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

kobus.eloff@up.ac.za, www@up.ac.za/phyto

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

1

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

2

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

3

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/description

Cowan 1999 Plant products as antimicrobial agentsGibbons 2004 Anti-staphylococcal plant natural productsLevy and Marshall 2004 Antibacterial resistance worldwide:causes, challenges and

responsesRios 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 2006 Plant extracts used to manage bacterial, fungal and parasitic

4

McGaw infections in southern AfricaAbad et al. 2007 Active antifungal substances from natural sourcesvan Vuuren 2008 Antimicrobial activity of South African medicinal plants

Svetaz et al. 2010Value 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.

5

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

6

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)

7

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)

Hardly any other plant water extracts in our laboratory had any antimicrobial activity

In a review of the literature 1989-1999 only 14/157 water extracts were listed

(Srivastava et al., 2000). In another survey on the antibacterial activity South African

plants used for medicinal purposes only 4/27 water plant extracts were active (Rabe

& van Staden, 1997). In our experience water extracts with antimicrobial activity

may contain tannins that would inhibit microbial growth but would not be useful

potential antibiotics or prophylactic growth enhancers.

4.2 Selection of plant material to investigate

There are different ways to select the plants that are to be investigated. Cos et al

(2006) identified the following: random selection of species and then chemical

screening, random selection of species for bioassays, following up on publications

on biological activities and using ethnomedicinal leads. Other approaches could be

to search on a taxonomic basis investigating families or genera with high activity

(Eloff, 1998c).

8

Because traditional healers usually only have water available as extractant and the

antimicrobial activity of aqueous extracts are very low this means that traditional

leads may not be useful to find compounds that can become antibiotics.

The Phytomedicine Programme has determined the MIC of acetone leaf extracts of

trees that have been used traditionally based on information on herbarium sheets

(Arnold et al., 2002). There were no differences in antibacterial activity between trees

used traditionally and randomly selected. There were no differences in the average

for all the pathogens with MICs of 0.94 & 0.95 mg/ml. For the trees not used

medicinally 67 out of 93 extracts (i.e. 9.0%) had average MICs <0.16 mg/ml against

6 bacteria and 2 fungi. For the trees used medicinally the MIC for 30 out of 53 (i.e.

6.3%) extracts had an average MIC of <0.16 mg/ml 30/53 against the same bacteria

and fungi. The distribution in activity against four important nosocomial pathogens

indicated that nearly all extracts had an MIC of 2.5 mg/ml or lower (Fig. 2).

S aureus Ent.faec. E.coli Pseud0

2

4

6

8

10

12

14

16

18

MIC categories trees used medicinally

0.02

0.04

0.08

0.16

0.32

0.64

1.28

2.50

5.00

test organisms

num

ber

9

S aureus Ent.faec. E.coli Pseud0

5

10

15

20

25

30

MIC categories trees not used medicinally

0.02

0.04

0.08

0.16

0.32

0.64

1.28

2.50

5.00

test organisms

num

ber

Figure 2 The number of extracts that had MICs of 0.02 to 5 mg/ml against

Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas

aeruginosa for trees that were used medicinally (top) and trees not used medicinally

(bottom).

It must be stressed that there were no indication for what the trees were used. If we

investigated only trees with an established traditional use of the leaves against

microbial infections, the results may have differed. Svetaz et al. (2010) investigated

the antifungal activity of 327 plant species from 7 Latin American countries against

11 human opportunistic and pathogenic fungi. They found a much higher hit rate

among extracts of plants used to treat fungal infections than of plants not used

traditionally. The number of hits was also higher against dermophytes infections that

could easily be diagnosed by traditional healers than against yeast or Aspergillus

infections where diagnosis would not be as easy.

