The occurrence and profiling of naphthoquinones in ethnobotanically selected plants

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Chapter 2 Literature review

An introduction to Euclea natalensis

The family Ebenaceae consists of about 500 species which is widespread in the tropics and subtropics. In southern Africa two genera are found namely Diospyros and Euclea. There are sixteen Euclea species to be found in southern Africa, with Euclea natalensis A.DC. occurring in the Eastern Cape, KwaZulu-Natal and Swaziland (Jordaan, 2003). E. natalensis is a shrub or small to medium size tree (Fig. 1.3.a) which grows in coastal and inland forests and also in the bushveld. The leave arrangement of Euclea species is very variable and may be opposite to sub-opposite or alternate to whorled even on the same plant. E. natalensis has alternate leaves that are elliptic to obovate-oblong, glossy dark green above and densely covered with woolly hairs below. The margins of the leaves appear wavy as shown in Fig.
1.3.b (Van Wyk & Van Wyk, 1997).

Traditional uses

According to Van Wyk & Van Wyk, (1997) the roots of the tree has been traditionally used for dying palm-mats, while decoctions of the roots have numerous medicinal applications as a purgative, analgesic and for its anti-inflammatory properties. The twigs are used as toothbrushes in oral hygiene (Stander & Van Wyk, 1991) and according to Sparg et al. (2000) the extracts are used to treat urinary infections and showed good activity against schistosomiasis. The Tonga people use the root for the relief of toothache and headache, while the Zulu people used the roots as a purgative and also for abdominal complaints. The Shangaan people apply the powdered root bark to skin lesions in leprosy and take it internally for ancylotomiasis (Watt & Breyer-Brandwijk, 1962).

Phytochemistry

The amount of research that has been done on this species is relatively small. The publications (24 in total) are mostly on the chemical constituents of E. natalensis. Stander and Van Wyk (1991) reported on the use of the root as toothbrushes and speculated that the naphthoquinones in the roots are responsible for the activity against Streptococcus species. There are four publications on the antimycobacterial activity of napthoquinones isolated from E. natalensis (Lall & Meyer, 1999 & 2001; Lall et al., 2003 & 2005). Weigenand et al. (2004) reported on the antibacterial activity of naphthoquinones and triterpenoids from the roots of E. natalensis. The compounds isolated from Euclea species are given in Table 2.1. In addition two compounds, neodiospyrin and 5-hydroxy-4-methoxy-2-naphthaldehyde, have been isolated recently (Van der Kooy, 2003) from this species.

Occurrence and profiling of 7-methyljuglone in plants

The occurrence of the naphthoquinones studied during this work is widely reported in the Ebenaceae family (Van der Vijver & Gerritsma, 1976; Mallavadhani et al.,1998). There are also reports that 7-methyljuglone occurs in some Drosera spp. (Caniato et al., 1989) and one report that it occurs in thrips where it is used in a defensive secretion (Susuki et al., 1995). No other species were reported to contain these naphthoquinones. The structurally similar plumbagin (methyl group on carbon 2) however occurs far more widely in different plant species. Plumbagin occurs in Plumbago spp. (Kapadia et al., 2005), Drosera spp. (Marczak et al., 2005), Diospyros spp. (Evans et al., 1998) and even in the Venus flytrap (Dionaea muscipula) (Tokunaga et al., 2004). Juglone (lacking the methyl group) occurs predominantly in Juglans spp. (Lee et al., 1969). This would give an indication that these structurally similar compounds are produced from different biosynthetic pathways. These molecules are also the parent molecules of a large number of dimers (including diospyrin), trimers and tetramers.

