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Sustainable instrumental thin-layer chromatography-based methodology for standardization of neuroprotective components in propolis collected from India

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Abstract

Bee propolis is a heterogeneous mixture of phytoactive metabolites with reported antioxidant, anti-inflammatory, and neuroprotective properties. The chemical composition is highly variable, complex and presents the greatest challenge for standardization. So, the study was aimed at standardization of selected phytochemicals in propolis collected from a region in India (IP). A thin-layer chromatography (TLC) method was developed for five markers with prior evidence of neuroprotective activity using orthogonal detection and subsequently validated as per the International Council for Harmonisation ICH Q2 (R1) guidelines. The methodology was assessed for greenness using different metrices and then applied to marketed samples. Furthermore, antioxidant and anticholinesterase activity were evaluated directly using TLC‒bioautography technique. The method was developed using toluene‒ethyl acetate‒formic acid (7.4:2.6:0.5, V/V) as the mobile phase, with RF values of 0.235, 0.353, 0.552, 0.606, and 0.655 for luteolin, p-coumaric acid, chrysin, galangin, and pinocembrin, respectively. The optimized method was found to be precise (%RSD ≤ 2.0%), accurate (90‒110%), linear over the concentration ranges (R2 ≥ 0.995), sensitive and robust. Pinocembrin (2.30 ± 0.12% w/w) and galangin (5.78 ± 0.30% w/w) were found in the highest concentration. TLC‒bioautography analysis confirmed the contribution of the selected phenolics in anticholinesterase and antioxidant activity. The analytical eco-score value of 76, analytical greenness calculator (AGREE) score of 0.62 and other assessed analytical pictograms indicated the methodology developed had low environmental impact. Overall, a simple, rapid, precise, and green TLC analytical methodology has been developed for IP for quality control and preliminary screening of neuroprotective activity.

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Abbreviations

IP:

Indian propolis

TLC:

Thin-layer chromatography

UV–Visible:

Ultraviolet visible

MS:

Mass spectrometry

ESI:

Electrospray ionization

AChE:

Acetylcholinesterase

Aβ:

Amyloid-beta

US FDA:

US Food and Drug Administration

λmax :

Lambda max

R F :

Retardation factor

ICH:

International Council for Harmonisation

NEMI:

National Environmental Methods Index

NFPA:

National Fire Protection Association

GAPI:

Green Analytical Procedure Index

AGREE:

Analytical greenness calculator

NP:

Natural product

ASR:

Anisaldehyde sulphuric acid reagent

DPPH:

2,2-Diphenyl-1-picryl hydrazyl

DTNB:

5,5′-Dithiobis-2-nitrobenzoic acid

ATCI:

Acetylthiocholine iodide

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Acknowledgements

The authors express their gratitude to the All-India Council for Technical Education (AICTE) for granting Sandeep Sankaran financial support via the AICTE Doctoral Fellowship Scheme (ADF). The authors would like to thank Mr. Amol Kadam, Dr. Sangram Patil, and the Centre of Food Testing Laboratories, Pune for assistance in MS work.

Funding

This research did not receive any specific grant from funding agencies for carrying out this work.

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Authors and Affiliations

  1. Department of Quality Assurance Techniques, Poona College of Pharmacy, Bharati Vidyapeeth (Deemed to be) University, Pune, Maharashtra, 411038, India

    Sandeep Sankaran & Anushka Bakore

  2. Department of Pharmaceutical Chemistry, Poona College of Pharmacy, Bharati Vidyapeeth (Deemed to be) University, Pune, Maharashtra, 411038, India

    Rahul Dubey & Sathiyanarayanan Lohidasan

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  1. Sandeep Sankaran
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Correspondence to Sathiyanarayanan Lohidasan.

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Sankaran, S., Dubey, R., Bakore, A. et al. Sustainable instrumental thin-layer chromatography-based methodology for standardization of neuroprotective components in propolis collected from India. JPC-J Planar Chromat 37, 233–245 (2024). https://doi.org/10.1007/s00764-024-00307-x

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