Acetyl-cholinesterase Enzyme Inhibitory Effect of Calophyllum species

Purpose: To search for new acetylcholinesterase enzyme inhibitors from Calopyllum species. Methods: Six stem bark extracts of Calophyllum inophyllum, C. soulattri, C. teysmannii, C. lowii, C. benjaminum and C. javanicum were subjected to anti-cholinesterase analysis against acetylcholinesterase (AChE) enzyme using Ellman’s method. Results: Most of the extracts showed promising inhibitory activity against AChE at concentrations of 100 µg/mL, with the methanol extract of C. inophyllum demonstrating the strongest inhibitory effect of 81.28 % followed by the methanol extract of C. benjaminum with 74.32 % inhibition. The methanol extracts of Calophyllum inophyllum and Calophyllum bejaminum also showed significant inhibitory activity towards the acetylcholinesterase (AChE) enzyme. Conclusion: Calophyllum species is capable of yielding potential lead compounds for the development for acetyl-cholinesterase enzyme inhibition drugs.


INTRODUCTION
Alzheimer's disease (AD) which is a neurodegenerative disease commonly affecting elderly people is the result of an irreversible degeneration of cholinergic neurons. Patients lose their memory and cognitive abilities. Rapid hydrolysis by acetylcholinesterase (AChE) enzyme decreases the level of acetylcholine. Therefore, treatment of AD is based on the inhibition of AChE in order to improve cholinergic neurotransmission. Tacrine, a well-known AChE enzyme inhibitor elicits severe side effects to patients such as nausea, insomnia and salivation [1]. Hence, new inhibitors for the disease must be discovered. We chose the species of Calophyllum, for our pharmacognosy investigation since these plants are traditionally used as antiseptics, astringents, expectorants, diuretic, purgatives and analgesics [2]. Previous investigations on some Calophyllum species have shown them to possess potential pharmaceutical usage such as they indicated cytotoxicity [3], anti-HIV [4], anti-leishmanial [5] The stem bark of the Calophyllum species (except for C. soulattri) were extracted using conventional extraction method in which the solvent was added and removed in batches. The dried and powdered samples were extracted in n-hexane (Hex), ethyl acetate (EA) and methanol (MeOH) in a polarity-increasing order. The solvent was decanted and replaced with new solvent after 3 days. The extracts were dried under reduced pressure using a rotary evaporator to yield the hexane, ethyl acetate and methanol extracts, respectively.
The air-dried stem bark of Calophyllum soulattri was ground to a fine powder and extracted successively in a Soxhlet apparatus with nhexane (60-80 °C, 3 × 2 L), dichloromethane (40 °C, 3 × 2 L), ethyl acetate (77 °C, 3 × 2 L) and methanol (65 °C, 3 × 2 L) for 24 h. The extracts were evaporated to dryness under vacuum to give n-hexane, ethyl acetate and methanol extracts. The weights of the extracts obtained from the six Calophyllum species are tabulated in Table 1.
Hydrolysis of ATCI was monitored by observation of the formation of the yellow 5-thio-2nitrobenzoate anion, a result of the reaction of DTNB with thiocholines which is catalyzed by the enzyme at a wavelength of 412 nm. Every well was initially filled with 210 μL (0.15 mM/well) DTNB solution. Samples of 20 μL volume (100 µg/mL for crude extracts and serial dilutions for pure compounds -100, 50, 25, 12.5, 6.25 µM) was added in triplicates and the control wells were filled with 20 μL (0.10 M, pH 7.4) of phosphate buffer, followed by 20 μL (0.037 U/mL per well) of AChE. The plate was then incubated for 10 minutes at 37 °C. Thereafter, 20 μL (0.25 μM/well) of ATCI were added into each well to initiate the hydrolysis reaction. The plate was immediately shaken for 2 sec and measured at 412 nm at 25 °C for 10 times consecutively. The experiments were carried out in triplicate. Tacrine (Sigma) was used as the reference drug. The percentage of inhibition was calculated as in Eq 1.

Statistical analysis
The data are expressed as mean ± standard deviation (SD). Statistical analysis was carried out by paired t-test. The level of significance used was p < 0.05. The statistical software used was Graph Pad Prism 5.

RESULTS
Non-aqueous extracts of six Calophyllum species, Calophyllum inophyllum, C. soulattri, C. teysmannii, C. lowii, C. benjaminum and C. javanicum were evaluated for their anti-AChE activity by Ellman's method. Results of the percentage inhibition of AChE are summarized in Table 2 and Figure 1. Overall, these extracts of Calopyllum spp. showed promising inhibitory effects against AChE at concentration of 100 µg/mL. Among the extracts, the methanolic extract of C. inophyllum demonstrated the strongest inhibitory activity with 81.28 % inhibition. This is followed by the methanolic extract of C. benjaminum and the hexane extract of C. soulattri which gave 74.32 % and 70.45 % inhibitions, respectively. All the percentage of inhibition extracts were statistically different at p < 0.05 from control.

DISCUSSION
The treatment of AD is mainly focused on the improvement of cholinergic neurotransmission by inhibiting AChE, which is the enzyme that catalyzes the hydrolysis of acetylcholine (ACh). Cholinesterase inhibitors reduce the degradation of ACh, which is a neurotransmitter in the synapses of the nervous system. In other words, AChE inhibitors diminish the formation of amyloid fibrils hence increases the neuroprotective activity. The hydrolysis reaction takes place by nucleophilic attack on the carbonyl carbon followed by acylation of the enzyme and the release of choline. It is followed by hydrolysis of the acylated enzyme to produce acetic acid and  The AChE inhibition activities of Calophyllum species showed no correlation with the polarity of the solvents, which are used to extract the plant samples. For C. inophyllum, C. benjaminum and C. javanicum, the activities of the enzyme inhibition indicated a positive correlation to the polarity of the solvent used to extract the plants. The inhibition effects of these plants are directly proportional to the polarity of the non-aqueous extracts. In contrast, the activities of the nonaqueous extracts of C. soulattri and C. lowii are inversely proportional to the polarity of their extracts. Besides this, C. teysmannii showed no correlation with AChE inhibition.
To the best of our knowledge, the anti-AChE activity of Calophyllum species has not previously been investigated. This is therefore the first report and the outcome of our work has shown the first data on anti-AChE activity of this genus. Calophyllum species have been previously reported for the presence of diverse secondary metabolites such as xanthones [10], coumarins [11], triterpenes [12] and chromanones [13]. AChE inhibitory effects, as described, are predicted to be due to the plant metabolites mentioned. Consequently, a further study on the detailed phytoconstituent investigation of these potential plant extracts is worthwhile to obtain the plant metabolites which correspond to or are responsible for the anti-AChE activities.

CONCLUSION
The methanol extracts of Calophyllum inophyllum and Calophyllum bejaminum show significant inhibitory effects against acetylcholinesterase (AChE). Calophyllum species can yield potential lead compounds for the development of acetylcholinesterase enzyme inhibition drugs.