**2. Extraction**

108 Fungicides for Plant and Animal Diseases

transmission electron microscopy (TEM) also will be discussed. Fig. 1 shows the various

•Plant sample collection

•Disc diffusion method

•Extraction and standardization

•Determination of the Minimal

Inhibitory Concentration

•Determination of Minimum Fungicidal Concentration

•Scanning Electron Microscopy

•Transmission electron microscopy

Fig. 1. Various steps involved in the development and evaluation of fungicidal property of

medicinal plants

**Extraction**

*In vitro* **antifungal testing**

*In situ* **antifungal activity**

steps involved in the evaluation of the medicinal plant's for fungicidal properties.

First steps in the process of screening medicinal plants for antifungal activity is extraction. Extraction is the separation of medicinally active portions of plant tissues using selective solvents through standard procedures. Such extraction techniques separate the soluble plant metabolites and leave behind the insoluble cellular marc. The products so obtained from plants are relatively complex mixtures of metabolites, in liquid or semisolid state or in dry powder form, and are intended for oral or external use. These include classes of preparations known as decoctions, infusions, fluid extracts, tinctures, pilular (semisolid) extracts or powdered extracts (Sukhdev et al., 2008). The basic operations of extraction include steps, such as pre-washing, drying of plant materials or freeze drying, grinding to obtain a homogenous sample and often improving the kinetics of analytic extraction and also increasing the contact of sample surface with the solvent system. Proper actions must be taken to assure that potential active constituents are not lost, distorted or destroyed during the preparation of the extract from plant samples.

The general techniques (Fig. 2) of medicinal plant extraction include maceration (Fig. 3), infusion, percolation, digestion, decoction, hot continuous extraction (Soxhlet). Recently, modern extraction methods have been developed which includes microwave-assisted extraction (MAE), ultrasound extraction (sonication) and supercritical fluid extraction (SFE) (Table 1).

Fig. 2. Conventional and modern extraction methods

Screening Methods in the Study of Fungicidal Property of Medicinal Plants 111

Methanol, ethanol, or mixture of alcohol and water

Room

applicable Not applicable 250–450 atm

**Time required** 3–18 hr 1 hr 3-4 days 30–100 min 10–40 min 20–40 min

Depending on

Phrompittayarat et al*.,*2007; Sasidharan et al.,2008a; Cunha et al., 2004; Woisky et al., 1998

Table 1. A brief summary of the experimental conditions for various methods of extraction

As the target compounds may be non-polar to polar and thermally labile, the suitability of the methods of extraction must be considered. The choice of solvent depends on several factors including the characteristics of the constituents being extracted, cost and environmental issues. SFE has been used for many years for the extraction of volatile components on an industrial scale. An important advantage of applying SFE to the extraction of active compounds from medicinal plants is that degradation as a result of lengthy exposure to elevated temperatures and atmospheric oxygen are avoided. Using MAE, the microwave energy is used for solution heating and results in significant reduction of extraction time (usually in less than 30 min) compared with conventional liquid–solid extraction methods in which a relatively long extraction time (typically 3–48 h) is required. Another advantage of MAE is that it enables a significant reduction in the consumption of organic solvents, typically less than 40 mL,

compared with the 100–500 mL required in Soxhlet extraction (Huie, 2002).

Not

Zygmunt & Namiesnik, 2003; Huie, 2002; Luque de Castro et al., 2000; Liu & Wai, 2001

the sample size **Supercritical Fluid extraction (SFE)** 

temperature 40–100 80–150 80–200

Carbon dioxide or carbon dioxide with modifiers, such as methanol

**Microwave assisted extraction (MAE)** 

Methanol, ethanol, or mixture of alcohol and water

Depending on if it is closed or opened vessel extraction

applicable 20–50 20–30

Zygmunt & Namiesnik, 2003; Huie, 2002; Camel, 2000; Pan et al., 2001; Pan et al., 2002 Fang et al., 2000

**Pressurized Liquid Extraction, (PLE)** 

Methanol

10–20 bar

Ong et al., 2000; Ong & Apandi, 2001; Lee et al., 2002; Ong, 2002; Ong & Len, 2003a; 2003b; Choi et al., 2003;

**Soxhlet** 

Methanol, ethanol, or mixture of

alcohol and water

Depending on solvent used

Not applicable

**Common solvents used** 

**Temperature** 

**(oC)** 

**Pressure applied** 

**Volume of solvent required (ml)** 

**References** 

for plants material

**extraction Sonification Maceration** 

Methanol, ethanol, or mixture of alcohol and water

Can be heated

Not

Zygmunt & Namiesnik, 2003; Huie, 2002

150–200 50–100

Zygmunt & Namiesnik, 2003; Huie, 2002

Fig. 3. An example of maceration method using *Euphorbia hirta* sample where organicsolvent extraction was performed by soaking 100g of powdered dried plant material in methanol (1.0 L) through occasional shaking and stirring for 7 days. The whole extract was then filtered and the solvent was evaporated to dryness *in vacuo* using a rotary evaporator at 40-50ºC to afford a paste mass.

