Acetylcholinesterase Inhibitor From Tabernaemontana pandacaqui Flowers

Abstract

The inhibition of acetylcholinesterase (AChE) is still considered a strategy for the treatment of Alzheimer’s disease. The aim of this study was the search for potential drugs from natural sources which can inhibit AChE. The methanol extract of fresh flowers of Tabernaemontana pandacaqui was partitioned with n-hexane and ethyl acetate. All extracts were evaluated for AChE inhibitory activity. The ethyl acetate fraction, which showed the strongest AChE inhibitory activity, was fractionated using various chromatographic techniques, leading to the isolation of 6 compounds (1-6), which were identified mainly by spectroscopic techniques; this is the first report of these compounds from T. pandacaqui flowers. Astragalin (6) was the major active constituent. The structure-AChE inhibitory activity relationship of 6 and its derivatives was studied. The results suggest that T. pandacaqui flowers and its flavonoid compounds could be potentially used for the treatment of Alzheimer’s disease.

Recently, the strategy for the treatment of Alzheimer’s disease involves the inhibition of acetylcholinesterase (AChE). ^{1 -4} Some potent AChE drugs derived from natural sources, such as galanthamine and huperzine A, have been used for the treatment of Alzheimer’s disease, but these drugs also show many side effects in patients. ^5,6 Thus, there is a need to search for compounds with anti-AChE activity with a minimum of these undesirable effects. Plants belonging to the genus Tabernaemontana (Apocynaceae), such as Tabernaemontana pandacaqui, have a worldwide distribution, including parts of the America, Africa, Asia, and Australia. ⁷ These plants are used in traditional medicine and for other purposes for the treatment of sore throat, hypertension, inflammatory, and abdominal pain. ^{8 -10} Many constituents of Tabernaemontana species have already been identified and monoterpene indole alkaloids have been shown to be present in several species. ⁸ However, the chemical constituents and AChE inhibitory activity of T. pandacaqui flowers have not been studied before. We now report the AChE inhibitory constituents from T. pandacaqui flower, as well as their structure-AChE inhibitory relationship.

The methanol extract of T. pandacaqui flowers {TPF(M)} was fractionated successively with n-hexane and ethyl acetate to afford n-hexane (TPF(M)-H), ethyl acetate (TPF(M)-EA), and aqueous residue fractions (TPF(M)-W). The modified Ellman’s assay was used to evaluate the AChE inhibitory activity of these fractions. The ethyl acetate fraction showed the strongest AChE inhibitory activity, with an inhibition at 5 mg/mL of 35.4% ± 0.14% (Figure 1). The ethyl acetate fraction was then subjected to silica gel 60 (Merck) quick column chromatography (QCC) using a gradient system of n-hexane-ethyl acetate and ethyl acetate-methanol to give 7 fractions (A1-A7). Fraction A2 was purified by column chromatography using a gradient system of n-hexane-ethyl acetate to give 1 and 2. Purification of fraction A3 (4.45 g) by repeated column chromatography and recrystallization yielded 3 and 4. Methanol was added to fraction A5 to give 2 subfractions (B1 and B2). Subfraction B1 was identified as compound 5. Purification of soluble subfraction B2 gave 8 subfractions (C1-C8). Subfraction C6 was also purified by silica gel 60 column chromatography to yield compound 6. The structures of all isolated compounds were elucidated on the basis of spectroscopic analysis. By comparing the spectroscopic and physical data with the literature values, their structures were identified as 7α-hydroxyindoleninevoacangine (1), ^11,12 β-sitosterol (2), ^13,14 p-anisic acid (3), ^15,16 ursolic acid (4), ^13,14,17 sitosterol-3-O-β-d-glucopyranoside (5), ^13,18 and astragalin (6). ^14,19 The chemical constituents from T. pandacaqui flowers are reported for the first time.

Figure 1

Anti-acetylcholinesterase activity of methanol extract and other fractions.

A number of flavonoid constituents have been reported to be AChE inhibitors, such as quercitrin, tiliroside, 3-methoxyquercetin, and quercetin from Agrimonia pilosa, ²⁰ and catechin and epigallocatechin gallate from Camellia sinensis. ²¹ Therefore, a major constituent, astragalin (6) and its derivatives, including kaempferol (7), quercetin (8), myricetin (9), and apigenin (10), was also evaluated for their AChE inhibitory activity using the modified Ellman method to study the structure-activity relationship.

