3 Biotech

, 9:37 | Cite as

Effect of different extraction techniques on total phenolic and flavonoid contents, and antioxidant activity of betelvine and quantification of its phenolic constituents by validated HPTLC method

  • Suryasnata Das
  • Asit Ray
  • Noohi Nasim
  • Sanghamitra Nayak
  • Sujata MohantyEmail author
Original Article


The objective of the study is to analyze the effect of different extracting methods on the polyphenolic content and antioxidant activities in Piper betle leaves. In the present research, P. betle leaf extract was prepared by sonication, Soxhlet and maceration methods using acetone (100%, v/v). The efficiency of the extraction methods was estimated by quantifying the total phenolic content (TPC) by the Folin–Ciocalteu method and total flavonoid content (TFC) by AlCl3 colorometric methods, and antioxidant power of the various extracts was determined by DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical scavenging assay. Thin-layer chromatography (TLC) bioautography was carried out to identify antioxidants, and their amount was determined by the newly developed high-performance thin-layer chromatography (HPTLC) method. DPPH free radical scavenging capacity of the different extracts from strongest to weakest was as follows: ascorbic acid (4.27 µg/mL) > sonication (5.35 µg/mL) >, maceration (5.53 µg/mL)>, soxhlet extraction (5.83 µg/mL). Same trend was also observed for the ABTS radical scavenging capacity. Similarly, findings of this study also showed that sonication extract possessed highest phenolic and flavonoid contents followed by maceration and Soxhlet extraction. In addition, important bioactive phenolic constituents which contribute largely towards antioxidant potential such as eugenol and eugenol acetate were quantified using HPTLC (high-performance thin-layer chromatography) method. The average percent recovery of eugenol and eugenol acetate was found to be 97.28% and 98.04%, respectively. The LOD (limit of detection) and LOQ (limit of quantification) for eugenol were 5 and 15 ng/spot, whereas that of eugenol acetate were 10 and 30 ng/spot. The HPTLC densitometric determination also supported the results of antioxidant assays by revealing the presence of higher amount of identified antioxidants in sonication followed by maceration and Soxhlet extraction. The developed HPTLC chromatogram profile may be used as a reference for the standardization of P. betle leaf extracts.


HPTLC DPPH Eugenol Phenolic Flavonoid 



The authors are grateful to Prof. Dr. S.C. Si, Dean, Centre for Biotechnology, and Prof. Dr. M.R. Nayak, President, Siksha ‘O’ Anusandhan (Deemed to be University), Odisha, India, for providing facilities and encouragement throughout.


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Copyright information

© King Abdulaziz City for Science and Technology 2019

Authors and Affiliations

  • Suryasnata Das
    • 1
  • Asit Ray
    • 1
  • Noohi Nasim
    • 1
  • Sanghamitra Nayak
    • 1
  • Sujata Mohanty
    • 1
    • 2
    Email author
  1. 1.Centre for Biotechnology, School of Pharmaceutical SciencesSiksha ‘O’ Anusandhan (Deemed to be University)BhubaneswarIndia
  2. 2.PG Department of BiotechnologyRama Devi Women’s UniversityBhubaneswarIndia

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