HPTLC and HPTLC-coupled Bioassays for Bioprocess Control of Medicinal Plant In Vitro Cultures from the Balkan region

An in vitro germplasm collection has been established for the delivery of biologically active compounds of medicinal and aromatic plants of the Balkans. Representatives of the species Pulsatilla montana, Pulsatilla slaviankae, Pulsatilla halleri and Clinopodium vulgare have been subjected to a media optimization study by means of alteration of plant growth regulators treatments. The aim of this study was to demonstrate the suitability of HPTLC phytochemical fingerprints combined with the assessment of antioxidant (DPPH and β-Caroteen/Linolenic Acid Bleaching HPTLC assay) as well as optimized Xanthine Oxidase [1] and Lipase [2] inhibiting HPTLC assays (enzymatic bioautography). For Pulsatilla species, a mixture of ethyl acetate, formic acid, acetic acid and water (68/8/6/18) and for Clinopodium vulgare a slight modification (68/6/8/18) has been identified for optimal separation. Chlorogenic acid and rutin were applied as references. Total polyphenolic content [3] of the cultures was assessed spectrophotometrically as a sum parameter for one class of target secondary metabolites. Phytochemical fingerprints with adequate derivatisation (Flavonoids and Anisaldehyde-Sulfuric acid) demonstrate visual differences in number and nature of in vitro formed substances as well as differences in colour fluorescence at 366nm or visible zones in white light. Compared to control vouchers from ex situ samples exhibiting also yellow zones, in vitro cultures of Pulsatilla sp. show only a large number of blue phenolcarbonic acids zones. In vitro cultures of C. vulgare exhibit also two faint yellow zones in in vitro samples, one at the Rf value of rutin. Inhibition zones of oxidant and enzyme reactions corresponding to phytochemical zones at the comparable Rf heights render valuable rapid and cost effective data and facilitates the control and optimization of the plant in vitro culture processes. C. vulgare culture showed compounds with Xanthine Oxidase and Lipase inhibition. In conclusion, the adapted HPTLC separation for Pulsatilla sp and Clinopodium vulgare combined with rapid activity screening demonstrate the potential of HPTLC and its hyphenated bioassay methods as a relatively easy and feasible in-process control tool. The obtainable data is the basis for further optimization.

Acknowledgements: Swiss National Science Foundation in the Framework in the Bulgarian-Swiss Research Program (BSRP, grant No. IZEBZO_142989; DO2 – 1153); World Federation of Sciences – Bulgarian National Scholarship Programme for young researchers.

Keywords: HPTLC, bioprocess control, bioassay-coupled-HPTLC, bioautography, in vitro plant culture.

References:

[1] Ramallo IA, Zacchino SA, Furlan RLE. A Rapid TLC Autographic Method for the Detection of Xanthine Oxidase Inhibitors and Superoxide Scavengers. Phytochem Anal 2006; 17: 15 – 19

[2] Hassan AMS. TLC Bioautographic Method for Detecting Lipase Inhibitors. Phytochem Anal 2012; 23: 405 – 407

[3] Saeed N, Khan MR, Shabbir M. Antioxidant activity, total phenolic and total flavonoid contents of whole plant extracts Torilis leptophylla L BMC Complement Altern Med 2012; 12: 221 – 233