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Chapter 2 :Literature review on the therapeutic use of plant compounds
Natural product drug discovery
For decades, natural products have been a wellspring of drugs and drug leads. When you have no idea where to begin in a drug discovery programme, nature is a good starting point. According to a survey by Newman and co-workers (1997) of the National Cancer Institute, 61% of the 877 small molecule new chemical entities introduced as drugs worldwide during 1981–2002 can be traced to or were inspired by natural products (Newman et al., 2003). These include natural products (6%), natural product derivatives (27%), synthetic compounds with natural-product-derived pharmacophores (5%), and synthetic compounds designed on the basis of knowledge gained from a natural product (i.e. a natural product mimic, 23%).
Two shortcomings of natural products are the difficulty in chemical derivatization and the small quantities available from nature. Natural products will not solve all the problems because in many cases, natural products may have negative side effects or insufficient biological activity. Synthetic chemists must investigate modification of lead compounds. It makes a lot of sense to be guided by natural products, and derivatives with validated biological relevance.
In a field that has been ravaged by herbivores, some plants, although they are without protective structures, are untouched. Perhaps they are distasteful or toxic and are therefore protected against natural enemies. Very well preserved plants may be chemically interesting. Extra-organismal interactions make up a chemical web that keeps the environment working the way it does. With the techniques now available, chemical ecology, which is the study of the chemical interactions between organisms, is poised to look at nature in a new way. To understand biotic interactions at a molecular level is both a great opportunity and a major challenge for chemists.
Chemical relevance is revealed by chemical profiling. Crude extracts are analyzed by high-performance liquid chromatography with mass spectrometric, light scattering, and ultraviolet detection. Mass spectrometry gives the molecular weight and structural information. Light scattering estimates the amount of material represented by each peak, because a minimum amount is needed to build good libraries that can be used for years. UV absorption gives additional insight on the compound’s structure.
Data are fed to internally developed software that compares all the peaks in an extract with all the peaks that the software has seen. At the end, the software ranks the extracts on the basis of the number of new peaks. The high-ranking extracts will very probably contain new compounds and will be taken further to purification and structure elucidation. The technique is useful in determining whether a collection of biological materials is chemically interesting. Typically, only 10 to 20% of the initially acquired biological samples qualify for further processing by this profiling step.
Secondary metabolites
Fig 2.1 shows the interaction between primary and secondary plant metabolism. Secondary metabolites are molecules that are not necessary for the growth and reproduction of a plant, but may serve some role in herbivore deterrence due to astringency, or they may act as phytoalexins, killing bacteria that the plant recognizes as a threat. Secondary compounds are often involved in key interactions between plants and their abiotic and biotic environments (Facchini et al., 2000).
Throughout history secondary metabolites of plants have been utilized by humanity. There are approximately four major classes of secondary compounds that are significant to humans. These classes are the flavonoids, alkaloids, phenylpropanoids and terpenoids (Edwards and Gatehouse, 1999).
Flavonoids
The flavonoids are a large group of natural products widespread in higher plants, and are also found in some lower plants including algae. The flavonoids are phenolic compounds possessing 15 carbon atoms and comprise two benzene rings joined by a linear three carbon chain.
Flavonoids constitute one of the most characteristic classes of compounds in higher plants. Many flavonoids are easily recognized as flower pigments in most angiosperm families (flowering plants). However, their occurrence is not restricted to flowers but includes all parts of the plant. The chemical structure of flavonoids is based on a C15 skeleton with a chromane ring being a second aromatic ring B in position 2, 3 or 4.
The oxygen bridge involving the central carbon atom (C2) of the 3C-chain occurs in a rather limited number of cases, where the resulting heterocyclic ring is of the furan type. Various subgroups of flavonoids are classified according to the substitution patterns of ring C. Both the oxidation state of the heterocyclic ring and the position of ring B are important in the classification.
Flavonoids are low molecular weight substances found in all vascular plants. In the broad sense they are virtually universal plant pigments. The anthocyanidins are responsible for flower colour in the majority of angiosperms, but colourless flavonoids are also widespread and abundant. They are phenylbenzopyrones with an assortment of basic structures usually found conjugated to sugars although the forms have been identified in nature. Flavonoids occur in several structurally and biosynthetically related classes and are important constituents of the human diet, being derived largely from fruits, vegetables, nuts, seeds, stems and flowers (Harborne, 1977).
