An overview of antifungal activities of natural products from fungi and plants
In early fifties of the twentieth century the agrochemical industry provided agriculture with a vast array of chemicals for crop protection, including fungicides. Random synthesis, biological screening and empirical optimization yielded many effective compounds (Cremlyn, 1991). Whereas it is generally acknowledged that the use of pesticides has large benefits to farmers, the present use of pesticides in agriculture also causes negative environmental (and health-related) effects to society. For example, during and after application of pesticides a substantial amount of it could end up in soil, ground- and surface water or air. These negative effects demands for an effective policy. Such policies have been initiated, both at the level of the individual Member States of the European Union and at the level of the European Union itself (Oppenheimer & Donnelly, 1997). Their selectivity between target organisms and plants is mainly based on differences in uptake. The more recently developed protective chemicals are more potent in terms of dose required to control the pest or disease, and in distinguishing between target and non-target organisms. They usually have a specific mode of action. Since selective compounds are specific site inhibitors in the metabolism of target organisms, the risk of developing resistance is high. This has occurred for a number of fungal plant pathogens (Delp, 1988). Although pathogenic microorganisms are mainly controlled chemically, the use of synthetic compounds is limited due to several undesirable aspects, which include carcinogenicity, teratogenicity, acute toxicity and the requirement of an extended degradation period with consequent development of environmental pollution problems. The new awareness of modern consumers about these problems has created a “green” consumer profile that demands the absence of synthetic chemicals in food production and preservation together with extended shelf life of the majority of food products. Fungal infections remain a therapeutic problem in many fields despite the availability of a number of treatments. Such diseases in humans have markedly increased during the past ten years, especially in immunocompromised patients. Consequently, up to 10% of hospital acquired systemic infections are caused by fungi. Altogether this forces the scientific community, agro–industry and pharmaceutical companies to search for natural compounds that will satisfy consumer requirements (Harvey, 2008). Furthermore, there is growing concern about chemicals for protection because of their undesirable side effects in humans, other target organisms and their behavior and fate in the environment (Jespers, 1994).
The total number of all known natural products is around one million, including both bioactive and inactive compounds, plants metabolites 600000, fungal metabolites 8600, microbial metabolites recognized until now is around 50000. It is an obvious question, where is the border in the diversity of natural products? The general needs of the human society are continuously increasing. We need every new compounds which may be useful for the human society. More food, new drugs, and other goods are highly necessary for the benefit of humankind. The only question is the existence of sufficient natural and technical resources to fulfill these demands. Fortunately, in the area of the research of bioactive microbial products it seems that the ever expanding scientific and technical possibilities are increasing together with the continuously widening needs of the human therapy, veterinary and agriculture. The problem really is not whether we would be able to discover further new useful microbial compounds, but rather how can we optimize and quickly and effectively apply the chances derived from the new discoveries. How can we pick up and use effectively the proverbial needle found in the haystack (Berdy, 2005). However, screening of more than a million substances in the last decade has resulted in the introduction of only a very limited number of compounds with novel modes of action and resistance. This explains the renewed interest of the chemical industry in natural compounds with a variety of unique characteristics, waiting to be exploited. Natural products derived from plants and fungi have traditionally been used in ethnomedicine. Throughout the development of both Western and Eastern civilizations, whole plants, fungi, their parts, derived compounds and extracts have functioned as sources of food and medicine, symbolic articles in religious and social ceremonies, and remedies to modify behavior. Plant and fungal extracts and compounds containing physiologically active biochemicals have immense potential for producing new agents of great benefit to mankind. In this context, systematic screening of secondary metabolites of folk herbs and fungi may result in the discovery of novel and effective antimicrobial compounds (Hussain et al., 2011). Recently, interest has been growing in natural products due to their availability, fewer side effects and less toxicity as well as better biodegradability when compared to other available antimicrobial agents and preservatives. Thus, plants and mushroom may offer great potential and hope. Consequently, natural products are attracting the attention of scientists because they are cheaper, safer, eco-friendly and within the reach of the current medical community. This paper gives an overview on the activity of plant and fungi derived extracts as well as their constituents against a wide variety of microfungi, methodology and potential uses (Figure 1.).
