HPTLC Method for Propolis Quality and Bioactivity Screening (2024)

Introduction

Bee propolis is a valuable yet often neglected therapeutic resource made up of a combination of plant resins gathered during foraging, mixed with the bees’ own salivary secretions deposited in the beehives. The chemical composition is highly heterogeneous and depends on the vegetation in and around the hive, climatic conditions, and the bee species. Various analytical techniques have been used to evaluate the quality of propolis, including the use of high-end instruments in combination with chemometric modeling for deriving the complete chemical profile. However, these methods are costly and hard to replicate in quality control labs. A more feasible approach is to standardize based on markers that correlate with the specific biological activity of that propolis variant. The present study was therefore designed to focus on fingerprint profiling for identifying the propolis type, screening for the antioxidant and anticholinesterase components directly on the plate through a new developed, validated and sustainable HPTLC methodology.

To identify the propolis type, a simplified, rapid, low-cost, low-environmental impact, and easily adoptable analytical methodology was developed, extending to the standardization of selected neuroprotective components in Indian propolis. The versatility of HPTLC, with various derivatizing reagents and orthogonal detection capabilities, allows for increased applications. With the advent of thin-layer chromatography-effect directed analysis, it enables direct screening on the TLC plate, establishing preliminary evidence of the biological activities. Thus, this HPTLC method is valuable for rapid chemical profiling and simultaneous screening of antioxidant and anticholinesterase activities of Indian propolis. Also, educating beekeepers about its medicinal value can help them generate additional revenue.

Standard solutions

Stock solutions (1.0mg/mL) are prepared in methanol, except dimethyl sulfoxide was used for initial solubilization of chrysin. The subsequent working solutions are prepared in methanol, i.e., chrysin (0.10mg/mL), p-coumaric acid (0.05mg/mL), pinocembrin (0.10mg/mL), luteolin (0.10mg/mL), and galangin (0.20mg/mL).

Sample preparation

Indian propolis extracts and the marketed samples (2.0mg/mL or 3.0mg/mL) are prepared by weighing 20.0mg or 30.0mg and dissolving in 10.0mL of ethanol. The samples are sonicated, centrifuged and filtered before TLC analysis.

Chromatogram layer

HPTLC plates silica gel 60 F₂₅₄ (Merck), 20x10cm are used.

Sample application

1.0-10.0μL of standard solutions (7-point calibration) and 2.0 and 5.0μL of sample solutions are applied as bands with the Linomat5 (with N2). Plate layout: 15 tracks, band length 6.0mm, distance from left plate edge 15.0mm, track distance 11.4mm, distance from the lower edge 8.0mm.

Chromatography

Plates are developed in the twin-trough chamber with chamber saturation for 30min (with filter paper) and development with toluene ‒ ethyl acetate ‒ formic acid 74:26:5(V/V) to the migration distance of 80mm (from the lower edge), followed by drying for 5min.

Post-chromatographic derivatization

The developed plate is first heated at 110°C for 2min and then placed in the immersion device containing Natural product reagent (NP or 2-aminoethyl diphenylborinate – 1% (W/V) in ethyl acetate). The developed plate is immersed in anisaldehyde sulfuric acid reagent (ASR – prepared fresh by combining 1.0mL p-anisaldehyde with 20.0mL glacial acetic acid, followed by 170mL methanol and 10.0mL concentrated sulfuric acid) and then heated at 100°C for 5min. The developed plate is immersed in Ferric chloride solution (FeCl3 – 2 %(W/V) in methanol) and then heated for 2min at 110°C.

Note: The derivatization was conducted on three different developed plates.