While several members of the flavonoid family are known to possess antiviral and anti-inflammatory properties, vasculo-protector and anti-thrombotic action, spasmolytic activity, estrogenic actions, antioxidant and liver protecting effects (Middleton and Kandaswami, 1994), very little was known before 1989 on the effects of this class of compounds on the central nervous system (CNS). Some flavonoids, like quercetin and gossypin, have recently been shown to possess sedative and analgesic effects (Picq et al., 1991). Another flavonoid, and biflavonoid derivatives, isolated from Ginkgo biloba have been shown to increase blood flow (Danser et al., 1933), and reduce neuronal oxidative metabolism (Quisumbing, 1951).
Triterpenoids
Triterpenoids have been shown to have antibacterial activity and a number of triterpenes have been isolated from plants (Rogers, 1998; Angeh, 2005). Rogers (1998) isolated seven novel triterpenoids from Combretum erythrophyllum and Angeh (2005) isolated two new triterpenoids. Terpenes can occur as monoterpenes, diterpenes, triterpenes, and tetraterpenes (C10, C20, C30 and C40 respectively) as well as hemiterpenes (C5) and sesquiterpenes (C15). When they contain additional elements, usually oxygen, they are termed terpenoids. They differ from fatty acids in that they contain extensive branching and are cyclized. Examples of common terpenoids are menthol and camphor (monoterpenes), farnesol and artemisinin (sesquiterpenoids). Artemisinin and its derivative, α-arteether, find current use as antimalarials.
Triterpenoids are non-steroidal secondary metabolites. The physiological function of these compounds is generally believed to be a chemical defense against pathogens and herbivores. Throughout the plant and animal kingdom, terpenoids are known to have a wide range of functions. They can act as defensive substances in plants (allomones) and animals, they can be used by plants to deter herbivores or to inform conspecifics or attract natural enemies of herbivores (synomones). Plant hormones are often derivatives of terpenoids, such as cytokinins, gibberellins and abscisic acid. It is therefore expected that triterpenoids should act against certain pathogens causing human and animal diseases (Mahato and Sen, 1997). Although medicinal use of this class of compounds is rather limited, possibly due to their hydrophobic nature, recent work in this regard indicates their great potential as drugs.
Moreover, despite the remarkable diversity already known to exist, new variants continue to emerge (Mahato et al., 1992).Terpenoids are active against bacteria (Taylor et al., 1996), fungi (Suresh et al., 1997) and viruses (Xu et al., 1996). Their mechanism of action is not fully understood. Capsaicin, a constituent of chili peppers, is bactericidal to Helicobacter pylori, although possibly detrimental to the human gastric mucosa (Jones et al., 1997). Another terpenoid called petalostemumol, isolated from the prairie clover (Dalea sp.) showed excellent activity against Bacillus subtilis and Staphylococcus aureus as well as Candida albicans (Cowan, 1999).
Terpenoids isolated from Combretum species include jessic acid and methyl jessate from Combretum elaeagnoides; imberbic acid from Combretum imberbe (Mahato et al., 1992); combregenin, combre- glucoside, arjungenin and arjunglucoside from Combretum nigricans (Jossang et al., 1996) and arjunolic and mollic acid from Combretum leprosum (Facundo et al., 1993), and many more which have been cited above.
