Bioautography is mainly known as a research strategy hyphenated with planar chromatography techniques aimed to detect bioactive chemical classes characterizing plant extracts, pointing out their efficacy directly on the chromatographic support used for their separation. Therefore,thin layer chromatographic (TLC) bioautography gives the opportunity to point out at the same time a semi-quantitative chemical fingerprinting and the bioactivity of the main involved chemical classes in plant extracts (1). Our research group set uppharmaceutical biology studiesdeveloping this particular techniqueemploying high performing thin layer chromatography plates (HP-TLC) to obtain the best separation of the chemical classes together with the best evidence of their possible biological activity,driving further in-depth investigations toward a more focused chemical identification and quantification, and a wider biological activity profile of officinal plant extracts. We optimizedHP-TLC bioautographyfor antimicrobial (antibacterial and antifungal) and antioxidant properties [DPPH, 1,1-diphenyl-2-picrylhydrazyl; and ABTS, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) assays]of different plant extractsevidencingthe chemical classes really involved in the bioactivity (2).Our research strategy of plant extracts is planned to have HP-TLC bioautography as preliminary data giving us the opportunity 1. to point out the chemical compounds with antibacterial, antifungal, antioxidant capacity; 2. to focus on the chemical isolation and identification of fractions characterized by specific bioactive molecules; 3. to check biological capacities ofpure single compounds, or combinations, assaying possible synergic interactions; 4. to explore other biological capacities -i.e. cytotoxic, mutagen and mutagen-preventive activity, etc. - related to those preliminary evaluated through HP-TLC bioautography. In light of these premises, some researches in progress pointing out the role of HP-TLC bioautography in the pharmaceutical biology profile of our studies are shown below: -Essential oils obtained from plant crude drugs of different geographical origin (for e.g. Cryptocaryamassoia, Lauraceae, from Indonesian regions; Piper aduncuum, Piperaceae; Croton lechleri, Euphorbiaceae from Amazonian Ecuador). Cryptocaryamassoia(Lauraceae) essential oil evidenced on HP-TLC platesrelevant antimicrobial activity related tomassoia lactones,as single pure compounds,versus Gram negative strains (Klebsiellaoxytoca) andyeasts (Candida albicans). These data suggested to test pure compounds mixtures to evaluate their possible synergic potential. The result of combining benzylbenzoate and benzyl salicatewas a specific efficacy against Gram positive bacteria (Enterococcus faecalis). The evidences will also drive the research towards in-depth investigations (cytotoxicity, mutagenicity) of the active isolated compounds and their different qualitative and quantitative combinations. The case of Piper aduncum (Piperaceae) essential oil, characterized by high abundance of dillapiole(45.92%), highlighted this compound as the main responsible of highly selective antibacterial activity against Staphylococcus aureus.Amazonian Croton lechleri essential oil evidenced antibacterial activity against Escherichia colion HP-TLC mainly due to sesquicineole (17.29%).Caryophyllene oxide (1.24%) and 1,10-di-epi-cubenol (4.75%) showed instead the best antioxidant capacity with HP-TLC-DPPH assay. -126 different plant extracts from industrial (agro-food) and agricultural by-products investigated through HP-TLC bioautography for achieving preliminary suggestions about their possible role as source of bioactive molecules. In this case ethanol, chloroform and acetone extracts have been assayed for matching chemical fingerprinting and biological activity, giving preliminary but clear suggestions about the most promising extracts in terms of bioactivity potential and composition. Among the bioactive extracts, those most interesting were ethanolones obtained from agro-food by-products ofMalusdomestica and Juglansregia, and from agricultural wastes of Allium sativum. In conclusion, HP-TLC bioautographyrepresent an important tool to quickly join on chromatographic supporta preliminary chemical fingerprinting and important biological evidences of officinal plant extracts. 1) I.M. Choma, E.M. Grzelak (2011) J. Chromatogr. A, 1218, 2684–2691 2) D. Rossi, A. Guerrini, G. Paganetto, G. Bernacchia, F. Conforti, G. Statti, S. Maietti, I. Poppi, M. Tacchini, G. Sacchetti (2013) FoodChem, 139, 439–447
HP-TLC bioautographic assay as a preliminary research tool to match chemical and biological properties of officinal plant extracts