4.3 Determining antimicrobial activity

Many scientists have used agar diffusion techniques to determine antibacterial

activity. This technique is relatively simple and is useful if the activity of a known

compound such as an antibiotic is to be determined. Briefly, microbes are freshly

10

inoculated into the growth medium in a Petri dish and then either a hole is made into

the agar and filled with the test solution or a disk containing the test solution is

placed on the agar. The active compound(s) diffusing from the area applied, leads

to growth inhibition along a concentration gradient. There is a linear relationship

between the logarithm of the concentration of the antimicrobial compound and the

zone of microbial inhibition (Hewitt and Vincent, 1998). The following factors

influence the inhibition zone size: concentration of antibiotic, volume of test solution,

density of the inoculum, duration and temperature of diffusion phase before

incubation, thickness of the agar medium, composition of the medium and incubation

temperature (Hewitt and Vincent, 1998). Most authors apparently never addressed

these factors.

What is even more important is that if there are compounds with different polarities in

the extract, the non-polar compounds will diffuse very slowly or even precipitate in

the aqueous agar medium. It therefore becomes impossible to compare activities

between different plant extracts accurately. As shown in section 4.2, 89% of papers

identified by Google scholar have the word agar somewhere in the text. When the

same terms (Africa antimicrobial plant) are searched in all databases of Web of

Science only 26% (54/206) listed manuscripts contained the word agar. Manuscripts

containing the word agar do not necessarily mean that agar diffusion techniques

were used, but it probably shows that journals accredited by Thomson Reuters do

not accept papers using agar diffusion techniques in plant antimicrobial studies. Van

Vuuren (2008) evaluated the incidence of using different methods of determining

antimicrobial activity.

Eisa et al (2000) investigated the antibacterial activity of Dichrostachys cineraria of

methanol and water extracts on 8 different bacteria using agar diffusion (results in

mm diameter) and serial dilution (results in minimum inhibitory activity in mg/ml)

assays. We analysed his data and found that there is no predictability between the

two sets of results with a correlation coefficient of 0.0553 (Figure 3). This may

explain why high quality journals do not accept agar diffusion assays for plant

extracts. Agar dilution assays, where different concentrations of the extract, fraction

or compound is incorporated in the agar growth medium overcomes the diffusion

problems. These assays are however much less sensitive than serial dilution assays

(Eloff, 1998b).

11

Figure 3 The correlation between MIC and zone of inhibition of methanol and water

extracts of Dichrostachys cineraria on different bacteria calculated from data of Eisa

et al (2000).

A microplate dilution assay was developed that gave robust and reproducible results

without the need for sophisticated apparatus, required a small quantity of extract,

and have been used worldwide with 672 citation (March 2013) by Google Scholar

(Eloff, 1998b). A slight modification was required to apply this method to fungi

(Masoko et al., 2007)

In essence extracts are made up to a concentration of 10 mg/ml in acetone and two-

fold serial dilutions are made in 100 µl of water. To this 100 µl of an actively growing

bacterial culture is added. Because this is such a high inoculum there is no lag

phase in the growth phase and the MIC results are insensitive to the cell

concentration of the bacteria. There was no difference when a I % inoculum of S.

aureus cultures in Müller-Hinton broth was incubated at 37°C for 1, 3, 6, and 24

hours and a 50% inoculum used in the assay. The microplates are sealed and grown

overnight under 100% humidity. It was not easy to determine the turbidity of the

cells with a microplate reader because cells clump at the bottom of the well with

some test organisms. Precipitation of compounds present in extracts of certain

plants and the green colour of the extracts at high concentrations also made it

difficult to see unambiguously where growth occurred with a microplate reader. To

12

detect growth addition of 40 µl of 0.2 mg/ml tetrazolium violet (INT) led to the best

results because bacterial changed the colourless INT to a red formazan (Eloff,

1998b). By registering the lowest concentration where growth was inhibited i.e.

where there was a decrease in the colour gave the MIC (Fig. 4)

Figure 4 Illustration of using tetrazolium violet to determine MIC of different plant

extracts in a serial dilution assay, highest concentration at the top. Extracts in lanes

3 and 8 were the most active and in lanes 5 and 11 the least active. The extracts

where microbial growth is inhibited led to a reduction or absence in colour.