A major flavonoid glucoside constituent, kaemferol-3-O-β-d-glucopyranoside or astragalin (6), isolated from the ethyl acetate fraction, was evaluated for AChE inhibitory activity and shown to have good activity producing 41.3% ± 0.5% inhibition at 100 µg/mL (Table 1). As this active constituent might play an important role in the treatment of Alzheimer’s disease, it was of great interest to study the structure-AChE activity relationship of flavonoid 6 and its derivatives. All flavonoids showed good inhibitory activity on AChE at 100 µg/mL (Table 1).

Table 1

Acetylcholinesterase Inhibitory Activity of Astragalin and Its Derivatives.

Conc. (μg/mL)	Acetylcholinesterase inhibitory activity (%) a
	6	7	8	9	10	Galanthamine b
1.56	7.4 ± 0.3	0.8 ± 0.3	1.2 ± 0.1	3.2 ± 0.3	1.7 ± 0.2	44.7 ± 0.3
3.13	11.1 ± 0.3	3.5 ± 0.5	3.9 ± 0.1	6.9 ± 0.4	4.4 ± 0.3	58.7 ± 0.3
6.25	15.4 ± 0.1	6.8 ± 0.4	5.4 ± 0.4	9.4 ± 0.3	7.6 ± 0.2	74.6 ± 0.1
12.50	21.0 ± 0.5	11.4 ± 0.2	8.5 ± 0.2	14.3 ± 0.6	10.0 ± 0.3	84.3 ± 0.4
25.00	25.8 ± 0.4	16.8 ± 0.3	15.9 ± 0.1	26.1 ± 0.1	17.7 ± 0.5	93.3 ± 0.2
50.00	32.5 ± 0.3	25.2 ± 0.4	26.6 ± 0.1	47.2 ± 0.2	31.1 ± 0.1	97.7 ± 0.4
100.00	41.3 ± 0.5C	32.8 ± 0.3D	41.3 ± 0.4C	68.6 ± 0.4A	46.5 ± 0.8B	99.8 ± 0.3

Data followed by different letters indicate statistically significant differences (P < 0.05).

^aAll values are mean ± SD based on 3 replicates.

^bStandard drug.

Based on the structure-activity relationship analysis, it was found that the effect of a free hydroxyl group at position 3 in the flavonoid moiety on AChE was observed. The isolated 3-O-β-d-glucopyranosyl flavonoid 6 was 1.3-fold more active than the aglycone 7 (32.8% ± 0.3%). With the absence of a hydroxyl group at position 3, apigenin (10, 46.5% ± 0.8%) was 1.1- and 1.4-fold more active than compounds 6 and 7, respectively. The results indicated that the absence of a hydroxyl group at position 3 on the 5,7,4′-trihydroxyflavonoids moiety caused an increase in AChE properties. In order to see the influence of the number of hydroxyl groups on ring B of the flavonoid moiety for AChE inhibitory activity, 3′,4′,5′-trihydroxyflavonoid 9 was found to be 1.7- and 2.1-fold more active than the dihydroxy 8 and monohydroxy 7, respectively. From these results, it was found that the number of hydroxyl groups on ring B of the flavonoid moiety is important for high AChE inhibitory activity. The structure-activity relationship of the isolated flavonoid and its derivatives is shown in Figure 2.

Figure 2

Structure-activity relationship of isolated flavonoid and its derivatives.

The inhibition of AChE is still considered an important strategy for the treatment of Alzheimer’s disease. The aim of this study was to search for new drugs from natural sources. Tabernaemontana pandacaqui flowers were found to contain constituents which have AChE inhibitory activity. In particular, the flavonoid glucoside astragalin (6), which is the major active constituent, showed good inhibitory activity on AChE. Tabernaemontana pandacaqui flowers and its flavonoid compounds should be further explored in the possible treatment of Alzheimer’s disease.

Experimental

General Experimental Procedures

All the organic solvents and chemicals used in this study were of analytical grade. ¹H and ¹³C NMR spectra were recorded on a Bruker AVANCE 400 spectrometer operating at 400 and 100 MHz, respectively. Unless indicated otherwise, column chromatography was carried out using Merck silica gel 60 (finer than 0.063 mm) and Pharmacia Sephadex LH-20. For Thin layer chromatography (TLC), Merck precoated silica gel 60 F254 plates were used. Reversed phase column chromatography was performed on Merck silica gel 60 RP-18. Compounds on TLC were detected under UV light and spraying with anisaldehyde-H₂SO₄ reagent followed by heating.

