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Isolation and characterization of α-amylase inhibitor from Leucas aspera (Willd) Link: α-amylase assay combined with FPLC chromatography for expedited identification

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Abstract

The aim of the present study was to isolate and characterize a proteinaceous α-amylase inhibitor from the whole plant extract of Leucas aspera (Willd) Link. The proteins were further purified by fast and reliable ion-exchange chromatography. A ~ 28 kDa protein from L. aspera inhibited the activity of fungal α-amylase by 90% at 80:1 (inhibitor:enzyme) ratio. The inhibition activity was examined in various α-amylases and its enhanced inhibition activity was witnessed. The activity of the inhibitor on α-amylase was stable and high at pH 6–7 and at temperatures of 30–50 °C. The high-resolution α-amylase inhibition assay/FPLC-MS-SPE platform allowed identification of 28 kDa protein with high purification fold as the α-amylase inhibitor in L. aspera and peptides were matched with highest score of alpha-amylase/trypsin inhibitor of Zea mays. In conclusion, results here obtained suggested that the primary metabolites (proteins) in L. aspera are mainly responsible for its versatile biological and pharmacological activities.

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Abbreviations

FPLC:

Fast protein liquid chromatography

BSA:

Bovine serum albumin

pI:

Isoelectric point

CBB:

Coomassie brilliant blue

MS:

Mass spectrometry

PMF:

Peptide mass fingerprints

SPE:

Solid-phase extraction

MWCO:

Molecular weight cutoff

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Acknowledgement

This work was supported by a grant and facility from SRM University, Chennai, India. We thank Dr. M. Vairamani, Dean, School of Bioengineering, SRM University, Chennai, India for his timely help and we also thank Dr. Jagadeshwar Reddy, CSIR, Lucknow, India for his assistance.

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    Authors and Affiliations

    1. Department of Biotechnology, School of Bioengineering, SRM University, Kattankulathur, Tamil Nadu, 603203, India

      Christopher Meera, Sakthivel Meenakumari & Raman Pachaiappan

    2. Graduate Institute of Environmental Engineering, National Central University, Chung-Li, Taoyuan County, 320, Taiwan

      Munusamy Thirumavalavan

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    1. Christopher Meera
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    2. Sakthivel Meenakumari
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    Correspondence to Munusamy Thirumavalavan or Raman Pachaiappan.

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    Munusamy Thirumavalavan and Raman Pachaiappan have contributed equally to this work.

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    Meera, C., Meenakumari, S., Thirumavalavan, M. et al. Isolation and characterization of α-amylase inhibitor from Leucas aspera (Willd) Link: α-amylase assay combined with FPLC chromatography for expedited identification. J. Plant Biochem. Biotechnol. 26, 346–355 (2017). https://doi.org/10.1007/s13562-017-0397-7

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