for the methanol extract and a MIC of 0.6 ?g/ml for ciprofloxacin (positive control). ? The MIC assay results of A. phylicoides provided preliminary evidence to support its use for the treatment of boils, sores, bad acne, infected wounds and cuts. 39 Chapter 5 ? Anti-oxidant activity 5.1 Introduction Free radicals such as hydrogen peroxide (H2O2), superoxide radicals (O2?) ?and hydroxyl radicals (OH?) are unstable, highly reactive molecules. They are produced as a normal consequence of biochemical processes in the body and as a result of increased exposure to environmental and/or dietary xenobiotics. Free radicals are major contributors to degenerative diseases of aging such as cancer, cardiovascular disease, cataracts, immune system decline and brain dysfunction (Lee et al., 2003; Atoui et al., 2005). Anti-oxidants are free radical scavengers that inhibit the oxidative mechanisms which lead to degenerative diseases. Mammalian cells possess defence mechanisms (anti-oxidant enzymes and small molecule anti-oxidants) for the detoxification of free radicals. The anti-oxidant enzymes include superoxide dismutase (SOD), which dismutes superoxide to hydrogen peroxide and oxygen; catalase (CAT), which converts H2O2 into H2O and oxygen and gluthathione peroxidase (GPX), which destroys toxic peroxides. In addition, small molecule anti-oxidants are available in vivo (gluthathione, bilirubin and melatonin) or obtained from the diet (?-tocopherol, ?-carotene, Vitamin A and E, flavonoids, zinc and selenium) (Lee et al., 2003). When an imbalance in oxidant and anti-oxidant processes exists (oxidative stress), the excess free radicals induce peroxidation of polyunsaturated fatty acids in the cell membrane lipid bi- layer. This causes a chain reaction of lipid peroxidation, thus damaging the cellular membrane and causing further oxidation of the membranes lipids and proteins. Subsequently, cell contents, including DNA are damaged. This free radical damage precedes the degenerative diseases (Cook and Samman, 1996; Gramza and Korczak, 2005). Traditionally, carotene, Vitamin E and C are referred to as dietary anti-oxidants. In recent years there has been particular interest in the anti-oxidant activity and health benefits of other phytochemicals. 40 Tea is an important source of dietary polyphenols. The types and amounts of polyphenols present in tea will differ dependant on the variety of leaf, growing environment, processing, manufacturing, particle size of ground tea-leaves and infusion preparation (Gramza and Korczak, 2005). The results of many studies showed the potential anti-oxidant properties of polyphenols (K?hk?nen et al., 1999; Miliauskas et al., 2004; Aoshima et al., 2007; Conforti et al., 2008). However, the relationship between the total phenolic content and anti-oxidant activity are in-conclusive. Some authors have found a correlation between the total phenolic content and anti-oxidant activity while others found no relationship. K?hk?nen et al. (1999) and Conforti et al. (2008) found no correlation between the total phenolic content and anti- oxidant activity, and have concluded that the difference in anti-oxidant activity is attributed to the type of phenolic constituents and not the amount of phenolic constituents. However, other authors confirm that the higher the polyphenol content, the higher the anti-oxidant activity (Miliauskas et al., 2004; Aoshima et al., 2007). The growing interest in the anti-oxidant activity of tea and the potential health benefits led to the study of the anti-oxidant activity of commercially available teas with A. phylicoides. The aims of this study were to: ? confirm that A. phylicoides possess anti-oxidant activity. ? compare the anti-oxidant activity of A. phylicoides to that of other commercially available teas. 5.2 Materials and methods The anti-oxidant activities of rooibos tea (green and fermented), green tea, Ceylon (black) tea and honeybush tea were compared to A. phylicoides (Bush tea) using the 2,2-diphenyl-1- picrylhydrazyl (DPPH?) free radical scavenging assay. Rooibos, green, Ceylon and honeybush tea were purchased at a local supermarket. Green rooibos tea was sourced from Rooibos Ltd in Clanwilliam and a pooled sample of A. phylicoides (Bush tea) (Figure 5.1) was used in the analysis. The botanical and common names of the samples tested are listed in Table 5.1. 41 Figure 5.1: Commercial packs of teas tested for anti-oxidant activity. Table 5.1: Botanical names and common names of samples tested in DPPH? assay. Botanical name Common name Aspalathus linearis Green rooibos tea Aspalathus linearis (fermented) Rooibos tea Athrixia phylicoides Bush tea (Zulu tea) Camellia sinesis Green tea Camellia sinesis (fermented) Black tea (Ceylon) Cyclopia intermedia Honeybush tea 5.2.1 Principle of the method DPPH? is a stable free radical with a dark violet colour. Anti-oxidant compounds donate electrons to DPPH?, resulting in decolourization from deep violet to light yellow. The degree of decolourization is proportionate to the number of electrons accepted by the DPPH? and is measured with a UV/VIS light spectrophotometer (Miliauskus et al., 2004). 5.2.2 Protocol A 96 ?M DPPH? solution was prepared in HPLC grade methanol and stored at 4 ?C in the dark. Tea extracts were dissolved in dimethyl sulfoxide (DMSO) to obtain a stock solution of 10 mg/ml. For the first dilution, 50 ?l of the stock solution was added to 950 ?l of DMSO to 42 obtain a concentration of 500 ?g/ml, but following the addition of the DPPH?, there was a final concentration of 100 ?g/ml in the well. Six serial dilutions (1:1) were performed thereafter using DMSO. Using a 96-well microtitre plate, 50 ?l of the stock solutions and serial dilutions was plated out in triplicate from rows B to G. In row A and H, 50 ?l of DMSO was added, which acted as a negative control. In columns 2, 4, 6, 8, 10 & 12 HPLC grade methanol (200 ?l) was added and in columns 1, 3, 5, 7, 9 & 11 DPPH? (200 ?l) was added. L-Ascorbic acid (Vitamin C) was used as a positive reference. The plate was shaken for 2 minutes and left to stand for 30 minutes at room temperature. Thereafter, the absorbance was read at 550 nm using an UV-spectrophotometer and the percentage decolourization (the percentage free radical scavenging activity of the test compound) was calculated. The IC50 values were calculated using Enzfitter? software. Each experiment was done in triplicate and the mean and standard deviation (s.d.) reported. 5.3 Results and discussion All the aqueous extracts showed free radical scavenging activity ranging from 9.64 ?g/ml to 29.26 ?g/ml (Figure 5.2). The results of the in vitro assay showed that green tea possessed the highest anti-oxidative activity (IC50 = 9.64 ? 0.96 ?g/ml) and A. phylicoides (Bush tea) (IC50 = 14.01 ? 2.68 ?g/ml) had greater anti-oxidant properties than fermented rooibos tea, honeybush tea, green rooibos tea and Ceylon tea (IC50 > 18.00 ?g/ml). A. phylicoides was at least 2.5 times less active than the reference compound, Vitamin C (L-Ascorbic acid). The anti-oxidant activity of the aqueous tea extracts tested in this study using the DPPH? assay decreased in the order: green > A. phylicoides (Bush) > green rooibos > honeybush > fermented rooibos > Ceylon (black) (Figure 5.2). These results are in agreement with a previous study in which the DPPH? free radical scavenging method was utilised to determine the anti-oxidant activity of green, rooibos (fermented, unfermented and semi-fermented), black and oolong tea extracts (concentration of tea extracts: 50 mg/100 ml). The anti-oxidant activity and percentage inhibition was as follows: green (90.8%) > unfermented rooibos 43 (86.6%) > fermented rooibos (83.4%) > semi-fermented rooibos (81.9%) > black (81.7%) > oolong (71.2%) (von Gadow et al., 1997). Vi ta m in C Gr ee n A. p hy lic oi de s ( Bu sh ) Gr ee n ro oi bo s Ho ne yb us h Ro oi bo s Bl ac k ( Ce ylo n) 0 10 20 30 40 5.69 ?0.43 18.01 ?4.06 18.02 ?4.27 25.83 ?2.39 29.26 ?2.68 9.64 ?0.96 14.01 ?2.68 A nt i-o xi da nt a ct iv ity IC 50 ( ?g /m l) Figure 5.2: In vitro anti-oxidant activity of aqueous tea extracts. In another study, the DPPH? assay was utilized to determine the anti-oxidant activity of fruits, vegetables and teas and the amount of anti-oxidants needed to decrease the initial DPPH? absorbance by 50% (EC50) was determined. It was concluded from the study that in general, honeybush tea (EC50 ranges from > 8000 to > 1000 ?g/ml) exhibited lower anti-oxidant activity than green (EC50 ranges from 120 to 320 ?g/ml), rooibos (EC50 ranges from 620 to 1100 ?g/ml) and black tea (EC50 ranges from 230 to 750 ?g/ml) (du Toit et al., 2001). The results from this study deviated with regard to anti-oxidant activity of honeybush tea from the du Toit et al. (2001) study (Figure 5.2). Honeybush tea (IC50 18.02 ? 