Phytochemical study of Illicium angustisepalum and its biological activities

Abstract

Sixteen compounds, including two new natural products (1 and 2), were obtained from the twigs of Illicium angustisepalum. The structures were elucidated based on NMR, MS, IR data and optical rotation values. Compounds 4, 5, 6 and 8 displayed moderate antibacterial activities against clinical isolates; compounds 4, 5, 8, 9 and 15 protected neural cells against oxidative stress; and compounds 10 and 14 exhibited anti-acetylcholinesterase activity.

Graphical abstract

The isolation of sixteen compounds (1—16) from the twigs of Illicium angustisepalum was reported, including the new structures of 3-O-benzoyl-myrcenediol (1) and 3,6-dimethyl-3-hydroxy-tetrahydro-2H-pyran-2-one (2). The isolated compounds were evaluated in a battery of in vitro bioassay models for cytotoxic, antimicrobial, neuroprotective and anti-acetylcholinesterase activities.

1. Introduction

The genus Illicium L., commonly known as star anise or anise tree, comprises of over thirty species to form one of the earliest evolutionary branches of the angiosperms¹. The genus was previously classified as the only member of the Illiciaceae family; but later, the Angiosperm Phylogeny Group (APG) III system of 2009 included Illicium in the Schisandraceae without recognizing Illiciaceae as a distinct family². Represented by evergreen trees and shrubs disjunctively distributed in North America, Mexico, Peru, the West Indies and Eastern Asia, with the highest concentration of species in Northern Myanmar and Southern China, the genus is known to contain unique secondary metabolites, such as terpenoids, phenylpropanoids, lignans and benzoquinones³. The fruit part of several Illicium species is particularly rich in sesquiterpenes of the secoprezizaane, anislactone, and allocedrane types³.

While plants, such as Illicium verum (the common star anise) which is well known for use as a spice and medicinal plant, and Illicium parviflorum as a garden plant, Illicium angustisepalum A.C. Smith is a lesser known species endemic to Southern China. Until now, only two reports concerning its chemical composition (abietane diterpenes and a prezizaane sesquiterpene)4, 5 and one paper describing anti-inflammatory and analgesic effects of the crude extract⁶ can be found in literature. Herein, we report the isolation of sixteen compounds from the twigs of I. angustisepalum (1—16, Fig. 1), including the new structures of 3-O-benzoyl-myrcenediol (1) and 3,6-dimethyl-3-hydroxy-tetrahydro-2H-pyran-2-one (2). The isolated compounds were evaluated in a battery of in vitro bioassay models for cytotoxic, antimicrobial, neuroprotective and anti-acetylcholinesterase activities.

Figure 1.

Sixteen compounds isolated from the twigs of I. angustisepalum (1—16), including the new structures of 3-O-benzoyl-myrcenediol (1) and 3,6-dimethyl-3-hydroxy-tetrahydro-2H-pyran-2-one (2).

