Alzheimer’s disease (AD) is a progressive and complex neurodegenerative disorder of the brain. Pathophysiology of the disease suggests that deposition of amyloid-β, accumulation of ᶩ-protein, release of inflammatory mediators and decreasing label of acetylcholine in the cholinergic system could be the possible reason for enhancing neurodegenerative condition of the AD brain (Mukherjee et al., 2007b). It is well known that patients with AD were deficient of acetylcholine, neuro transmitter that navigates the memory process of central nervous system. This fact has led to develop the cholinergic hypothesis, which focuses impairment of cognitive function in AD related disorder. The specific events of AD caused due to termination of the neurologic impulses at cholinergic synapses (White et al., 1977). It escalates to chemist to build a suitable cholinesterase inhibitor as potential model compounds for the treatment of several central nervous system disorders including AD. Synthetic cholinesterase inhibitors such as tacrine, donepezil, rivastigmine and natural origin galantamine have been approved for symptomatic treatment for the mild to moderate form of the AD. These inhibitors are primarily nature of competitive or non-competitive type for true acetyl cholinesterase (AChE) and pseudo butyryl cholinesterase (BChE) or for both isoforms. Searching for alternatives, chemical entity of natural origin and its derivatives have been tested scientifically and employed successfully in the treatment of cognitive disorders (Mukherjee and Houghton, 2009; Mukherjee et al., 2007a).

Cinnamomum zeylanicum L. (C. zeylanicum) is the oldest known spice, widely employed in several cultural practices as flavoring, perfumery, beverages and medicines (Mateos-Martín et al., 2012). It grows up to 6 - 8 meters height in the wild region. The parts of the plant including rhizomes, fruits/berries, leaves, seeds, barks, flowers, roots, whole plant, gall and bulb are explored for several medicinal properties. Bark of cinnamon is used as antiemetic, antidiarrheal, antiflatulent and general stimulant (Laekeman, 2011). Leaves of the species have showed potent medicinal properties like analgesic, antiseptic, antispasmodic, aphrodisiac, astringent, carminative, homeostatic and insecticidal property (Ranasinghe et al., 2013; Trajano et al., 2010). C. zeylanicum of Iranian origin has been reported for its AChE inhibition activity (Gholamhoseinian et al., 2009). Similarly, fruit of the genus cinnamomum (C. cassia) have also been reported for its potent anticholinesterase activity (Boga et al., 2011). Cinnamon oil possesses hot taste with a spicy odor (O’Shea et al., 2012). Cinnamon oil obtained from leaves and bark has been widely used in food industry for its antimicrobial property (Mallavarapu et al., 1995). It exhibits wide range of pharmacological activities like antioxidant, antimicrobial, antidiabetic and antihypertensive and vasorelaxant effects (Nyadjeu et al., 2011). Trans-cinnamyl acetate is the major phytoconstituents found in fruits, flowers, and fruit stalks. Eugenol, trans-caryophyllene, linalool, eugenol acetate, benzyl benzoate, α-pinene, β-pinene, β-caryophyllene, p-cymene, camphor and cinnamyl acetate were the most prevalent compound present in oil of C. zeylanicum leaves. However, another literature has been reported eugenol and cinnamaldehyde as major chemical constituents of cinnamon oil (Trajano et al., 2010). Eugenol (4-hydroxy-3-methoxy allylbenzene) (Fig. 1), a major phenolic active constituent was reported in oil of cinnamomum species. It possesses antioxidant, antiseptic, carminative, analgesic and neuroprotective activities (Jaganathan and Supriyanto, 2012; Prasad and Muralidhara, 2013). Isoeugenol, an isomer of eugenol, imparts flavor characteristic (Kermasha et al., 1994; Prasad and Muralidhara, 2013). Several spices have been explored for their AChE/BChE inhibitory property from our laboratory (Bhadra et al., 2011; Mukherjee et al., 2007a; Satheesh et al., 2009). To continue search of bioactive compounds from spices of Indian origin have led to investigate the cholinesterase inhibition activity of C. zeylanicum, to explore their therapeutic efficacy in neuronal disorder.

In this work, hydro-distillation process was successfully applied to extract the cinnamon oil from cinnamon leaves. It was transparent, yellow colored with smell of spice. % yield of the oil was found to be 3.4% (v/w).

TLC bio-autographic assay was carried out to evaluate the cholinesterase inhibitory activity of standardised plant extract. It was found that methanol extracts of C. zeylanicum leaves (1 mg/ml) shows positive inhibitory activity on TLC plate. The result further confirmed with false positive reaction, which does not correspond with positive inhibitory reaction. These positive inhibitory activities indicated number of white spot over yellow background of TLC.