4.27 ?g/ml) exhibited greater anti-oxidant activity than both rooibos and black tea and lower anti-oxidant activity than green tea. 44 According to McGaw and co-authors (2007), the decoction (50 g of dried aerial parts boiled in 1.5 L water for 15 min) and the infusion (1.5 L of boiling water poured over 50 g of dried aerial parts and allowed to stand for 15 min) of A. phylicoides revealed higher anti-oxidant activity than A. elata in the Trolox? equivalent anti-oxidant capacity (TEAC) assay. In the same study the anti-oxidant activity of the A. phylicoides decoction was higher than rooibos (TEAC content: 0.269 ? 0.015 vs. 0.257 ? 0.001) and the anti-oxidant activity of the A. phylicoides infusion was similar to that of rooibos (TEAC content: 0.248 ? 0.012 vs. 0.257 ? 0.001). The comparative results from the McGaw et al. (2007) study deviates from the results obtained in the DPPH? assay, in which A. phylicoides (Bush tea) displayed greater anti-oxidant activity than fermented rooibos tea. The difference in results can be attributed to the different methods utilised to determine antioxidant activity. However, the McGaw et al. (2007) study supports the conclusion that A. phylicoides (Bush tea) possesses some anti-oxidant activity. In another study, the anti-oxidant DPPH? scavenging activity of the ethanol extract of A. phylicoides, harvested in Venda in the Limpopo Province (South Africa) in mid-July was investigated. The concentration of the extract required to scavenge 50% DPPH? (EC50) was calculated. The results revealed that the ethanol extract displayed some degree of anti-oxidant activity (EC50 = 10.64 ? 0.08 ?g/ml) (Mavundza et al., 2007). These results further supports the conclusion that A. phylicoides (Bush tea) possesses some anti-oxidant activity. The difference in results between the aqueous extract from this study (IC50 = 14.01 ? 2.68 ?g/ml) and the ethanol extract (EC50 = 10.64 ? 0.08 ?g/ml) from the Mavundza et al. (2007) study can be attributed to the difference in extraction solvents used, locations from where samples were collected and the time of collection of samples. Previous studies have shown the potential anti-oxidant properties of polyphenols (K?hk?nen et al., 1999; Miliauskas et al., 2004; Aoshima et al., 2007; Conforti et al., 2008). Polyphenols have been found in A. phylicoides, however, no consistent correlation between the anti-oxidant activity and total phenolic content could be found (McGaw et al., 2007). According to Manzocco and co-authors (1998), tea exhibits a significant health protecting activity due to its high flavonoid content. Flavonoids are the most abundant compounds in 45 fresh tea leaves and extracts. Flavonoids have long been recognised to have strong anti- oxidant activity and are therefore considered to be responsible for the anticarcinogenic and antimutagenic properties of tea, as well as its protective action against cardiovascular disease. However, it is well known that the tea manufacturing processes can greatly affect the content of flavonoids. Tea flavonoids are very reactive species, which can easily undergo enzymatic and chemical reactions, which may be responsible for changes in the anti-oxidant properties of the product (Manzocco et al., 1998). The anti-oxidant activity of the commercially available teas assessed in this study, are in agreement with the results of previous studies, in which the effects of manufacturing processes on anti-oxidant activity of tea extracts were investigated (von Gadow et al., 1997; Manzocco et al., 1998). Green tea and green rooibos tea are produced from fresh leaves, preventing oxidation of flavonoids and therefore exhibit stronger anti-oxidant activity than black tea and fermented rooibos tea, respectively (Figure 5.2). Both black tea and fermented rooibos tea are subjected to chemical modifications during processing. The greater free radical scavenging activity of A. phylicoides compared to black and fermented rooibos tea can be attributed to the fact that A. phylicoides has not been subjected to any processing. It is not known how A. phylicoides free radical scavenging activity will be affected if it is subjected to manufacturing processes like other commercially available teas. The literature revealed that A. phylicoides is used by the local people as a medicinal product and as a beverage (van Wyk and Gericke, 2000). A recent survey conducted in a rural area in Limpopo and three black urban areas in Gauteng revealed that A. phylicoides is still a very popular beverage in the rural area and is known and utilised by some urban residents as a tea. It was further indicated by urban residents that A. phylicoides would be purchased to be enjoyed as a beverage if it was commercially available (Rampedi and Olivier, 2005). The favourable anti-oxidant property of A. phylicoides, in comparison to other commercially available teas (Figure 5.2) supports its traditional use as a beverage. 46 5.4 Conclusions ? A. phylicoides (bush tea) exhibited anti-oxidant activity (IC50 = 14.01 ? 2.68 ?g/ml) against the free radical scavenging (DPPH?) assay. ? A. phylicoides (bush tea) anti-oxidant activity was greater than unfermented rooibos and Ceylon (black) tea (IC50 > 25.00 ?g/ml); but comparable to green rooibos and honeybush tea ((IC50 = 18.01 ? 4.06 ?g/ml; 18.02 ? 4.27 ?g/ml, respectively), but less than green tea (IC50 = 9.64 ? 0.96 ?g/ml). ? The in vitro anti-oxidant results of A. phylicoides against the free radical scavenging (DPPH?) assay provided preliminary evidence to support its use of as a health- promoting beverage. 47 Chapter 6 ? Antimalarial activity 6.1 Introduction As recent as the early 1900?s, human malaria was endemic across every continent except Antarctica (Girard et al., 2007). Despite intense efforts to control and eradicate malaria, the disease continues to be one of the greatest health problems facing Africa. The WHO estimated that at least 247 million cases of malaria occurred in 2008 with nearly one million deaths, mostly among children living in Africa (WHO, 2010). Malaria, transmitted by the bite of an infected female Anopheles mosquito is caused by four species of Plasmodium, which are P. falciparum, P. vivax, P. ovale and P. malariae (Kaur et al., 2009). P. falciparum is the most dangerous of the human malaria parasites and is responsible for the majority of infections in Africa (Pillay et al., 2008). The life cycle of a malaria parasite is presented in Figure 6.1. Symptoms appear seven days or more (usually 10? 15 days) after exposure and include fever, headache, chills and vomiting (WHO, 2010). Although a number of advances have been made towards the understanding of the disease, the increasing prevalence of drug-resistant strains of P. falciparum to standard antimalarial drugs necessitates a continuous effort to search for new antimalarial drugs with new modes of action, used alone or in combination. History reveals that the majority of antimalarial drugs have been derived from medicinal plants. These include the quinoline-based antimalarials such as quinine as well as artemisinin and its derivatives (Clarkson et al., 2004; Bourdy et al., 2008). Since the majority of antimalarials originated from plants, an investigation of the chemical components of traditional medicinal plants could lead to the development of new antimalarial drugs. South Africa with its rich diversity of plants is an ideal place to screen plants for antiplasmodial activity (van Wyk, 2008). The mortality of malaria and therefore the pressing need to discover new antimalarials substantiates the investigation of A. phylicoides for antiplasmodial activity. 48 Figure 6.1: The life cycle of the malaria parasite (http://www.emro.who.int/rbm/Images/MalariaLifeCycle-1.gif). The aim of this study was to investigate the essential oil and methanol extract of the indigenous plant A. phylicoides for potential antiplasmodial activity. 6.2 Materials and methods 6.2.1 Parasite suspension The parasites were grown continuously in culture medium and maintained at a 5 ? 10% haematocrit. The culture medium consists of Roswell Park Memorial Institute media (RPMI- 1640), HEPES buffer (N-2-hydroxyethylpiperazine-N?-2-ethanesulfonic acid), D-glucose, hypoxathine, gentamicin, 5% (w/v) NaHCO3 and 10% (v/v) heat-inactivated human plasma (Desjardin et al., 1979). The stock culture was adjusted to a haematocrit of 1% and parasitemia of 0.5% for addition to the microtitre plates. 6.2.2 Tritrated hypoxanthine incorporation assay 49 The antimalarial activity of the methanol extract and essential oil of A. phylicoides was assessed in vitro on the chloroquine-resistant Plasmodium falciparum (FCR-3) strain using the tritrated hypoxanthine incorporation assay (Desjardin et al., 1979; van Zyl et al., 2006) in collaboration with Dr RL van Zyl, Department of Pharmacy and Pharmacology, University of Witwatersrand. 