Phenolic acid analysis and biological activity of methanolic extracts of some medicinal plants against some phytopathogenic fungi
A Singh, S Singh, B Sarma, U Singh, R Srivastava, K Singh
antifungal, phenolic acids
A Singh, S Singh, B Sarma, U Singh, R Srivastava, K Singh. Phenolic acid analysis and biological activity of methanolic extracts of some medicinal plants against some phytopathogenic fungi. The Internet Journal of Alternative Medicine. 2008 Volume 6 Number 2.
About three quarters of the world population rely mainly on plants and plant extracts for health care. The methanolic extracts of some potential medicinal plants such as
Extracts of several plants are highly effective against parasitic as well as saprophytic microbes. It is estimated that around 70,000 plant species, from lichens to trees, have been used at one or the other time for medicinal purposes (1). The demands of medicinal plants by the modern pharmaceutical industries has also increased manifold (2). The medicinal plants occupy a significant place in modern medicine for some important drugs, although synthetic drugs and antibiotics brought about a revolution in controlling different diseases (3). The anti-infectional compounds show broad-spectrum bioactivity against bacteria, fungi, protocists, protozoans, viruses, yeasts, etc. (4).
The objective of this research was to see whether methanolic extracts of these plants are antifungal. High performance liquid chromatographic (HPLC) analysis was also done to see the phenolic profile as some of the phenolic acids are antimicrobial and some others are potential agents for human health. The results are presented here.
Materials and methods
Collection and extraction of medicinal plant material
The raw material (root and aerial parts) of
The crude extracts of
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.
Sample preparation for the analysis of phenolic compounds
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 (Rt) 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.
Results and Discussion
Comparative analysis of antifungal activity
Crude extracts of
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
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,