Sandeep Sankaran*, PhD Scholar from the Department of Quality Assurance Techniques at Poona College of Pharmacy, BVDU, carried out his research work focusing on the systematic evaluation of the chemical profile and its correlation to neuroprotective activity for Indian bee propolis. The research team under the supervision of Dr Sathiyanarayanan worked comprehensively on deriving the chemical profile of Indian propolis extracts through the HPTLC fingerprinting methodology developed inhouse, extending to marker-based standardization and HPTLC-effect-directed analysis.
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 (1.0 mg/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 (0.10 mg/mL), p-coumaric acid (0.05 mg/mL), pinocembrin (0.10 mg/mL), luteolin (0.10 mg/mL), and galangin (0.20 mg/mL).
Sample preparation
Indian propolis extracts and the marketed samples (2.0 mg/mL or 3.0 mg/mL) are prepared by weighing 20.0 mg or 30.0 mg and dissolving in 10.0 mL of ethanol. The samples are sonicated, centrifuged and filtered before TLC analysis.
Chromatogram layer
HPTLC plates silica gel 60 F254 (Merck), 20 x 10 cm are used.
Sample application
1.0-10.0 μL of standard solutions (7-point calibration) and 2.0 and 5.0 μL of sample solutions are applied as bands with the Linomat 5 (with N2). Plate layout: 15 tracks, band length 6.0 mm, distance from left plate edge 15.0 mm, track distance 11.4 mm, distance from the lower edge 8.0 mm.
Chromatography
Plates are developed in the twin-trough chamber with chamber saturation for 30 min (with filter paper) and development with toluene ‒ ethyl acetate ‒ formic acid 74:26:5 (V/V) to the migration distance of 80 mm (from the lower edge), followed by drying for 5 min.
Post-chromatographic derivatization
The developed plate is first heated at 110 °C for 2 min 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 1.0 mL p-anisaldehyde with 20.0 mL glacial acetic acid, followed by 170 mL methanol and 10.0 mL concentrated sulfuric acid) and then heated at 100 °C for 5 min. The developed plate is immersed in Ferric chloride solution (FeCl3 – 2 % (W/V) in methanol) and then heated for 2 min 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 30 min. 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) (1 mM DTNB and 1 mM ATCI in buffer A) until the plate was saturated, dried for 5 min and then around 3-4 mL of acetylcholinesterase enzyme solution (Electrophorus electricus – AChE – 3 U/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 Visualizer 2 in UV 254 nm, UV 366 nm, and white light.
Densitometry
Absorbance measurement is performed with the TLC Scanner 3 and visionCATS at 268 nm (chrysin), 297 nm (p-coumaric acid and pinocembrin) and 352 nm (luteolin and galangin), slit dimension 5.00 mm x 0.45 mm, scanning speed 20 mm/s, spectra scanned from 200 to 450 nm.
Mass spectrometry
The selected bands are eluted with the TLC-MS Interface 2 at a flow rate of 0.5 mL/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 RF 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 RF 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.