6.2.3 Principle of the method The human malaria parasite P. falciparum was exposed to different concentrations of the test compounds for a single cycle of parasite growth (48 hours). The in vitro uptake of [3H]- hypoxathine by malaria parasites was used as an indicator of parasite growth. Hypoxanthine is required by the parasites for their DNA synthesis and is only supplied by the human host. The standard antimalarial, quinine was used as a positive control. 6.2.4 Protocol The methanol extract was dissolved in DMSO to make a stock solution of 10 mg/ml, subsequent dilutions made in experimental medium (RMPI-1640, glucose and HEPES buffer). The essential oil (10 ?l) was diluted with 90 ?l of DMSO to make a stock solution of 10%; whilst quinine (control) was dissolved in the experimental medium. For each dilution, 25 ?l (extract or control) or 2 ?l (essential oil) of the various dilutions were plated out in triplicate in a 96-well plate. To ensure a total of 25 ?l per well, 23 ?l experimental medium was added to the wells containing essential oils. The parasite suspension 200 ?l was then added to each well. Untreated parasites in eight wells and uninfected red blood cells in four wells were used as controls. After preparation, the plates were placed in a humidified airtight candle jar with a gaseous atmosphere of 5% O2, 5% CO2 and 90% N2. The candle jar was then incubated at 37 ?C for 24 hours. Following incubation, 25 ?l of [3H]-hypoxanthine was added to each well. The plate was then returned to the candle jar and incubated for a further 18 hours at 37 ?C. After the second incubation, parasitic 3H-DNA was harvested on glass fiber filter mats with a cell 50 harvester. The filter mats were dried and the incorporated [3H]-hypoxanthine was counted with a liquid scintillation counter. The inhibitory effects of the test compounds were expressed as percentage parasite growth of untreated parasitised and erythrocyte controls. The percentages were plotted against their respective concentrations and the log sigmoid dose-response curve was constructed using the Enzfitter? software. The IC50 values (the concentration required to kill 50% parasite) determined from the log sigmoid dose-response curve was calculated as a measure of the antimalarial activity of the solvent extract and essential oil. The experiment was repeated four times for each test compound and the mean and standard deviation reported. 6.3 Results and discussion The methanol extract of A. phylicoides displayed no significant in vitro activity against the chloroquine-resistant P. falciparum (FCR-3) strain (IC50 value = 83.489 ? 5.482 ?g/ml). However, the essential oil showed promising in vitro activity against the chloroquine-resistant P. falciparum (FCR-3) strain (IC50 value = 1.006 ? 0.06 ?g/ml) when compared to the control quinine (0.034 ? 0.002 ?g/m) (Table 6.1). Table 6.1: In vitro antimalarial activity of A. phylicoides. Sample Antimalarial activity: IC50 ? s.d. (?g/ml) n Methanol extract 83.489 ? 5.482 4 Essential oil 1.006 ? 0.06 4 Quinine 0.034 ? 0.002 6 Malaria is a major health problem and new treatments are urgently needed to curb and eradicate this endemic. As a result antimalarial studies have been performed on several plants indigenous to South Africa (Prozesky et al., 2001; Clarkson et al., 2004). To date no data has been published on the antimalarial activity of A. phylicoides. The antimalarial activity in this study varied greatly between the essential oil and methanol extract (Table 6.1). The essential oil showed good antimalarial activity (1.006 ? 0.06 ?g/ml). 51 According to Clarkson and co-authors (2004), solvent extracts with an IC50 value ?10 ?g/ml were classified as having promising antimalarial activity. Therefore it can be concluded that the methanol extract exhibited poor antimalarial activity (IC50 value = 83.489 ? 5.482 ?g/ml). Physical properties of essential oils, including low density (?0.94 g/ml) and the ability to readily diffuse across cell membranes (Boyom et al., 2003) may partly explain why the essential oil is more active than the methanol extract. It has been reported that sesquiterpenoids and their derivatives possess numerous biological properties, including antimalarial activity (Boyom et al., 2003; Kamatou et al., 2005). In a study on antimalarial use of the volatile oil from leaves of Virola surinamensis (Rol.) Warb, nerolidol (an acyclic oxygenated sesquiterpene) was identified as one of the active constituents (Lopes et al., 1999). ?-Pinene (IC50 = 1.2 ? 0.2 ?M) and (E & Z)?(?)-nerolidol (IC50 = 0.9 ? 0.3 ?M) have been reported to have potent antimalarial activity (van Zyl et al., 2006). In literature, the antimalarial activity of essential oils of other plant species have been reported frequently. Kamatou and co-authors (2005) reported that the essential oils of Salvia species displayed good antimalarial activity against the chloroquine-resistant P. falciparum (FCR-3) strain (IC50 values ranged from 1.23 ? 0.31 to 4.38 ? 1.07 ?g/ml). Lippia multiflora exhibited similar antimalarial activity against the chloroquine-resistant (FcB1) and chloroquine-sensitive (F32) strains of P. falciparum (Valentin et al., 1995). Nerolidol and linalool were thought to be the constituents responsible for the antimalarial activity of the essential oil of L. multiflora. Boyom et al. (2003) investigated the antimalarial activity of H. crispiflorus against P. falciparum and found that the oils were also active (IC50 = 2.0 ?g/ml). Athrixia phylicoides essential oil samples studied consists of the following major sesquiterpenoids and their derivatives in varying quantities: caryophyllene oxide (1.0 ? 34.2%); spathulenol (1.5 ? 12.6%); germacrene D (trace amounts ? 13.5%); ?-caryophyllene (0.2 ? 11.9%) (Figure 3.3). The acyclic oxygenated sesquiterpene, (E)-nerolidol is also present in trace amounts in some samples. In addition, the monoterpene ?-pinene (0.8 ? 14.8%), which has previously, exhibited antimalarial activity (IC50 = 1.2 ? 0.2 ?M) (van Zyl et al., 2006) is also present (Table 3.1). From the literature review it can be concluded that these 52 constituents of the essential oil could contribute to the overall antimalarial activity of A. phylicoides. These preliminary studies on the antimalarial activity of the essential oils can contribute towards the discovery of agents which may assist with prophylaxis of malaria. However, further in vitro and in vivo studies are required to understand the mechanism of action of the essential oil. 6.4 Conclusions ? The essential oil of A. phylicoides exhibits antimalarial activities against the chloroquine-resistant Plasmodium falciparum (FCR-3) strain (IC50 = 1.006 ? 0.06 ?g/ml). ? The methanol extract of A. phylicodes exhibits poor antimalarial activities against chloroquine-resistant Plasmodium falciparum (FCR-3) strain (IC50 = 83.489 ? 5.482 ?g/ml). 53 Chapter 7 ? Anti-inflammatory activity 7.1 Introduction Inflammation is the body?s reaction in response to injury, infections, chemicals and poisons and is characterized by redness, swelling, heat and pain. The inflammatory response has survival value but if inappropriately deployed, as occurs in many diseases, may be deleterious. For this reason it is essential to understand the process of inflammation and the mediators responsible for this process (Dale and Haylett, 2004). Two important groups of mediators responsible for the inflammatory response are prostaglandins and leukotrienes. The pathway by which prostaglandins are formed, can be inhibited by non-steroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen and aspirin that inhibit the cyclooxygenase enzyme (Figure 7.1). These drugs are commonly utilized for the management of diseases associated with inflammation such as rheumatoid arthritis, oesteoarthritis, acute bursitis and tendonitis (Dale and Haylett, 2004). The use of NSAIDs for the management of these diseases provides more substrate for the alternate pathway of the arachidonic acid casade (Figure 7.1), in which the 5-lipoxygenase enzyme converts arachidonic acid into biologically active leukotrienes. Leukotrienes are the major pathophysiological mediators of the inflammatory response, since they are more potent than the prostagladins with regard to the increase in vascular permeability, adhesion of leukocytes to the vessel wall and oedema production. Leukotrienes mediate the inflammatory response, which can lead to various diseases, such as asthma, inflammatory bowel disease and allergic rhinitis. It is therefore essential to discover compounds that can inhibit the 5- lipoxygenase enzyme. A study conducted on South African medicinal plants revealed that some plants such as the aqueous extract of Melianthus comosus (IC50 = 13.84 ppm) and the essential oil of Ballota africana (IC50 = 29.99 ppm) exhibited 5-lipoxygenase inhibitory activity when compared to the control, nordihydroguaiaretic acid (NDGA) (IC50 = 5.0 ppm) (Frum and Viljoen, 2006). 