Which extractant should be used for the screening and isolation of antimicrobial components from plants?
Introduction
The World Health Organisation estimates that 80% of the people living in developing countries almost exclusively use traditional medicine. Medicinal plants form the principle component of traditional medicine. This means that in the order of 3300 million people use medicinal plants on a regular basis. Medicinal plants used in traditional medicine should therefore be studied for safety and efficacy (Farnsworth, 1994).
Medicinal components from plants also play an important role in conventional western medicine. In 1984, at least 25% of the prescription drugs issued in the USA and Canada were derived from or modelled after plant natural products (Farnsworth, 1984). In 1985, Farnsworth et al. identified 119 secondary plant metabolites that are used globally as drugs. It has been estimated that 14–28% of higher plant species are used medicinally, that only 15% of all angiosperms have been investigated chemically and that 74% of pharmacologically-active plant derived components were discovered after following up on ethnomedical use of the plant (Farnsworth and Soejarto, 1991). Southern Africa contains ∼10% of the worlds plant diversity, but relatively little chemical work has been done on medicinal plants from this region. Of the 300 plants investigated by Noristan, 31% showed marked activity, 48% were moderately active and 21% were inactive (Fourie et al., 1992).
The numbers of resistant strains of microbial pathogens are growing since penicillin resistant and multiresistant pneumococci caused a major problem in South African hospitals in 1977. Berkowitz (1995)calls the emerging of drug resistant bacteria a medical catastrophe. Leggiadro (1995)stated that effective regimens may not be available to treat some enterococcal isolates and that it is critically important to develop new antimicrobial compounds for these and other organisms before we enter the post-antibiotic era. New compounds inhibiting micro-organisms, such as benzoin and emetine have been isolated from plants (Cox, 1994). The antimicrobial compounds from plants may inhibit bacteria by a different mechanism than the presently used antibiotics and may have clinical value in treatment of resistant microbial strains.
For these reasons, there has been a substantial increase in the number of papers where authors screened plants for antimicrobial properties. From these papers it is clear that authors use different extractants varying from 80% ethanol (Vlietinck et al., 1995), methanol (Taylor et al., 1995), petroleum ether, chloroform, ethanol, methanol and water (Salie et al., 1996). After reviewing the re-awakening of pharmacognosy Cordell (1993)concluded that `There is clearly substantial room for improvement in the extraction methodologies, given that there are a variety of techniques that could be used to prepare extracts. Farnsworth in Balick (1994), states that the biggest problem in drug development with plants is answering a very simple question: what kind of extract should we test?
Various solvents have been used to extract plant metabolites. Many scientists employ soxhlet extraction of dried plant material using solvents with increasing polarity, e.g. ether, petroleum ether, chloroform, ethyl acetate and ethanol. This works well for compounds that can withstand the temperature of the boiling solvent, but can not be used for thermolabile compounds. The problem can be overcome by extracting under reduced pressure, but it is difficult.
The choice of solvent depends also on what is intended with the extract. If extraction is to screen plants for antimicrobial components, the effect of the extractant on subsequent separation procedures is not important, but the extractant should not inhibit the bioassay procedure. If the plant material is extracted to isolate chemical components without using bioassay, toxicity of the solvent is not important because the solvent can be removed before subsequent isolation procedures.
In order to find an extractant that would be optimally useful both in the screening and isolation of antimicrobial components from plants, I decided to compare a number of extractants. After a scrutiny of the literature to determine which extractants were used, I initially compared ethanol used by the National Cancer Institute in the USA (Suffness and Douros, 1979), a 1:1 mixture of methanol and methylene dichloride (Balick, 1991) and a one-phase mixture of methanol, chloroform and water (12:5:3) (Bieleski and Turner, 1966).
Due to problems encountered with the subsequent treatment of extracts (Section 4), the following extractants were compared: acetone, ethanol (EtOH), methanol (MeOH), methylene dichloride (MDC), a mixture of chloroform, methanol, water (12:5:3) (MCW) and water. Preliminary work with dimethylsulfoxide, frequently used in microbial studies, was abandoned due to the high boiling point 189°C and because it was also more toxic to Staphylococcus aureus than acetone in the bioassay used (results not shown).