One should take into account that adding the 100 µl of the bacterial culture to the

100 µl in the series of diluted wells also decreases the concentration the cells were

subjected to. If one started with a 10 mg/ml extract the MIC in lanes 5 and 11 would

be 0.125 mg/ml.

4.4 Determining number of antibacterial compounds

13

In examining plant extracts for the discovery of compounds with interesting activity it

is also very useful to know how many different compounds are present in different

plant extracts or fractions. Bioautography detects biological activity of separated

compounds on a chromatogram. There are three ways in which this can be done.

The first is by the agar overlay technique where an inoculated agar solution is

poured over a chromatogram and then incubated. In theory one should be able to

see where microbial growth is inhibited. In our hands this is a very messy

procedure. Especially the separated polar compounds tended to dissolve in the agar

growth medium and move it from its original position. Another technique is to blot

the chromatogram with sterile filter paper and then place this on an agar culture of

the microorganism. This also did not work well in our experience.

Our approach was based on that of Begue and Klein (1972). We remove the

solvents from chromatograms and then spray the chromatogram with a 10 times

concentrated culture of the organism in growth medium directly on the thin layer

chromatogram. After incubating overnight under 100% relative humidity, the

chromatogram is sprayed with 2 mg/ml INT in methanol. This technique worked well

with many microbes but only if the eluents for the thin layer chromatography were

volatile enough to be removed by a cold stream of air within a short period. We have

developed eluents (Kotze and Eloff, 2002) that worked well. With a slight variation

the method also worked very well for fungal pathogens (Masoko and Eloff, 2005).

An example of a bioautograms is presented in Fig. 6.1

5. Results of random screening of large number of species

5.1 Problems with results in the literature

Many scientists and funding agencies have become disillusioned with screening

plants for antimicrobial activities. If one compares the number of publications on

antibacterial activities of plants with the number of successful products that have

resulted it seems screening for antimicrobial compounds in plants is an exercise in

futility. Much has been promised and little delivered.

Some of the reasons for the current situation are: .

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Many wide screening processes in the past have failed miserably to deliver

worthwhile new pharmaceutical or herbal products especially in the field of

antimicrobial activity. The failure may be due to one or more of the following reasons:

1. The traditional agar diffusion bioassay method to determine antimicrobial activity

does not work well with plant extracts consequently comparable quantitative results

are seldom produced

2. Scientists have been focussing on the wrong plant species by investigating plants

used traditionally to treat infections. Because all aqueous plant extracts that we have

tested to date have very little activity in our assays, we currently believe that these

plants may have been effective through other mechanisms and not by inhibiting

microbial growths directly.

3. Because relatively simple methods were involved many inexperienced scientists

jumped on the band wagon

3. Inefficient extractants may have been used

4. There has been very limited follow up on results of screening publications

because it requires substantial expertise of a different kind

5. Journals have not been critical enough in evaluating manuscripts before

publication even stating that an extract with an MIC of 7 mg/ml was active (Fabry et

al., 1998). There appears to be reasonable consensus that only MICs of 100 µg/ml

or lower should be considered active for extracts (Eloff, 2004) and MICs of 10 µg/ml

should be considered active for pure compounds (Rios and Recio, 2005).

6. Our approach to random screening

Selection of plants to work on

In our experience plants used traditionally to treat infections are not more active than

randomly selected plant species. With the new Benefit Sharing and Access Control

legislation in South Africa to prevent biopiracy there are administrative complications

15

in in investigating plants used traditionally. We therefore selected random screening

of plants as our approach.

It is practically impossible to screen all the c. 24000 southern African angiosperm

species. Even collecting and screening one representative of each of the c. 2300

genera (Arnold and de Wet, 1993) will be a mammoth task, but it may be feasible to

screen good representative members of the 224 families. One spin off of such an

approach would be that it would be possible to determine which plant families or

genera had the highest activity. High activity in a family should then be followed up

by investigating representatives of genera and eventually species.