2. Results and discussion

Compound 1 was obtained as a colorless amorphous powder. The HR-ESI-MS of 1 displayed a quasi-molecular ion [M+H]⁺ at m/z 275.1569 (for C₁₇H₂₃O₃; Calcd. 275.1647), corresponding to the molecular formula of C₁₇H₂₂O₃ with seven indices of hydrogen deficiency. The IR broad stretch at 3429 cm^—1 suggested the presence of hydroxyl and a broad stretch at 1713 cm^—1 was consistent with the presence of benzoyl carbonyl moiety. The ¹H NMR spectrum (Table 1) displayed signals for olefinic protons at δ_H 6.34 (dd, J = 11.0, 17.7, H-7), 5.16 (d, J = 17.7, H-8a), 5.02 (br. s, H-8b), 5.0, (br. s, H-9) and two methyl groups at δ_H 1.22 (s, H₃-1/10). The ¹³C NMR and DEPT-135 NMR spectra of 1 (Table 1) exhibited 17 carbons signals corresponding to two methyl, four methylenes (two olefinic and two aliphatic), seven methines (five aromatic, an olefinic and an aliphatic carbons), an oxygenated tertiary carbon, a carbonyl carbon and two quaternary carbons. The presence of two double bonds accounted for two degrees of unsaturation; the remaining five were ascribed to an aromatic ring and a carbonyl group. In the ¹H—¹H COSY spectrum, signal at δ_H 7.61 (t, J = 7.3 Hz, H-5′) displayed correlations with the overlapped signal at δ_H 7.49 (H-4′/6′), which further exhibited correlation with an overlapped signal at δ_H 8.07 (dd, J = 7.5, 1.3 Hz, H-3′/7′), suggesting the presence of an unsubstituted aromatic ring. Correlations between H₂-4 (δ_H 1.87/2.02, m) and H-3 (δ_H 5.09, dd, J = 2.3, 10.1 Hz) and H₂-5 (δ_H 2.23, m) were observed. In addition, observation of COSY correlations between δ_H 6.34 (H-7), δ_H 5.16 (H-8a) and δ_H 5.02 (H-8b) led to establishing the connectivity of the skeleton. Aided by HMBC data, structure 1 was proposed as depicted. It is noteworthy that both the methine proton at δ_H 5.09 (H-3) and aromatic protons at δ_H 8.07 (H-3′/7′) displayed long-range correlations with C-1′ carbonyl carbon (δ_C 168.0). This piece of evidence was helpful in locating an ester group between the aromatic ring and the monoterpene portion of the molecule. The hydroxyl group was assignable to C-2 (δ_C 72.9) based on HMBC correlations with H₃-1, H-3 and H₃-10. Finally, the location of the two terminal double bonds was assigned based on the HMBC long-range correlations between H₂-8a/8b and C-6/C-7, as well as between H₂-9 and C-5/C-6/C-7. Comparison of the NMR data with literature values suggested close similarity of the aliphatic portion of 1 to myrcene-2,3-diol7, 8, 9, 10. Taking all evidence together, the planar structure of 1 was determined to be 3-O-benzoyl-2-methyl-6-methyleneoct-7-ene-2,3-diol and given a trivial name of 3-O-benzoyl-myrcenediol. The small value of optical rotation (${[\alpha ]}_{\mathrm{D}}^{25}$ + 1.4°; c 0.07, MeOH) suggested it was a racemic mixture of the 3R and 3S enantiomers. To the best of our knowledge, compound 1 is a new natural product.

Table 1.

¹H NMR and ¹³C NMR spectral data for compound 1.

Position	δH	δC
1	1.22, s	25.5, CH3
2	–	72.9, C
3	5.09, dd (J = 2.3, 10.1)	81.3, CH
4	4a, 2.02, m (overlapped)	29.3, CH2
	4b, 1.87, m (overlapped)
5	2.23, m (overlapped)	29.6, CH2
6	–	147.2, C
7	6.34, dd (J = 11.0, 17.7)	139.7, CH
8	8a, 5.16, d (J = 17.7)	113.7, CH2
	8b, 5.02, m (overlapped)
9	5.0, m (overlapped)	116.7, CH2
10	1.22, s	26.4, CH3
1′	–	168.0, C
2′	–	131.6, C
3′	8.07, dd (J = 7.5, 1.3)	130.6, CH
4′	7.49, t (J = 7.5)	129.6, CH
5′	7.61, t (J = 7.3)	134.3, CH
6′	7.49, t (J = 7.5)	129.6, CH
7′	8.07, dd (J = 7.5, 1.3)	130.6, CH

^aData were measured in MeOD-d₄ at 400 MHz for ¹H NMR and 100 MHz for ¹³C NMR. The δ values are expressed in ppm and the coupling constants (J) in Hz. The assignments were made on the basis of DEPT, ¹H—¹H COSY, HSQC and HMBC results.