Declaration
Acknowledgements
Summary
Table of Contents
List of Figures
List of Tables
List of Abbreviations
Chapter 1 Introduction
1.1 Introduction
1.2 Antimicrobial resistance
1.2.1 Antiviral resistance
1.2.2 Antibiotic resistance
1.2.3 Factors contributing to development of resistance to antimicrobial drugs
1.2.3.1 Failure to use narrow-spectrum antibacterial drugs
1.2.3.2 Colonization pressure in hospitals
1.2.3.3 Length of hospital and ICU stays
1.2.3.4 Antibiotic misuse in agriculture
1.2.3.5 The FAAIR initiative
1.3 Viruses and viral diseases
1.3.1 Structure of viruses
1.3.2 HIV and AIDS
1.3.3 Other viral infections
Chapter 2 Literature review on the therapeutic use of plant compounds
2.1 Natural product drug discovery
2.2 Secondary metabolites
2.2.1 Flavonoids
2.2.2 Triterpenoids
2.2.3 Glycosides
2.2.3.1 Classification of glycosides
2.2.4 Coumarins
2.2.5 Plant-derived drugs employed in Western medicine
2.3 A brief history of pharmacology
2.3.1 Historical development
2.3.2 The herbal approach to viral infection
2.3.3 Some plants with antiviral and antibacterial activities
2.4 Study of medicinal plants
2.5 Work done on the Combretaceae family
2.5.1 The Combretaceae family
2.5.2 Taxonomy
2.5.3 Evaluation of the antibacterial activity of different species
2.5.4 Combretum erythrophyllum
2.5.5 Combretum woodii
2.5.6 Combretum microphyllum
2.5.7 Unpublished work on other members of Combretaceae
2.5.8 Combretum apiculatum
2.5.9 Combretum paniculatum
2.5.9.1 Ethnomedical information on C. paniculatum
2.5.9.2 Description of C. paniculatum
2.5.9.3 Previous work on C. paniculatum
2.6 Hypothesis
2.7 Aim of study
2.8 Objectives
Chapter 3: Materials and Methods
3.1 Plant collection
3.2 Preparation and extraction of plant material
3.3 Analysis by thin layer chromatography (TLC)
3.4 Bioassay-guided isolation
3.5 Solvent/Solvent fractionation
3.6 Chromatography
3.6.1 Amberlite XAD-16
3.6.2 Chromatotron
3.7 High Pressure Liquid Chromatography (HPLC)
3.8 Analysis and concentration of fractions
3.9 Antiviral activity
3.9.1 University of Pretoria method
3.9.1.1 Cell culture
3.9.1.2 Virus
3.9.1.3 Determination of the antiviral efficacy of the extract
3.9.1.4 Determination of the cytotoxicity of the extracts (MTT assay)
3.9.2 Hans-Knöll Institute (HKI) method
3.9.2.1 Cytotoxicity test to determine the maximum tolerated dose (CC10) of the test compound in HeLa, MDCK and Vero cell monolayers
3.9.2.2 Determination of the antiviral efficacy of the test compounds by means of inhibition of the cytopathic effect (CPE)
3.10 Antibacterial activity
3.10.1 Microdilution assay for MIC determination
3.10.2 Total activity
3.10.3 Bioautography
3.11 Antifungal activity of extracts
3.12 Agar diffusion method for antibacterial and antifungal activity
3.13 Antioxidant activity
3.14 Anti-inflammatory activity
3.14.1 Enzyme assay
Chapter 4: Selection of the best extractant for the plant material
4.1 Introduction
4.2 Extraction
4.3 Results
4.4 Discussion
Chapter 5: Determination of antiviral, antimicrobial, cytotoxic and antioxidant activities of extracts
5.1 Introduction
5.2 Methods
5.2.1 Antiviral activity
5.2.2 Antibacterial activity
5.2.3 Bioautography
5.2.4 Antioxidant activity
5.2.5 Cytotoxicity (MTT assay)
5.3 Results
5.3.1 Antiviral and cytotoxic activities of extracts
5.3.2 Antibacterial activity
5.3.3 Bioautography of extracts
5.3.4 Antioxidant activity of extracts
5.4 Discussion
5.5 Conclusion
Chapter 6: Preliminary isolation study
6.1 Introduction
6.2 Methods
6.2.1 Extraction and preliminary column chromatography
6.2.2 Solvent/solvent fractionation of root bark
6.2.3 Bioactivity testing
6.3 Results and Discussion
6.4 Conclusion
Chapter 7: Isolation of compounds from C. paniculatum leaves
7.1 Introduction
7.2 Methods
7.2.1 Extraction and isolation
7.3 Results and Discussion
7.4 Conclusion
Chapter 8: Determination of chemical structures, biological activities and cytotoxicity of isolated compounds
8.1 Introduction
8.2 Structure elucidation
8.3 Biological activity and cytotoxicity
8.4 Results and Discussion
8.4.1 Identification of isolated compounds
8.4.2 Biological activity of isolated compounds
8.4.2.1 Antiviral activity
8.4.2.2 Antibacterial and antifungal activities
8.4.2.3 Cytotoxic activity of isolated compounds
8.5 Conclusion
Chapter 9: General Conclusions
9.1 Introduction
9.2 Selection of the best extractant for the plant material
9.3 Determination of antimicrobial, cytotoxic and antioxidant activities of extracts
9.4 Preliminary isolation study
9.5 Isolation of antibacterial compounds
9.5.5 Determination of chemical structures, biological assays and cytotoxicity of compounds References
Appendix
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