Chemical characterization and anticholinesterase effects of essential oils derived from Salvia species

Abstract Inhibitory effect of Salvia species herbal preparations on acetylcholinesterase (AChE) and butylcholinesterase (BuChE) activitiy may contribute to regulation of cognitive performance and impaired cholinergic functions in patients with Alzheimer’s disease. This functional role of Salvia species and their components makes the investigations on Salvia valuable in medicine-related plant research. Within this work it was aimed to investigate the in vitro anti-cholinesterase effect of essential oils derived from ten Salvia species, which grow in Turkey. The chemical composition of essential oils were characterized by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS), respectively. Results showed that all of the essential oils exhibited AChE inhibitory activity. S. pseudeuphratica, S. hydrangea and S. divaricata essential oils demonstrated the most potent AChE inhibitory effect [50% inhibition concentration (IC50) = 26.00 ± 2.00 μg/mL, 40.0 ± 4.00, 64.68 ± 4.16, respectively]. The essential oil of S. pseudeuphratica demonstrated the highest inhibitory activity against AChE and BuChE among the tested Salvia essential oils. Evidences from the our study augment the importance of essential oils obtained from Salvia species and may support utilization of Salvia species for symptomatic treatment of Alzheimer disease.


Introduction
Enzymes are important biochemical targets for treatment of several diseases and nearly 30% of all drugs in clinical use show their therapeutic effect by inhibition of enzymes (1). Beside synthetic drugs, plants and natural product-based therapeutics are an important potential source for treatment of a wide spectrum of pathologies. Ethnobotanical use of various plants may be either important in demonstrating the efficacy of natural products in treatment of diseases or a pathfinder in studies of drug-effect mechanism. Nowadays, as an enzyme inhibitor, natural compounds have a wide range of usage on many pathological conditions from analgesia to the symptomatic theraphy of Alzheimer's Disease (AD). Main treatment strategy for AD is restoration of decreased brain neuromediator acetylcholine levels with inhibition of acetylcholinesterase (AChE) and butrylcholinesterase (BuChE) enzymes. For treatment of AD, researchers have focused on screening the phytochemical contents and inhibitory effects of chemical contents of different plant fractions on enzyme activities. Because of their reputed inhibitory effect on cholinesterases Salvia L. species are an attractive research topic (2,3).
The number of species known is now 100, demonstrating that Turkey is a major center of diversity for the genus in Asia (4). S. ballsiana, S. cyanescens, S. divaricata, S. kronenburgii, S. nydeggeri and S. pseudeuphratica are species endemic to Turkey (5). Both S. ballsiana and S. pseudeuphratica are also local species. The main chemical constituents like flavonoids, polyphenols, monoterpenes, diterpenes and triterpenes of Salvia species have been subjected to phytochemical studies published in several reviews (6)(7)(8)(9)(10).
Salvia species are used in traditional medicine all around the world and their essential oils possess animicrobial, antioxidant, antidiabetic, antimutagenic, antitumor gas (0.8 mL/min). GC oven temperature was kept at 60°C for 10 minutes and programmed to 220°C at a rate of 4°C/ min, and kept constant at 220°C for 10 minutes and then programmed to 240°C at a rate of 1°C/min. Split ratio was adjusted at 40:1. The injector temperature was set at 250°C. The GC-MS analysis was conducted at 70 eV with a mass range of 35-450 m/z and a scanning velocity of 3.46 scan/second.

GC analysis
The GC analysis was carried out using an Agilent 6890N GC system. FID detector temperature was 300°C. To obtain the same elution order with GC-MS, simultaneous auto-injection was done on a duplicate of the same column applying the same operational conditions. Relative percentage amounts of the separated compounds were calculated from FID chromatograms. The analysis results are given in Table 2.

Identification of components
Identification of the essential oil components were carried out by comparison of their relative retention times with those of authentic samples or by comparison of their relative retention index (RRI) to series of n-alkanes. Computer matching against commercial (Wiley GC/MS Library, MassFinder 3 Library) (15,16) and in-house 'Başer Library of Essential Oil Constituents' built up by genuine compounds and components of known oils, as well as MS literature data (17,18), was used for the identification.

