The raw material (root and aerial parts) of Saraca indica, Withania somnifera and Bacopa monnieri were collected from different fields. The dried plant parts were powdered and extracted separately with methanol:sterile water (1:1) using soxhlet apparatus for 48 h. The solvent was distilled at lower temperature under reduced pressure in rotary flash evaporator and concentrated on water bath to get the crude extract. The extracts were stored in desiccators for further experiments.

The crude extracts of S. indica, W. somnifera and B. monnieri were used in the present experiment. Alternaria cajani, Helminthosporium sp., Bipolaris sp., Curvularia lunata and Fusarium sp. were isolated from respective infected plant parts on potato dextrose agar (PDA) (peeled potato 250 g, dextrose 20 g, agar 15 g, distilled water 1 L) medium. The cultures were further purified by single spore isolation technique and maintained at 25 ± 2 ° C on PDA slants. Seven to ten-day-old cultures were used in the experiment.

Stock solutions (5000 µg/ml) of the crude extracts were prepared by dissolving 5 mg of the extract in 1 ml of distilled water. Required concentrations (1000, 2000, 3000, and 4000 µg/ml) were prepared from each stock solution by diluting with distilled water. One drop (30-35 µl) from each concentration was placed on grease-free glass slides. Fungal spores (200-300) were picked up from 7-10-day-old cultures with sterilized inoculation needle and mixed in solutions of different concentrations of the three extracts separately. The slides were placed in moist chambers made by placing two sterile filter papers each on the lid and base of the petri plates. They were incubated at 25 ± 2 ° C for 24 h. Germination was observed after mixing a drop of cotton blue prepared in lactophenol on every slide containing fungal spores under binocular microscope (Nikon, Japan Type 102). Spores mixed in only sterile distilled water served as control. All the experiments were conducted in triplicate.

The phenolic acids were analysed through High Performance Liquid Chromatography (HPLC) as per the method of Singh et al. (₆). The samples of each plant were prepared separately. One gram of each extract was macerated and suspended in 5 ml ethanol:water (80:20; v/v). The collected samples were subjected to ultrasonication (Branson Sonifier, Danbury, CT, USA) for 15 min at 4°C followed by centrifugation at 12500 x g for 15 min. The clear supernatant was subjected to charcoal treatment for the removal of pigments. The residue was re-extracted twice with the same extracting solution and the supernatant was pooled prior to evaporation under vacuum (Buchi Rotavapor Re Type, Labco, India; Ambala Cantt. India). Dried extracts were resuspended in 1.0 ml HPLC grade methanol by vortexing and filtered through ultra membrane filter (pore size 0.45 µm: Millipore) before HPLC analysis.

Quantitative analysis of the sample was performed according to the method of Singh et al. (₆). The HPLC system (Shimadzu Corporation, Kyoto, Japan) was equipped with two Shimadzu LC-10 ATVP reciprocating pumps, a variable Shimadzu SPD-10 AVP UV-VIS detector and a Rheodyne Model 7725 injector with a loop size of 20 µl. The peak area was calculated with a Winchrom integrator. Reverse-phase chromatographic analysis was carried out in isocratic conditions using a C-18 reverse phase column (250 x 4.6 mm i.d., particle size 5 µm, Luna 5µ C-18 (₂); phenomenex, Torrance, CA, USA) at 25°C. Running conditions included: injection volume 5µl; mobile phase, methanol: 0.4% acetic acid (80: 20 v/v); flow rate 1 ml/min; and detection at 290 nm. Samples were filtered through an ultra membrane filter (pore size 0.45 µm; E-Merck, Darmstadt, Germany) prior to injection in the sample loop. Benzoic, gallic, ferulic, catechin and tannic acids were used as internal and external standards. Phenolic acids present in each sample were identified by comparing chromatographic peaks with the retention time (R_t) of individual standards and further confirmed by co-injection with isolated standards. The amount of each phenolic acid is expressed as micrograms per gram of fresh weight unless otherwise stated.

