The use of plants against microbial infections

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CHAPTER 3 Preliminary screening for antifungal activity of six selected plant species

 Introduction

Antimicrobials are compounds that at low concentrations exert an action against microorganisms and exhibit therapeutic toxicity towards them (Goodyear and Threlfall 2004). These can be any substances of natural, synthetic or semi-synthetic origin that may be used to kill microorganisms including bacteria, fungi, protozoa and viruses (Yazaki 2004). The antimicrobial activity of different plant extracts can be detected by observing the growth of various microorganisms that have been placed in contact with extracts of the plants. If the plant extracts inhibit the growth of the test organism, and general toxic effects are not present, then the plant can potentially be used to combat diseases caused by the pathogens. The antimicrobial activities of plant extracts have formed the basis of many applications, including raw and processed food preservation, pharmaceuticals, alternative medicine and natural therapies (Lis-Balchin and Deans 1997, Reynolds 1996).
There are several assays that can be used to determine antimicrobial activity in plant extracts, including agar diffusion, bioautography (direct, contact and overlay) and microplate assays (serial dilution assay). The agar diffusion assay is, in general, only suitable for aqueous extracts and can also be used to test up to six extracts per Petri dish against a single microorganism. However, the diffusion method is not suitable for testing non-polar samples or samples that do not easily diffuse into the agar (Cos et al. 2006).
The bioautography assay is used to detect active compounds in a crude plant extract (Cos et al. 2006). An inoculated layer of agar is poured over a developed thin layer chromatography (TLC) plate, and lack of bacterial or fungal growth in certain areas identifies the presence and location of antibacterial compounds on the TLC plate. On the other hand, TLC plates can also be sprayed with a fine suspension of bacteria or fungi and then sprayed with an indicator tetrazolium salt. The inhibition of fungal growth by compounds separated on the TLC plate is visible as white spots against a deep red background (Begue and Kline 1972). In the direct bioautography technique, the microorganism sprayed on the TLC plates will grow directly on the chromatograms, while in contact bioautography, the antimicrobial compounds are transferred from the TLC plate to an inoculated agar plate through direct contact. In overlay bioautography the agar is applied directly on the TLC plates and this can also be used with microorganisms that grow slowly (Hamburger and Cordell 1987, Rahalison et al. 1991). The advantage of using bioautography is that it can locate separated active compounds easily and also supports a quick search for antimicrobial agents through bioassay-guided isolation.
However, there are problems associated with the assay, for example TLC eluent solvents with low volatility such as n-butanol (BUOH) and ammonia need to be allowed to evaporate completely so that they cannot inhibit the growth of the microorganism (Cos et al. 2006). The time taken for this may increase the risk of decomposition of active compounds.
In serial dilution assays, plant extract is mixed with water or broth in 96-well microplates and then fungal or bacterial cultures are added to the wells. The minimum inhibitory concentration (MIC) is recorded as the lowest concentration of plant extract resulting in inhibition of fungal growth, shown by a reduction in the red colour of the tetrazolium salt added as an indicator.
Dilution techniques require a homogenous dispersion of the sample in water. They are used to determine, principally, the MIC values of an extract or pure compound. In the liquid dilution method, turbidity is often taken as an indication of growth, so where the sample is inactive against the microorganism tested, the liquid will appear turbid (Rios et al. 1998). The assay is quick, and works well with different microorganisms and non-aqueous extracts from different plant species. Moreover, it gives precise, reproducible results and requires just a small volume of extract to determine the minimal inhibitory concentration (MIC) for each bacterial test species against each plant extract or isolated compound. It suffers from one major drawback in that some compounds present in plant extracts may precipitate in the presence of the bacterial growth medium, making it difficult or impossible to use turbidity as a measure of microbial growth. This problem was resolved by adding p-iodonitrotetrazolium to the extract and microbial suspension. In the presence of microbial growth this compound is changed to a violet-coloured formazan (Eloff 1998b).
The above-mentioned assays differ in principle, and antimicrobial assay results are in general influenced by the type of assays used (Cos et al. 2006). It is necessary for bioassays to be as simple as possible; in this way sufficiently large numbers of different tests may be performed so that many biological properties can be screened (Hostettman 1999). In this chapter, serial dilution and bioautography assays will be used to determine antimicrobial activity of the six plant species under investigation.

Materials and methods

Fungal strains

The seven test fungal species, Aspergillus niger, A. parasiticus, Colletotrichum gloeosporioides, Trichoderma harzianum, Penicillium expansum, P. janthinellum and Fusarium oxysporum, were obtained from the Department of Microbiology and Plant Pathology at the University of Pretoria. These fungi are among the most important pathogenic fungi of economic significance to plants. Fungal strains were maintained on Potato Dextrose (PD) agar. Fungal cultures were subcultured (1% inoculum) in PD broth at 35°C for at least two to four days before being used in the screening assays.