In order to test antifungal activities of natural products derived from plants and fungi few conventional and non-conventional methods were applied. Method is selected depending on the characteristics of extracts and compounds tested. A various number of plant, animal and human fungal pathogens were used. Growth cultures are conducted under optimal physical conditions for individual species. The growth of fungi was assessed visually or instrumentally. Two replicates were done for each compounds and the experiment was repeated two times.
Agar diffusion method is suitable for testing of antifungal activity of hydrophilic compounds which easily could be dispread trough to the agar medium. The compounds investigated were mixed with 0.01% Tween 20 surfactant and dissolved in molten MA medium. The fungal species were cultured for 7 days on Malt agar medium. Micromycetes were inoculated in the centre of Petri dishes and incubated for 21 days at 25° C. Mycelial growth was observed every 7 days and compared with the control. The commercial fungicide was used as a positive control (Ishii, 1995). The minimum inhibitory concentration (MIC) of compounds was determined when it achieved a complete stop in the growth of mycelium.
Microdilution method is suitable for testing small quantities of extracts, fractions or components, simultaneously in many different concentrations. In order to investigate the antifungal activity of the compounds, a modified microdilution technique was used (Hanel & Raether, 1988; Daouk et al., 1995). The fungal spores were washed from the surface of agar plates and adjusted with sterile saline to a concentration of approximately 1.0 x 105 in a final volume of 100 μl per well. Minimum inhibitory concentration (MIC) determinations were performed by a serial dilution technique using 96-well microtiter plates. The compounds investigated were dissolved in 5% DMSO solution containing 0.1% Tween 80 (v/v) (1 mg/ml) and added in broth Malt medium with inoculum. The microplates were incubated at Rotary shaker (160 rpm) for 72 h at 28 °C. The lowest concentrations without visible growth (at the binocular microscope) were defined as MICs. The fungicidal concentrations (MFCs) were determined by serial subcultivation of a 2 μl of tested compounds dissolved in medium and inoculated for 72 h, into microtiter plates containing 100 μl of broth per well and further incubation 72 h at 28 °C. The lowest concentration with no visible growth was defined as MFC indicating 99.5% killing of the original inoculum. DMSO was used as a negative control, commercial fungicides were used as positive controls.
Microatmosphere test, a slightly modified agar disc diffusion method, is perfectly suitable for the estimation of essential oil activity in vapor phase (Zollo et al., 1998). The assay was performed using mushroom pathogenic fungi. Petri dishes were filled with malt agar (MA), and then seeded with a 7 day-old mycelial culture of the tested fungi. The Petri dishes were then inverted and the determined amount of essential oils impregnated on sterile filter paper discs (4 mm) attached to the inverted lid (1 disc per lid). The Petri dishes were wrapped with parafilm along the rim, inverted and incubated for 21 days at 25 °C in an incubator. The results are presented as the diameter of the microorganism growth inhibition zone, or as the essential oil minimal inhibitory quantity (MIQ), which inhibits the total growth of microorganism. Commercial fungicides were tested as a control.
Bioautography is widely used for the examination of extracts. When the solvent evaporates, the broth and microorganisms are applied on the chromatographer paper or plates, and after the incubation time, growth is scrutinized. No growth is observed on the active spot components. Simultaneously, the components of the extract are eluted and identified. Different volumes of the mycelium extracts and pure compounds were dissolved in appropriate solutions. Ten micro liters of each sample were applied on TLC plates and sprayed either with freshly prepared fungal suspensions in nutrient broth (TSB). The plates were incubated for 18 h at 37°C and then sprayed with aqueous sol. 3% of p-iodonitrotetrazolium violet and stored for another 3 h. After this period plates were sprayed with 70% EtOH to stop fungal growth and were incubated for 36 h at 27 °C. White inhibition zones on a pinkish background were indicative of antimicrobial activity of tested extracts or compounds. The widths of these zones (mm) are the measure of efficiency and presented as minimal inhibitory concentration (MIC) (Pacher et al., 2001). As positive controls commercial fungicides have been used.