The most demanding part of this project would be to collect the plant material. If

roots, bark, leaves and flowers are to be sampled, this would vastly complicate the

project. It would quadruple the number of analyses; it would require getting

permission from nature conservation and landowners to collect plant material; it

would require collecting during the flowering season; it would rule out the possibility

of collecting plants in a botanical garden.

To limit this project to feasible dimensions we decided to focus on tree species and

to examine only leaves and twigs, because this is a renewable resource. All of our

plant materials were accessed from Botanical Gardens because it is easy to collect

leaves from the same tree at a later stage to confirm results or expand work. This

may also reduce the onerous task of collecting voucher specimens if the botanical

garden’s herbarium contained voucher specimens.

According to Coats Palgrave (2005) there are 112 families containing 236 genera

and 2839 species of trees in southern Africa. This list includes trees occurring south

of the Zambezi and Cunene rivers as well as alien species that have become

naturalized in southern Africa. We planned to screen at least one species of each

genus, subgenus or section of the trees available in Botanical Gardens and to

analyse all the data.

For practical reasons we decided to use only properly dried material. In some cases

at least, dried material retains its biological activity for many decades (Eloff, 1999).

Furthermore we ensure that material is properly collected to limit the effect that

microbial infection of leaves could have. Leaves are only picked when they are dry,

16

collected in open mesh orange bags hung in the shade to limit photo-oxidative

damage. Leaves are ground to a fine powder and stored in closed glass containers.

5.3 Novelty of our approach

Many scientists have focused on looking for compounds that can be used as single

substance pharmaceuticals. We have found substantial evidence for synergistic

antimicrobial effects in plant extracts. We believe that there is a reasonable chance

to develop anti-infective extracts rather than isolate single compounds that can be

patented. The novelty of our approach is that

1. We have a good basis for collecting plants in association with the South African

National Biodiversity Institute SANBI.

2. We use validated techniques for a variety of bioassays with a wide range of

expertise within our group.

3. We focus on extracts with a higher percentage of success. This does not rule out

the less likely possibility that novel pharmaceuticals may be discovered, but that is

not our main aim.

4. We try to develop low-level technology that can be applied where it is needed

most.

5.4 Methods used

The procedures used are explained in more details elsewhere (Eloff and McGaw,

2006). Leaf powder was extracted with acetone, made up to 10 mg/ml and then the

antibacterial activity is determined (See section 4). The test organisms used were

the four most important nosocomial pathogens Staphylococcus aureus (ATCC

29213), Escherichia coli (ATCC 27853), Enterococcus faecalis (ATCC 29212) and

Pseudomonas aeruginosa (ATCC 25922) as well as methicillin resistant

Staphylococcus aureus, Mycobacterium smegmatis (a non-pathogenic species of the

TB causing genus). Two fungi Candida albicans and Cryptococcus neoformans

were also used.

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5.5 Some preliminary results

Many plant extracts with exciting activities were discovered. Many extracts had

exciting activities against Mycobacterium smegmatis (Table 1) and Cryptococcus

neoformans (Table 2).

Table 1 Minimal inhibitory activities of acetone leaf extracts of 324 tree species

against Mycobacterium smegmatis

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MIC number cumulative % total0.02 15 15 50.03 7 22 7

Table 2 Minimal inhibitory activities of acetone leaf extracts of 707 tree species

against Cryptococcus neoformans

In both cases several extracts had MICs lower than 0.08 mg/ml and no extract had

an activity higher than 2.5 mg/ml.

Before the current serial dilution microplate assays were developed Vlietinck et al

(1995) investigated 100 Rwandese medicinal plants used by traditional healers to

treat infections using agar diffusion and agar dilution assays. They found that 45% of

the extracts were active against Staphylococcus aureus, 2% against Escherichia

coli, 16% against Pseudomonas aeruginosa, 7% against Candida albicans, 80%

against Microsporum canis and 60% against Trichophyton mentagrophytes. It is a

pity that this important work published in an excellent journal cannot really be

compared with MIC data. It does however show that the 80% ethanol in water

extracts had lower activity against Gram-negative organisms.