GUERRINI, Alessandra;TACCHINI, Massimo;GRANDINI, Alessandro;SPAGNOLETTI, Antonella;MARESCA, Immacolata;ROSSI, Damiano;MAIETTI, Silvia;SACCHETTI, Gianni
2014
Abstract
Bioautography is mainly known as a research strategy hyphenated with planar chromatography techniques aimed to detect bioactive chemical classes characterizing plant extracts, pointing out their efficacy directly on the chromatographic support used for their separation. Therefore,thin layer chromatographic (TLC) bioautography gives the opportunity to point out at the same time a semi-quantitative chemical fingerprinting and the bioactivity of the main involved chemical classes in plant extracts (1). Our research group set uppharmaceutical biology studiesdeveloping this particular techniqueemploying high performing thin layer chromatography plates (HP-TLC) to obtain the best separation of the chemical classes together with the best evidence of their possible biological activity,driving further in-depth investigations toward a more focused chemical identification and quantification, and a wider biological activity profile of officinal plant extracts. We optimizedHP-TLC bioautographyfor antimicrobial (antibacterial and antifungal) and antioxidant properties [DPPH, 1,1-diphenyl-2-picrylhydrazyl; and ABTS, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) assays]of different plant extractsevidencingthe chemical classes really involved in the bioactivity (2).Our research strategy of plant extracts is planned to have HP-TLC bioautography as preliminary data giving us the opportunity 1. to point out the chemical compounds with antibacterial, antifungal, antioxidant capacity; 2. to focus on the chemical isolation and identification of fractions characterized by specific bioactive molecules; 3. to check biological capacities ofpure single compounds, or combinations, assaying possible synergic interactions; 4. to explore other biological capacities -i.e. cytotoxic, mutagen and mutagen-preventive activity, etc. - related to those preliminary evaluated through HP-TLC bioautography. In light of these premises, some researches in progress pointing out the role of HP-TLC bioautography in the pharmaceutical biology profile of our studies are shown below: -Essential oils obtained from plant crude drugs of different geographical origin (for e.g. Cryptocaryamassoia, Lauraceae, from Indonesian regions; Piper aduncuum, Piperaceae; Croton lechleri, Euphorbiaceae from Amazonian Ecuador). Cryptocaryamassoia(Lauraceae) essential oil evidenced on HP-TLC platesrelevant antimicrobial activity related tomassoia lactones,as single pure compounds,versus Gram negative strains (Klebsiellaoxytoca) andyeasts (Candida albicans). These data suggested to test pure compounds mixtures to evaluate their possible synergic potential. The result of combining benzylbenzoate and benzyl salicatewas a specific efficacy against Gram positive bacteria (Enterococcus faecalis). The evidences will also drive the research towards in-depth investigations (cytotoxicity, mutagenicity) of the active isolated compounds and their different qualitative and quantitative combinations. The case of Piper aduncum (Piperaceae) essential oil, characterized by high abundance of dillapiole(45.92%), highlighted this compound as the main responsible of highly selective antibacterial activity against Staphylococcus aureus.Amazonian Croton lechleri essential oil evidenced antibacterial activity against Escherichia colion HP-TLC mainly due to sesquicineole (17.29%).Caryophyllene oxide (1.24%) and 1,10-di-epi-cubenol (4.75%) showed instead the best antioxidant capacity with HP-TLC-DPPH assay. -126 different plant extracts from industrial (agro-food) and agricultural by-products investigated through HP-TLC bioautography for achieving preliminary suggestions about their possible role as source of bioactive molecules. In this case ethanol, chloroform and acetone extracts have been assayed for matching chemical fingerprinting and biological activity, giving preliminary but clear suggestions about the most promising extracts in terms of bioactivity potential and composition. Among the bioactive extracts, those most interesting were ethanolones obtained from agro-food by-products ofMalusdomestica and Juglansregia, and from agricultural wastes of Allium sativum. In conclusion, HP-TLC bioautographyrepresent an important tool to quickly join on chromatographic supporta preliminary chemical fingerprinting and important biological evidences of officinal plant extracts. 1) I.M. Choma, E.M. Grzelak (2011) J. Chromatogr. A, 1218, 2684–2691 2) D. Rossi, A. Guerrini, G. Paganetto, G. Bernacchia, F. Conforti, G. Statti, S. Maietti, I. Poppi, M. Tacchini, G. Sacchetti (2013) FoodChem, 139, 439–447I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