3. Antifungal activity against plant pathogens
3.1. Fungal extracts and metabolites
The fungi constitute a very large group of organisms that are found everywhere and are of great importance to life on earth and to human society. This is mainly due to the many interactions among fungi and other organisms. Most fungi produce a wide variety of secondary metabolites with biological activity. A wealth of literature exist on the discovery and potential use of mycotics in agriculture (Berdy, 1980; Guterson, 1990). However, their exploitation in agriculture remained limited, because widespread application in crops might simultaneously select for resistance to these mycotics in human pathogens (Isono, 1990). The concept that substances derived from one living organism may aﬀect another organsm is old. Some of the secondary metabolites that occur in fungi are fairly widespread, but many are conﬁned to a few species. Hence, screening of further fungi species usually leads to the discovery of new bioactive secondary metabolites. The broad diversity of the fungi, as well as their easy acquisition makes them especially interesting for natural products screening program. Among fungal species, the various microscopic (filamentous) fungi (ascomycetes, fungi imperfecti,
While herbs are rather commonly used in the Western hemisphere, medicinal use of mushrooms, which has a long tradition in Asian countries, has also slightly increased in Europe during the last few decades. Although there has been extensive research on properties of medicinal mushrooms, their true potential is yet to be revealed. A number of compounds possessing significant antimicrobial activity have been isolated from polypore fungi. They provide a rich variety of active secondary metabolites and polysaccharides. Medicinal mushrooms such as
3.2. Plant extracts and metabolites
With increasing acceptance of traditional plants as an alternative form of health care the search for active compounds in plants becomes very important. Medicinal and aromatic plants have been employed for many centuries and they are mentioned in folklore from ancient times. After the advent of antibiotics in the 1950s, the use of plant derivatives as antimicrobials become virtually nonexistent to be rediscovered, as well as other alternative forms of medical treatments in the late 1990s (Cowan, 1999). There are several approaches to choosing sources of natural products for the discovery of potential antifungal compounds. One of approach is to investigate whole extracts of potential antifungal plants. Other approaches are to obtain biological material, which has not previously been studied for fractionation and testing, or some other sources. One strategy is to use ethnobotanical and/or chemical ecology clues to select which plants to sample (Duke et al., 2000). Here at first we will discuss the antifungal activities of plants extracts and after that some secondary metabolites derived from plants.
3.2.1. Plant extracts
There are many reports concerning the antifungal activity of plant extracts, but we will mention only a few. Ushiki et al., (1996) found that root extracts from twelve medicinal plants displayed antimicrobial activity against certain pathogens of soil-borne plant diseases. Among these plants,
An ethanol extract of
Genuine mosses constitute a large group of nonvascular higher plants, consisting of about 14 000 species. Generally, bryophytes are not damaged by microorganisms, insects, snails, slugs, and other small mammals. Up to date, over several hundred new compounds have been isolated from bryophytes and their structures elucidated (Veljić et al., 2009). In spite of a number of secondary metabolites identified from various mosses, the chemical profiles of most species are insufficiently known or even unknown. Secondary metabolites from mosses, identified so far, include terpenoids, flavonoids and bibenzyls, and also derivatives of fatty acids. Mosses rich in flavonoids has been found to possess strong antimicrobial activity (Veljić et al., 2009). An ethanol extract of bryophyte,
3.2.2. Plant secondary metabolites
Plants produce a diverse array of secondary metabolites, many of which have antifungal activity. Some of these compounds exist in healthy plants in biologically active forms. Others, such as cyanogenic glycosides and glucosinolates, occur as inactive precursors and are activated in response to tissue damage or pathogen attack. This activation often involves plant enzymes, which are released as a result of breakdown in cell integrity. Compounds belonging to the latter category are still regarded as constitutive because they are immediately derived from preexisting constituents (Mansfield, 1983). A large number of plant compounds have been reported to have antifungal activity. Well known examples include; flavonoids (Ćirić et al., 2011; Karioti et al., 2011; Weidenbörner & Jha 1997), lactones (Djeddi et al., 2007; Janaćković et al., 2002; Skaltsa et al., 2000a, 2000b; Vajs et al., 1999, 2004), proteins (Giudici et al., 2000), sulfur compounds (Ilić et al., 2012), cyanogenic glycosides and glucosinolates (Osbourn, 1996) and essential oils (Daouk et al., 1995; Džamić et al., 2010; Garg & Siddiqui, 1992; Glamočlija et al., 2006a, 2006b, 2009; Marinković et al., 2002; Mishra & Dubey, 1994; Müller-Riedau et al., 1995; Rančić et al., 2005; Shimoni et al, 1993; Soković, 2001, 2002, 2008a, 2008b, 2009a, 2009b, 2009c; Stojković et al., 2011b, 2011b; Thompson, 1989).
