In vitro culture of Cistus creticus subsp. creticus – a source of biological active compounds

Abstract

Cistus creticus subsp. creticus is a native plant of the Mediterranean region and it has been used since ancient times for its medicinal properties. Labdane type diterpenes are predominant compounds in leaves and stems of the C. creticus subsp. creticus, as well as in resin, while other terpenes, polyphenols and flavonoids contribute to chemical profile of this species. In vitro plant tissue culture is an attractive alternative approach to the traditional methods of plantations, as it offers a controlled supply of biochemicals independent of plant availability and more consistent product quality. In order to obtain applicable production of secondary metabolites of interest we have implemented in vitro culture of several clones of C. creticus, which were previously morphogenetically described. The ethanol extracts of obtained in vitro cultures were analyzed by GC-FID and GC-MS, and were characterized concerning the total phenolic and flavonoid contents. Labdane diterpenes were the most abundant compounds in above ground parts extracts, but absent from the root extracts. Head-space GC-MS was used to determine the profile of volatile compounds in the atmosphere of the culture vessels. α pinene, β pinene, and camphene were the most abundant ones. PTR-MS was used for further quantification of representative volatile compounds in the atmosphere of the culture vessels. Finally, the biological activity of obtained ethanol extract was studied. Microdilution method was used for investigation of antibacterial and antifungal activity. Almost all tested extract against human pathogenic bacteria showed better antibacterial activity than streptomycin, and much better antibacterial activity than ampicillin. All tested extracts also exhibited slightly higher or similar antifungal potential as bifonazole, and showed much better antifungal effect than ketoconazole. Antioxidant activity was determined by using DPPH (2,2-Diphenyl-1-picrylhydrazyl) test, while cytotoxic activity was previously demonstrated by sulforhodamine B (SRB) assay.