Chapter 1 Introduction
1.1 General background and introduction
1.1.1 Occurrence and treatment of Mycobacterium tuberculosis
1.1.2 Natural product chemistry
1.1.3 Organic synthesis
1.1.4 Stability and solubility of naphthoquinones
1.1.5 Toxicity of naphthoquinones
1.1.6 Structure-activity relationship
1.1.7 Mode of action studies
1.2 Objectives of this study
1.3 Structure of thesis
1.4 References
Chapter 2 Literature review
2.1 An introduction to Euclea natalensis
2.1.1 Traditional uses
2.1.2 Phytochemistry
2.2 Occurrence and profiling of 7-methyljuglone in plants
2.3 Chemistry and biological activity of naphthoquinones
2.3.1 Synthesis of naphthoquinones
2.3.2 Biological activity of naphthoquinones
2.3.3 Mode of action of naphthoquinones
2.4 References
Chapter 3 The occurrence and profiling of naphthoquinones in ethnobotanically selected plants
3.1 Introduction
3.2 Materials and methods
3.2.1 Plant material
3.2.2 Preparation of extracts
3.2.3 Profiling with TLC
3.2.4 Profiling with HPLC
3.2.5 Profiling with NMR
3.2.6 Fingerprinting Drosera capensis
3.3 Results
3.3.1 Profiling with TLC
3.3.2 Profiling with HPLC
3.3.3 Profiling with NMR
3.3.4 Fingerprinting Drosera capensis
3.4 Discussion and conclusions
3.5 References
Chapter 4 Synthesis of 7-methyljuglone and diospyrin
4.1 Introduction
4.2 Materials and methods
4.2.1 Materials
4.2.2 Methods
4.2.2.1 Synthesis of 7-methyljuglone
4.2.2.1.1 Effect of different quantities of reagents on 7-methyljuglone formation
4.2.2.1.2 Effect of different quantities of catalyst on 7-methyljuglone formation
4.2.2.1.3 Influence of temperature on 7-methyljuglone formation
4.2.2.1.4 Effect of altering reaction times
4.2.2.2 Epoxidation of 7-methyljuglone
4.2.2.2.1 Effect of reaction time on epoxide formation
4.2.2.2.2 Influence of time before acidification
4.2.2.2.3 Effect of amount of acid on epoxide formation
4.2.2.3 Synthesis of diospyrin
4.2.2.3.1 Oxidative dimerisation of 7-methyljuglone
4.2.2.3.2 Buffered reaction between hydroquinone and 7-methyljuglone
4.2.2.3.3 Epoxide condensation
4.2.2.3.3.1. Addition of an Bronsted-Lowry acid to the epoxide
4.2.2.3.3.2 Addition of an Lewis acid and steam distillation
4.3 Results
4.3.1 Synthesis of 7-methyljuglone
4.3.1.1 Effect of different quantities of reagents on 7-methyljuglone formation
4.3.1.2 Effect of different catalyst ratios
4.2.1.3 Influence of temperature on 7-methyljuglone formation
4.1.2.4 Effect of altering stirring times
4.3.2 Epoxidation of 7-methyljuglone
4.3.2.1 Influence of reaction time on epoxide formation
4.3.2.2 Effect of time before acidification
4.3.2.3 Effect of the amount of acid
4.3.3 Synthesis of diospyrin
4.3.3.1 Oxidative dimerisation of 7-methyljuglone
4.3.3.2 Buffered reaction between hydroquinone and 7-methyljuglone
4.3.2.3 Epoxide condensation
4.3.2.3.1 Addition of Bronsted-Lowry acid
4.3.2.3.2 Addition of Lewis acid and steam distillation
4.4 Discussion and conclusions
4.5 References
Chapter 5 Stability of naphthoquinones
5.1 Introduction
5.2 Materials and methods
5.2.1 Materials
5.2.2 Methods
5.2.2.1 Stability in dimethylsulfoxide
5.2.2.2 Stability in BACTEC buffer solution
5.2.2.3 Stability in vero cell toxicity bioassay buffer
5.2.2.4 Stability in 20% DMSO/Agar mixture
5.2.2.5 Stability in broth used for mode of action studies
5.3 Results
5.3.1 Stability in dimethylsulfoxide
5.3.2 Stability in BACTEC buffer solution
5.3.3 Stability in vero cell toxicity bioassay buffer
5.3.4 Stability in 20% DMSO/Agar mixture
5.3.5 Stability in broth used for mode of action studies
5.4 Discussion and conclusions
5.5 References
Chapter 6 Toxicity of naphthoquinones
6.1 Introduction
6.2 Materials and methods
6.2.1 Materials
6.2.1.1 Culturing of Vero monkey kidney cells
6.2.1.2 Toxicity of 7-methyljuglone and diospyrin in mice
6.2.1.3 Toxicity of 7-methyljuglone in Musca domestica
6.2.2 Methods
6.2.2.1 Determination of the IC50 of naphthoquinones on vero cells
6.2.2.2 Toxicity of 7-methyljuglone and diospyrin in mice
6.2.2.3 Toxicity of 7-methyljuglone in Musca domestica
6.3 Results
6.3.1 Determination of the IC50 of naphthoquinones on vero cells
6.3.2 Toxicity of 7-methyljuglone and diospyrin in mice
6.3.3 Toxicity of 7-methyljuglone in Musca domestica
6.4 Discussion and conclusions
6.5 References
Chapter 7 Structure-activity relationship of naphthoquinones
7.1 Introduction
7.2 Materials and methods
7.2.1 MIC determination
7.2.2 Toxicity bioassay
7.3 Results
7.3.1 MIC and toxicity determination
7.3.2 Structure-activity relationship
7.4 Discussion and conclusions
7.5 References
Chapter 8 The mode of action of naphthoquinones in Mycobacterium smegmatis
8.1 Introduction
8.2 Materials and methods
8.2.1 Activity against M. smegmatis
8.2.2 M. smegamatis cultures
8.2.3 Extraction of M. smegmatis cells
8.2.4 HPLC analysis
8.3 Results
8.3.1 Activity against M. smegmatis
8.3.2 M. smegamatis cultures
8.3.3 HPLC analysis
8.4 Discussion and conclusions
8.5 References
Chapter 9 General Discussion and Conclusions
Acknowledgements and Publications