22.214.171.124. Essential oils
Essential oils from aromatic and medicinal plants have been known since antiquity to possess biological activity and constitute one of the most investigated groups of secondary metabolites. With growing interest in their use in the pharmaceutical and agrochemical industries, systematic examination of oils for these properties has become increasingly important. Over the last hundred years antimicrobial properties of common spice oils have been demonstrated (Bullerman et al., 1977) and many studies have been made on antifungal activities of essential oils (Daouk et al., 1995; Garg & Siddiqui, 1992; Glamočlija, 2006b, 2009; Kalemba & Kunicka, 2003; Mishra & Dubey, 1994; Müller-Riedau et al., 1995; Shimoni et al, 1993; Soković, 2001; Thompson, 1989). Thus, Maruzzela & Balter (1959) found that, out of 119 spice oils tested, 100 essential oils possessed an antagonistic effect to at least one of twelve phytopathogenic fungi and 50 of these compounds showed wide spectrum activity against all fungi tested. The essential oil of
Essential oils and their components from three spices of
Essential oils isolated from the aerial parts during the flowering and vegetative phases, roots and seeds of the plant
Among a variety of oils tested
126.96.36.199.1. Essential oils components
We will also discuss here the antifungal activities of essential oil components. Eugenol has exhibited very strong antifungal activity against
Essential oil components (β-caryophyllene, β-caryophyllene oxide, α-pinene, cadinene, linalool) from eight
It can be seen that growth of the tested fungi responded diversely to the essential oils and their components, which indicates that different components may have different modes of action or that the metabolism of some fungi is able to overcome the effect of the oil or adapt to it. Terepenic compounds inhibit electron transport, proton translocation, phosphorylation steps and other enzyme-dependent reactions or act on the cell membrane. Their antifungal activity will depend on the chemical composition of the cell wall and on the structure of the terpenoid molecules. Terpenic hydrocarbons are water insoluble and revealed poor activity, while among the water soluble compounds vanilin, piperonal and camphor, were not remarkably active, whereas the non-aromatic ester borneol acetate showed antiseptic effects. Aliphatic alcohols, such as linalool or citronellol and ketones like pipertone or carvone exhibited antifungal properties. Phenol compounds showed very strong antifungal activity in spite of their relative low capacity to dissolve in water (Knobloch et al., 1988). The most active terpenoids were found among phenols, followed by aldehydes and ketones, alcohols and hydrocarbons. Thymol and carvacrol were the most effective compounds which causing total inhibition of oxidative phosphorylation. The ability of terpenoids to inhibit the reactions described above arises both from lipophilic properties, which enables them to dissolve in the cytoplasmic membrane, and from their functional groups, which interfere with enzyme structure (Griffin et al., 1999; Knobloch et al., 1988; Shelef, 1983; Soković, 2001). Studies of antimicrobial activity of essential oils and their components showed that, terpene acetates and hydrocarbons tended to be relatively inactive, regardless of their structural type, and that this inactivity appears to be closely related to their limited hydrogen bonding capacity and water solubility. Ketones, aldehydes and alcohols showed activity but with differing specificity that was not always defined by the functional group present but was associated with hydrogen-bonding parameters in all cases (Griffin et al., 1999). Our results concerning the antifungal activity of many essential oils and their components indicate different efficacy. Also, the modes of action of essential oils differ among fungal species. The strong antifungal activity of some oils (
Recently, Kalemba & Kunicka (2003) reviewed the classical methods commonly used to evaluate the antibacterial and antifungal activities of essential oils, including the agar diffusion method (paper disc and well), the dilution method (agar and liquid broth) and turbidimetric and impedimetric monitoring of microorganism growth in the presence of these oils. Besides drawing conclusions about factors that influence the antimicrobial activity of essential oils
