Collection and extraction of medicinal plant material

The raw material of medicinal plants such as, Tectona grandis, Shilajit and Valeriana wallachi were collected from different regions of India. Voucher specimens deposited at Institute of Bioengineering and Biological Sciences, Varanasi, India for future reference.

The dried powdered of plant materials (roots and aerial parts) were extracted separately with methanol: sterile water (1:1) using soxhlet apparatus for 48 hrs. The solvent was distilled off at lower temperature under reduced pressure in rotory flash evaporator and concentrated on water bath to get the crude extract which is stored in dessicator for future use.

Antifungal activity

Three different medicinal crude extract which showed in vitro antifungal activity against some plant pathogens such as Alternaria cajani, Helminthosporium sp., Bipolaris sp., Curvularia lunata and Fusarium sp., were used in the present experiment. Test fungi were isolated on potato dextrose agar (PDA) (peeled potato 250 g, dextrose 20 g, agar 15 g, distilled water 1 L) medium from their respective hosts collected from experimental farm of Banaras Hindu University, Varanasi, India. The cultures were further purified by single spore isolation technique and maintained at 25±2 °C on PDA slants. 7-10 days old culture were used in the experiment.

Stock solution (5000 µg/mL) of the crude extract were prepared by dissolving 5 mg of the culture in 1 ml of distilled water. Required concentrations (1000, 2000, 3000, 4000 and 5000 µg/mL) were prepared from each stock solution by diluting with distilled water. One drop (40 µL) from each concentration was placed on grease-free glass slides. Fungal spores (200-300) were picked up from 7-10 days old culture with sterilized inoculation needle and mixed in solution of the fraction of different concentrations separately. The slides were placed in moist chambers made by placing two sterile filter papers each on the lid and base of the petriplates. The slides with spores were then incubated at 25±2 °C for 24 hr. Germination was observed after staining with cotton blue prepared in lactophenol under binocular microscope (Nikon, Japan Type 102). Spores mixed in sterile distilled water only served as control. All the experiments were conducted in triplicate.

Sample Preparation of phenolic compounds

The phenolic acids were extracted as per the method of Singh et al. 17 . Three crude extracts of Tectona grandis, Shilajeet and Valeriana wallachi were collected from different places of India. One gram of each extract was macerated and suspended in 5 ml methanol-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 12 500 x g for 15 min. The clear supernatant was subjected to charcoal treatment. 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 extract were resuspended in 1.0 ml high-performance liquid chromatography (HPLC)-grade methanol by vortexing and filtered through ultra membrane filter (pore size 0.45 µm: Millipore) before HPLC analysis.

HPLC analysis

Quantitative analysis of the sample was performed according to the method of Singh et al. 17 . 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 mm. Samples were filtered through an ultra membrane filter (pore size 0.45 µm; E-Merck, Darmstadt, Germany) prior to injection in the sample loop. Tannic, gallic, caffeic, ferulic, benzoic, cinnamic, capachin and salicylic 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.

Comparative analysis of antifungal activity

Crude extract of Tectona grandis, Shilajit and Valeriana wallachi were tested against phtopathogenic fungi such as Alternaria cajani, Helminthosporium sp., Bipolaris sp., Curvularia lunata and Fusarium sp Alternaria cajani, Curvularia lunata, Fusarium sp., Bipolaris sp. and Helminthosporium sp. at concentrations of 1000, 2000, 3000, 4000 and 5000 µg/mL. The effects of the different concentrations of crude extracts on five different phytopathogenic fungi are presented in Table 1 and Fig. 1a, b,c, d, e.

Table 1: Effect of medicinal extract of Tectona grandis, Shilajeet and Valeriana wallachi on spore germination of some pathogenic fungi

The methanolic extract, on the other hand, inhibited growth of the test fungi to varying degrees. Concentrations at 1000 and 2000 µg/mL brought minimal inhibition (Valeriana wallachi showed 13.21% and 15.38 % against Bipolaris) against test fungi (Table 1). Based on the inhibition profiles, Bipolaris sp. and Fusarium sp. were least sensitive to all the three extracts, while Curvularia lunata was less inhibited by Tectona grandis and Valeriana wallachi. A considerable reduction in the sporulation was also recorded. The methanolic extract tested at 5000 µg/mL against a number of pathogenic fungi were found effective at higher concentrations. Among the five fungi tested, the extract of Valeriana wallachi was found to be most effective and evinced excellent inhibitory activity against Helminthosporium sp. (96.15%) followed by Shilajit extract against Alternaria cajani (95.12 %), Helminthosporium (95.00 %) and Curvularia lunata (93.33%) at the concentration of 5000 µg/mL. Also at concentrations of 3000 and 4000 µg/mL, Shilajit exhibited better antifungal activity against Alternaria cajani and Helminthosporium sp. The extract of Tectona grandis showed 90.00% and 86.84% inhibition growth against Alternaria cajani and Helminthosporium. It is revealed from the above statement, that higher concentration of the methanolic extract impart maximal antifungal activity (Table 1 and Fig. 1)

