ELOFF JN and MCGAW LJ (2014) Using African plant biodiversity to combat microbial infections pp 163-173 in GURIB-FAKIM A (Ed) Novel Plant Bioresources: Applications in Food Medicine and Cosmetics. John Wiley DOI: 10.1002/9781118460566.ch12
Chapter: Using African plant biodiversity to combat microbial infections
JN Eloff and LJ McGaw
Phytomedicine Programme, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort 0110, South Africa
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Table of Contents
Introduction and problem statement
Commercial use of African medicinal plants in the herbal medicine industry
Why is there such a difference in product development for antimicrobials versus other medicinal applications?
Methods used in developing useful products
Results of random screening of large number of species
Our approach to random screening
Activity of compounds isolated against Staphylococcus aureus
Discovering antifungal compounds from natural products.
Review papers focussing on antimicrobial activity of plants from Africa
Promising new approaches
The potential of using African medicinal plants as extracts.
Conclusions
References
Abstract
Despite many thousands of publications investigating the antibiotic activity of plant extracts and the wide-spread use of African medicinal plants to treat animal and human microbial infections no single entity commercial antimicrobial product has yet been developed from plants. This is in contrast to many commercial medicinal products that have been developed from plants for other diseases. After an extensive survey of the literature, it appears that plants combat infections by using synergistic interactions between different compounds and not single highly active compounds. The random screening of acetone leaf extracts of more than 700 tree species yielded many extracts with high activities. Although the chance of developing single entity antibiotics seems elusive, there are examples where plant extracts can be used to deliver highly effective products that can compete with current commercially used antimicrobial agents. By manipulating extracts the biological activity can be enhanced and patentable products can be developed.
Keywords
Antifungal, antimicrobial, synergistic activity, method development, commercial product
1. Introduction and problem statement
Microbial infections have a major effect on human health especially in rural parts of Africa where antibiotics are not freely available or too expensive. It also has a very important effect on the quality of life though microbial infections of animals and plants produced for food.
People have been using plants to treat ailments for many years. In a 60 000 years old Neanderthal Shanidar cave burial site in present day Iraq, pollen was found of 8 plant species, seven of these species including an Ephedra species are still used as medicinal plants around the world (Solecki, 2007). In South Africa Khoi San rock paintings include 8 clearly defined medicinal plant species including Aloe and Harpagaphyton spp. For thousands of years plants were the only resources that people had to combat diseases. In the training of doctors, knowledge of plants was so important that Galen once stated: ““The doctor who does not know his plants should quit the profession”.
In the 16th century Paracelsus started using chemicals and for a long time two systems were in use. In days when people still accepted the spontaneous development of life, it was very difficult to understand certain diseases until microorganisms were discovered and Louis Pasteur developed the germ theory of infectious diseases. A giant step in the battle between plant medicine and chemical medicine was taken when Paul Ehrlich discovered salvarsan as a “magic bullet” against organisms causing syphilis. This was an enormous improvement compared to the mercury salt treatment of syphilis that led to horrible side effects such as losing hair and teeth. The most remarkably change away from using herbal medicines however came after the discovery and commercialization of penicillin.
It is not widely known that many ancient cultures, in Greece and India have already used moulds to treat infections. There have been other people who have discovered antibiotic activity of fungi on bacteria before the well-known serendipitous discovery of Alexander Fleming in 1928 that a Penicillium notatum infection on one of his Staphylococcus plates inhibited the growth of the bacteria. The penicillin in his culture was unstable and the yield was very low (1 part per million).
Ten years later the biochemists Florey, Chang and Heatly in England were able to produce a stable penicillin. During the second world war scientists in the United States succeeded in getting higher yielding Penicillium strains and developing fermentation technology to produce large quantities of penicillin in time to save thousands of lives of soldiers with infected wounds. The discovery of antibiotics had a major influence on the development of the pharmaceutical industry and the downgrading of the herbal medicine industry.