54 Phospholipase A2 Cyclooxygenase (COX) 5-Lipoxygenase PGI2 TXA2 PGF2? PGD2 PGE2 LTC4 LTB4 LTD4 Figure 7.1: Inflammatory cascade, where PG is prostaglandins, LT is leukotrienes, TXA2- thromboxane and HPETE is hydroperoxyeicosatetraenoic acid (Dale and Haylett, 2004). It was reported that a decoction or strong tea of A. phylicoides leaves has been used in traditional medicine by Sotho speaking people to treat sore feet, and the leaves are chewed by Sotho and Xhosa speaking people to treat sore throats (Table 1.2) (Watt and Breyer- Brandwijk, 1962; Roberts, 1990; Hutchings et al., 1996). These traditional uses imply that the indigenous plant may possess anti-inflammatory activity. The aim of this study was to determine whether the non-volatile compounds and the essential oil of A. phylicoides possess anti-inflammatory activity by specifically inhibiting the 5- lipoxygenase enzyme. PHOSPHOLIPIDS ARACHIDONIC ACID CYCLIC ENDOPEROXIDES 5-HPETE 55 7.2 Materials and methods The anti-inflammatory activity of the essential oil and methanol extract of A. phylicoides was performed in vitro using the 5-lipoxygenase assay of Sicar et al. (1983) as modified by Evans (1987). 7.2.1 Principle of the method 5-Lipoxygenase is known to catalyse the oxidation of unsaturated fatty acids containing 1,4- pentadiene structures. In the body, arachidionic acid (biological substrate) is oxidized to hydroperoxyeicosatetraenoic acid (HPETE?s) by the 5-lipoxygenase. In the in vitro assay, linoleic acid is used as the substrate for 5-lipoxygenase enzyme. Linoleic acid is oxidized in vitro to a conjugate diene by 5-lipoxygenase, which absorbs at 234 nm. 5-Lipoxygenase activity is evaluated by the spectrophotometric measurement of the conjugated diene at 234 nm. The initial reaction rate was measured with the UV-VIS spectrophotometer and the decrease of this initial reaction rate is a measure of the sample?s inhibitory activity (Baylac and Racine, 2003). 7.2.2 Protocol The test compounds were dissolved in DMSO and Tween? 20 to achieve a starting concentration of 100 ?g/ml. The potassium phosphate buffer (pH 6.3) was prepared by mixing 50 ml of 0.1 M potassium dihydrogen phosphate and 9.7 ml of 0.1 M sodium hydroxide. In a 3 ml cuvette maintained at 25 ?C in a thermostated bath, 10 ?l of the test compound dissolved in DMSO and Tween? 20 was mixed with 2.95 ml of pre-warmed potassium phosphate buffer (pH 6.3) and 45 ?l of linoleic acid (? 99%) from Fluka. Thereafter 100 U of the 5-lipoxygenase (enzyme) diluted in an equal volume of ice-cold potassium phosphate buffer (pH 6.3) (stored at 4 ?C), was added to the cuvette to initiate the enzymatic reaction. The same mixture was prepared as above, but only with DMSO and Tween? 20 (no test sample) to act as a negative control. 56 The inhibitory activity of the test compounds on the enzyme was assessed by recording the absorbance using a UV-VIS spectrophotometer at 234 nm, of the conjugated diene formed from linoleic acid. The initial reaction rate was determined from the slope of the straight line portion of the curve. The percentage inhibition of enzyme activity was calculated by comparison with the negative control. The concentration that gave 50% inhibition (IC50) was calculated using Enzfitter? software. Nordihydroguaiaretic acid was used as a positive control in this assay. 7.3 Results and discussion Results obtained for the 5-lipoxygenase assay for the methanol extract and essential oil of A. phylicoides were quite contrasting. The essential oil displayed in vitro 5-lipoxygenase inhibitory activity with an IC50 value of 25.68 ?g/ml, whereas the methanol extract was found to be inactive at the starting concentration of 100 ?g/ml (IC50 > 100 ?g/ml). The positive control, NDGA inhibited enzyme activity at an IC50 value of 5.0 ?g/ml. Only one test was performed for both the essential oil and methanol extract due to insufficient quantities of samples remaining after the completion of all other assays. An essential oil with a 10 ? IC50 ? 30 ppm (= ?g/ml) is defined as a good 5-lipoxygenase inhibitor (Baylac and Racine, 2003). Therefore, it can be concluded that the essential oil of A. phylicoides is a good 5-lipoxygenase inhibitor (IC50 = 25.68 ?g/ml). Essential oil constituents, terpenic hydrocarbons (d-limonene, 10 ? IC50 ? 30 ppm (= ?g/ml) and ?-pinene, 31 ? IC50 ? 50 ppm (= ?g/ml)) and sesquiterene hydrocarbons (?-caryophyllene and germacrene D, 10 ?IC50 ? 30 ppm (= ?g/ml)) exhibited inhibitory activity against the 5- lipoxygenase enzyme (Baylac and Racine, 2003). These compounds could have contributed to the overall anti-inflammatory activity of A. phylicoides since they were present in varying concentrations in most samples studied (limonene: 0.7% to 4.0%; ?-pinene: 0.8% to 14.8%; ?-caryophyllene: 0.2% to 11.9%; germacrene D: trace amounts to 13.5%) (Table 3.1). Traditionally the leaves of A. phylicoides are chewed for the treatment of sore throats and a decoction or strong tea of A. phylicoides is used for bathing of sore feet (Table 1.2). The 57 chewing of the plant can result in the release of the essential oils from the plant. The in vitro result obtained from this study confirms that the essential oil of A. phylicoides inhibits the 5- lipoxygenase enzyme, providing scientific evidence to justify its traditional use. It is important to note that the absence of 5-lipoxygenase inhibitory activity of the methanol extract of A. phylicoides does not necessarily imply that the extract is devoid of any anti- inflammatory activity. The activity may be observed at concentrations greater than 100 ?g/ml as assayed in this study. The inflammatory cascade (Figure 7.1) is very complex and the extract may exert pharmacological effects on other target sites of the cascade, such as the cyclooxygenase enzyme or phospholipase A2 enzyme. Therefore further studies (e.g. COX-1, COX-2 and phospholipase A2 inhibition studies) are required to confirm whether or not the extract of A. phylicoides displays inhibitory activity at any of the other target sites of the inflammatory cascade. 7.4 Conclusions ? The essential oil exhibited inhibitory activity against the 5-lipoxygenase enzyme (IC50 = 25.68 ?g/ml). ? The methanol extract was inactive against the 5-lipoxygenase enzyme at a concentration of 100 ?g/ml. ? The in vitro anti-inflammatory results of the essential oil of A. phylicoides against the 5-lipoxygenase enzyme, provided preliminary evidence to support its use as a treatment of sore feet and sore throats. 58 Chapter 8 ? Cytotoxicity 8.1 Introduction Herbal remedies are rapidly gaining popularity throughout the world as a result of dissatisfaction with conventional medicines. It is a widely held belief that herbal preparations are "natural" and are therefore intrinsically harmless (Bateman et al., 1998). In South Africa, traditional or herbal remedies form a significant part of day-to-day living. While many traditional remedies may be of benefit, some may have little or no effect, and others may be cytotoxic. Cytotoxicity is a measure of toxicity of a substance to living cells of the human body. Cytotoxicity of herbal remedies may be attributed to several factors such as hepatotoxicity of main constituents, contamination of preparations by heavy metals or micro- organisms, and adverse reactions due to age, and genetic and concomitant disease characteristics of the user (Bateman et al., 1998). Some classical examples of plants that are toxic to humans include foxglove, poison hemlock and aconite (Wittstock and Gershenzon, 2002). In South Africa, the process for registration and use of conventional medicines is clearly defined and all medicines must have proven quality, safety and efficacy. However traditional remedies are not subjected to the same process. Therefore, the potential toxicity of traditional remedies becomes an important consideration when studying the traditional use of plants. Very little information is available in the literature on the safety and toxicity of A. phylicoides, in contrast to other commercially available teas such as rooibos tea and honeybush tea. A number of studies have addressed aspects of safety and toxicity of rooibos and honeybush, for example, it was reported that in a ten week study, chronic consumption of rooibos and honeybush by rats did not cause any adverse effects in the kidney or liver (Marnewick et al., 2003). Several studies have also addressed tea-drug interactions (Jang et al., 2004; Mertens- Talcott et al., 2006; Matsuda et al., 2007). The lack of toxicity data for the volatile oils of A. phylicoides and the pharmacological activities displayed in the previous Chapters prompted the investigation of the cytotoxicity properties to determine whether A. phylicoides is safe for consumption. 