The Trypsin Inhibitory Activity of Vigna unguiculata ssp. Sesquipedalis (Hawari Mae) Seeds Grown in Sri Lanka was Evaluated
YN Wickramaratne
Department of Pharmacy, Faculty of Allied Health Sciences, General Sir John Kotelawala Defence University, Sri Lanka.
SAADSD Peiris
Department of Pharmacy, Faculty of Allied Health Sciences, General Sir John Kotelawala Defence University, Sri Lanka.
DMHSK Doranegoda
Department of Pharmacy, Faculty of Allied Health Sciences, General Sir John Kotelawala Defence University, Sri Lanka.
AALT Ampemohotti
Faculty of Graduate Sciences, General Sir John Kotelawala Defence University, Sri Lanka.
FASS Pillay
Faculty of Graduate Sciences, General Sir John Kotelawala Defence University, Sri Lanka.
KDKP Kumari
Department of Basic Sciences, Faculty of Allied Health Sciences, General Sir John Kotelawala Defence University, Sri Lanka.
ARN Silva *
Department of Basic Sciences, Faculty of Allied Health Sciences, General Sir John Kotelawala Defence University, Sri Lanka.
*Author to whom correspondence should be addressed.
Abstract
Aims: This study aimed to screen the entire seed sample of Vigna unguiculata ssp sesquipedalis (Hawari mae) for serine protease inhibitory activity. Additionally, the study explored the impact of various factors such as temperature, pH, metal ions, detergent, oxidizing and reducing agents on the inhibitory activity.
Methodology: A batch of Hawari Mae seeds was obtained from the Plant Genetic Resources Center in Sri Lanka. A concentration gradient of aqueous seed extracts was tested for Serine Inhibitory Activity (SIA). To carry out the SIA assay, seed extracts were mixed with trypsin in phosphoric acid buffer (pH 7.6) and casein was added as the substrate. The samples were then incubated at 37°C and trichloroacetic acid was used to stop the reaction. The supernatants were checked for absorbance at 280 nm and the percentage of serine inhibitory activity (SIA%) was calculated. All experiments were conducted three times to ensure accuracy. The inhibitory activities were tested under various conditions, including different temperatures, pH levels, metal ions, detergents, and oxidizing and reducing agents.
Results: The crude extract containing 10% of the substance showed the highest level of trypsin inhibitory activity at 96.03±0.005%. This particular extract was selected for further analysis. The study revealed that the enzyme inhibition activity was highest at a temperature of 37°C (95.42±0.006%) and a pH of 7.6 (95.71±0.003%). It was observed that all tested metal ions, except Cu2+, significantly reduced (P<0.05) the trypsin inhibitory activity in Hawari mae. The presence of the detergent Triton X-100 did not significantly affect (P>0.05) the trypsin inhibitory activity of Hawari mae. However, the presence of DMSO and dithiothreitol significantly (P<0.05) reduced its activity.
Conclusion: Significant trypsin inhibitory activity was observed in the seed extract of Hawari mae. Further studies on the effects of various physio-chemical parameters can aid in the purification and isolation of trypsin inhibitors.
Keywords: Proteases, trypsin inhibitors, legumes, Vigna unguiculate, Hawari mae