It is interesting that in the excellent paper of Svetaz et al (2010) investigating the

MICs of 80% ethanol extracts of 327 plant species against 11 fungal pathogens only

10 out of the 1067 MICs reported in tables had MICs lower than 62.5 µg/ml. Only

about 1% of the assays led to MICs lower than 62.5 µg/ml compared to 54% of our

extracts against Cryptococcus neoformans. This may be due to a high sensitivity of

the C. neoformans strain we used, to the effect of different extractants or to other

differences in the methods used.

7. Activity of compounds isolated against Staphylococcus aureus

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24 699 0.04 2025 706 0.04 2126 55 0.04 2227 204 0.06 1

Gibbons (2004) wrote an excellent review on the use of compounds isolated from

plants to combat S. aureus infections. There is no single chemical entity derived

from a plant that is used clinically as an antibacterial. Lewis and Ausubel stated that

many if not most of the 100,000 small molecule compounds in plants have

antimicrobial activity. Many of these compounds have very weak activity, frequently

orders of magnitude lower than antibiotics obtained from fungi or bacteria.

Gibbons’ review of more than 150 papers published between 1995 and 2003 show

that 116 single chemical entities (SCE) have been isolated with MICs lower than 64

µg/ml against different Staphylococci.. He provided the structures of 116 different

compounds that have been isolated during this period and discussed it under

headings of monoterpenes, sesquiterpenes, diterpenes, triterpenes,

phenylpropoanoids and stilbenoids, simple phenols and tropolones, flavonoids,

alkaloids, polyketides and polyynes, sulphur containing products and

acylphloroglucinols. Many of these compounds had activities lower than 1 µg/ml.

Some were more active against methicillin resistant S. aureus than against S.

aureus.

The question arises why have many of these compounds not been developed to

become new antimicrobials especially since the first vancomycin resistant S. aureus

has been discovered in 2002. One reason may be that the scientists working in this

field may not determine the cytotoxicity of the isolated compounds. If a compound is

active against many diverse organisms it may contain a general metabolic toxin that

would be ytoxic to human or animal cells as well. A second stumbling block is the

ADME pharmacokinetic parameters, i.e. absorption, distribution, metabolism and

excretion that affect the clinical usefulness require a totally different expertise. Many

compounds may be too difficult to synthesize de novo, and partial synthesis from a

plant based precursor may be too expensive it it depends on isolation from plant

material that is not widely available. The most important reason is probably that

pharmaceutical companies have developed and invested heavily in fermentation

technology and developing products from microbial sources.

8. Discovering antifungal compounds from natural products.

20

Plants was the most important group investigated for antifungal activity in Journal of

Natural Products during the last decade with 41% of publications, followed by fungi

(34%), marine organisms (5%) and bacteria (5%) (Sortino et al., 2012). There has

been an increase in the number of papers published on antifungal compounds ion

two of the leading journals in this field Journal of Ethnopharmacology and Planta

Medica. Between 1981 and 1990 there were 40 papers followed by 139 papers in

the next decade and 223 papers published between 2001 and 2010 (Sortino et al.,

2012)

Sortino et al., (2012) also analysed the antifungal studies registered in the

NAPRALERT database between 1975 and 2005 to determine the families to which

the c. 2000 species investigated belong. The most studied families were

Asteraceae, Fabaceae, Lamiaceae, Euphorbiaceae, Ranunculaceae and Myrtaceae.

They concluded that the selection of species was correlated with the abundance of

species in the families and the accessability. What was more interesting is that the

families with the largest percentage of active species from species investigated

were: Solanaceae (57%), Poaceae (56%), Rutaceae (38%), Brassicaceae (37.5%)

and Lamiaceae (35%). Sortino et al., (2012) also provided the structures of 89

antifungal compounds isolated.