Plant Material

The flowers of T. pandacaqui (Apocynaceae) were purchased from a local market in Saensuk district, Chonburi province, Thailand during the month of May 2014. A voucher specimen has been deposited at the Faculty of Science, Burapha University.

Preparation of the Extract and Fractionation

The fresh powdered flowers of T. pandacaqui (2.0 kg) were immersed in methanol at room temperature and the extracts filtered. The solvent was then removed under reduced pressure at 50°C to give the dry methanol extract (150 g), which was dissolved in methanol-water (4:1) and extracted successively with equal volumes of n-hexane and ethyl acetate. Each fraction was then concentrated under reduced pressure at 50°C to yield the hexane, ethyl acetate, and residual fractions. The methanol extract and its fractions were freeze-dried and then refrigerated until further use.

Isolation of Active Compound From Tabernaemontana pandacaqui Flowers

The ethyl acetate fraction of TPF(M) (7.02 g) was subjected to silica gel 60 (Merck) QCC using a gradient system of hexane-ethyl acetate and ethyl acetate-methanol to give 7 fractions (A1-A7). Fraction A2 (309.1 mg) was purified by column chromatography using a gradient system of n-hexane-ethyl acetate to give 1 (1.6 mg) and 2 (1.2 mg). Purification of fraction A3 (4.45 g) by repeated column chromatography using a gradient system of n-hexane-dichloromethane and recrystallization from methanol yielded 3 (3.4 mg) and 4 (7.8 mg). Methanol was added to fraction A5 (1.25 g) to give insoluble and soluble subfractions (B1 and B2). Subfraction B1 was identified as compound 5 (7.1 mg). Purification of soluble subfraction B2 by Sephadex LH-20 column chromatography using isocratic conditions of methanol gave 8 subfractions (C1-C8). Subfraction C6 was also purified by silica gel 60 column chromatography using isocratic conditions of 1.0% methanol in ethyl acetate to yield 10.6 mg of compound 6. The structures of all isolated compounds were elucidated on the basis of spectroscopic analysis and also by comparison of the ¹H NMR and ¹³C NMR spectral data with those reported in the literature. Compounds 1 to 6 were identified as 7α-hydroxyindoleninevoacangine, ^11,12 β-sitosterol, ^13,14 p-anisic acid, ^15,16 ursolic acid, ^13,14,17 sitosterol-3-O-β-d-glucopyranoside, ^13,18 and astragalin, ^14,19 respectively.

Acetylcholinesterase Inhibitory Activity

Acetylcholinesterase inhibition was determined spectrophotometrically using acetylthiocholine iodide (ATCI) as substrate, by modifying the method of Ellman. ²² Briefly, in a 96-well plate, 150 µL of 10 mM phosphate buffer (pH 8.0), 20 µL of a solution of AChE (4.0 U/mL in 10 mM phosphate buffer, pH 8.0), and 10 µL of the test compound solution dissolved in dimethyl sulfoxide (DMSO) were mixed and incubated at room temperature for 15 minutes. The reaction was started by adding 20 µL of either a solution of 5 mM 5,5′-dithiobis-(2-nitrobenzoic acid) or DTNB in 10 mM phosphate buffer (pH 8.0), containing 0.1% bovine serum albumin and 5 mM ATCI in 10 mM phosphate buffer, pH 8.0 (3:1). The hydrolysis of acetylthiocholine was determined by monitoring the formation of the yellow 5-thio-2-nitrobenzoate anion as the product of the reaction with DTNB and thiocholines, catalyzed by enzymes at the wavelength of 405 nm using an EPOCH-2 microplate reader spectrophotometer and the absorbance was measured after 5 minutes of incubation at room temperature. Galanthamine was used as a reference standard. The percentage of inhibition was calculated by comparing the rate of enzymatic hydrolysis of ATCI for the sample to that of the blank (DMSO in buffer), which was calculated by the following equation: inhibition (%) = {[(A – B) – (C – D)]/(A – B)} × 100, where A is the activity of the enzyme without the inhibitor; B is the control of A without the inhibitor and enzyme; and C and D are the activities of the inhibitors with and without AChE, respectively. All experiments were carried out in triplicate.

Statistical Analysis

All analyses in this study were performed in 3 replicates. Minitab software version 18 was used for statistical analyses. Tukey’s tests were used to determine the variations between the means. Differences at 5% (P < 0.05) level were considered as significant.