Determination of AChE activity and BuChE activity
Different concentrations of the test samples were initially prepared in methanol. Essential oils tested at final concentration range 5-80 μg/mL and six different concentrations. Twenty micro litre of AChE enzyme (1 U/mL), 10 μL sample added to 2.4 mL buffer, the mixture was and anticholinesterase properties. Biological studies performed especially with essential oils of S. lavandulaefolia ve S. officinalis have demonstrated that essential oils have potential for improvement mood and mental functions (8,11). This species are known as 'adaçayı' in Turkey and consumed as a hot drink. In addition, many Salvia species are used for food flavoring, as well as in cosmetics, perfumery and the pharmaceutical industry (7,12).
Although there are numerous studies on the chemistry of Salvia essential oils (13,14), up to date there are no reports on the chemical characterization of S. ballsiana, S. nydeggeri, S. pachystachys, S. pseudeuphratica and S. russellii essential oils . In addition, the inhibitory effect of essential oils obtained from Salvia species namely Salvia ballsiana, S. cyanescens, S. divaricata, S. hydrangea, S. kronenburgii, S. macrochlamys, S. nydeggeri, S. pachystachys, S. pseudeuphratica and S. russellii) collected from Turkey were investigated for the first time for their AChE and BuChE activity.

Plant material
Aerial parts of the plant materials were collected from different regions of Turkey. Voucher specimens were kept at the Laboratory of Plant Systematics & Taxonomy, Department of Biology, ODTU. Information concerning the plant material is given in Table 1.

Isolation and analysis of the essential oils
The dried aerial parts were hydrodistilled for 3 hours using a Clevenger-type apparatus. The yields were calculated on a dry weight basis (v/w). Yields are given in Table 1.

GC-MS analysis
The GC-MS analysis was carried out with an Agilent 5975 GC-MSD system. Innowax FSC column (60 m × 0.25 mm, 0.25 μm film thickness) was used with helium as carrier  Table 2. the chemical composition of the essential oils of Salvia species.  A control mixture and blank was prepared by using 10 μL of methanol instead of the oil sample, with all other procedures similar to those used in the case of the sample mixture. Absorbances were measured at 412 nm and 37°C using polystyrol cuvets using a specrophotometer (Shimadzu, UV-1700) as previously reported (19,20). Experiments were repeated in triplicates. Experimental data were calculated using the Microsoft Excel. All data are presented as mean ± standard deviation (SD) in Table 3 and Table 4 and of all The percent inhibition of enzymes activities (I%) was calculated by using the equation :   A total of twenty-six compounds were characterized at S. pseudeuphratica essential oilwhich were representing 96.0% of the total oil. This oil was characterized with a relatively high content of camphor (53.6%). The other main component was found as 1,8-cineole (17.4%).
In our study, it has been demonstrated that there is a qualitative and quantitative difference between investigated Salvia species among constituents of essential oils. The differences about chemical compositions of Salvia species evaluated in our study and the Salvia species investigated at either in our work or previous studies in the literature may be related with source of the plant, individual genetic variability, collection time of the plant, the proportions of distilled parts, variations in biosynthetic pathways and metabolism (25).

