**3.4 Results**

130 Fungicides for Plant and Animal Diseases

humans and plants. Several molecules obtained from the natural environment are studied and described in bibliography with antimycotic activity. Several extracts are tested for antifungal activities like crude extracts or isolated constituents like, essential oils, terpenoids, saponins,

Aromatic plants have been widely used in folk medicine. About three quarter of the world's population relies on plants and plant extracts for healthcare (Parekh & Chanda, 2007). Several plants have been used in folklore medicine in Portugal (Pina-Vaz et al., 2004; Figueiredo et al., 2008). Spices have been used with primary purpose of enhancing the flavor

Plants generally produce many secondary metabolites with antifungal and microbicide activity (Bobbarala et al., 2009). According to the WHO (World Health Organization), plants would be the best source for obtaining a variety of drugs and a possible way to treat diseases caused by multidrug resistant microorganisms (Bhattacharjee et al., 2006). The medicinal value of plants lies in some chemical substances that produce a definite physiological action on the human body. The most important of these bioactive compounds of plants are alkaloids, flavanoids, tannins and phenolic compounds (Edeoga et al., 2005; Panghal et al., 2011). Some medicinal plants exert strong antifungal properties and could be conveniently used as a promising alternative source for presently problematic antifungal

Many commercially drugs used in modern medicine was initially used in crude form in traditional or folk healing practices. Benefits of using plant extracts are that they are relatively safer than synthetic alternatives, offering profound therapeutic benefits and more

The genus *Thymus* (Lamiaceae), widely distributed on the Iberian Peninsula, is a taxonomically complex group of aromatic plants traditionally used for medicinal purposes because of their antiseptic, antispasmodic and antitussive properties (Pina-Vaz et al., 2004). Previous studies on the antimicrobial activity of the essential oils of some *Thymus* spp., most of them possessing a large amount of phenolic monoterpenes, showed activity against fungi

Screening of medicinal plants for antimicrobial activities and phytochemicals is important for finding potential new compounds for therapeutic use (Duraipandiyan et al., 2006).

The main objective of this study was to investigate the inhibitory effects of *Thymus* and

Fungal strain was obtained from the collection of pathogenic fungi maintained in the University of Trás-os-Montes and Alto Douro, Vila Real, Portugal. Subcultivations on Petri dishes and other manipulations with the strain were carried out in the Bio Security Level 2 (BSL 2) laboratory. The evaluation of antifungal capacity was done by the method of mycelial growth (Zhang et al., 2006). The fungus used in the assays was the fungi *Aspergillus fumigatus*. The mold was grown on potato dextrose agar (PDA). The solution was mixed with PDA culture media respectively to give a series of 5, 10, 20, and 25 mg/mL concentrations of culture media containing the compounds described above. The media

of foods rather than their medicinal and antioxidant properties (Souza et al., 2005).

treatment in many areas with respect to their natural origin (Zabka et al., 2011).

affordable treatment (Panghal et al., 2011).

*Mentha* extracts against *Aspergillus fumigatus*.

(Pina-Vaz et al., 2004).

**3.3 Methodology** 

phenolic compounds, alkaloids, peptides and proteins (Abad et al., 2007).

Effect of four different concentrations (5 mg/mL, 10 mg/mL, 20 mg/mL and 25 mg/mL) of *Thymus* and *Mentha* extract plants was tested against *Aspergillus fumigatus*. Antifungal activity was assayed and data on effect of plant extracts on the growth of *Aspergillus fumigatus* in the third, fifth and seventh day is presented in Table 4. The data revealed that reduction in growth of *Aspergillus fumigatus* was observed with extracts of *Thymus* and *Mentha*.


aAll Values are mean of three replicates.

Table 4. Inhibition effect of plant extracts on *Aspergillus fumigatus* in four different concentrations.

The results indicated that *Thymus mastichina* exhibited antifungal activity against the tested *Aspergillus fumigatus* at two different concentrations of 20 mg/mL and 25 mg/mL. The highest antifungal activity was exhibited at 25 mg/mL in *Thymus*. The percent of inhibition were statistically significant with different concentrations in *Thymus*. The lowest concentration of *Thymus mastichina* did not show any activity against *A. fumigates* in the 3 days, while the other two higher concentrations showed good antifungal activity.

*In Vitro* Multiplication of Aromatic and Medicinal Plants and Fungicide Activity 133

accepted level. Nonetheless, combinations of spices and other antimicrobial barriers could enhance the food shelf stability and microbial safety even in moderated levels (Pandit & Shelef 1994; Brull & Coote, 1999; Souza et al., 2005). In the other way, the use of aromatic plants as remedies in folk medicine, provide a good reason to investigate them scientifically as potential sources of new plant drugs. It is important to prove which plant extracts have a biological activity on some specific medical conditions, *e.g.* antimicrobial and antifungal

It was possible the establishment of a micropropagation protocol in order to multiplicate and maintain *in vitro* the aromatic and medicinal plants, to have enough material to use in

Considering the fact that *in vitro* cannot be directly extrapolated to *ex vitro* effects the results suggests that, the use of plant extracts such as *Thymus* and *Mentha* against *Aspergillus sp.* has potential as a topical antifungal agent as they offer a cheap and effective module for

Our results showed that extracts from *Thymus and Mentha* may be particularly useful against *Aspergillus fumigatus*. These results may justify the popular use of these aromatic plants.

