Research at UAF - Novel Bioactive Compounds (Peptides/ proteins) from Potensive Medicinal Plants under Stress Conditions
Principal Investigator with phone, fax and email address:
Dr. Muhammad Shahid, 041- 9200161-9/3309 Cell # 0333 6629271
Fax # +92 41 9200764,
Duration: Three year
Cost: 6 Million
Funding Agency: HEC

Progress Reports

Antifungal activity of N. sativa seedling of fungal stress against A. niger at dpi 1 to 6 by disc diffusion method

Abstract: Ultimate objective of the present project is to find novel antifungal compounds including peptides and proteins with commercial potential in pharmaceutical and agricultural utility as biocontrol.

  1. To explore the local species of potential medicinal plants with bioactivity (antifungal, antibacterial, antioxidant and antitermitic etc) under stress conditions for the induction of bioactive compounds.
  2. Characterization of the “Lead Compounds” including peptides/proteins.

Methodology: Antifungal peptides and proteins hold promise in combating fungal and bacterial infections. Local species of potential medicinal plants will be screened for bioactive properties. Extracts of the following plants will be investigated by disc diffusion and microtiter plate reader based method and antitermitic activity byAntitermitic sugarcane strip bioassay under different abiotic and biotic stress after germination  at laboratory scale. Complete biochemical characterization will be screened  for the optimal induction of antimicrobial compounds. Extraction will be prepared in buffer different chromatographic and electrophoretic carried out to check the extent of purity and mass. Characterization like proteinase K, trypsin treatment, heat denaturation, effect of ionic concentration and effect of pH on the peptide/protein will be performed. The homology of the sequenced peptide will be compared by BLAST search engine and also the prediction of the functional class of the novel plants proteins by a statistical learning method by using web SVMProt.

Antibacterial activity of N. sativa seedling of salt stress (100 mM) against S.aureus at dpi 1 to 6 by disc diffusion method

Results and Discussion:
Biochemical analysis of  of Nigella sativa seedlings germinated under biotic and abiotic stress
Different biochemical parameters were determined in seed extracts of the plants to find out inherent potential of the medicinal plants under stress conditions induced by fungus and salt. The results of different metabolically important enzymes and compounds proteases, peroxidase, catalase, superoxide dimutase, amylase, total soluble sugars, ascorbic acid and  antioxidant compounds (total phenolics content) showed a wide change in profile of enzymes.
The minimum inhibitory concentration (MIC) of Nigella was studied by microdilution method. The sample extracts of seedlings both of fungal and salt stress in the dpi 4 showed maximum activity. Nigella sativa showed strong antifungal activity against A. niger, strong antibacterial activity against S. aureus then E. coli. The lowest MIC value of Nigella sativa against A. niger is 3.53 mg/mL and highest MIC value is 226.25 mg/mL. The lowest MIC value of Nigella sativa against S. aureus is 3.53 mg/mL and highest MIC value is 56.56 mg/mL while against E. coli  is 3.53 mg/mL and highest MIC value is 226.25 mg/mL. The antimicrobial activity of organic and aqueous extracts from fruits, leaves and roots of Tribulus terrestris L., was examined against 11 species of pathogenic and non-pathogenic microorganisms: Staphylococcus aureus, Bacillus subtilis, Bacillus cereus, Corynebacterium diphtheriae, Escherichia coli, Proteus vulgaris, Serratia marcescens, Salmonella typhimurium, Klebsiella pneumoniae, Pseudomonas aeruginosa and Candida albicans using microdilution method in 96 multiwell microtiter plates. The most active extract against both Gram-negative and Gram-positive bacteria was ethanol extract from the fruits with a minimal inhibitory concentration (MIC) value of 0.15 mg/mL against B. subtilis, B. cereus, P. vulgaris and C. diphtheriae. In addition, the same extract from the same plant part demonstrated the strongest antifungal activity against C. albicans with an MIC value of 0.15 mg/mL. (Firas et al., 2008). The lowest MIC value of Nigella sativa against S. aureus is 3.66 mg/mL and highest MIC value is 58.62 mg/mL while against E.coli  is 3.66 mg/mL and highest MIC value is 234.00 mg/mL The antidermatophyte activity of ether extract of Nigella sativa seed and its active principle thymoquinone was tested against eight species of dermatophytes. The diameter of fungal colonies and the percentage inhibition of the fungal growth at each dilution were determined, taking those of the controls as 100%. The MICs of the ether extract of Nigella sativa and thymoquinone were between 10 and 40 and 0.125 and 0.25 mg/mL, respectively, while those of griseofulvin ranged from 0.00095 to 0.0155 mg/mL. These results denote the potentiality of Nigella sativa as a source for antidermatophyte drugs and support its use in folk medicine for the treatment of fungal skin infections  ( Aljabre et al., 2005).
Conclusions: The present study showed antifungal and antibacterial activity and also strong antioxidant activity. The most promising antimicrobial properties indicating the potential for discovery of antibacterial and antifungal principles. Further phytochemical studies are required to determine the types of compounds responsible for the antibacterial and antifungal effects of these species. The results also indicate that scientific studies carried out on medicinal plants having traditional claims of effectiveness might warrant fruitful results.
The inductions played an important role in the satire up the gene of bioactive peptides/proteins by biotic and abiotic stress. Further studies are recommended for exploration of these plants.
Recommendations: The biotic and abiotic stress stire up many biomolecules by cascade mechanism. The bioactivity also increased by such factors. So it is recommended if we want to hyperexpress these bioactive compound especially protein peptides  and other battery of lead compound will must be produced under stress condition due to higher express under these factors.