9. Review papers focussing on antimicrobial activity of plants from Africa

Vlietinck et al.,(1995) investigated the antimicrobial activity of 100 plant species from

Rwanda. The methods used at that stage was still agar diffusion and agar dilution

assays. In 2008 an issue of the Journal of Ethnopharmacology was dedicated to

review papers research done in South Africa. A paper by van Vuuren (2008) listed

the contributions of many authors in this area and also discussed methods and

evidence for investigating synergism. Eloff et al., (2008) provided information on the

compounds isolated from members of the Combretaceae in different countries in

Africa and the biological activities. Eloff and McGaw (2006, 2008) provided some

results obtained in the Phytomedicine Programme and focused on methods uses

including methods to be applied in determining cellular toxicity and in vivo animal

experiments. . In Cameroon an excellent paper evaluated the activity of some natural

products against bacteria expressing a multidrug-resistant phenotype (Kuete et al.,

21

2011). They found that some natural products are substrates of efflux pumps acting

in resistant strains of four bacteria but that terpenoids in general were not substrates.

`

10.Promising new approaches

There has been a strong development of new methods that can aid in the

identification of active compounds. Queiros et al., (2012) provides an excellent

summary of recent developments especially in hyphenated techniques and

metabolomics. In an older paper Lewis and Ausubel (2006) discusses some

interesting possibilities for example using extracts from plants that were stimulated to

synthesize antibacterials by pathogen attack or treatment with immune elicitors such

as salicylic acid. They also discuss the hypothesis that plants use a different

strategy for controlling infections by acting in combination. This echoes our

conclusion after isolating many active compounds from active fractions only to

discover that the activity is much lower than what we expected.

One way in which such a synergistic combination can work is by resistance

modifying agents (RMAs). Examples of these RMAs are methicillin-resistance

reversing agents and modulators of multidrug resistance (MDR) where efflux

transporters are involved. It appears that most MDR inhibitors are large molecules

with a high degree of lipophilicity (Gibbons, 2004). We have been thought that the

efficacy of aqueous extracts in healing patients of traditional healers may be related

to other compounds enhancing the activity of antibacterial compounds with a low

activity or that some compounds may affect the efflux pumps in bacteria. The high

lipophilicity of these compounds makes this idea less attractive.

Another idea that extracts may target bacterial virulence rather than bacterial growth

(Lewis and Ausubel, 2006) could be an attractive explanation for the apparent

efficacy of aqueous extracts. These authors identified three potential aspects to be

considered for future research.

Find a viable new lead

It is important that a viable lead must have specificity to be selectively toxic against

the pathogen. The drug industry has developed excellent new antibiotics against

22

Gram-positive species i.e. Zyvox, Synercid (two protein synthesis inhibitors) and

Cubicin (a membrane acting inhibitor) (Lewis and Ausubel, 2006).

Gram-negative bacteria have developed a sophisticated permeability barrier with a

hydrophilic lipopolysaccharide outer membrane. This limits the entry of most drugs

that are hydrophobic or amphipathic compounds. The drug industry has failed to find

new broad spectrum compounds against Gram-negative species. The

fluoroquinolines the last class of broad spectrum antibiotics were discovered 40

years ago.

It therefore seems a viable option to search for natural products with activity against

Gram-negative bacteria. As stated above there is also an urgent need to find new

antifungal drugs.

In both cases it appears to be important to determine the mechanism of activity. If a

novel mechanism of activity is found, the compound may have much promise using

in combination with existing antibiotics.

The second approach would be to search for MDR inhibitors that could be used to

inhibit the Gram-negative resistance –nodulation-cell division efflux pump. This

excretes amphipathic compounds from Gram-negative bacteria. If such compounds

that are non-toxic are found, it would be a great advantage.

The third possibility is to find compounds that would block pathogen virulence. This

would be a challenging exercise. An interesting model has been developed to infect

the free living nematode Caenorhabditis elegans with bacteria and then see if a

compound or extract can cure the nematode from the bacteria by decreasing the

virulence.

