Plant Material

The bark and leaves of Cz were collected from Forest Research Institute, Dehradun in October/November 2006. Freshly collected plant parts were shade-dried at room temperature for 10–15 days. Dried bark and leaf samples were separately crushed and ground into fine powder with mortar and pestle.

Preparation of extracts

Powdered plant materials were sequentially extracted with different solvents in a Soxhlet apparatus for 8 h according to the method described elsewhere [21]. The solvents used for extraction included petroleum ether (PE), benzene (BZ), chloroform (CH), ethyl acetate (EA), acetone (AC), ethanol (ET) and water (AQ). The respective extracts were filtered and dried under reduced pressure using rotary evaporator to yield solid/semisolid residues. The residues were lyophilized to get dry solid mass.

Phytochemical analysis

Qualitative phytochemical analysis of Cz bark and leaf extracts was done as follows:

Tannins

20 mg extract was dissolved in 2 ml distilled water and filtered. 2 ml FeCl₃ was added to the filtrate, blue-black precipitate indicated the presence of tannins [22].

Alkaloids

20 mg extract was dissolved in 2 ml distilled water and filtered. To the filtrate, 2–4 drops of 1% HCl was added and steam was passed through it. To the 1 ml of this solution 6 drops of Wagner's reagent was added. Brownish-red precipitate indicated the presence of alkaloids [22].

Saponins

To 0.5 ml of the filtrate obtained in alkaloids test 5 ml distilled water was added. Frothing persistence indicated the presence of saponins [22].

Flavonoids

20 mg extract was dissolved in 10 ml ethanol and filtered. 0.5 ml conc. HCl and magnesium ribbon were added to 2 ml filtrate. Development of pink-tomato red color indicated the presence of flavonoids [22].

Terpenoids

Salkovski test was performed using a small amount of extract solution. To this solution 5 drops of conc. H₂SO₄ and 1 ml Chloroform were added. Change of yellow colour into red indicated the presence of terpenoids [23].

Phenols/polyphenols

A small amount of material was extracted in ethanol and evaporated to dryness. Residue was dissolved in distilled water and 0.5 ml Folin-ciocalteau reagent was added followed by 2 ml 20% Na₂CO₃ solution. Development of bluish colour indicated the presence of phenols [24].

Test moulds

A. solani and C. lunata were isolated from soil on potato dextrose agar (PDA) plates. These dematiaceous moulds were grown and maintained on PDA slants at 28 ± 1°C. Following incubation for five days, the cultures were either utilized for test or stored at 4 ± 1°C for further use. The organisms were subcultured once in every fifteen days and the purity of the cultures was checked regularly under microscope.

Bioactivity testing

For the study of antimould activity, several concentrations of extracts (50, 100 and 500 μg/ml) were prepared in 5% DMSO. Bioactivity of the extracts was determined by counting spore (conidia) germination at various concentrations of extracts after 5 h of incubation through hanging drop technique [25]. Briefly, the spores were isolated from PDA slants and transferred in 1 ml of each extract prepared in 5% DMSO. 50 μl extract containing spores was transferred at the centre of glass coverslip and number of spores was counted with the help of microscope. The coverslips containing spore suspension were placed on different depression slides containing a drop of water. The slides along with hanging spore suspension were incubated for 5 h at 28 ± 1°C. The spores were further counted under microscope to evaluate the effect of extracts on germination. Blank test showed that 5% (v/v) DMSO used in the preparations of the test solutions does not affect the spore germination of test organisms. All experiments were carried out in triplicate and data were expressed as mean ± SEM. The bar diagrams were prepared using Graphpad Prism software.

Phytochemical Analysis

Inhibition of spore germination by different plant extracts

The inhibition of spore germination of two different species of moulds namely A. solani and C. lunata by extracts of Cz bark and leaves was examined at three different concentrations (50, 100 and 500 μg/ml). Both of the mould species were found sensitive to the treatment with these extracts. Results of antimould activity of bark and leaf extracts on A. solani are shown in Figures 1 and 2 while their effects on C. lunata are depicted in Figures 3 and 4.