Quantification of fungal inoculum

For quantification of fungi, the haemocytometer cell-counting method described by Aberkane et al. (2002) with some modifications was used for counting the number of cells for each fungal culture. The inoculum of each isolate was prepared by first growing the fungus on PD agar slants for 7 days at 35ºC. The slant was rubbed carefully with a sterile cotton swab and transferred to a sterile tube with fresh PD broth (50 ml). The sterile tubes were then shaken for five minutes and appropriate dilutions were made in order to determine the number of cells by microscopic enumeration using a haemocytometer (Neubauer chamber; Merck S.A.). The final inoculum size was adjusted to approximately 1.0×10⁶ cells/ml. To confirm the inoculum adjustment, 100 ml of serial dilutions of the conidial suspensions was spread onto PD agar plates. The plates were incubated at 35ºC and observation of the presence of fungal growth was done daily. The colonies were counted after the observation of visible growth and used to calculate the corresponding cells/ml.

Bioassays for antifungal activity

Dilution method

The serial microplate dilution method of Eloff (1998b), modified for antifungal activity testing by Masoko et al. (2005), was used to determine the MIC values for plant extracts of B. buceras, B. salicina, H. caffrum, O. ventosa and V. infausta. The plant extracts were tested in triplicate in each assay, and the assays were repeated once in their entirety to confirm results. Residues of different extracts were dissolved in acetone to a concentration of 10 mg/ml. The plant extracts (100 ml) were serially diluted 50% with water in 96 well microtitre plates (Eloff 1998c), and 100 ml of fungal culture was added to each well. Amphotericin B was used as the reference antibiotic and 100% acetone as the negative control. As an indicator of growth, 40 ml of 0.2 mg/ml p-iodonitrotetrazolium violet (INT) dissolved in water was added to the microplate wells. The covered microplates were incubated for three to five days at 35ºC at 100% relative humidity after sealing in a plastic bag to minimize fungal contamination in the laboratory. The MIC was recorded as the lowest concentration of the extract that inhibited antifungal growth. The colourless tetrazolium salt acts as an electron acceptor and was reduced to a red-coloured formazan product by biologically active organisms (Eloff 1998b). Where fungal growth is inhibited, the solution in the well remains clear or shows a marked reduction in intensity of colour after incubation with INT.
In order to determine which plants can be used for further testing, not only the MIC value is important, but also the total activity. Since the MIC value is inversely related to the quantity of antifungal compounds present, the quantity of antifungal compounds present was calculated by dividing the quantity extracted in milligrams from 1g leaves by the MIC value in mg/ml. The total activity is used to determine to what volume an extract from 1 g of plant material can be diluted and still inhibit the growth of the test organism (Eloff 1999). It can also be used to evaluate losses during isolation of active compounds and the presence of synergism (Eloff 2004).
The total activity can be calculated as:
Total activity = Quantity of material in mg extracted from 1 g of plant material /Minimum inhibitory concentration (mg/ml)
In the case of bioassay guided fractionation, the total activity in the crude extract and fractions can be calculated by dividing the mass in mg in the fraction with the MIC in mg/ml. Total activity in this case [x ml/fraction] provides an indication of the volume to which the crude extract or fraction can be diluted and still kill the microorganism.

Bioautography

TLC plates (10 × 10 cm) were loaded with 100 mg of each of the extracts with a micropipette. The prepared plates were each run using different mobile systems: CEF, BEA and EMW. The chromatograms were dried at room temperature under a stream of air overnight or up to five days until the remaining solvent were removed. Fungal cultures were grown on Potato Dextrose agar for 3 to 5 days. Cultures were transferred into PD broth from agar with sterile swabs. The developed TLC plates were sprayed with concentrated suspension containing c.1.0 × 10⁶ cells/ml of actively growing fungi. The plates were sprayed until they were wet, incubated overnight and then sprayed with a 2 mg/ml solution of p-iodonitrotetrazolium violet and further incubated overnight or longer at 35ºC in a clean chamber at 100% relative humidity in the dark. White areas indicated where reduction of INT to the coloured formazan did not take place due to the presence of compounds that inhibited the growth of the tested fungi. The plates were sealed in plastic to prevent the spreading of the fungi in the laboratory and to retain the humidity and then scanned to produce a record of the results.