Compound 2 was obtained as an oil. The HR-ESI-MS quasi-molecular ion [M+H]⁺ at m/z 145.0866 (Calcd. for C₇H₁₃O₃, 145.0861) indicated a molecular formula of C₇H₁₂O₃ with two indices of hydrogen deficiency. The IR absorption at 1779 cm^—1 suggested the presence of a δ-lactone. The ¹³C NMR and DEPT-135 NMR spectra (in methanol-d₄, Table 2) displayed seven carbons, including two CH₃ (C-7, δ_C 21.8; C-8, δ_C 29.8), two CH₂ (C-4, δ_C 44.4; C-5, δ_C 43.8), a CH (C-6, δ_C 75.6), a tertiary oxygenated carbon (C-3, δ_C 68.5) and a carbonyl carbon (C-2, δ_C 174.0). The ¹H—¹H COSY spectrum revealed correlations between H₂-4 and H₂-5, suggesting that the two methylene groups are adjacent to each other. The COSY data also correlated H-6 (δ_H 4.77, m) with H₃-7 and H₂-5. It is noteworthy that the carbonyl carbon at δ_C 174.0 (C-2) displayed HMBC correlation with H-6 at δ_H 4.77. Compound 2 was thus elucidated to be 3,6-dimethyl-3-hydroxy-tetrahydro-2H-pyran-2-one based on available evidence. Its stereochemistry has not been determined. Nevertheless, it is noted that, in a similar structure [3-hydroxy-3-methyl-6-(3-methylbut-3-enyl)tetrahydro-2H-pyran-2-one]¹¹, the 3β-methyl group resonates at δ_H 2.1, whereas the methyl group in 2 resonates δ_H 1.3. Such a discrepancy may be due to a reversed stereochemistry at C-3 or C-6. To the best of our knowledge, compound 2 is a new natural product.

Table 2.

¹H NMR and ¹³C NMR spectral data for compound 2.

Position	δH	δC
2	–	174.0, C
3	–	68.5, C
4	2.50—2.57, m	44.4, CH2
5	5a, 1.64, dd (J = 7.1, 11.8)	43.8, CH2
	5b, 1.90, dd (J = 1.8, 11.8)
6	4.77, m	75.6, CH
7	1.37, d (J = 6.4)	21.8, CH3
8	1.30, s	29.8, CH3

^aData were measured in MeOD-d₄ at 400 MHz for ¹H NMR and 100 MHz for ¹³C NMR. The δ values are expressed in ppm and the coupling constants (J) in Hz. The assignments were made on the basis of DEPT, ¹H—¹H COSY, HSQC and HMBC results.

The structures of other compounds isolated from I. angustisepalum were determined by detailed examination of their spectroscopic data and comparison with published values. They were identified to be majusanic acids B (3) and C (4)¹², angustanoic acids E (5), F (6), G (8)⁴, angustanol (7)⁴, (—)-τ-muurolol (9)¹³, clovane-2β,9α-diol (10)¹⁴, angustisepalin (11)⁵, majucin (12)¹⁵, 6β-hydroxy-4-stigmasten-3-one (13)¹⁶, thymol (14)¹⁷, 2,6-dimethoxychavicol (6-methoxyeugenol, 15)¹⁸ and (+)-taxifolin (dihydroquercetin, 16)¹⁹. With the exception of 3—8 and 11, other compounds are reported from I. angustisepalum for the first time.

In order to establish the biological profile of I. angustisepalum and the isolates, they were evaluated in a battery of testing models as follows.

2.1. Cytotoxicity study

All compounds were tested in three cell lines, i.e. MDA-MB-435 human melanoma cancer cells, MDA-MB-231 human breast cancer cells and OVCAR3 human ovarian cancer cells, using vinblastine as positive control. None of the compound displayed cytotoxic activity (IC₅₀>25 µg/mL). These results were consistent with the non-cytotoxic property previously reported for angustanoic acids B and E, angustanol and other similar compounds12, 20.

2.2. Antimicrobial activity evaluation

Isolated compounds from I. angustisepalum were tested against clinical isolates of antibiotic resistant strains. Among them, angustanoic acid E (5) was the most active against Escherichia coli BW25113 ∆TolC (MIC 21 µmol/L), Staphylococcus aureus USA 300 (MIC 42 µmol/L), and S. aureus MSSA 476 (MIC 42 µmol/L). It was also active against Bacillus anthracis sterne and Bacillus cereus 14579 with MIC of 21 µmol/L, but was inactive against Acinetobacter calcoaceticus. On the other hand, majusanic acid C (4) and angustanoic acids F (6) and G (8) displayed MIC of 75, 75, and 150 µmol/L, respectively, against E. coli BW25113 ∆TolC. In the literature, angustanoic acids (e.g. angustanoic acid F)²¹ and majusanic acids (e.g. majusanic acids B, D, E and F)²² have shown to possess antiviral activity against Coxsackie B viruses.