Fig. 3. An example of maceration method using *Euphorbia hirta* sample where organicsolvent extraction was performed by soaking 100g of powdered dried plant material in methanol (1.0 L) through occasional shaking and stirring for 7 days. The whole extract was then filtered and the solvent was evaporated to dryness *in vacuo* using a rotary evaporator at

40-50ºC to afford a paste mass.


Table 1. A brief summary of the experimental conditions for various methods of extraction for plants material

As the target compounds may be non-polar to polar and thermally labile, the suitability of the methods of extraction must be considered. The choice of solvent depends on several factors including the characteristics of the constituents being extracted, cost and environmental issues. SFE has been used for many years for the extraction of volatile components on an industrial scale. An important advantage of applying SFE to the extraction of active compounds from medicinal plants is that degradation as a result of lengthy exposure to elevated temperatures and atmospheric oxygen are avoided. Using MAE, the microwave energy is used for solution heating and results in significant reduction of extraction time (usually in less than 30 min) compared with conventional liquid–solid extraction methods in which a relatively long extraction time (typically 3–48 h) is required. Another advantage of MAE is that it enables a significant reduction in the consumption of organic solvents, typically less than 40 mL, compared with the 100–500 mL required in Soxhlet extraction (Huie, 2002).

Screening Methods in the Study of Fungicidal Property of Medicinal Plants 113

reached 1.5 cm. Results will be expressed as the percentage of hyphal growth inhibited (Gamliel et al., 1989). Concentration response curves will be prepared in which the percentage of hyphal growth inhibition is plotted against concentration mg/mL. The concentration required to give 50% inhibition of hyphal growth IC50 will be calculated from the regression

Electron microscopy (EM) is one of the many methods available for visual inspection of fungal strains. The effects of potential antifungal extracts from natural sources can also be evaluated by using the EM methods. Hence in this section the microscopical techniques such as Scanning (SEM) and Transmission (TEM) electron microscopy on the *in situ* antifungal

After treatment with plant extract, scanning electron microscopy SEM observation will be carried out on fungal strains. First of all, the plate containing 25 mL PDA medium will be seeded with 1 mL of the fungal conidial spore suspension containing 105 spores per mL from a 120-h-old culture. The extract 1mL, at the concentration of IC50 (obtained from the hyphal growth inhibition test), is then dropped onto the inoculated agar and will be further

to ten mm segments will be cut from cultures growing on potato dextrose plates at various time intervals 1, 2, 3, 4, 5, 6, and 7 days for SEM examination (Sasidharan et al., 2008b). The specimen then placed on double-stick adhesive tabs on a planchette and the planchette placed in a petri plate. In a fume hood, a vial cap containing 2% osmium tetroxide in water will be placed in an unoccupied quadrant of the plate. After being covered, the plate will be sealed with parafilm, and vapor fixation of the sample proceeded for 1 h. Once the sample is

on to the "peltier-cooled" stage of the freeze dryer, and freeze drying of the specimen will be proceeded for 10 h. Finally, the freeze dried specimen will be sputter coated with 5–10 nm gold before viewing in the SEM. The SEM is advantageous over several other microscopy methods as it is three-dimensional and almost the whole of the specimen is sharply focused. Furthermore, besides having a combination of higher magnification, larger depth of focus and greater resolution, the preparation of samples is also relatively easier, compared to the TEM method (Sasidharan et al., 2010). From the SEM micrograph (Fig. 4) we can observe the

Further confirmation of SEM finding can be obtained from TEM study. To study the antifungal activity through TEM method the hyphal specimens (1×3 mm2, with approximately 1 mm thickness of underlying agar blocks) of test fungal strains will be excised from the margin of actively growing SDA culture treated with plant extract using a sterilized razor blade. The specimens are then fixed with modified Karnovsky's fixative (Karnivsky, 1965) consisting of 2% (v/v) glutaraldehyde and 2% (v/v) paraformaldehyde in 0.05 M sodium cacodylate buffer solution (pH 7.2) at 4°C overnight. Subsequently, the fixed specimens are washed with the solution three times for 10 min each. The specimens were then will be post-fixed in the solution with 1% (w/v) osmium tetroxide at 4°C for 2 h and then will be washed briefly with

vapor fixed, the planchette will be plunged into slushy nitrogen -210

changes caused by the plant extract on fungal surface.

**4.2 Transmission electron microscopy (TEM)** 

C. A vehicle-treated culture can be used as a control. Five

C and then transferred

equation. Miconazole can be used as a positive control.

**4.** *In situ* **antifungal activity** 

activity by plant extract will be discussed.

**4.1 Scanning electron microscopy** 

incubated for another 7 days at 28
