Analytical and Bioanalytical Chemistry

, Volume 409, Issue 14, pp 3645–3655 | Cite as

Enzyme-assisted supercritical fluid extraction: an alternative and green technology for non-extractable polyphenols

  • Muhammad Mushtaq
  • Bushra Sultana
  • Sumia Akram
  • Farooq Anwar
  • Ahmad Adnan
  • Syed S. H. Rizvi
Research Paper


This contribution proposes an enzyme-assisted eco-friendly process for the extraction of non-extractable polyphenols (NEPPs) from black tea leftover (BTLO), an underutilized tea waste. BTLO hydrolyzed with various enzyme formulations was extracted using supercritical carbon dioxide and ethanol as co-solvent (SC-CO2 + EtOH). A conventional solvent extraction (CSE) was performed using EtOH + H2O (80:20, v/v) for comparison purposes. The results revealed that hydrolysis of BTLO with 2.9% (w/w) kemzyme at 45 °C and pH 5.4 for 98 min improved the liberation of NEPPs offering 5-fold higher extract yield (g/100 g) as compared with non-treated BTLO. In vitro antioxidant evaluation and LC-MS characterization of extracts revealed the presence of phenolic acids (mainly caffeic and para-coumaric acid) of high antioxidant value. Scanning electron micrograph of the hydrolyzed BTLO samples indicated noteworthy changes in the ultrastructure of BTLO. Moreover, polyphenol extracts obtained by SC-CO2 + EtOH extraction were found to be cleaner and richer in polyphenols as compared to CSE. The devised enzyme-assisted SC-CO2 + EtOH extraction process in the present work can be explored as an effective biotechnological mean for the optimal recovery of antioxidant polyphenols.

Graphical abstract

Enzymatic pretreatment can effectively liberate non-extractable polyphenols (NEPPs) while hydrolyzing the cellulosic and hemicellulosic framework of black tea left overs (BTLO)


BTLO Green extraction Non-extractable polyphenols Enzymatic hydrolysis Scanning electron micrograph SC-CO2 



The work presentment in this paper was financially sponsored by Higher Education Commission (HEC), Pakistan, under the International Research Support Initiative Program (IRSIP); PIN: IRSIP 24 PS 17.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Supplementary material

216_2017_309_MOESM1_ESM.pdf (397 kb)
ESM 1 (PDF 396 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Muhammad Mushtaq
    • 1
    • 2
    • 3
  • Bushra Sultana
    • 1
  • Sumia Akram
    • 1
    • 2
  • Farooq Anwar
    • 4
  • Ahmad Adnan
    • 3
  • Syed S. H. Rizvi
    • 2
  1. 1.Department of ChemistryUniversity of AgricultureFaisalabadPakistan
  2. 2.Food ScienceCornell UniversityIthacaUSA
  3. 3.Department of ChemistryGC UniversityLahorePakistan
  4. 4.Department of ChemistryUniversity of SargodhaSargodhaPakistan

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