Very soon after the discovery of penicillin, the development of resistant bacteria was discovered in London (Levy and Marshall, 2004). Due to misuse of antibiotics there is such a development of resistance that some authors have warned that we are entering the post-antibiotic era. Recently Britain's chief medical officer, Sally Davies, stated that the issue should be added to the list of national emergencies (Kåhrström, 2013). The world health organization have identified several levels of drug resistant Mycobacterium tuberculosis strains i.e. multiple drug resistant (MDR), extensively drug resistant (XDR) and even total drug resistant (TDR) strains discovered in India, Italy and Iran (Mahr, 2013). Dr Shelly Batra, President of a New-Delhi based non-governmental organization that fights TB stated that “We are on the brink of another epidemic, and it has no treatment. If TDR spreads, we will go back to the Dark Ages” (Mahr, 2013).
Despite substantial research efforts by the pharmaceutical industry, no new class of broad-spectrum antibiotics were developed after the fluoroquinolones nearly 50 years ago (Lewis and Ausubel, 2006). There are many problems in finding new antibiotics active against Gram-negative bacteria. Abad et al (2007) made the following statement: “In the past few decades, a worldwide increase in the incidence of fungal infections has been observed as well as a rise in the resistance of some species of fungus to different fungicidals used in medicinal practice. Fungi are one of the most neglected pathogens, as demonstrated by the fact that the amphotericin B, a polyene antibiotic discovered as long ago as 1956, is still used as a “gold standard” for antifungal therapy. The last two decades have witnessed a dramatic rise in the incidence of life threatening systemic fungal infections.”
Many pharmaceutical products are based on plant products. In a survey in the 1970s it was found that 25% of prescription medicines in the USA are based on compounds originally discovered from plants (Farnsworth and Morris, 1976). Some of the most important anti-malarial pharmaceuticals are plant based and several anticancer pharmaceuticals are based on compounds isolated from plants.
There have been many review papers written on this topic. Some of the papers we consulted to prepare this paper are shown in Table 1
Reference / date / Title/descriptionCowan / 1999 / Plant products as antimicrobial agents
Gibbons / 2004 / Anti-staphylococcal plant natural products
Levy and Marshall / 2004 / Antibacterial resistance worldwide:causes, challenges and responses
Rios and Recio / 2005 / Medicinal plants and antimicrobial activity
Cos et al. / 2006 / Anti-infective potential of natural products: How to develop a stronger in vitro ‘proof of concept’
Lewis and Ausubel / 2006 / Prospects for plant-derived antibacterials
Eloff and McGaw / 2006 / Plant extracts used to manage bacterial, fungal and parasitic infections in southern Africa
Abad et al. / 2007 / Active antifungal substances from natural sources
van Vuuren / 2008 / Antimicrobial activity of South African medicinal plants
Svetaz et al. / 2010 / Value of ethnomedicinal information for the discovery of plants with antifungal properties. A survey among seven Latin American countries
Kuete et al. / 2011 / Antibacterial activity of some natural products against bacteria expressing a multi-resistant phenotype
Queiros et al. / 2012 / Modern approaches in the search for new active compounds from crude extracts of natural sources
Sorting et al. / 2012 / The role of natural products in the discovery of new anti-infective agents with emphasis on antifungal compounds
Cragg et al. / 2012 / Natural products in drug discovery: recent advances
2. Commercial use of African medicinal plants in the herbal medicine industry
Single chemical entities as pharmaceuticals have taken the place of herbal medicines for the treatment of many serious diseases since the wide-spread use of antibiotics in the 1950s. There has however been resurgence in the use of formal herbal medicines to treat less serious illnesses and for maintaining health. Africa has missed out in the growth in this industry because the trade in African medicinal plants to the developed world is very low. Therefore this limits job and wealth creation by growing, beneficiating and exporting of herbal medicines.
One of the major constraints for trade in African medicinal plants was identified at the Medicinal Plants Forum for Commonwealth Africa held in Cape Town in 2000, was the lack of suitable technical specifications and quality control standards (Brendler et al., 2010). This conclusion was justified when van Wyk and Wink (2004) analysed the origin of commercialized herbal medicines. The percentage of commercialized medicinal plants was much lower from southern hemisphere countries where the traditional knowledge was transferred orally than from Europe, India and Asia where traditional knowledge has been written down for many centuries. Africa including the Indian Ocean islands contains about 60 000 plant species (Klopper et al., 2007) and only 83 species are used as herbal medicines in the developed world compared to the 434 herbal medicines commercialized from the 13600 species occurring naturally in Europe. This clearly shows that there is a tremendous opportunity to develop many more herbal medicines from Africa.