Our results showed that the MICs for essential oils and their components are generally higher, in disc-diffusion assays and with diffusion methods than with the microdilution method. Poor activity can be explained by low water solubility of the oil and its components, which limits diffusion through the agar medium in the disc diffusion and agar diffusion methods. Only more water-soluble compounds, such as 1,8-cineole, diffuse into the agar. The hydrocarbon components either remain on the surface of the medium or evaporate. This could be the reason for better results obtained using the microdilution method. Also, essential oil and their components showed greater antifungal activity when diluted in Tween 80. Both MICs and MFCs were lower in the microdilution than in the diffusion method, especially when Tween 80 was employed. Non-ionic emulsifiers, such as Tween 20 or 80, are relatively inactive when tested alone and have been widely reported as useful emulsifying agents (Soković, 2001; Soković et al., 2002; Soković & van Griensven, 2006a). We observed that some oils and compounds acted not only as fungicidal agents but also inhibited sporulation of different fungi (Glamočlija et al., 2006a, 2006b; 2007; Soković & van Griensven, 2006a). Treatment was not only effective in solution or by contact, but even in a vapor treatment their were very effective enabling fungal growth to be inhibited by a smaller amount of essential oil while also acting as a potent inhibitor of sporulation. Vapor concentration and the duration of exposure are important. The gaseous contact activity was demonstrated primarily by the maximum vapor concentration at an early stage of incubation. Maintaining a high vapor concentration for long periods of time appeared to be unnecessary. Essential oil vapors might serve to control proliferation of moulds that are now treated with other sanitizing agents. Oils and their components have high vapor pressures and are relatively volatile. Solutions and emulsions used in the form of sprays with or without a carrier therefore represent the preferred form in which these agents should be applied to large areas of casing soil surface with minimal effort. Also evaporation by heating could be considered. An additional advantage of the volatile of essential oils is that no or only little residue will be left on the product after treatment. Our own experience leads us to propose the use of the microdilution method, carried out in microtiter trays, which involves a low workloads for a larger number of replicates and the use of small volumes of test substance and growth medium (Soković 2001, Soković et al., 2006a, 2009b). On other hand, for investigation of some plants and fungal extracts and separation of fractions and highly pure compounds bioautographic methods on TLC plates were recommended. This qualitative techniques will only give an idea of the presence or absence of substances with antimicrobial activity in very small amounts. The method was useful for screening plants and fungi for antimicrobial activity and for the bioassay-guided isolation of natural antimicrobial compounds. Bioautography allows easy localization of activity even a matrix as complex as that derived from natural products. Comparison chromatograms developed under identical conditions and visualized using suitable chromogen reagents can provide useful information about the nature of active compounds (Figure 3.) (Rančić et al., 2006, Ćirić, 2010).
Considerable changes in legislation have been made and there are increasing consumer trends for more natural alternatives to chemical fungicides (Brul & Coote, 1999). The use of essential oils is particularly advisable because herbs and spices are common plant additives. Among the natural antimicrobials that were tested in our laboratory the essential oils of Oregano and Thyme, as well as their components, carvacrol and thymol were the most promising. Addition of various plant derived antimicrobials in combination should improve both the spectrum of activity and the extent of inhibition due to synergistic effects. Thus, combination of these compounds might have even higher potential. The use of essential oils is limited, and possible reasons for this may be their strong smell and taste when used at effective doses (Skandamis & Nychas, 2000). Although the majority of essential oils are classified as Generally Recognized As Safe (GRAS) (Kabara, 1991), their use in foods as preservatives is often limited due to flavor considerations, as effective antimicrobial doses may exceed organoleptically acceptable levels. Therefore, there is an increasing demand for accurate knowledge of the minimum inhibitory (effective) concentrations (MIC) of essential oils to enable a balance between sensory acceptability and antimicrobial efficacy (Lambert et al., 2001).
