Extracts of salvia species : relation to potential cognitive therapy
BACKGROUND: Dementia is a neurodegenerative disease of the brain associated with cognitive and memory impairments. Despite recognition of several types of dementia, the Alzheimer type is the most studied and understood. The cholinergic theory of Alzheimer's disease led to the development of licensed drugs based on the inhibition of the enzyme acetylcholinesterase. Extracts of Salvia (sage) species have been reported to have cholinergic activities relevant to the treatment of Alzheimer's disease. AIMS: Lack of information on a chemical fingerprint of the extracts responsible for inhibition of the enzymes butyrylcholinesterase and acetylcholinesterase prompted this in vitro investigation of sage species for anti-cholinesterase activity. Cholinergic receptor binding activity, inhibition of ß-secretase, and a pro-inflammatory cytokine suppressive activity of extracts of sage species were also studied as relevant treatment targets. METHODS: The extracts were obtained by methods of supercritical fluid extraction using 1,1,1,2-tetrafluoroethane (Phytosol A) and steam distillation. Dose-dependant inhibition of human cholinesterases by the extracts and constituents was determined using the method of Ellman, while inhibition of ß-secretase via a fluorometric method. The nicotinic acetylcholine receptors binding activity was measured as an amount of [3H]-nicotine displaced from human acetylcholine receptors, whereas the muscarinic activity was assessed using the displacement of [3H]-scopolamine. Determination of interleukin 8 inhibitory activity by the extracts was performed via a quantitative sandwich enzyme immunoassay using a commercially available kit. RESULTS: Inhibition of butyrylcholinesterase by the Phytosol extracts of S. apiana, S. fruticosa and S. officinalis var. purpurea was non-competitive. In contrast, inhibition of acetylcholinesterase by S. officinalis var. purpurea oil was competitive. S. corrugata extract was the most potent inhibitor of acetylcholinesterase with an IC50 value of 0.009±0.004 mg ml", while S. officinalis var. purpurea oil was the most active inhibitor of butyrylcholinesterase with an IC50 value of 0.015±0.004 mg ml''. Time dependent increase in inhibition of butyrylcholinesterase by steam distilled oils of S. fruticosa and S. officinalis var. "purpurea" was also evident. IC50 values decreased from 0.15±0.007 and 0.14±0.007 mg ml-1 with 5 minutes to 0.035±0.016 and 0.06±0.018 mg ml-1 with 90 minutes incubation time respectively. Phytosol A extracts were more potent than steam distilled oils with respect to anti-cholinesterase activity. Minor synergy in inhibition of bovine acetylcholinesterase was apparent in 1,8-cineole/a-pinene and 1,8- cineole/caryophyllene oxide combinations, whereas a combination of camphor and 1,8- cineole was antagonistic. Oil of S. apiana displaced [3H]-nicotine from human nicotinic acetylcholine receptors and [3H]-scopolamine from muscarinic acetylcholine receptors in a dose dependent manner with IC50 values of 0.02 mg ml" and IC50 0.1 mg ml" respectively. This oil also showed a modest suppression of interleukin 8 secretions from goblet cells. None of the tested oils and constituents had anti-ß secretase activity. CONCLUSION: These findings demonstrate that the cholinergic activity of the extracts results from a complex interaction between their constituents. Thus, inhibition of acetylcholinesterase is mainly due to the activity of the main constituents with some degree of synergy, whereas anti-butyrylcholinesterase activity is down to major synergistic interactions and identification of a chemical fingerprint responsible for the overall activity is therefore challenging. A synergistic combination of extracts or their standardised fractions with multiple activities is may be a candidate for clinical trials in Alzheimer's disease.