There is another possibility i.e. by investigating the effect of plant extracts on the

adherence of bacteria (Katsikogianni and Missirlis, 2004). If compounds in a plant

extract inhibits the adherence of microorganisms that may explain why aqueous

plants extracts that have hardly any direct antibacterial activity may be effective

when used by traditional healers to treat patients.

23

10 The potential of using African medicinal plants as extracts.

It appears that much of the work done to date has stopped before determining the

safety of extracts or compounds or the efficacy of the product in animal or field

experiments. Furthermore in the human herbal product market it appears that

marketing is much more important than activity of the product. This has led to

frustration where the human herbal medicine market was not interested in

developing products with higher activity.

Consequently we decided to focus on developing products that could be used in the

animal and plant production industry. We have found that there is slower

development of resistance against complex plant extracts than against single

compound antibiotics. Additionally there is a growing market for organically produced

foods. It therefore seems as if there is a good motivation for investigating the use of

plant extracts in animal and plant production. The increase in activity by potentizing

extracts and an application in plant production and another in animal production will

be discussed.

There are challenges in applying plant extracts as therapeutic products. The use of

the plant material should be on a sustainable basis. That is why we focus only on

using tree leaves. There may be variation in activity based on the season, genetic

factors and environmental factors. Quality control could be difficult. The long term

use would probably involve selection of chemotypes with a higher activity and then

production of the trees as a source of the leaves to ensure consistent quality. The

use of leaf extracts may be cheaper than using chemical control and it would

probably be safer to the environment and may deliver “organically grown” products.

10.1 Increasing activity of extracts by low cost potentizing of extracts

By investigating the chromatogram and bioautogram in Figure 1 it is clear that the

hexane and di-isopropylether and water extracts did not extract the active

24

compounds from leaves of Combretum woodii. By extracting leaves with hexane or

water prior to extracting it with acetone, it is possible to remove a number of inactive

compounds leading to a higher activity per mass unit in the extracts. The C. woodii

extract also had excellent antioxidant activity and the activity against coccidiosis

were evaluated in a poultry experiment (Naidoo et al., 2008). Unfortunately the

extract was toxic to the poultry.

By using such an approach to remove inactive components, we have been able to

develop a grape seed extract with nearly double the antioxidant activity of the best

product on the market leading to a South African patent and a commercial product

Biooxidin.

10.2 Example of a plant extract with higher activity than commercial fungicides against plant fungal pathogens

Eloff, Angeh and McGaw (2006) found that an extract of Melianthus comosus Vahl

growing widely in southern Africa had excellent activity against animal fungal

pathogens, but the toxicity to animals would have complicated the development of a

product.

The extracts also had an excellent activity against 10 plant fungal pathogens

investigated (Rhizoctonia solani, Fusarium oxysporum, Penicillium janthinelum,

Penicillium expansum, Colletotrichum glocosponicales, Trichoderma harzianum,

Pythium ultimum, Phytophthora nicotiana, Aspergillus niger, and Aspergillus

parasiticus). The extract contained one major antifungal compound and this

compound was isolated and characterized as 3-hydroxy-12-oleanen-30-oic acid.

By selective extraction and solvent fractionation the activity of the extract could be

increased leading to an extract with an average MIC of 0.066 mg/mL against all ten

fungal pathogens. If the MIC values of 0.16 mg/mL against Penicillium expansum

and Aspergillus niger were ignored, the average MIC for the other fungi was 0.04

mg/ml. The acetone extract did not lose activity after storage at room temperature for

a month. The dried extract was slightly soluble in water and ethanol, reasonably

soluble in ethyl acetate and highly soluble in acetone. The potentised extract had a

25

higher antifungal activity than six commercially used fungicides against some

important plant fungal pathogens. In a limited field trial it gave a much better result

than a commercial fungicide even though it was used at a quarter of the dose of the

commercial fungicide.

This shows that plant extracts have the potential to deliver products that can control

fungal infections.

10.3 Example of a plant extract with similar activity than a commercial fungicide

against the animal fungal pathogen Aspergillus fumigatus.