188.8.131.52.2. Morphophysiological changes in fungi due to inhibition activity by essential oils
Dematiceous fungi are characterized by the presence of the dark brown pigment – melanin within their cell wall structure. Melanins are negatively charged, hydrophobic biopolymers of high molecular weights. They are typically brown or black and formed by the oxidative polymerization of phenol or indolic compounds in organisms from all biological kingdoms, including fungi. Fungal melanins are usually found in the cell walls of spores, sclerotia, mycelia or fruiting bodies. They enable fungi to survive adverse environmental conditions by protecting them from oxygen free radicals, UV radiation and wall-degrading enzymes produced by antagonist microbes (Butler et al., 2001).
Many human pathogenic fungi contain melanin within their cell wall structure (e.g.
Flavonoids are another group of secondary metabolites with great antifungal potential. Besides other biological activities, have been shown to be active against microorganisms. At least in some cases their presence might serve as a chemical barrier to invading microorganisms. Since they are natural compounds and possess highly specific antimicrobial activity, flavonoids may be an alternative to conventional fungicides in the control of plant diseases caused by fungi. Twenty-five flavonoids were examined for their effect on the mycelial growth of a crop pathogen,
Since the individual unsubstituted flavonoids showed strong antifungal activity, various mixtures have been tested against fungi occurring on grain to enhance the fungicidal potential of each substances by anticipating synergy. In general a combination of flavones and flavanones in different proportions was most effective. However, it was interesting that a mixture containing flavonoid molecules with one methoxy group and several hydroxy groups in general exhibited higher activity than a mixture containing only hydroxylated flavonoids. It becomes obvious that combinations of several suitable flavonoids (depending on the number, kind and location of the substitutents) may result in even greater increase in antifungal potential. Consequently, lower active concentrations may make flavonoids more attractive as natural protectants (Silva et al., 1998; Weidenbörner & Jha, 1997). Concerning the antifungal activity of flavonoid glycosides, it should be noted that no substantial effect could be detected (Weidenbörner & Jha, 1997). Krauze-Baranowska et al., (1999) found that cupressuflavone and 4'-O-methylcupressuflavone, isolated from leaves of
The antifungal activities of many phenol compounds isolated from Holm Oak
184.108.40.206. Sesquiterpene lactones
Sesquiterpene lactones are natural products present in many families of plants, but mostly distributed in the family Compositae. They display a wide spectrum of biological activity, one of the most important of which is antifungal activity. The general mechanism of action is considered to be alkylation of biological nucleophiles such as cysteine (cys) and glutathione or sulfhydryl-containing systems, phosphofructoki-nase and glycogen synthetase by a,b-unsaturated carbonyl structures in a Michael-type addition (Koukoulitsa et al., 2005). This group of compounds was analyzed for potential antifungal activity,
The fungicidal activities of 36 natural and synthetic sesquiterpene lactones with guaianolide, trans-germacranolide, cis-germacranolide, medampolide, and eudesmanolide carbon skeletons were evaluated against the phytopathogenic fungi
The biological activity of sesquiterpene lactones is generally attributed to the alkylating property of the -methylene--lactone moiety. Moreover, the presence of other alkylating sites (epoxides and conjugated carbonyl groups) may enhance their biological activities. Lipophilicity seems to play an important role in antifungal activity. Since the chemical composition of fungal cells walls is highly lipophilic, they generally provide a strong barriers against penetration of hydrophobic compounds and transport of polar compounds through the outer lipid layer. According to Skaltsa et al., (2000a) an inverse relationship exists between polarity and antifungal activity for sesquiterpene lactones in general. Their polarity decreases in the order eudesmanolides elemanolides germacranolides. Some of the differences between the responses of
220.127.116.11. Other compounds tested
Inhibition of certain thiol-containing enzymes in microorganisms by the rapid reaction of thiosulfinates with thiol groups was assumed to be the main mechanism involved in the antimicrobial effect of allicin. The mode of action of allicin on the fungal cell has not yet been elucidated but it is assumed to act on thiol enzymes as in other microorganisms. Other requirements such as molecular accessibility and lipophilicity seem to play an important role for in their antifungal activity (Yamada & Azuma, 1997). Antifungal activities of allicin and related organo-sulfur products obtained by microwave-assisted transformation of allicin in ethanol were studied in our laboratory against eight fungi:
18.104.22.168. Synergistic effect of different natural compounds and fungicides
Fungi that are pathogenic to plants which produce antimicrobial compounds often have greater tolerance to these natural compounds
4. Antifungal activity against animal and human pathogens
Among animal and human pathogens, dermatomycetes are the main cause of dermatomycoses (infections of the hair, skin, and nails), superficial infections that are not life threatening but are chronic and cause considerable morbidity. The unpleasant side effects of therapy including nausea, abdominal pain and itching, and its toxicity, can limits its therapeutic use in many cases (Shin & Lim, 2004).