59 The aim of this study was to: ? evaluate the cytotoxic properties of the essential oil and methanol extract of A. phylicoides. 8.2 Materials and methods 8.2.1 Microculture tetrazolium cellular viability assay The cytoxicity properties of the methanol extract and essential oil of A. phylicoides was assessed on human kidney epithelial cells using the microculture tetrazolium cellular viability assay, in collaboration with Dr RL van Zyl, Department of Pharmacy and Pharmacology, University of Witwatersrand (Mosmann, 1983; van Zyl et al., 2006). 8.2.2 Principle of the method 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) is a tetrazolium dye which is used to measure the cellular conversion of the dye into an insoluble formazan product by active mitochondria of living cells. The conversion of MTT to the insoluble compound is monitored by a shift in absorbance. The amount of formazan produced is directly proportional to the number of active cells. The MTT assay evaluates cellular viability in the presence of a test compound. 8.2.3 Protocol The human kidney epithelial cells were maintained in culture medium consisting of HAM F10, 5% (v/v) foetal calf serum (FCS) and 0.5 mg/ml gentamicin sulphate. HAM F10 solution consists of HAM F10 medium (9.38 g) and NaHCO3 (1.18 g) in a total volume of one litre of sterile MilliQ? water. The culture medium was replaced three times a week. Once the cells reached confluency, they were trypsinised with 4 ml of 0.25% Trypsin containing EDTA (0.1%) to obtain a single cell suspension. The cells were centrifuged at 1500 rpm for 5 minutes to remove the trypsin. Once the cells were resuspended in experimental medium, 1 ml was used to seed a new culture and the remaining was used in the MTT assay. The experimental medium was prepared the same way as the culture medium except no gentamicin sulphate was included. The trypsinised cell suspension (50 ?l) was stained with an equal 60 volume of Trypan blue (0.2% w/v) and the number of cells were counted using a haemocytometer. The experimental medium was used to adjust the cell suspension (? 95% cell viability) to 0.25 million cells/ml. The methanol extract were dissolved in DMSO to make a stock solution of 10 mg/ml. Subsequent dilutions were made in experimental medium. The essential oil (10 ?l) was diluted with 90 ?l of DMSO to make a stock solution of 10% and subsequent dilutions were made in DMSO. Quinine (negative control) was dissolved in the experimental medium. Kidney epithelial cell suspension (180 ?l) was plated out in a 96-well plate and incubated at 37 ?C for six hours to allow the cells to adhere to the well. For each dilution, 20 ?l (extract or control) or 2 ?l (essential oil) of the various dilutions were plated out in triplicate in a 96-well plate. To ensure a total of 200 ?l per well, 18 ?l experimental medium was added to the wells containing essential oils. Each plate contained 10 wells for the untreated cell control and two wells for the blank cell-free control. The plates were incubated in 5% CO2 at 37 ?C. After 44 hours of incubation 40 ?l of MTT (12mM) was added to each well and re-incubated for further four hours. The supernatant (180 ?l) was removed from each well and replaced with 150 ?l of DMSO to stop the reaction and to solubilise the formazan crystals. The plates were shaken for four minutes and the absorbance was read at a test wavelength of 540 nm and a reference wavelength of 690 nm. The results were expressed as percentage cellular viability of the drug and cell-free controls. The percentage cellular viability data was plotted against their respective concentrations and log sigmoid dose-response curves were generated by the Enzfitter? software. The concentration which inhibits 50% of cellular growth (IC50 value) was determined from the log sigmoid dose-response profile. The experiment was repeated four times and the mean and standard deviation (s.d.) calculated. Quinine, a plant -derived compound considered to be relatively safe, was used as a negative control. The safety index relates the dose of the drug required to produce a desired effect to that which causes an undesired effect. The toxicity of the methanol extract and essential oil was compared to their antimalarial activity, using the safety index Equation 1. 61 Equation 1 8.3 Results and discussion The toxicity of the methanol extract and essential oil of A. phylicoides was assessed using the MTT assay and the results obtained are presented in Table 8.1. The essential oil proved to be more toxic than the methanol extract. The safety indices of the samples and control were calculated and are presented in Table 8.1. The safety index of the control, quinine (SI = 4001.74) was appreciably higher than the methanol extract (SI = 2.28) and the essential oil (SI = 1.77). The low safety indices of the methanol extract and the essential oil implied that they have low degrees of selectivity for P. falciparum. Table 8.1: In vitro cytotoxic properties of A. phylicoides. Sample Toxicity: IC50 ? s.d. (?g/ml) n Antimalarial activity IC50 ? s.d. (?g/ml) Safety index (SI) Methanol extract 189.893 ? 12.658 4 83.489 ? 5.482 2.28 Essential oil 1.776 ? 0.2298 4 1.006 ? 0.06 1.77 Quinine 136.059 ? 4.057 6 0.034 ? 0.002 4001.74 The cytotoxicity of the extract of A. phylicoides had been investigated in two in vitro studies and one in vivo study. McGaw et al. (2007) investigated the cytotoxicity of the A. phylicoides against the Vero monkey kidney cell line (MTT assay) and brine shrimp eggs (Solis et al., 1993 method) and the concentration required to kill 50% of cells (LC50) was calculated. It was found that the ethanol extract displayed low toxicity (Vero cell: LC50 = 252 ?g/ml and Brine shrimp: LC50 = 394 ?g/ml) and the aqueous preparations (i.e. aqueous extract, decoction and infusion) had no effect on either assay method. In another study using the sodium 3?-[1- (phenylaminocarbonyl)-3,4-tetrazolium]-bis-(4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT) colorimetric assay, the cytotoxicity of the ethanol extract against Vero cells lines was investigated. The extract displayed low toxicity (IC50 = 107.8 ? 0.13 ?g/ml) Safety Index = Toxicity Activity 62 (Mavundza et al., 2010). Chellan et al. (2008) investigated the effect of high doses of aqueous extract of A. phylicoides (30 to 180 mg) on Wistar rats for a period of three months. No mortality or morbidity was reported. Histopathology showed no signs of any extract induced toxicity in the liver, kidneys or gastrointestinal tract and all laboratory results (serum alkaline phosphatase, creatinine and urea levels) were normal. No data has been published on the cytotoxicity of the essential oils of A. phylicoides. The essential oil is about 77 times more toxic than the control, quinine and has a low safety index of 1.77. The essential oil composition in Table 3.1 was evaluated to identify compounds possibly contributing to the toxic effects noted. Caryophyllene oxide has been reported to have cytotoxic activity (LC50 values range from 147 to 351 ?M) (Sibanda et al., 2004). The cytotoxicity of P. serrulata essential oil can be partly explained by the presence of ?- humulene, caryophyllene oxide, ?-elemene and ?-elemene (Jie et al., 2007). In a study conducted by van Zyl et al. (2006), it was reported that ?-pinene (IC50 = 172.2 ? 13.4 ?M), ?- pinene (IC50 = 166.7 ? 19.6 ?M), (E- & Z-)-(?)-nerolidol (IC50 = 5.5 ? 1.2 ?M) and 1,8- cineole (IC50 = 69.1 ? 9.4 ?M) displayed some degree of toxicity. Caryophyllene oxide, ?- pinene and ?-pinene have been identified as major constituents of the essential oil (Figure 3.3). Nerolidol, ?-humulene and 1,8-cineole are either present in trace amounts or very small percentages in some of the samples studied (Table 3.1). It can therefore be assumed that caryophyllene oxide, ?-pinene and ?-pinene together with the minor constituents (nerolidol, ?