Phytochemical Composition , Antiradical and Anticholinesterase Potentials of Centaurea alba and Centaurea jacea Volatile Oils

This paper reports on the phytochemical composition as well as antiradical and anticholinesterase potential of volatile oils isolated from Centaurea alba and Centaurea jacea, from Croatia. The volatile components, obtained by hydrodistillation, were determined by GC and GC-MS analyses. A total of 18 compounds were identified in C. alba volatile oil with hexadecanoic acid, germacrene D and tetradecanoic acid as main compounds. A total of 29 compounds were identified in C. jacea volatile oil, with epi-bicyclosesquiphellandrene, aromadendrene and hexadecanoic acid as a major compounds. The tested volatile oils showed low DPPH inhibition potential as well as low to moderate antiAChE potential and low antiBuChE potential.


INTRODUCTION
HE genus Centaurea L. is one of the richest genera in the Asteraceae family.According to today's concept, genus contains about 250 species which have the main spreading area in the Mediterranean region. [1,2]8][9][10][11][12][13] Plants are good source of volatile components that are important in biological and ecological terms.These compounds could be good antioxidants or cholinesterase inhibitors.Antioxidants are compounds that delay autoxidation by inhibiting formation of free radicals or by interrupting propagation of the free radicals.Free radicals alter the structure and function of substances in the body and in high concentrations they can damage proteins, lipids and DNA.The brain is particularly susceptible to oxidative damage.There is substantial evidence that oxidative damage to the brain is an early event in Alzheimer's disease (AD).Many biochemical mechanisms have been proposed to explain the neuropathology of AD but cholinergic hypothesis is the most accepted theory nowadays.It postulates that at least some of the cognitive decline experienced by patients with AD results from a deficiency of acetylcholine, or cholinergic neurotransmission. [14]Neutralizing free radicals and inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) has become an important and persuasive therapeutic strategy against AD. [15][18] we report here the results of phytochemical composition as well as antiradical and anticholinesterase potential of Centaurea alba L. and Centaurea jacea L. volatile oils from Croatia.
According to our knowledge, this is the first report about chemical composition of Centaurea alba L. and the second report about chemical composition of Centaurea jacea L. volatile oil, but the first one for the plant from Croatia.The internal database of compounds was created during previous analyses from authentic compounds obtained commercially and from more than thousand volatile oils obtained during our previous studies.The percentages of components were calculated as mean values from the GC and GC-MS peak areas.

Antiradical Potential of Tested Extracts
Antiradical potential of volatile oils isolated from C. alba and C. jacea was performed by DPPH (2,2-diphenyl-1-picrylhydrazyl radical scavenging method) method of Brand-Williams et al. [22] .The DPPH radical scavenging activities of the tested extracts were calculated according to formula: % inhibition = [(A0 -Asample) / A0] × 100, where A0 was absorbance of the DPPH ethanol solution measured at the beginning and Asample was absorbance of the sample measured after 60 min.The results were expressed as percentage inhibition of DPPH or IC50 (amount of antioxidant required for 50 % decrease in initial DPPH concentration).Ascorbic acid (vitamin C) was used as positive control.The experiment was performed in triplicate and the results were expressed as mean ± standard deviations.

Anticholinesterase Potential of Volatile Oils
Antiacetylcholinesterase (antiAChE) and antibutyrylcholinesterase (antiBuChE) potential of volatile oils isolated from C. alba and C. jacea were carried out by a slightly modified Ellman method. [23]A run consisted of 180 µL of phosphate buffer (0.1 M, pH 8), 10 µL of DTNB (at a final concentration of 0.3 mM prepared in 0.1 M phosphate buffer pH 7 with 0.12 M sodium bicarbonate added for stability), 10 µL of sample solution (dissolved in 80 % EtOH), and 10 µL of AChE/BChE solution (with final concentration 0.03 U mL -1 ).
Reactants were mixed in a 96-well plate wells and reaction was initiated by adding 10 µL of acetylthiocholine iodide / butyrylthiocholine iodide (ATChI / BTChI, to reach a final concentration of 0.5 mM).As a negative control, 80 % EtOH was used instead of sample solution.Non-enzymatic hydrolysis was also monitored by measurement of two blank runs for each run.All spectrophotometric measurements were performed at 405 nm and at room temperature for 6 min periods.The results are expressed as percentage inhibition of enzyme activity.The experiment was performed in triplicate and the results were expressed as mean ± standard deviations.