Compound-activity relationship for oils components against fungus organisms must be

However, in order to evaluate possible clinical application in food microbiology and therapy

Further phytochemical studies are required to determine the types of compounds

Authors are grateful to Professor Mariana Sottomayor from IBMC- Institute for Molecular and Cell Biology for providing seeds for *in vitro* establishment of *Catharanthus* roseus. The authors also like to thank to Carina Alves, Luís Silva, Sandra Cabo and Tatiana Louçano,

Abad, M.J.; Ansuategui, M. & Bermejo, P (2007). Active antifungal substances from natural

Afonso, MLR. & McMurtrie, M. (1991). Plantas do Algarve Lisboa, Portugal: Serviço Nacional de Parques, Reservas e Conservação da Natureza, ISSN 0870 - 2977 Angelini, L.G.; Carpanese, G.; Cioni, P. L.; Morelli, I.; Macchia, M. & Flamini, G. (2003).

Essential oil from mediterranean Lamiaceae as weed germination inhibitors. *Journal of Agricultural and Food Chemistry,*Vol*.* 51, Nº.21, pp. 6158-6164, ISSN 0021-8561 Bandeira, J. M.; Lima, C. S.; Rubin, S.; Vaz Ribeiro, M.; Falqueto, A. R.; Peters, J. A. & Braga,

E. J. (2007). Diferentes tipos de vedações dos frascos e concentrações de sacarose na

properties (Tomczykowa et al., 2008).

therapeutic and/or preventive purposes.

of aspergillosis, further studies needed to be made.

responsible for the antifungal effects of these species.

students of University of Trás-os-Montes and Alto Douro.

sources. *ARKIVOC*, Nº.7, pp.116-145, ISSN 1424-6376

future studies of antifungal activity and of genetic variability.

elucidated to explain its antifungal activity (Tomczykowa et al., 2008).

**4. Conclusion** 

**5. Acknowledgment** 

**6. References** 

Among the species tested, *Mentha* was less active. No enhancing effect was observed for *Mentha* extract against *Aspergillus fumigatus* at higher concentrations (20 mg/mL and 25 mg/mL) while the lowest concentrations i.e. 5 mg/mL, 10 mg/mL showed some inhibition activity against the mold strain. The percent of inhibition were statistically significant with different concentrations in *Mentha*.

None of the above concentrations completely inhibited the test fungus. The percent of inhibition ranged from 0.9 to 19.1%.

#### **3.5 Discussion**

Multi-drug resistance is a medical problem in world-wide and has therefore led researchers in the search for new antimicrobial drugs or resistance, particularly from natural resources (Sharma et al., 2005; Moghaddam et al., 2010). Recently, various natural products or synthetic compounds have been reported to increase the antifungal activity (Duraipandiyan et al., 2006; Bobbarala et al., 2009; Moghaddam et al., 2010; Pai et al., 2010).

Antifungal activity was exhibited by different concentrations extracts. The chronological age of the plant, percentage humidity of the harvested material, the method of extraction were possible sources of variation for the bioactivity of the extracts (Panghal et al., 2011).

The results presented indicate different spectrum of antifungal activity of the two extracts.

The antifungal activity of *Thymus mastichina* extract against the mentioned fungi was dosedependent and increased with the increase in the plant extract concentrations. It also supports the earlier investigations of other authors (Bobbarala et al., 2009; Moghaddam et al., 2010). Previous studies have shown that *Thymus* possess antimicrobial activity (Pinto et al., 2006; Figueiredo et al., 2008).

In the other way, it was revealed in this study, that the antifungal activity of *Mentha* was enhanced in low concentrations of the extracts.

Therefore, this study suggests that plant extracts of screened plants could be helpful in treating diseases in plants caused by *Aspergillus fumigatus*.

However, there is little information about *Thymus* and *Mentha* and their derivatives in the fungal cell in order to promote fungistatic or fungicide effect (Pina-Vaz et al., 2004; Figueiredo et al., 2008). They have been empirically used as antimicrobial agents, but the mechanisms of action are still unknown (Pinto et al., 2006). Generally, inhibitory action of natural products on fungi involves cytoplasm granulation, cytoplasmic membrane lesion, and inactivation and/or inhibition of intercellular and extracellular enzymes (Cowan, 1999; Pinto et al., 2006) and might be due to various compounds, including terpenoids, phenolics and alkaloids. These compounds jointly or independently, exert different levels of antifungal effect culminating with mycelium germination inhibition (Cowan, 1999). Also, it is reported that plant lytic enzymes act in the fungal cell wall causing breakage of β-1,3 glycan, β-1,6 glycan and chitin polymers (Brull & Coote, 1999). The antimicrobial action of the aqueous extracts could be attributed to the anionic components such as thiocyanate, nitrate, chlorides and sulphates besides other water soluble components which are naturally occurring in the plant material (Darout et al., 2000).

Use of aromatic plants as microbial growth inhibitor in foods is often limited because of flavor considerations as effective antimicrobial dose may exceed the organoleptically accepted level. Nonetheless, combinations of spices and other antimicrobial barriers could enhance the food shelf stability and microbial safety even in moderated levels (Pandit & Shelef 1994; Brull & Coote, 1999; Souza et al., 2005). In the other way, the use of aromatic plants as remedies in folk medicine, provide a good reason to investigate them scientifically as potential sources of new plant drugs. It is important to prove which plant extracts have a biological activity on some specific medical conditions, *e.g.* antimicrobial and antifungal properties (Tomczykowa et al., 2008).
