An α‐amylase inhibitor (EC 3.2.1.1) was purified by buffer extraction, ammonium sulfate fractionation, CM‐cellulose, and sephadex G‐75 chromatography from the soaked seeds of Mucuna pruriens. The molecular weight determined by gel permeation chromatography on Sephadex G‐100 and SDS‐PAGE, both in the presence and absence of 2‐mercaptoethanol, was found to be 27.24 kDa and 25.6 kDa, respectively. The purified Mucuna pruriens amylase inhibitor showed a specific inhibitor activity of 61.18, fold purity of 36.68, and the yield obtained was 14.01%. The purified amylase inhibitor was found to be heat‐stable and retained 80.50% activity at 65°C. Inhibitor was found to have pH optima of 6.9. Hundred percent zone of inhibition was observed when added on the plated organisms of purified inhibitor. Purified amylase inhibitor was found to inhibit the activity of human salivary α‐amylase. Inhibitory activity of α‐amylase inhibitor against mammalian amylases could suggest its potential in treatment of diabetes and cure of nutritional problems, which results in obesity. Practical applications Purified amylase inhibitor was found to inhibit the activity of human salivary α‐amylase. The potential of this inhibitory activity from α‐amylase inhibitors, especially in the mammalian α‐amylase, could play an important role in the management of nutritional and diabetes‐related disorders. Mucuna, an underutilized legume found in tropical region and also cultivated as food by various tribal's in Asia and Africa can be used as a potential source for extraction of these beneficiary protease inhibitors, which in turn finds its applications in various human therapeutic and/or disorder management.
A Bowman-Birk protease, i.e., Mucuna pruriens trypsin inhibitor (MPTI), was purified from the seeds by 55.702-fold and revealed a single trypsin inhibitor on a zymogram with a specific activity of 202.31 TIU/mg of protein. On sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) under non-reducing conditions, the protease trypsin inhibitor fraction [i.e., trypsin inhibitor non-reducing (TINR)] exhibited molecular weights of 74 and 37 kDa, and under reducing conditions [i.e., trypsin inhibitor reducing (TIR)], 37 and 18 kDa. TINR-37 revealed protease inhibitor activity on native PAGE and 37 and 18 kDa protein bands on SDS–PAGE. TINR-74 showed peaks corresponding to 18.695, 37.39, 56.085, and 74.78 kDa on ultra-performance liquid chromatography (UPLC) coupled with electrospray ionization/quadrupole time-of-flight-mass spectrometry (ESI/QTOF-MS). Similarly, TINR-37 displayed 18.695 and 37.39 kDa peaks. Furthermore, TIR-37 and TIR-18 exhibited peaks corresponding to 37.39 and 18.695 kDa. Multiple peaks observed by the UPLC-ESI/QTOF analysis revealed the multimeric association, confirming the characteristic and functional features of Bowman-Birk inhibitors (BBIs). The multimeric association helps to achieve more stability, thus enhancing their functional efficiency. MPTI was found to be a competitive inhibitor which again suggested that it belongs to the BBI family of inhibitors, displayed an inhibitor constant of 1.3 × 10–6 M, and further demonstrates potent anti-inflammatory activity. The study provided a comprehensive basis for the identification of multimeric associates and their therapeutic potential, which could elaborate the stability and functional efficiency of the MPTI in the native state from M. pruriens.
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