Various extract of medicinal plants have shown inhibitory effects against phytopathogenic fungi in vitro 17 . The antimicrobial activities of various plants have been reported by many researchers 18 . Tectona grandis sawdust extract exhibited the growth of Aspergillus niger 4 . Endophtic isolated from Tectona grandis could produce inhibitory substances effective against Bacillus subtilis, Staphylococcus aureus and Escherichia coli and Candida albicans in vitro 19 . A lipophilic crude extract of Valeriana capense exhibited antifungal activity against the plant pathogenic fungus Cladosporium cucumerinum 20 .

HPLC analysis

Recent researches indicate that the polyphenols, being secondary metabolites, are present in rich amount in several plants. Many of them possess antioxidant, anti-inflammatory and several others therapeutic properties. The HPLC fingerprints of the crude extracts of Tectona grandis, Shilajeet and Valeriana wallachi showed four types of the Phenolic acids i.e. Tannic acid, gallic acid, Ferulic acid and caffeic acid that are present in varying amount (Table 2 and Fig. 2). Although a primary objective of carrying out HPLC may be to standardize dosage, more information may be obtained during the course of a run, if appropriate detection hardware and software are used.

Table 2: Phenolic acid in the crude extract of Tectona grandis, Shilajit and Valeriana wallachi

The HPLC ‘fingerprint' (Fig. 2) of the methanolic extract of Tectona grandis, Shilajit and Valeriana wallachi show major peaks at the retention times (min.) of 2.61, 2.92, 3.72 and 3.48 at a wavelength of 290 nm. Out of the three extracts, Shilajit showed maximum amount of tannic acid (319.33 µg/g) followed by gallic acid (20.76 µg/g) and ferulic acid (37.55 µg/g) (Fig. 2b). Valeriana wallachi also revealed three types of Phenolic acids in which tannic acid (285.90 µg/g) was present in maximal amount whereas gallic acid and caffeic acid were in trace (Fig. 2c) Tannic acid (176.71 µg/g) was also maximum in Tectona grandis followed by gallic acid (17.48 µg/gm) and ferulic acid (7.27 µg/g) which were present in minimal amount. HPLC analysis of the samples revealed wide-variability in their Phenolic acid content (Fig. 2a). The results of the antifungal activity of the various crude extracts were in agreement with the uses of the extract of Tectona grandis, Shilajit and Valeriana wallachi in traditional medicine. The rhizome and aerial parts of the plants appeared to be a potential source of broad spectrum antibiotics.

According to Bauer and Tittel 21 and Springfield et al., 22 , HPLC fingerprinting is the best way for chemical characterization, and therefore this study also established HPLC fingerprint for the active phenolic acids that can act as antioxidant, antifungal, antibacterial and anti-inflammatory. diverse pharmacological activities have been accredited to phenolic acids for instance, gallic acid has anti-inflammatory 23 , antibacterial 24 , caffeic acid with anti inflammatory 13 , antibacterial, antifungal 24 ; ferulic acid with anti-inflammatory 13 , antifungal 25 ; cinnamic acid with antifungal 25 , anthelmintic 15 , natural protection against infections by pathogenic microorganisms 26 ; salicylic acid with antipyretic and antiinflammatory 27 , externally used as antiseptic, antifungal and for various skin conditions 15 .

Validated HPLC method was developed for Valeriana officinalis and this method was validated with respect to system suitability, specificity, accuracy, precision, linerarity and ruggedness 28 . For the quantitative analysis of valerenic acid, HPLC method of Hansel and Schulz was adopted 29 . Validated HPLC method was developed for Valeriana officinalis and this method was validated with respect to system suitability, specificity, accuracy, precision, linearity, and ruggedness 28 . Valepotriates are considered as one of the main groups of compounds responsible for the sedative activity of Valeriana. For the quantitative determination of valepotriates a direct spectrophotometric scanning on TLC plates was compared with HPLC 30 . In Tectona grandis, centrifugal partition chromatography was used to isolate the active compound such as deoxylapachol and tectoquinine that indicated fungal cell wall stress 4 . The actively ingredients are called valepotriates, research has confirmed that these have a calming effect on agitated people, but are also a stimulant in cause of fatique 31 . The leaves of the swietenia macrophylla plant possess antidiabetic, anti-inflammatory, antipyretic, analgesic, antispasmodic, antimicrobial, cathartia and expectorant activities 32 .

Acknowledgements

The authors thank the Institute of Bioengineering and Biological Sciences, Varanasi, India for financing the research project.