- humulene and 1,8-cineole) synergistically contribute to the cytotoxicity of the essential oil. The cytotoxicity of the essential oil was compared to the antimalarial results in Table 6.1. The results from the MTT assay suggest that the antimalarial activity (Table 6.1) of the essential oil may be due to general cytotoxic effects and this was further supported by the low safety index of the essential oil (SI = 1.77). This implied that the essential oil had a greater affinity for human kidney epithelial cells than for P. falciparum. The extensive traditional use of A. phylicoides aqueous extracts as a tea and medicine (Table 1.2) and the lack of adverse effects published in the literature (van Wyk and Gericke, 2000; Olivier and De Jager, 2005; Rampedi and Olivier, 2005), may be an indication of the lack of 63 toxicity of this species. This hypothesis that A. phylicoides is unlikely to be toxic was supported by the studies conducted by McGaw et al. (2007) and Chellan et al. (2008). However, further in vivo testing is needed to confirm the safety profile of A. phylicoides. 8.4 Conclusions ? The essential oil of A. phylicoides (IC50 = 1.776 ? 0.2298 ?g/ml) is more toxic than the methanol extract (IC50 = 189.893 ? 12.658 ?g/ml). ? Both the essential oil (SI = 1.77) and methanol extract (SI = 2.28) of A. phylicoides have low safety indices. 64 Chapter 9 ? General Conclusions and Recommendations 9.1 Summary For centuries eastern countries have been using herbal remedies in the form of teas to treat infections, ailments and diseases, however, in recent years herbal teas have gained popularity in western countries for their health-related properties, including antimicrobial activity and anti-oxidant activity (Almajano et al., 2008; Chan et al., 2010). One of the most beneficial effects of tea is its anti-oxidant activity. Anti-oxidants play a role in the prevention of cardiovascular disease, cancers and neurodegenerative diseases (Kris-Etherton et al., 2002). As such research on teas and their effects on human health have intensified. This report focused on the health-related properties as well as the toxicity of A. phylicoides. The results of the in vitro biological activities and the compounds identified in this study are given in Table 9.1. The methanol extract of A. phylicoides was found to be more active against the micro-organisms tested compared to the essential oil; whereas the essential oil displayed greater antimalarial and anti-inflammatory activity compared to the methanol extract (Table 9.1). The essential oil was also found to be more toxic when compared to the methanol extract of A. phylicoides (Table 9.1). The aqueous extract of A. phylicoides exhibited anti- oxidant activity comparable to that of commercially available teas (green, green rooibos, honeybush, rooibos and Ceylon) (Table 9.1). Seven major compounds (>10%) were identified in the essential oil and one bioactive compound was identified in the methanol extract of A. phylicoides. The bioactive compound was found to be 24 to 51 times more active against micro-organisms tested compared to the methanol extract of A. phylicoides (Table 9.1). This report is the first consolidated report in which the essential oil composition and the biological activities of both the essential oil and methanol extract of A. phylicoides has been investigated. It has revealed that Athrixia phylicoides possesses antimicrobial, anti-oxidant anti-inflammatory and antimalarial activity. As such this report paves the way for the successful marketing of A. phylicoides against the commonly known rooibos and honeybush tea. 65 9.2 Recommendations for future work ? It is recommended that a seasonal and geographical variation study is performed on A. phylicoides to explain the variations in essential oil composition noted in the samples studied. ? The chemotypic variation be investigated for solvent extracts using liquid chromatography and HPLC. ? Since the anti-inflammatory activity was only performed on one sample, it is recommended that the assay be repeated to confirm the results obtained in this report. ? A study on the anti-oxidant activity of the essential oil and methanol extract should be performed to obtain a complete anti-oxidant profile of A. phylicoides. ? Since the aqueous extract was only tested for anti-oxidant activity, to obtain a complete profile the other assays conducted in this study should be done. ? Studies should be performed to identify the active compounds responsible for anti- inflammatory and antimalarial activity, and toxicity of the essential oil of A. phylicoides and for the anti-oxidant activity of the aqueous extract of A. phylicoides. 66 Table 9.1: Summary of the phytochemistry and in vitro biological activities of the essential oils and extracts of A. phylicoides. Antimicrobial activity MIC range (mg/ml) Anti-oxidant activity (?g/ml) Antimalarial activity (?g/ml) Anti- inflammatory activity (?g/ml) Cytotoxicity (?g/ml) Compounds identified A. phylicoides essential oil Gram +ve bacteria: 3 to >32. Gram ?ve bacteria: 12 to >32. Yeasts: 4 to 6. ND 1.006 ? 0.06 25.68 1.776 ? 0.2298 Major compounds (>10%): caryophyllene oxide, spathulenol, germacrene D, ?-caryophyllene, ?-pinene, ?-pinene and myrcene A. phylicoides methanol extract Gram +ve bacteria: 1 to 4. Gram ?ve bacteria: 3 to >6. Yeasts: 1.5 to 2. ND 83.489 ? 5.482 >100 189.893 ? 12.658 (4- Hydroxyphenyl) propyl coumaroate (4- Hydroxyphenyl) propyl coumaroate Gram +ve bacteria: 0.0195 Gram ?ve bacteria: 0.125 Yeasts: 0.078 ND ND ND ND ND Aqueous tea extracts ND Green: 9.64 ? 0.96 A. phylicoides: 14.01 ? 2.68 Green rooibos: 18.01 ? 4.06 Honeybush: 18.02 ? 4.27 Rooibos: 25.83 ? 2.39 Ceylon: 29.26 ? 2.68 ND ND ND ND ND: Not determined; Gram +ve: Gram-positive; Gram ?ve: Gram-negative 67 References Almajano, M.P., Carbo, R., Jimenez, J.A.L., Gordon, M.H. 2008. Anti-oxidant and antimicrobial activities of tea infusions. Food Chemistry 108: 55-63 Aoshima, H., Hirata, S., Ayabe, S. 2007. Antioxidative and anti-hydrogen peroxide activities of various herbal teas. Food Chemistry 103: 617-622. Atoui, A.K., Mansouri, A., Boskou, G., Kefalas, P. 2005. Tea and herbal infusions: Their antioxidant activity and phenolic profile. Food Chemistry 89: 27-36. Avato P., Fortunato, I.M., Ruta, C., D?Eila, R. 2005. Glandular hairs in micropropagated plants of Salvia officinalis L. Plant Science 169: 29-36. Bakkali, F., Averbeck, S., Averbeck, D., Idomar, M. 2008. Biological effects of essential oils - A review. Food and Chemical Toxicology 46: 446-475. Bateman, J., Chapman, R.D., Simpson, D. 1998. Possible toxicity of herbal remedies. Scott Medical Journal 43: 7-15. Baylac, S. and Racine, P. 2003. Inhibition of 5-lipoxygenase by essential oils and other natural fragrant extracts. The International Journal of Aromatherapy 13: 138-142. Bohlmann, F. and Zdero, C. 1977. Nuene Norkauren- und thymol-derivate aus Athrixia- arten. Phytochemistry 16: 1773-1776. Bohlmann, F., Wallmeyer, M., Jakupovic, J. 1982. A new seco-labdane derivative from Athrixia elata. Phytochemistry 21: 1806-1807. Bourdy, G., Willcox, M.L., Ginsburg, H., Rasoanaivo, Ph., Graz, B., Deharo, E. 2008. Ethnopharmacology and malaria: New hypothetical leads or old efficient antimalarials? International Journal of Parasitology 38: 33-41. Boyom, F.F., Ngouana, V., Zollo, P.H.A., Menut, C., Bessiere, J.M., Gut, J., Rosethal, P.J. 2003. Composition and anti-plasmodial activities of essential oils from some Cameroonian medicinal plants. Phytochemistry 64: 1269-1275. Chan, E.W.C., Lim, Y.Y., Chong, K.L., Tan, J.B.L., Wong, S.K. 2010. Anti-oxidant properties of tropical and temperate herbal teas. Journal of Food Composition and Analysis 23: 185-189. Chellan, N., De Beer, D., Muller, C., Joubert, E., Louw, J. 2008. A toxicological assessment of Athrixia phylicoides aqueous extract following sub-chronic ingestion in a rat model. Human and Experimental Toxicology 27: 819-825. 68 Chiang, M.T., Bittner, M., Silva, M., Mondaca, A., Zemelman, R., Sammes, P.G. 1982. A prenylated coumarin with antimicrobial activity from Haplopappus multifolius. Phytochemistry 21: 2753-2755. Clark, A.M., 1996. Natural products as a resource for new drugs. Pharmaceutical Research 13: 1133-1140. Clarkson, C., Maharaj, V.J., Crouch, N.R., Grace, O.M., Pillay, P., Matsabisa, M.G., Bhagwandin, N., Smith, P.J., Folb, P.I. 2004. In vitro antiplasmodial activity of medicinal plants native to or naturalized in South Africa. Journal of Ethnopharmacology 92: 177-191. Conforti, F., Sosa, S., Marrelli, M., Menichini, F., Statti, G.A., Uzunov, D., Tubaro, A., Menichini, F., Loggia R.D. 2008. In vivo anti-inflammatory and in vitro antioxidant activities of Mediterranean dietary plants. Journal of Ethnopharmacology 116: 144-151. Cook, N.C. and Samman, S. 1996. Flavonoids ? Chemistry, metabolism, cardioprotective effects, and dietary sources. Nutritional Biochemistry 7: 66-76. Dale, M.M. and Haylett, D.G. 2004. Pharmacology Condensed. Churchill Livingstone Publications. De Smet, P.A.G.M. 1997. The role of plant-derived drugs and herbal medicines in healthcare. Drugs 54: 801-840. Delaquis, P.J., Stanich, K., Girard, B., Mazza, G. 2002. Antimicrobial activity of individual and mixed fractions of dill, cilantro, coriander and eucalyptus essential oils. International Journal of Food Microbiology 74: 101-109. Desjardins, R.E., Canfield, C.J., Haynes, J.D., Chulay, J.D. 1979. Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrobial Agents and Chemotherapy 16: 710-718. Du Toit, R., Volsteedt, Y., Apostolides, Z. 2001. Comparison of the anti-oxidant content of fruits, vegetables and teas as vitamin C equivalents. Toxicology 166: 63-69. Eloff, J.N. 1998. A sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extracts for bacteria. Planta Medica 64: 711-713. 69 Eldeen, I.M.S., Elgorashi, E.E., Mulholland, D.A., van Staden, J. 2006. Anolignan B: a bioactive compound from the roots of Terminalia sericea. Journal of Ethnopharmacology 103: 135-138. Esposito, S., Leonoe, S., Petta, E., Noviello, S., Ianniello, F. 2009. Treatment options for skin and soft tissue infections caused by methicillin-resistant Staphylococcus aureus: oral vs. parenteral; home vs. hospital. International Journal of Antimicrobial Agents 34: S30-S35. Evans, A.T. 1987. Actions of cannabis constituents on enzymes of arachidonate metabolism anti-inflammatory potential. Biochemical Pharmacology 36: 2035-2037. Fabry, W., Okemo, P.O., Ansorg, R. 1998. Antibacterial activity of East African medicinal plants. Journal of Ethnopharmacology 60: 79-84. Fennell, C.W., Lindsey, K.L., McGaw, L.J., Sparg, S.G., Stafford, G.I., Elgorasti, E.E., Grace, O.M., van Staden, J. 2004. Assessing African medicinal plants for efficacy and safety: pharmacological screening and toxicology. Journal of Ethnopharmacology 94: 205-217. Frum, Y. and Viljoen, A.M. 2006. In vitro 5-lipoxygenase and anti-oxidant activity of South African medicinal plants commonly used topically for skin diseases. Skin Pharmacology and Physiology 19: 329-335. http://www.emro.who.int/rbm/Images/MalariaLifeCycle-1.gif (28 June 2010) Gramza, A. and Korczak, J. 2005. Tea constituents (Camellia sinensis L.) as antioxidants in lipid systems. Trends in Food Science and Technology 16: 351-358. Gibbons S. 2004. Anti-staphylococcal plant natural products. Natural Products Report 21: 236-277. Gillespie, R.J., Eaton, D.R., Humphreys, D.A., Robinson, E.A. 1994. Atoms, molecules and reactions: An introduction to chemistry. Prentice-Hall, New Jersey. Girard, M.P., Reed, Z.H., Friede, M., Kieny, M.P. 2007. A review of human vaccine research and development: Malaria Vaccine 25: 1567-1580. Goth, A. 1945. The antibacterial properties of dicoumarol. Science 101: 384-385. Gulumian, M., Savolainen, K. 2008. Scientific investigations in African herbal remedies is an imperative and not an indulgence. Human and Experimental Toxicology 27: 791-792. 70 Hammer, K.A., Carson, C.F., Riley. T.V. 1999. Antimicrobial activity of essential oils and other plant extracts. Journal of Applied Microbiology 86: 985-990. Hernandez, T., Canales, M., Teran, B., Avila, O., Duran, A., Garcia, A.M., Hernandez, H., Angeles-Lopez, O., Fernandez-Araiza, G., Avila, G. 2007. Antimicrobial activity of essential oil and extracts of Cordia curassavica (Boraginaceace). Journal of Ethnopharmacology 111: 137-141. Hili, P. 2001. The antimicrobial properties of essential oils. Winter Press, Surrey, UK. Hlahla, L.N., Mudau, F.N., Mariga, I.K. 2010. Effecy of fermentation temperature and time on the chemical composition of bush tea (Athrixia phylicoides DC.). Journal of Medicinal Plants Research 4: 824-829. Hutchings, A., Scott, A.H., Lewis, G., Cunningham, A. 1996. Zulu medicinal plants: an inventory. University of Natal Press, Pietermaritzburg. Jang, E.H., Park, Y.C., Chung, W.G. 2004. Effects of dietary supplements on the induction and inhibition of cytochrome P450s protein expression in rats. Food Chemical Toxicology 42: 1749-1756. Jie, H., Tao, S., Jun, H., Shuangyang, C., Xiaoqiang, C., Guolin, Z. 2007. Chemical composition, cytotoxicity and antioxidant activity of the leaf essential oil of Photinia serrulata. Food Chemistry 103: 355-358. Joubert, E., Gelderblom, W.C.A., Louw, A., de Beer, D. 2008. South African herbal teas: Aspalathus linearis, Cyclopia spp. and Athrixia phylicoides ? A review. Journal of Ethnopharmacology 119: 376-412. K?hk?nen, M.P., Hopia, A.I., Vuorela, H.J., Rauha, J.P., Pihlaja, K., Kujala, T.S., Heinonen, M. 1999. Antioxidant activity of plant extracts containing phenolic compounds. Journal of Agricultural Food Chemistry 47: 3954-3962. Kamatou, G.P.P., Viljoen, A.M., Gono-Bwalya, A.B., van Zyl, R.L., van Vuuren, S.F., Lourens, A.C.U., Ba?er, K.H.C., Demirci, B., Lindsey, K.L., van Staden, J., Steenkamp, P. 2005. The in vitro pharmacological activities and a chemical investigation of three South African Salvia species. Journal of Ethnopharmacology 102: 382-390. 71 Kamatou, G.P.P., van Vuuren, S.F., van Heerden, F.R., Seaman, T., Viljoen, A.M. 2007. Antibacterial and antimycobacterial activities of South Africa Salvia species and isolated compound from S. chamelaeagnea. South African Journal of Botany 73: 552-557. Kamatou, G.P.P., van Zyl, R.L., van Vuuren, S.F., Figueiredo, A.C., Barroso, J.G., Pedro, L.G., Viljoen, A.M. 2008. Seasonal variation in essential oil composition, oil toxicity and the biological activity of solvent extracts of three South African Salvia species. South African Journal of Botany 74: 230-237. Kambizi, L., Sultana, N., Afolayan, A.J. 2004. Bioactive compounds isolated from Aloe ferox: A plant traditionally used for the treatment of sexually transmitted infections in the Eastern Cape, South Africa. Pharmaceutical Biology 42: 636-639. Kaur, K., Jain, M., Kaur, T., Jani, R. 2009. Antimalarials from nature. Bioorganic and Medicinal Chemistry 17: 3229-3256. Kim, N.S., Lee, D.S. 2004. Headspace solid-phase microextraction for characterization of fragrances of lemon verbena (Aloysia triphylla) by gas chromatography-mass spectrometry. Journal of Separation Science 27: 96-100. Kris-Etherton, P.M., Hecker, K.D., Bonanome, A., Coval, S.M., Binkoski, A.E., Hilpert, K.F. 2002. Bioactive compounds in food: Their role in the prevention of carsiovascular disease and cancer. American Journal of Medicine 113: 71S ? 88S. Kostova, I. 2005. Synthetic and natural coumarins as cytotoxic agents. Current Medicinal Chemistry Anticancer Agents 5: 29-46. Lalli, J.Y.Y., van Zyl, R.L., van Vuuren, S.F., Viljoen, A.M. 2008. In vitro biological activities of South African Pelargonium (Geraniaceae) species. South African Journal of Botany 74: 153-157. Lee, S.E., Hwang, H.J., Ha, J.S., Jeong, H.S., Kim, J.H. 2003. Screening of medicinal plant extracts for antioxidant activity. Life Sciences 73: 167-179. Lopes, N.P., Kato, M.J., Andrade, E.H.A., Maia, J.G.S., Yoshida, M., Planchart, A.R., Katzin, A.M. 1999. Antimalarial use of volatile oil from leaves of Virola surinamensis (Rol.) Warb. by Wai?pi Amazon Indians. Journal of Ethnopharmacology 67: 313-319. Mabogo, D.N.E. 1990. The ethobotany of the Vhavenda. MSc Dissertation, University of Pretoria, Pretoria, South Africa. 72 Mahboubi, M., and Haghi, G. 2008. Antimicrobial activity and chemical composition of Mentha pulegium L. essential oil. Journal of Ethnopharmacology 119: 325-327. Manzocco, L., Anese, M., Nicoli, M.C. 1998. Antioxidant properties of tea extracts as affected by processing. Journal of Technology 31: 694-698. Marnewick, J.L., Joubert, E., Swart, P., van der Westhuizen, F.H., Gelderblom W.C.A 2003. Modulation of hepatic drug metabolising enzymes and oxidative status by green and black (Camellia sinensis), rooibos (Aspalathus linearis) and honeybush (Cyclopia intermedia) teas in rats. Journal of Agriculture and Food Chemistry 51: 8113-8119. Marnewick, J.L., van der Westhuizen, F.H., Joubert, E., Swanevelder, S., Swart, P., Gelederblom, C.A. 2009. Chemoprotective properties of rooibos (Aspalathus linearis), honeybush (Cyclopia intermedia) herbal and green and black (Camellia sinensis) teas against cancer promotion induced by fumonisin B1 in rat liver. Food and Chemical Toxicology 47: 220-229. Mashimbye, M.J., Mudau, F.N., van Ree, T., Soundy, P. 2006. A new flavanol from Athrixia phylicoides. South African Journal of Chemistry 59: 1-2. Masika, P.J., Sultana, N., Afolayan, A.J. 2005. Isolation of two antibacterial compounds from the bark of Salix capensis. South African Journal of Botany 71: 441-443. Matsuda, K., Nishimura, Y., Kurata, N., Iwase, M., Yasuhara, H. 2007. Effects of continuous ingestion of herbal teas on intestinal CYP3A in the rat. Journal of Pharmacological Science 103: 214-221. Mavundza, E.J., Tshikalange, T.E., Mudau, F.N., Meyer, J.J.M. 2007. Evaluation of antibacterial and antioxidant activities of ethanol extracts of wild bush tea (Athrixia phylicoides). Journal of Medicinal and Aromatic Plant Science and Biotechnology 1: 240-242. Mavundza, E.J., Tshikalange, T.E., Lall, N., Mudau, F.N., Hussein, A.A. 2010. Abstract: Antioxidant activity and cytotoxicity of three flavonoids from Athrixia phylicoides ethanol extract. South African Journal of Botany 76: 398. Mbambezeli, G. 2005. Athrixia phylicoides DC., Kirstenbosch NBG. www.plantzafrica.com/plantab/athrixphyl.htm (10 April 2010). 