The result of 96-well microtiter plate methods for methanol extract, cinnamon oil, eugenol and standard galantamine has been shown in Table 1. The data value indicates the test and standard chemical compounds have the potential cholinesterase inhibitory activity. Cinnamon oil has shown potent AChE inhibition with IC₅₀ value of 45.88 ± 1.94 μg/ml than methanol extract having IC₅₀ value of 77.78 ± 0.03 μg/ml. However, AChE activities of cinnamon oil showed least activity than the standard eugenol compound (IC₅₀: 36.42 ± 1.22 μg/ml) used. Moreover, the reference compound galantamine have shown the stronger AChE activity (IC₅₀: 22.34 ± 0.56 μg/ml) than the standard eugenol, cinnamon oil and methanol extract. In contrast to BChE activity, the cinnamon oil (IC₅₀: 87.39 ± 1.60 μg/ml) have shown the slight potent enzyme activity than the methanol extract (IC₅₀: 88.62 ± 1.72 μg/ml) of C. zeylanicum.

Similarly, the standard biomarker compound eugenol have shown potent BChE activity with IC₅₀ value of 52.14 ± 1.05 μg/ml and found to be active than the cinnamon oil and methanol extract. The reference compound galantamine have shown IC₅₀ value 25.35 ± 1.02 μg/ml and possess as most active against BChE. Data value (Table 1) was also determining the cholinesterase enzyme affinity of the studied substances towards more AChE than BChE. Dose response curve (Figs. 2- 3) of the methanol extract, cinnamon oil, eugenol and galantamine was shown in increase in percentage of AChE and BChE inhibition with increase in concentration of test samples. The cinnamon oil from C. zeylanicum has exhibited potent AChE and BChE inhibition over other test compounds.

RP-HPLC method was carried out to know the purity of natural product extract (Ross, 1976). The complex mixture of the chemical compound in natural product was separated on the basis of the affinity towards stationary phase and mobile phase gradient. The label of eugenol in cinnamon oil was calculated from linear regression equation (y = 1.519065e + 007X + 1659936). The following equation showed a good agreement between peak area and used concentration (0.1 - 1.0 μg/ml) with a correlation coefficient, r² = 0.984. The identity of eugenol in cinnamon oil was affirmed by comparing the retention times of reference compound. Chromatogram of HPLC analysis showed retention time of eugenol in cinnamon oil was 15.81 min (Fig. 4a and b). The percentage content of eugenol in cinnamon oil was found to be 0.205 mg per ml.

In GC-MS analysis, seven chemical compounds were identified in cinnamon oil which comprising of 97.92% of the total cinnamon oil. Cinnamaldehyde (66.74%) and eugenol (22.91%) were found main components of cinnamon oil, followed by Triacontane (3.30%), Heptacosane (2.10%), Penta tri acontane (1.12%), Camphene (1.02%) and Tetratetracontane (0.73%) were other minor constituents (Table 2, Fig. 5).

C. zeylanicum oil of different region contains eugenol as major chemical constituent and found abundant amount in Lauraceae family (Rana et al., 2012). It has been reported that eugenol containing spices able to restore the normal behavior of an experimental animal against acrylamide induced oxidative stress (Prasad and Muralidhara, 2013). Thus, it prompted to investigate the standardized extract of C. zeylanicum leaves and its essential oil for anticholinergic activity.

The result of TLC bio-autographic/96-well microtiter plates confirmed the cinnamon leaves and oil has potential anticholinesterase activity. Cholinesterase inhibitors obtained from plant sources have been extensively studied for neurodegenerative disorder. These natural occurring compounds have provided convincing evidence of anticholinesterase activity in mild to moderate form of Alzheimer’s disease (Yang et al., 2003). It has been hypothesized that both AChE/BChE play critical role in maintaining chemical neurotransmitter (ACh) in the region of human brain. However, the ratio AChE/BChE label in certain region of brain is altered in due course of AD. These changes provide strong evidence of decline in label of acetylcholine in the region of dementia brain (Bhadra et al., 2012). This needs to design a suitable chemical substance which can optimize the therapeutic balance between AChE as well as BChE inhibition. The tested substances were shown more affinity towards AChE than BChE. This may be due to presence of chemical substance eugenol as constituent in methanol extract (Laekeman, 2011) and cinnamon oil. The incurred data clearly demonstrate the active principle contain tested substance have the potential cholinesterase activity and support the earlier evidence of claiming eugenol contain plant extract has possible anticholinesterase activity (Kumar et al., 2009).

The content of eugenol in cinnamon oil was standardized with RP-HPLC and identified through gas chromatography−mass spectrometry (GC−MS) technique. GC−MS technique is widely employed for identification and quantification of volatile compound present in essential oil (Bhadra et al., 2012). The variation of the different chemical compounds identified in cinnamon oil may be due to geographical location or soil condition (Rana et al., 2012). The identification of chemical compounds in cinnamon oil was corelated with data value of retention indices and mass spectra obtained from data library (Adams, 2001).

The present study highlights the eugenol as important constituent of C. zeylanicum leaf oil. Further, the studied plant has well characterised as potent cholinesterase inhibitory activity. Therefore, the underlying active principles of C. zeylanicum may have therapeutic value in treatment of neurodegenerative disease such as AD.