After determining the MIC of many tree leaf extracts against Cryptococcus

neoformans, we investigated the activity of extracts with good activity against

Aspergillus fumigatus. A. fumigatus is a fungal pathogen causing serious problems

in the poultry industry and also in immune-compromised humans. The acetone

extract of one of the species Loxostylus alata had good in vitro activity against A.

fumigatus (Suleiman et al., 2010). The toxicity of this extracts to poultry was

determined. At a dose of 300 mg/kg there were signs of toxicity but not at

concentrations up to 200 mg/kg. Young birds were infected with A. fumigatus and

treated with 50, 100 and 200 mg/kg as well as with 60 mg/kg ketoconazole the best

therapeutic drug. By measuring the pathology and presence of A. fumigatus a dose

related protection was obtained with the plant extract. The highest concentration

tested gave the same level of protection as the positive control ketoconazole

(Suleiman et al., 2012).

These results indicate that there is a good possibility of developing useful products

based on a plant extract and not a single chemical entity. Registering products may

still lead to complications if the use has environmental toxicity implications.

11 Conclusions

26

There has been a large number of publications investigating the antimicrobial activity

of plant extracts. Up to about one or two decades ago the methods used did not

make it possible to compare the activity of different plants.

11.1 Work on Gram-positive bacteria

Since newer methods have been developed there has been much work done

especially on Gram-positive bacteria and it is surprising that with so many highly

active compounds discovered, so little progress has been made in the development

of these single chemical entities to new drugs. One limitation in existing work on

Gram-positive organisms is that apparently not much work has been done on

determining the safety of these compounds and in doing in vivo efficacy studies. It

may also be very useful to evaluate the mechanism of activity of these promising

compounds. This work would require a multidisciplinary approach

Development of resistance in Gram-positive bacteria is frequently associated with

efflux pumps that remove antibiotics from the microbe cells. There has been

encouraging results in identifying compounds that can inhibit the efflux pump to such

a level that the microbe can be controlled. It is encouraging that there is much

evidence of MDR compounds than can reverse the development of resistance in

Gram-positive bacteria. Combination of older drugs with plant extracts may reverse

the resistance of many Gram-positive bacteria. There have been some new drugs

with very good activity developed recently.

The situation is not that positive with the Mycobacterium species causing

tuberculosis in many organisms. It is not clear if they are closer to the Gram-positive

of Gram-negative organisms. Some classified them acid-fast Gram-positive bacteria

due to their lack of an outer cell membrane while other authors consider them closer

to Gram-negative bacteria.

These organisms have an unusual, waxy coating on its cell surface (primarily

mycolic acid), which makes the cells impervious to Gram staining.

11.2 Gram-negative bacteria

27

It appears that there are many more difficulties in developing new drugs from plants

against Gram-negative bacteria. This has to do with the hydrophilic outer membrane

of Gram negative bacteria. Because most antibacterial compounds are non-polar or

amphipathic transport through the hydrophilic polysaccharide is difficult. A search for

compounds that would enhance the uptake of antibacterial compounds by Gram-

negative bacteria appears to be a useful endeavour.

One place to start would be to investigate plant extracts that have a good activity

against Gram-negative bacteria especially if the isolated active compounds have a

low activity.

Investigating factors that would decrease virulence of these bacteria may be a

difficult, but promising option (Sortino et al., 2012)

11.3 Antifungal compounds

From this review it is clear that there is a great need to develop antifungal

compounds especially with the increase of fungal infections of immunocompromised

patients.

The application of plant antifungal extracts to control fungal infections affecting plant

or animal productivity appears to be a promising area to investigate. Here evaluating

the safety of products and the mechanism of activity should be priority areas

requiring a multidisciplinary and collaborative approach.

Acknowledgements

This research was funded by the National Research Foundation and the Medical

Research Council of South Africa. The curators of several National Botanical

Gardens of the South African National Biodiversity Institute allowed us to collect

plant material in the botanical gardens.

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