RESULTS AND DISCUSSION
The volatile oils of two Centaurea species were analyzed by GC and GC-MS.According to my knowledge, this is the first report about chemical composition of Centaurea alba and the second report about chemical composition of Centaurea jacea volatile oil, but the first one for the plant from Croatia.A total of 18 compounds were identified in Centaurea alba volatile oil, representing 98.5 % of the total volatile oil composition, Table 1.This oil was dominated by nonterpene compounds (61.7 %), especially by nonterpene acids (31.7 %) with hexadecanoic acid (19.1 %) and tetradecanoic acid (11.7 %) as main compounds.Nonterpene hydrocarbons (10.8 %) and aldehydes (9.9 %) were also presented in remarquable quantity.Among them pentadecanal (9.6 %) was presented as the most common.Terpene compounds were presented with sesquiterpenes and sesquiterpenoids in quantity of 36.8 %.The main terpene compounds were germacrene D (14.3 %), hexahydrofarnesyl acetone (9.3 %) and ledol (5.8 %).Other terpene and nonterpene compounds were identified in quantity lower than 4 %.
A literature search showed that Centaurea species were poor in monoterpene and monoterpenoid compounds and rich in fatty acids as well as sesquiterpene and sesquiterpenoid compounds.[26][27][28][29][30][31][32][33][34] To date, several Centaurea volatile oils chemical analysis for plants from Croatia were performed.Hexadecanoic acid, germacrene D, β-caryophyllene and caryophyllene oxide were also the most abundant compounds.][36][37][38] Antiradical and anticholinesterase potential of C. alba and C. jacea volatile oils were also tested.Antiradical potential was tested by DPPH method, [22] while anti AChE and antiBuChE potential was tested by Ellman method, [23] Table 2. DPPH method is widely used for antiradical detection, while Ellman method is widely used for the detection of cholinesterase inhibitors.Both of used methods are sensitive, good repetitive and requeres a little sample material.
Results showed low DPPH inhibition potential for both of tested volatile oils (5.6 ± 0.3 % for C. alba and 4.9 ± 0.2 % for C. jacea) in tested concentration of 1 mg mL -1 (0.048 mg mL -1 in reaction system).For comparison, vitamin C as reference compound, showed 95.3 ± 1.7 % inhibition of DPPH, for the same tested concentration.Results for tested cholinesterase inhibition potential showed low to moderate AChE potential of tested oils (28.3 ± 2.8 % for C. alba and 11.7 ± 0.9 % for C. jacea) and low BuChE inhibition potential of tested oils (5.4 ± 0.9 % for C. alba and 2.6 ± 0.7 % for C. jacea) for tested concentration of 1 mg mL -1 (0.045 mg mL -1 in reaction system).For comparison, eserine as referent compound showed 92.2 ± 3.4 % inhibition of AChE and 77.9 ± 1.9 % inhibition of BuChE for tested concentration of 0.1 mg mL -1 .
Low DPPH inhibition potential could be connected to composition of these oils which does not contain components attributed to antiradical potential, like phenolic or monoterpenoid compounds. [39]Low to moderate anticholinesterase inhibition potential of tested volatile oils also could be connected to volatile oils composition or synergism among volatile oil constituents.Among volatile compounds identified as components of tested oils, pulegone, anethole, α-copaene, aromadendrene, ledol and caryophyllene oxide were tested on AChE, while anethole, αcopaene, ledol and caryophyllene oxide were tested on BuChE inhibition. [40]Among them, anethole and pulegone showed good AChE inhibition potential, while only anethole showed good BuChE potential.

CONCLUSION
The volatile oils of Centaurea alba and Centaurea jacea from Croatia were subjected to phytochemical composition as well as antiradical and anticholinesterase potentials.C. alba volatile oil was dominated by nonterpene compounds, especially by nonterpene acids, with hexadecanoic acid and tetradecanoic acid as main compound.Nonterpene hydrocarbons and aldehydes were also presented in remarquable quantity with pentadecanal as most abundant.Terpene compounds were presented with sesquiterpenes and sesquiterpenoids.The main terpene compounds were germacrene D, hexahydrofarnesyl acetone and ledol.C. jacea volatile oil was dominated by sesquiterpene compounds, with epi-bicyclosesquiphellandrene and aromadendrene as major compounds.β-Elemene and caryophyllene oxide were also identified in remarkable quantity.Nonterpene compounds were presented in lower quantity, with hexadecanoic acid as main compound.a) 4.9 ± 0.2 11.7 ± 0.9 2.6 ± 0.7 vitamin C (a) 95.3 ± 1.7 -eserine (b) -92.9 ± 3.4 77.9 ± 1.9 VO = volatile oil.

Table 1 .
Volatile oil composition of Centaurea alba and Centaurea jacea from Croatia.

Table 2 .
Antiradical and anticholinesterase potential of Centaurea alba and Centaurea jacea volatile oils.