Despite the advancements of science and technology, surprisingly the development of novel and efficient antifungal drugs is still lagging behind due to the very fact that fungi are also eukaryotic and have mechanisms similar to human beings. Hence it becomes very difficult to develop an antifungal agent that is more specific in targeting the fungi alone without any damage to human beings. For successful treatment of the disease, proper diagnosis of the disease is always essential. The treatment is chosen based on the infection site, etiological agent and penetration ability of the drug. The penetration ability and retention in the site of infection of the agent determines its efficacy and frequency of utility. Since the dermatomycetes reside in the stratum corneum especially within the keratinocytes, the antifungal agents should have a good penetrating ability (Lakshmipathy & Kannabiran, 2010). Therefore, the development of more effective and less toxic antifungal agents is required for the treatment of dermatomycosis. Essential oils play a great role in these investigations, the majority have good penetration possibilities the lipophilic properties of oil components might have also aided in the ability of the oil to penetrate the plasma membrane, strong antifungal activity and if they are used in active (MIC and MFC) concentration they are not harmful for animals and humans.
After reviewing of the results of the antifungal activity of essential oils and individual components
Extracts of seventeen microfungi (Alternaria alternata, Cladosporium cladosporioides,
5. Susceptibility of fungal species to tested compounds
In our investigation, the extracts of different plants exhibited inhibitory effect on the growth of micromycetes. Among the tested extracts one of
The human and food-borne pathogens are most frequently chosen for testing essential oil antimicrobial activity. Many laboratories deal with plant pathogens but fewer with animal pathogens. The essential oils which are the most tested compounds in our investigation of antifungal activity showed different effect on plant and human pathogens species. In our earlier investigation (Soković, 2001) essential oils in general exhibited higher antifungal activity against plant pathogen species (
6. A novel approach to solve the problem
Fungal disease is responsible for significant losses of global crop production every year, and thus has a major impact on the world’s agricultural productivity. Numerous strategies have been developed in attempts to minimize the losses caused by plant pathogens. Traditional approaches are based on the avoidance of sources of infection, vector management, modification of cultural practices, the use of resistant varieties obtained through conventional breeding, cross protection and chemical control. While these methods have been successful in some cases, indeed there is a need for new approaches. Furthermore some fungicides are being withdrawn from the market because of their undesirable effects on the environment. Whereas this information is interesting with regards to economic aspects, such as the share of fungicides costs in the output or in the variable costs, the information is less useful with regards to environmental aspects: i.e., the amounts spent on fungicides do say little about the types and quantities used. Over recent decade, producers have used synthetic fungicides as the main tool to control this problem. It has been estimated that over 23 million kg of these synthetic fungicides are used annually worldwide and it is generally accepted that production and marketing of plants would be not possible without their use (Martinez-Romero et al., 2008). New strategies for disease control are therefore urgently required. The development of novel control strategies for plant diseases is particularly important for pathogens that are difficult to control using existing methods.