Crude extracts of S. indica, W. somnifera and B. monnieri were tested against Alternaria cajani, Helminthosporium sp., Bipolaris sp., Curvularia lunata and Fusarium sp. at concentrations of 1000, 2000, 3000, 4000 and 5000 µg/ml (Fig. 1). The methanolic extract inhibited growth of the test fungi to varying degrees. All the three extracts, i.e. of S. indica, W. somnifera and B. monnieri, were highly effective at 5000 µg/ml. S. indica was highly inhibitory against Fusarium sp. (90%), A. cajani (89.47%), Helminthosporium sp. (83.87%) and C. lunata (85.37%). Similarly, W. somnifera extract was highly inhibitory against C. lunata (90.32%), Fusarium sp. (84.37%), and Bipolaris sp. (82.35%). Antifungal activity of B. monnieri was the least inhibitory compared to the other two plant extracts but highly inhibitory against C. lunata (83.33%) and A. cajani (79%) at 5000 µg/ml. The inhibitory effect was less at lower concentrations of the extracts. According to Joshi et al. (₇) methanolic extract of S. indica showed good fungicidal activity against Aspergillus fumigatus, A. niger, Penicillium frequentence, P. notatum and Botrytis cinerea. It also showed antimicrobial activity against Aspergillus flavus, A. niger and Candida albicans in a range of 75-1200 µg/ml (₈). S. indica also exhibited complete toxicity against Pythium debaryanum, Fusarium oxysporum, Rhizoctonia solani and Sclerotium rolfsii (₉). W. somnifera has also potential antibacterial activity against Staphyloccus aureus and Pseudomonas aeruginosa (₁₀). Its antifungal activity was demonstrated against A. flavus, F. oxysporum, F. verticilloides and antibacterial activity against Clvibacter michiganensis subsp. michiganensis (₁₁). The phytochemicals betulinic acid, wogonin and oroxindin from aerial part of B. monnieri were found effective against Altenaria alternata and Fusarium fusiformis (₁₂).

Figure 1

Recent researches indicate that phytophenols, being chief secondary metabolites, are present in rich amount in several plants. The HPLC fingerprints (Fig. 2 a, b and c) of the crude extracts of S. indica, W. somnifera and B. monnieri showed presence of several phenolic acids of which five were identified, i.e., benzoic, gallic, ferulic, catechin and tannic acids present in varying amounts (Table 1).

Figure 2

Table 1: Amount of phenolic acids in the crude extract of and

The retention times (min.) of the five phanolic acids were 5.67, 2.80, 3.77, 2.73 and 2.55, respectively, at a wavelength of 290 nm. Out of the three extracts, B. monnieri showed maximum amount of tannic acid (5380.89 µg/g) followed by W. somnifera (1467.24 µg/g). The amount of gallic acid was also maximum in B. monnieri (892.17 µg/g) compared to S. indica (16.016 µg/g). Out of the five phenolic acids, only catechin (0.663 µg/g), gallic (16.016 µg/g), ferulic (5.931 µg/g) and benzoic acids (987.44 µg/g) were detected in S. indica. Highest amount of catechin was recorded in W. somnifera (111.85 µg/g) followed by S. indica (0.663 µg/g). HPLC analysis of the samples revealed wide variability in their phenolic acid content (Fig. 2).

Figure 3

S. indica contains about 6% tannins, catechol, haematoxylin, ketosterol, saponin and organic calcium and iron compounds (₁₃). Saracin, a seed integument lectin from S. indica is highly specific for binding N-acetyl-neuraminyl-N-acetyllactosamine (₁₄). The enzymatic activity of S. indica was determined by HPLC and the bark of this plant extracts showed inhibitory effect on human immunodeficiency virus type 1 (HIV-1) protease (₁₅). In W. somnifera, the biological activities of withanolides, especially of the dominant withanolide A and withaferin A, were detected using HPLC analysis which were found to have anti-cancerous activity (₁₆, ₁₇). Similarly, Bandyopadhyay et al. (₁₈) detected the accumulation of withaferin-A and withanolide-D in roots of W. somnifera. HPTLC method has been developed for the estimation of withaferin-A and withanolide-A in different plant parts such as, leaf, root, stem and fruit of two morphotypes of Withania somnifera (₁₉). The pharmacological properties of B. monnieri were studied extensively which were attributed mainly to the presence of saponins called “bacosides” (₂₀). In B. monnieri, an HPLC method was developed for the quantitative determination of Bacoside A, the putative bioactive component, was found to be a mixture of saponins with bacoside A₃ (1), bacopaside II (2), jujubogenin isomer of bacopasaponin C (3) and bacopasaponin C (4) as major constituents (₂₁). Glycosides of the 20-deoxy derivatives of jujubogenin and pseudojujubogenin were selected from B. monnieri. The compounds were tested for their cytotoxicity, antileishmanial, antimalarial, antioxidant, and anti-inflammatory activities (₂₂). Looking into the previous reports as well as evidence from the present investigation it can be concluded that the methanolic extracts of B. monnieri, W. somnifera and S. indica are antifungal effective against a diverse group of fungi and therefore could be exploited for their use in agricultural crop protection.