Post-chromatographic bioautography

The developed plate is immersed into a 2,2-diphenyl-1-picryl hydrazyl solution (DPPH - 0.25 %(W/V) in methanol), stored in the dark for 30min. The yellow zones captured against purple background are an indicator of antioxidant components when visualized in white light. The Ellman assay protocol was used wherein the developed plate is first immersed in a solution of 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) and acetylthiocholine iodide (ATCI) (1mM DTNB and 1mM ATCI in buffer A) until the plate was saturated, dried for 5min and then around 3-4mL of acetylcholinesterase enzyme solution (Electrophorus electricus – AChE - 3U/mL) is sprayed onto the plate. The white band on the plate is an indicator of acetylcholinesterase inhibition.

Documentation

Images of the plate are captured with the TLC Visualizer2 in UV254nm, UV366nm, and white light.

Densitometry

Absorbance measurement is performed with the TLC Scanner3 and visionCATS at 268nm (chrysin), 297nm (p-coumaric acid and pinocembrin) and 352nm (luteolin and galangin), slit dimension 5.00mmx0.45mm, scanning speed 20mm/s, spectra scanned from 200 to 450nm.

Mass spectrometry

The selected bands are eluted with the TLC-MS Interface2 at a flow rate of 0.5mL/min with methanol (with 0.1 % formic acid) into an Electrospray ionization (ESI)-Triple Quadruple Mass Analyzer (Agilent 6460) in the negative ionization mode.

Results and discussion

The HPTLC fingerprint image of the various propolis extracts is shown, and the profiles are key indicators of the diversity in vegetation across different regions. The sample coded HAR was mainly of ‘O-type’ propolis due to the presence of flavonoids like chrysin, galangin, pinocembrin, as well as non-flavonoids like p-coumaric acid, matching the characteristic bands of the standard when derivatized with various reagents. Interestingly, the applicability of the method on two marketed products presented a similar fingerprint to that of the HAR extract.

The optimized method is found to be precise (%RSD ≤ 2.0 %), accurate (90‒110 %), linear over the concentration ranges (r2 ≥ 0.995), sensitive and robust resulting in the R_F values of 0.235, 0.353, 0.552, 0.606, and 0.655 for luteolin, p-coumaric acid, chrysin, galangin, and pinocembrin, respectively. Pinocembrin (2.30 ± 0.12 % W/W) and galangin (5.78 ± 0.30 % W/W) are found in the highest concentrations in the HAR sample. The m/z values of the molecular ion and fragment ions from the isolated sample bands matched those of the standards, further confirming the identity of the peaks. The bands with R_F values corresponding to chrysin, galangin, and pinocembrin showed strong antioxidant activity, as indicated by bright yellow zones against a purple background, while the white bands in the extract fingerprint that appeared along the plate following the Ellman’s assay are indicative of acetylcholinesterase inhibitors.

Thus, the developed analytical method with orthogonal capabilities can be universally applied to different propolis extracts and formulated propolis products as a quick screening method for fingerprint and neuroprotective profiling.

Acknowledgements

The authors would like to express their gratitude to Poona College of Pharmacy (Bharati Vidyapeeth Deemed to be University), Central Bee Research and Training Institute (CBRTI, Pune), All-India Council for Technical Education (AICTE), Anchrom Enterprises Pvt. Ltd. (Mumbai), Bee Basket Enterprises Pvt. Ltd and the Centre of Food Testing Laboratories, (Pune) for all the assistance and support in the work.

Literature

[1] Sankaran, S. et al. (2024) J Planar Chromat 37 (3), 233–245

[2] Bankova, V. et al. (2019) J Apic Res 58, 1–49

[3] Sankaran, S. et al. (2023) J Biol Active Prod Nat 13, 76–93.

Further information is available in the article published “Sustainable instrumental thin-layer chromatography-based methodology for standardization of neuroprotective components in propolis collected from India” J Planar Chromat 37, 233–245 (2024). https://doi.org/10.1007/s00764-024-00307-x or on request from the authors.

Contact

Sandeep Sankaran, Department of Quality Assurance Techniques, Poona College of Pharmacy, Bharati Vidyapeeth (Deemed to be) University, Pune, Maharashtra 411038, India, sandeepsss1992@gmail.com