More recently the scientific community has turned its attention to secondary metabolites from actinobacteria and its exploitation for various purposes which include therapeutic, environmental and industrial applications. With developing microbial resistance and need for safe and cost-effective antifungal drugs, screening of some other source, i.e. micro- and macro-fungi, mosses, for potential bioactive secondary metabolites becomes necessary. Particularly desirable is the discovery of novel prototype therapeutic agents representing new chemical classes, that operate by different modes of action compared to existing agents. We were developed suitable approves during the last several years in order to find new solutions in the aim to discover new antifungal drugs either by testing already existing medical compounds, compounds from natural sources such as plants, micro- and macrofungi, or by combination of chemical compounds with natural one. Researchers also strive to elucidate the underlying biology of fungal microorganism both
The majority of our results are focused on investigation of antifungal activity of natural products isolated from plants, especially essential oils. We used conventional research methods for testing of antifungal activity and introduced some modification for the corresponding class of compounds and microorganisms in which we operate. For example, for testing of compounds in small quantity, we recommend using a microdilution method, and microathmosphere method for testing of volatile compounds. Bioautogaphic method on TLC plates is suitable for testing of plant and fungi extracts and fractions. Considering the fact that the fungi (micro and macro) may be the source of new biologically active substances, special attention in our laboratory is given to the research of biological activities of fungal metabolites and their application in protection and treatment of diseases caused by fungi and environmental protection. The broad diversity of the fungi, as well as their easy acquisition make them especially interesting for natural products screening program. The fungi possess high capacity of bio-synthesizing various metabolites possessing different structural and pharmacological characteristics. Many medicinal and therapeutic properties are attributed to the presence of active substances in fungi. Some such compounds are investigated because their known triggering mechanisms important for fungi, while other compounds are tested blindly for their antifungal properties.
Finally, research should be kept up in order to uncover as much potentially interesting data as possible, including toxicity against animal or human cells, mechanisms of action, effects
7. Conclusion and future trends
Natural product-based fungicides are generally considered safer than synthetic herbicides, because of their relatively short environmental half-life and they are not harmful. The recent resurgence of interest in natural sources of bioactive compounds may, in part, be attributed to improved methods and instrumentation that has greatly reduced the time and effort required in natural product discovery programs. This interest is also associated with several other factors, including the realization that nature has already selected for very specific biological activities, that many natural compounds have yet to be discovered, and that the biological activities of relatively few of the known natural products have been characterized. All of the described and possible secondary metabolites have some kinds of inherent activity but in many cases these activities have not yet been discovered. Only the methods to detect their possible, perhaps until now unknown type of activity, has to be developed. There is no reason to suppose that the majority of the natural products including fungal metabolites should not exhibit some kind of biological function.
Hundreds of presently known bioactive metabolites originally was discovered as “inactive”, natural product and their activity was only discovered later, investigating them with new more specific methods, or reisolated them (sometimes from different species). In our days, in fact, there would not be any reason to talk about “bioactive” or “inactive” secondary metabolites and treat them separately. Moreover, the study of natural products may lead to the discovery of novel target sites, and/or new classes of chemistry that can be developed for pathogen management. Structural diversity has been, and still remains, an invaluable source of lead compounds in developing novel products. A recent study on complementary synthetic and natural products confirmed that the later generally have higher molecular weights than the former. Such diversity may be useful to the synthetic chemist in developing new classes of fungicides. One indirect and important benefit of the chemical composition and structural characteristics of natural products (the absence of "unnatural" ring structures and the low content of heavy atoms) is that most of them are rapidly degraded in the natural environment to benign products. In addition to their structural features, natural products tend to have different target sites from conventional fungicides.
Our results contribute to the development of safe, effective, and inexpensive formulations and processes to reduce the presence of pathogens. The antifungal compounds identified by us as the most active against major pathogens are candidates for future studies of synergism, compatibility and activity in different systems. Isolation and identification of natural active components may include a multitude of different extractions, chemical modifications, and increase knowledge of their mechanisms of action. As essential part of obtaining natural fungicides is the development of bioassays. The development of fungal resistance to synthetic drugs poses a serious long-term trait to plant, animal and human health and environmental requirements. This could also possess as significant financial issues. The advantage of natural products compared to synthetic is not only in their non toxic characteristics but also in low costs. Growing of medicinal and aromatic plants and fungi is well established and in most cases economically justified. Identification and isolation of active components from plants and fungi is also good elaborated. Natural products with antifungal activity usually operate in very small concentrations, especially essential oils, and the for further application small amount are needed. All together makes them relative cheap and available as antifungal agents.
The future of fungicide management will probably be significantly influenced by research on natural products. Modern instrumentation has simplified the isolation and identification of lead compounds from which fungicides will be derived. The reviewed studies clearly demonstrate that natural products from plants and fungi present great potential for medical procedures and for the food, cosmetic, agricultural and pharmaceutical industries.
The authors are grateful to the Ministry of Education and Science of Serbia for financial support (Grant number 173032).