3. Why is there such a difference in product development for antimicrobials versus other medicinal applications?
A search on Google Scholar using the terms “Africa antimicrobial plant” without citations or patents since 2000 yields about 16600 hits. Even using such a rough measure it is clear that many papers have been written investigating plants for antimicrobial activities. In a very large proportion of the publications the aim of the authors was probably to eventually identify compounds that could be used as new antibiotics. Many papers written unfortunately had limited if any value because the methods used were not acceptable and does not yield comparable results.
A common error bedevilling many publications in this field is that statements are made that a certain plant extract or compound has antibacterial or antifungal activity. If there is no quantitative measure attached, such a statement is meaningless. If the concentration is high enough, probably all compounds or extracts would be toxic to microbes. Nobody would think of sucrose as an antibiotic but high enough concentration will certainly kill microorganisms.
In many of these 16000 papers there are serious problems with the methods used. For example if “agar” is included in the search terms, Google Scholar yields 14800 hits. This may mean that 89% of papers delivered since 2000 probably used agar diffusion techniques. As discussed in section 4.3 below, this makes comparisons difficult if not impossible.
4. Methods used in developing useful products
4.1 Extraction of plant material
Plants contain at least 100 000 small compounds (Lewis and Ausubel, 2006) and many of these compounds would not readily be soluble in different extractants. One way of classifying these compounds is by the polarity. Non-polar or lipophilic compounds are soluble in non-polar solvents such as hexane or dichloromethane. The more polar or hydrophilic compounds are mainly soluble in polar solvents such as water or methanol. By using the wrong solvent, one may therefore miss active compounds. Some authors use mixtures of solvents to extract as many different compounds as possible in searching for plants with promising activities. Another approach is to use an intermediate polarity extractant that would extract compounds over a wide range of polarities. Among the commonly available solvents acetone would fulfil this role. The ability of acetone to mix with polar and non-polar solvents is important. Several extractants were investigated on a 5 point scale and given different weights (in brackets) for the quantity extracted (3) from Combretum erythrophyllum and Anthocleista grandiflora, the rate of extraction (3), number of compounds extracted (5), number of antibacterial compounds extracted (5), the toxicity to pathogens in subsequent bioassays (4) and ease of removal (5) and hazardous to use (2). The average values out of a potential 130 were acetone 102, methanol:chloroform:water (12:5:3) 81, methylene dichloride 79, methanol 71, ethanol 68 and water 47 (Eloff, 1988a). If only one solvent is used acetone was by far the best extractant based on these results.
Acetone also had the lowest toxicity for four fungi with the following minimum inhibitory concentration (MIC) acetone (51%) dimethylsulphoxide (45%), methanol (16-44%) and ethanol (30%) (Eloff et al., 2007). Acetone is also very useful when determining MICs of volatile oils or non-polar fractions during bioassay guided fractionation because it dissolves non-polar compounds and mixes with aqueous growth mediums.
When a serious of extractants were used to extract dried leaves of Combretum microphyllum (Kotze and Eloff, 2002) or Combretum woodii (Eloff et al, 2005) it was clear that the most polar (water) and most non-polar solvents (hexane) did not extract antimicrobial compounds (Fig.1)
Figure 1 Separation of 100 µg of compounds extracted from dried leaves of Combretum woodii by hexane, di-isopropylether, diethyl ether, methylene dichloride, ethylacetate, tetrahydrofurane, acetone ethanol, methanol and water separated with ethylacetate:methanol:water and sprayed with vanillin sulphuric acid top and with a culture of Staphylococcus aureus, incubated overnight and then sprayed with tetrazolium violet. Clear areas indicate where bacterial growth is inhibited. (Chromatograms from Eloff et al., 2005)