Effect of Cz bark extracts on the germination of spores from A. solani and C. lunata

The Cz bark extracts prepared in chloroform and acetone were found to be most active against germination of spores of A. solani as their lowest dose (50 μg/ml) was sufficient to prevent 100% germination (Fig. 1), while in case of C. lunata only acetone extract of bark could exert complete inhibitory effect (Fig. 3). The results demonstrated in Figure 1 reflected that at 50 μg/ml concentration, the extracts prepared into petroleum ether and ethanol exhibited about 90–95% activity against spores' germination of A. solani, whereas those prepared in water and benzene could inhibit this activity by about 50 and 60%, respectively. The inhibitory potential at all the three concentrations tested with extracts prepared into petroleum ether and chloroform were similar to those of ethanol and acetone, respectively. The bark extracts into petroleum ether, chloroform, acetone and ethanol completely arrested the spores germination of A. solani, whereas aqueous and benzene preparations could inhibit this activity by about 70 and 76%, respectively, at 100 μg/ml concentration. The highest concentration (500 μg/ml) of all the extracts tested caused complete (100%) inhibition of germination of A. solani spores. The inhibitory effect of these extracts from bark was found to be maximum with chloroform or acetone extract and minimum with aqueous preparation.

These extracts when tested against the germination of C. lunata spores displayed a different pattern (Fig. 3). The extract in acetone completely inhibited the germination of spores at minimum concentration tested (50 μg/ml). The ethanolic extract was almost equally active as it could show about 95% inhibition at this concentration. However, other extracts at this concentration could display inhibitory effect up to 35–80% only. The bark extracts at higher concentration (100 μg/ml) could exhibit 100% inhibition in the spores' germination of C. lunata only when prepared in benzene, acetone and ethanol. The ethanol extract showed almost similar inhibitory pattern with that of acetone extract at all the three concentrations tested. The treatment of C. lunata spores with highest concentration (500 μg/ml) of all the test extracts resulted in almost complete inhibition of germination. The Cz extract in acetone was most active against C. lunata while the aqueous extract proved to be relatively least active (Fig. 3).

Effect of Cz leaf extracts on the germination of spores from A. solani and C. lunata

Since the data obtained from above experiments conducted with the extracts of bark demonstrated significant fungicidal potential, the attempts were made to evaluate the antifungal property of the extracts of leaves prepared in the aforesaid solvents. The results obtained after treatment of A. solani spores with the extracts of leaves showed a different trend in inhibitory activity. The data presented in Figure 2 indicated that the lowest concentration (50 μg/ml) of leaves extracts prepared in ethanol and petroleum ether was most active and caused 100% inhibition followed by the acetone, chloroform benzene and aqueous extracts. However, all other extracts of leaves were able to inhibit completely the spores' germination of A. solani only at the highest concentration (500 μg/ml). At lower concentrations (50 and 100 μg/ml) all the extracts (excepting those prepared in petroleum ether and ethanol) registered only 40–80% and 60–95% inhibition, respectively. The aqueous extract from the leaves exhibited relatively lesser inhibitory activity at lower concentrations (Fig. 2). These extracts when tested against the germination of C. lunata spores, the lowest concentration of leaves extract prepared in petroleum ether could show complete (100%) inhibition followed by the extracts prepared in other solvents (Fig. 4). The inhibitory activity pattern of the extracts prepared in benzene and ethyl acetate were similar to those of chloroform and acetone, respectively, at all the three concentrations tested. The ethanol extract exhibited close proximity in the inhibitory efficacy with that of petroleum ether extract at all the concentrations tested (Fig. 4).

The complete inhibition (100%) of spore germination in both these moulds was observed with acetone extract of Cz bark (Figs. 1 and 3) as well as with petroleum ether and ethanol extracts of Cz leaf (Figs. 2 and 4) at the lowest concentration (50 μg/ml). At 100 μg/ml concentration all the extracts exhibited about 100% inhibition of spore germination of these two mould species. However, complete inhibition (100%) of spore germination was observed at a concentration of 500 μg/ml for all extracts from both the bark and leaf. About 40–50% reduction in spore germination was observed for these two moulds at the lowest test concentration (50 μg/ml) of aqueous extract.