73 McGaw, L.J., Steenkamp, V., Eloff, J.N. 2007. Evaluation of Athrixia bush tea for cytotoxicity, anti-oxidant activity, caffeine content and presence of pyrrolizidine alkaloids. Journal of Ethnopharmacology 110: 16-22. Mertens-Talcott, S.U., Zadezensky, I., De Castro, W.V., Derendorf, H., Butterweck, V. 2006. Grapefruit-drug interactions: can interactions with drugs be avoided? Journal of Clinical Pharmacology 46: 1390-1416. Miliauskas, G., Venskutonis, P.R., van Beek, T.A. 2004. Screening of radical scavenging activity of some medicinal and aromatic plant extracts. Food Chemistry 85: 231-237. M?ller, A., du Toit, E.S., Soundy, P., Olivier, J. 2006. Morphology and ultrastructure of glandular trichomes on field grown and micropropagated leaves of Athrixia phylicoides (Asteraceae). South African Journal of Plant and Soil 23: 302-304. Mudau, F.N., Soundy, P., du Toit, E.S., Olivier, J. 2006. Variation in polyphenolic content of Athrixia phylicoides (L.) (bush tea) with season and nitrogen application. South African Journal of Botany 72: 398-402. Muller, H. and Brackhagen, O. 2000. The role of natural products in drug discovery. Springer-Verlag, Berlin, Heidelberg, Germany. Mosmann, T. 1983. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. Journal of Immunological Methods 65: 55-63. Nyiligira, E., Viljoen, A.M., van Heerden, F.R., van Zyl, R.L., van Vuuren, S.F., Steenkamp, P.A. 2008. Phytochemistry and in vitro pharmacological activity of South African Vitex (Verbenaceae) species. Journal of Ethnopharmacology 119: 680-685. Olivier, J., De Jager, A. 2005. ? Etnobotaniese opname van ? inheemse Suid-Afrikaanse tee: Athrixia phylicoides. Suid-Afrikaanse Tydskrif van Natuurwetenskap en Tegnologie 24: 139-147. Olivier, J., Rampedi, I. 2008. Mountain tea: A new herbal tea from South Africa? Food Science and Technology 22: 47-49. 74 P?la-Pa?l, J., P?rez-Alonso, M.J., Velasco-Negueruela, A., P?la-Pa?l, R., Sanz, J., Conejero, Fco. 2001. Seasonal variation in chemical constituents of Santolina rosmarinifolia L. ssp. rosmarinifolia. Biochemical Systematics and Ecology 29: 663-672. Paraskeva, M.P., van Vuuren, S.F., van Zyl, R.L., Davids, H., Viljoen, A.M. 2008. The in vitro biological activity of selected South African Commiphora species. Journal of Ethnopharmacology 119: 325-327. Pillay, P., Maharaj, V.J., Smith, P.J. 2008. Investigating South African plants as a source of new antimalarials drugs. Journal of Ethnopharmacology 119: 438-454. Plowes, D.C.H. and Drummond, R.B. 1976. Wild flowers of Rhodesia. Longman, Salisbury. Prozesky, E.A., Meyer, J.J.M., Louw, A.I. 2001. In vitro antiplasmodial activity and cytotoxicity of ethnobotanically selected South African plants. Journal of Ethnopharmacology 76: 239-245. Putievsky, E., Ravid, U., Dudai, N. 1986. The influence of season and harvest frequency on the essential oil and the herbal yield from a pure clone of sage (Salvia officinalis) grown under cultivated conditions. Journal of Natural Products 49: 326-329. Rabe, C., Steenkamp, J.A., Joubert, E., Burger, J.F.W., Ferreira, D. 1994. Phenolic metabolites from Rooibos tea (Aspalathus linearis). Phytochemistry 35: 1559-1565 Rabe, T. and van Staden, J. 1997. Antibacterial activity of South African plants used for medicinal purposes. Journal of Ethnopharmacology 56: 81-87. Rampedi, I.T., Olivier, J. 2005. The use and commercial development potential of A. phylicoides: A multipurpose indigenous South African plant. Acta Academy 37: 165-183. Rates, S.M.K. 2001. Plants as source of drugs. Toxicon 39: 603-613. Roberts, M. 1990. Indigenous healing plants. Southern Book Publishers. Halfway House, South Africa. Sangwan, N.S., Farooqi, A.H.A,, Shabih, F., Sangwan, R.S. 2001. Regulation of essential oil production in plants. Plant Growth Regulation 34: 3-21. 75 Sibanda, S., Chigwanda, G., Poole, M., Gwebu, E.T., Noletto, J.A., Schmidt, J.M., Rea, A.I., Setzer, W.N. 2004. Composition and bioactivity of the essential oil of Heteropyxis dehniae from Zimbabwe. Journal of Ethnopharmacology 92: 107-111. Simmons, D. and Parsons, R.F. 1987. Seasonal variation in the volatile leaf oils of two Eucalyptus Species. Biochemical Systematics and Ecology 15: 209-215. Sircar, J.C., Shwender, C.F., Johnson, E.A. 1983. Soybean lipoxygenase inhibition by nonsteroidal anti-inflammatory drugs. Prostaglandins 25: 393-396. Smyth, T., Ramachandran, V.N., Smyth, W.F. 2009. A study of the antimicrobial activity of selected naturally occurring and synthetic coumarins. International Journal of Antimicrobial Agents 33: 421-426. Valentin, A., P?lissier, Y., Benoit, F., Marion, C., Kone, D., Mallie, M., Bastide J.M., Bessi?re, J.M. 1995. Composition and antimalarial activity in vitro of volatile components of Lippia multiflora. Phytochemistry 40: 1439-1442. van Vuuren, S.F., Viljoen, A.M., van Zyl, R.L., van Heerden, F.R., H?sn?, K., Ba?er, C. 2006. The antimicrobial, antimalarial and toxicity profiles of helihumulone leaf essential oil and extracts of Helichrysum cymosum (L.) D. Don subsp. cymosum. South African Journal of Botany 72: 287-290. van Vuuren, S.F., Viljoen, A.M, Ozek, T., Demirci, B., Ba?er, K.H.C. 2007. Seasonal and geographical variation of Heteropyxis natalensis essential oil and the effect thereof of the antimicrobial activity. South African Journal of Botany 73: 441- 448. van Vuuren, S.F. 2008. Antimicrobial activity of South African medicinal plants. Journal of Ethnopharmacology 119: 462-472. van Wyk, B.E., and Gericke, N. 2000. People?s plants ? A Guide to Useful Plants of South Africa. Briza Publications, Pretoria, South Africa. van Wyk, B.E., Van Oudtshoorn, B., Gericke, N. 2002. Medicinal Plants of South Africa. Briza Publications, Pretoria, South Africa. van Wyk, B.E., Wink, M. 2004. Medicinal plants of the world. Briza Publications, Pretoria, South Africa. van Wyk, B.E. 2008. A broad review of commercially important southern African medicinal plants. Journal of Ethnopharmacology 119: 342-355. 76 van Zyl, R.L., Seatlholo, S.T., van Vuuren, S.F., Viljoen, A.M. 2006 (Special edition). The biological activities of 20 nature identical essential oil constituents. Journal of Essential Oil Research 18: 129-133. Viljoen, A.M., Moolla, A., van Vuuren, S.F., H?sn?, K., Baser, C., Demirci, B., ?zek, T. 2006 (Special edition). A seasonal variation study of the chemical composition and antimicrobial activity of the essential oil of Agathosma ovata (Thunb.) Pillans (Rutaceae). Journal of Essential Oil Research 18: 30-36. Von Gadow, A., Joubert, E., Hansmann, C.F. 1997. Comparison of the antioxidant activity of rooibos tea (Aspalathus linearis) with green, oolong and black tea. Food Chemistry 60: 73-77. Watt, J.M. and Breyer-Brandwijk, M.G. 1962. The medicinal and poisonous plants of Southern and Eastern Africa. 2nd edition. Livingstone, Edinburgh. Weinmann, I. 1997. History of the development and application of coumarin and coumarin- related compounds. In: Coumarins: Biology, Applications and Modes of Action. Chichester, UK, Wiley Press. WHO (World Health Organization) 2002. Antimicrobial resistance ? Fact Sheet No.194. http://www.who.int/mediacentre/factsheets/fs194/en/index.html (4 April 2010). WHO (World Health Organization) 2008. Traditional Medicine ? Fact Sheet No. 134. http://www.who.int/mediacentre/factsheets/fs134/en/index.html (20 March 2010). WHO (World Health Organization) 2010. Malaria - Fact Sheet No. 94. http://www.who.int/mediacentre/factsheets/fs094/en/index.html (21 April 2010). Wittstock, U., Gershenzon, J. 2002. Constitutive plant toxins and their role in defense against herbivores and pathogens. Elsevier Science Ltd, Germany. 77 Appendix A: NMR data for the (4-hydroxyphenyl)propyl coumaroate. Table A.1: NMR data (in CDCl3 + CD3OD) of the compound isolated from A. phylicoides. CD3OD: methanol; CDCl3: chloroform; d: doublet; t: triplet, ?C: carbon shift; ?H: proton shift; J: coupling constant; Hz: hertz. * Assignment may be interchanged. Position ?C ?H 1 30.9* 2.62 (2H, t, J 7.5 Hz) 2 30.9 * 1.94 (2H, quintet, J 7.3 Hz) 3 64.2 4.14 (2H, t, J 6.5 Hz) 1? 132.6 2? 129.7 7.00 (2H, d, J 8.5 Hz) 3? 115.5 6.72 (2H, d, J 8.6) 4? 155.3 5? 115.5 6.72 (2H, d, J 8.6) 6? 129.7 7.00 (2H, d, J 8.5 Hz) 1? 126.3 2? 130.4 7.40 (2H, d, J 8.7 Hz) 3? 116.2 6.80 (2H, d, J 8.7 Hz) 4? 159.9 5? 116.2 6.80 (2H, d, J 8.7 Hz) 6? 130.4 7.40 (2H, d, J 8.7 Hz) ? 114.7 ? 145.6 7.56 (1H, d, J 16.0 Hz) =O 168.6 78 Figure A.1: The NMR carbon spectra (1H) for (4-hydroxyphenyl)propyl coumaroate. 79 Figure A.2: The NMR carbon spectra (13C) for (4-hydroxyphenyl)propyl coumaroate.