Inhibition effect of essential oils on cholinesterases
The AChE and BuChE inhibitory activity of essential oils derived from ten Salvia species has not been reported to date. This is the first study performed to demonstrate this activity. AChE and BuChE inhibitory activities of the essential oils are reported in Table 3 and Table 4. It is also known that Salvia species are used traditionally at various nervous system disorders (26). Previous studies demonstrated that Salvia essentials oils have potential therapeutic effects on mood and cognitive functions through cholinesterase inhibition which could be attributed to terpenes (11,27).
The main finding of this study was that all investigated Salvia essential oils inhibited AChE enzyme activity. However, Essential oils were less active than galantamin; the standart inhibitor of AChE enzyme. While essential oil of S. pseudeuphratica showed the highest inhibitory effect on AChE (IC 50 = 26 ± 2 μg/mL), S. cyanescens and S. pachystachys showed the lowest inhibitory effects on AChE at the same concentration (80 μg/mL) among the species investigated. Our results also confirmed previous findings about AChE inhibitory activity of Salvia species (26).
Main component of S. pseudeuphratica essential oil, generally atttirubuted to the biological activity, is a naturally occuring monoterpenoid, camphor with a relative biological activity of 53.6%. Whereas, S. cyanescens (0.3%) and S. pachystachys (0.4%) were the Salvia species with the lowest camphor content. Svalev et al. (28) have demonstrated that AChE enzyme inhibitory effect of essential oil derived from poor camphor containing Salvia officinalis individual components are given in Table 2 with their relative percentages.
Despite no camphor content, S. russelli have exhibited a high level of AChE inhibition and although main component of S. divaricata was 1,8 cineole, it did not demonstrate any BuChE inhibition. In spite the major factor that takes part in cholinesterase enzyme inhibition seems to be the main component of the essential oils, synergistic or antagonistic chemical interactions of essential oil components may play an important role.
Şenol and co-workers showed neither dichloromethane nor ethyl acetate extracts of the S. pseudeuphratica has inhibitory effect on AChE at a concentration of 100 μg/mL. AChE inhibitory effect of dichloromethane extract obtained from S.russellii (100 μg/mL) was 1.70 ± 0.85% and ethyl acetate extract of S. russellii (100 μg/mL) was 11.54 ± 0.34%. While dichloromethane extracts of S. pachystachys showed an inhibitory effect on AChE (2.34 ± 0.34%-100 μg/mL), ethyl acetate extract of S. pachystachys has no inhibitory effect on AChE activity at same concentration. However, relevant components of extracts were not reported in study of Senol et al. (34). Hence no inhibitory effect of S. pseudeuphratica ethyl acetate extracts on AChE activity was determined in the previous study, dual inhibitory effect of S. pseudeuphratica volatile fraction obtained in our work has showed the importance of investigation different fractions from the plants.
Evidences from our study augment the importance of essential oils obtained from Salvia species and support the utilization of Salvia species, especially the essential oil of S. pseudeuphratica, which has demonstrated the highest inhibitory activity against both enzymes, for symptomatic treatment of Alzheimer disease. Main constituents, camphor and 1,8-cineole are relevant compounds associated with cholinesterase inhibition. Contribution ratio of the active components effects cholinesterase activity in a important manner. Due to these dual efficacy on both cholinesterase enzymes, investigations on cholinesterase inhibitory effect of S. pseudeuphratica volatile fractions at in vivo studies can be worthwhile. was lower than essential oils rich in camphor content. These findings suggested that camphor may be responsible from inhibitory effect on AChE enzyme. Perry et al. (2) demonstrated that camphor is an uncompetitive reversible inhibitor of human erythrocyte AChE. Camphor, 1,8-cineole, and α-pinene inhibited the enzyme in a dose-dependent manner. When compared with standard drug physostigmine (IC 50 = 4.5 × 10 −8 M), the most active monoterpenes were 1,8-cineole (IC 50 = 0.67 mM), α-pinene (IC 50 = 0.63 mM) and camphor (4,7 mM), respectively. Inhibitory effect of camphor enantiomers on AChE activity was reported as (+)-camphor: 26.4%, (-)-camphor: 21.2% has been reported by Miyazawa et al. (29).
Other effective essential oils for AChE enzyme inhibition in our study were the essential oils obtained from S. hydrangea (40 ± 4%) and S. divaricata (64.68 ± 4.16%), respectively. While main component of Salvia hydrangea was camphor (46.9%), main component of S. divaricata was 1,8-cineol (30.9%). 1,8-cineole and α-pinene are two common monoterpenes in Salvia. AChE inhibitory activity of these Salvia species may be contributed with the presence of 1,8-cineole. Common property of forthcoming three Salvia with highest inhibitory effect on AChE enzyme was markedly rich camphor content among Salvia species investigated in our study. Combination of 1,8-cineole and camphor may result with either synergy or antagonism (30). Although Svalev et al. reported that Salvia species rich in 1,8-cineole, but not camphor, may provide oils with more potent cholinergic activities data obtained our study have showed that essential oils rich in camphor content also have inhibitory effect on cholinesterase activity (28). While camphor is known for therapeutic effects it should not be ignored that toxic effects of Salvia essential oils might be related with it (11,31).
In enzyme inhibition assays, the percentage inhibition effect on AChE activity of essential oils obtained from S. nydeggeri, S. russelli, S. ballsiana, S. macrochlamys, S. kronenburgii was higher than 40% at 80 μg/mL concentration.
BuChE activity, which is also responsible for the hydrolysis of ACh; may increase for the compensation of deteriorated AChE activity in AD. In addition to esterase function, BuChE have peptidase effect. Peptidase activity ameliorates formation of β-amyloid by degredation of amyloid precursor protein (APP) which can be determined in large amounts at brain tissue of Alzheimer Disease patients (32). Therefore, inhibition BuChE (in addition to AChE) seems to have a therapeutic impact in AD.
Another finding of this study was inhibition of BuChE by essential oils obtained from some investigated Salvia species inhibitory activity of the oils on BuChE varied among the studied species.