Fresh or dried plant material can be used as a source for secondary plant components. Most scientists working on the chemistry of secondary plant components have tended to use dried material for the following reasons: (i) There are fewer problems associated with the large scale extraction of dried plant material than with fresh material; (ii) the time delay between collecting plant material and processing it makes it difficult to work with fresh material because differences in water content may affect solubility or subsequent separation by liquid–liquid extraction; (iii) the secondary metabolic plant components should be relatively stable especially if it is to be used as an antimicrobial agent; (iv) many, if not most plants are used in the dried form [or as aqueous extract] by traditional healers.
The two plant species used in this study Anthocleista grandiflora (Burch.) Sond. (Loganiaceae) and Combretum erythrophyllum (Afzel. ex R. Br.) Gilg. (Combretaceae) were identified as plants containing antimicrobial activity (Dr T.G. Fourie, personal communication) after an extensive screening of South African plants by Noristan (Fourie et al., 1992).
The following parameters were investigated with the different extractants: the quantity extracted, the rate of extraction, the diversity of different compounds extracted, the diversity of inhibitory compounds extracted, the ease of subsequent handling of the extracts, the toxicity of the solvent in the bioassay process, the potential health hazard of the extractants. The different solvents were compared by grading on a five point weighted scale.
Section snippets
Plant material
The Noristan scientists discovered that Combretum erythrophyllum (Burch.) Sond. and Anthocleista grandiflora (Afzel. ex R. Br.) Gilg. had some antibiotic activity, but did not follow up the preliminary results. Leaf material was collected from a tree in the Pretoria National Botanic Gardens (C. erythrophyllum) and a tree on the campus of the University of Pretoria (A. grandiflora). Plants were identified by the plant labels on the trees and confirmed by Professor A.E. van Wyk, Pretoria
Drying of plant material
There was no difference in the toxicity level to the test organisms or the number of compounds extracted or Rf values for both plants when comparing freeze dried and leaves dried at room temperature. The toxicity decreased and the number of compounds separated by TLC changed when the leaves were dried overnight at 105°.
Quantity extracted with initial extractants
In initial experiments with the two plants, the plant material was not ground finely and extraction took place over 4, 6 and 24 h on a rotating shaker. MCW extracted the most
Conclusion
Because acetone dissolves many hydrophylic and lipophylic components from the two plants used, is miscible with water, is volatile and has a low toxicity to the bioassay used, it is a very useful extractant. Fractions collected from a chromatography column dry off within a relatively short period in a cold room.
In this study, the use of the solvent for screening and for the isolation of active components was examined. If the aim of the research is to extract components for preparative work only
Acknowledgements
This research was supported by the Faculty of Medicine Research Committee, University of Pretoria. Maryna Steinmann and Nataly Martini provided valuable technical assistance.
References (18)
- et al.
Separation and estimation of amino acids in crude plant extracts by thin-layer electrophoresis and chromatography
Analytical Biochemistry
(1966) The pharmacological evaluation of natural products-general and specific approaches to screening ethnopharmaceuticals
Journal of Ethnopharmacology
(1983)- et al.
Preliminary antimicrobial screening of four South African Asteraceae species
Journal of Ethnopharmacology
(1996) - et al.
Screening of selected medicinal plants of Nepal for antimicrobial activities
Journal of Ethnopharmacology
(1995) - et al.
Screening of hundred Rwandese medicinal plants for antimicrobial and antiviral properties
Journal of Ethnopharmacology
(1995) - Balick, M.J., 1994. Ethnobotany, drug development and biodiversity conservation-exploring the linkages. In: Prance,...
- Balick, M.J., 1991. Ethnobotany and the identification of therapeutic agents from the rainforest. In: Chadwick, D.J.,...
- et al.
The use of tetrazolium salts in bioautographic procedures
Journal of Chromatography
(1972) Antibiotic resistance in bacteria
Southern Medical Journal
(1995)