Results and discussion

Quantification of fungal inoculum

The number of fungal cells in the two diagonally opposite corner grids of the haemocytometer were counted and averaged. If the cell number was more than 100, a calculated volume of fresh broth was added to obtain an approximate average of 100 cells. Hence, the cell concentration for use in the bioassay was maintained at 100 × 10⁴ cells/ml = 1.0 × 10⁶ cells/ml. The same procedure was used for all other tested fungal species under study.

Microplate dilution assay

Plant pathogenic fungi were used as test organisms for testing antifungal activity of extracts of the six selected plant species (B. buceras, B. salicina, H. caffrum, O. ventosa, V. infausta and X. kraussiana). Extracts using solvents of different polarities (acetone, hexane, dichloromethane and methanol) were prepared from the six selected plants. Hexane, DCM and methanol extracts were re-dissolved in acetone since acetone was reported not to be toxic to microorganisms at the concentrations used in the assay (Masoko et al. 2007). The extracts were tested for antifungal activity against seven fungal species: Aspergillus niger, Aspergillus parasiticus, Colletotrichum gloeosporioides, Penicillium janthinellum, Penicillium expansum, Trichoderma harzianum and Fusarium oxysporum. The minimum inhibitory concentration (MIC) results presented in Table 3-1 indicate that plant pathogens are more susceptible than animal pathogens in this case. The plant extracts were tested in a preliminary screening test against two animal pathogens, Candida albicans and Cryptococcus neoformans, and the lowest MIC values obtained were 0.03 mg/ml.

CHAPTER 1  Medicinal Plants 
1.1 Introduction
1.2 Literature review
1. 2.1 Importance of medicinal plants
1.2.2 The use of plants against microbial infections
1.2.3 Fungi as pathogens
1.2.3.1 Antifungal drugs
1.2.4 Resistance of fungi
1.2.5 Food production and effects of fungal pathogens
1.2.6 Plants as antifungals
1.2.6.1 Previous related antimicrobial work in the Phytomedicine laboratory
1.2.7 Selection of plants for study
1.2.7.1 Ethnobotanical information on six selected species
1.2.7.2 Phytochemical data available on selected species
1.2.8 Motivation
1.2.9 Aim
1.2.10 Objectives
CHAPTER 2  Extraction and phytochemical investigation of selected plant species 
2.1 Introduction
2.2 Materials and methods .
2.2.1 Plant selection
2.2.2 Plant collection
2.2.3 Plant storage
2.2.4 Extraction Procedure
2.2.4.1 Laboratory extraction method
2.2.5 Phytochemical analysis
2.2.6 Retention factor (Rf) values of compounds
2.3 Results and discussion
2.3.1 Extraction using different solvents
2.3.2 Phytochemical analysis of extracts
2.3.2.1 TLC analysis of plant extracts for preliminary screening .
2.4 Conclusion
CHAPTER 3nPreliminary screening for antifungal activity of six selected plant species 
3.1 Introduction
3.2 Materials and methods
3.2.1 Fungal strains
3.2.1.1 Quantification of fungal inoculum
3.2.2 Bioassays for antifungal activity
3.2.2.1 Dilution method
3.2.2.2 Bioautography
3.3 Results and discussion
3.3.1 Quantification of fungal inoculum
3.3.2 Microplate dilution assay
3.3.3 Bioautography assay
3.4 Conclusion
CHAPTER 4 Antioxidant activity
4.1 Introduction
4.2 Materials and methods
4.3 Results and discussion
4.4 Conclusion
CHAPTER 5  Activity of crude leaf extracts of plant species against Aspergillus fumigatus 
5.1 Introduction
5.2 Materials and methods
5.3 Results and discussion
5.4 Conclusion
CHAPTER 6  Antifungal activity of Breonadia salicina leaf extracts 
6.1 Introduction
6.2 Materials and methods
6.3 Results and discussion
6.4 Conclusion
CHAPTER 7  Isolation of antifungal compounds from leaves of Breonadia salicina 
7.1. Introduction
7.3 Microplate dilution assay .
7.4 Bioautography assay
7.5 Results and discussion
7.5.1 TLC analysis
7.6 Conclusion
CHAPTER 8 Structure elucidation of four isolated compounds 
8.1 Introduction
8.2 Materials and methods
8.3 Results and discussion
8.4 Conclusion .
CHAPTER 9  Antifungal and antibacterial activity and cytotoxicity of isolated compounds
9.1 Introduction
9.2 Materials and methods
9.3 Results and discussion
9.4 Conclusion
CHAPTER 10 In vivo experiment: Plant extracts active against Penicillium species 
10.1 Introduction
10.2 Materials and methods
10.3. In vivo experiment
10.4 Results and discussion
10.5 Therapeutic Index
10.6 Conclusion
CHAPTER 11 Summary and conclusion 
CHAPTER 12 References 
Appendix
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