2.3. Neuroprotection activity study

Investigation of bioprotection was evaluated in pheochromocytoma PC12 cells against damages induced by 1-methyl-4-phenylpyridinium (MPP⁺). The most significant protection against MPP⁺ damage was exerted by angustanoic acid G (8) at 1.3 µmol/L, comparable to the positive control AOP-6A (at a concentration of 16 µmol/L). Moderate protective effect was also observed for majusanic acid C (4), angustanoic acid E (5), (—)-τ-muurolol (9) and 2,6-dimethoxychavicol (15), at concentration range between 5—30 µmol/L. The neuroprotection activity may be related to their antioxidant property. It is interesting to note that, while majucin itself did not show significant neurotropic effect, some of the related seco-prezi-zaanes (such as jiadifenolide) were found to promote neurite outgrowth in PC12 cells²³.

2.4. Acetylcholinesterase inhibitory activity evaluation

Using TLC bioautographic screening, both the volatile oil fraction and the EtOAc fraction of I. angustisepalum were active in inhibiting acetylcholinesterase activity. Thymol (14) was an active ingredient in the volatile fraction. The compound has been reported to exhibit anti-acetylcholinesterase activity (IC₅₀ 0.2 mg/mL)²⁴. When the isolated compounds were tested in the same system, 3,6-dimethyl-3-hydroxy-tetrahydro-2H-pyran-2-one (2), clovane-2β,9α-diol (10), angustisepalin (11), 2,6-dimethoxychavicol (15), and taxifolin (16) displayed inhibitory activity. Among these compounds, taxifolin has been shown to strongly inhibit acetylcholinesterase at an IC₅₀ of 30 nmol/L²⁵. The inhibitory activity was further demonstrated using the Ellman׳s method, in which 10 and 14 exhibited the best results with IC₅₀ values of 45 and 90 µmol/L, respectively.

3. Conclusions

The twigs of I. angustisepalum afforded sixteen compounds in the present phytochemical study. Several of these secondary metabolites displayed in vitro activities in anti-microbial, neuroprotection and anti-acetylcholinesterase test models. As a whole, I. angustisepalum is an under-exploited plant species; more in-depth studies are warranted in order to fully evaluate its potentials.

4. Experimental

4.1. General experimental procedures

All solvents used were analytical or HPLC grade. TLC: Merck aluminium backed sheets coated with 60F254 silica gel or 60F254 RP-silica gel; visualization by using a UV lamp (λ_max 254 nm), and spraying with Komarowsky reagent (a mixture of 2% 4-hydroxybenzaldehyde MeOH and 5% H₂SO₄/EtOH, 10:1 (v/v)), followed by heating. Open column chromatography: silica gel (SiO₂), MCI gel CPH20P (Supelco, Sigma--Aldrich, USA) or Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Sweden). For HPLC purification, a C18 semi-preparative HPLC column (Phenomenex C18 column, 250 mm ×10 mm, 5 mm) and a Shimadzu UFLC system were used; the UV detection wavelength and flow rate were set at 254 nm and 4 mL/min, respectively. Optical rotations: at Na D line; Perkin-Elmer 241 digital polarimeter using quartz cell with a path length of 100 mm. NMR Spectra: Bruker DPX-400 spectrometer; chemical shifts (δ) in ppm using residual solvent as the internal standard (DMSO-d₆: 2.50 ppm for ¹H NMR and 39.51 ppm for ¹³C NMR; CDCl₃: 7.24 ppm for ¹H NMR and 77.23 ppm for ¹³C NMR; methanol-d₄: 3.31, 4.78 ppm for ¹H NMR and 49.2 ppm for ¹³C NMR); coupling constants (J) in Hz. HR-ESI-MS: Shimadzu LC—MS-IT-TOF mass spectrometer.

4.2. Plant material

Twigs of I. angustisepalum were collected in spring 2011 from Lantau Island, Hong Kong (China) by one of the author (Ming Zhao) and authenticated by Miss Yuying Zong of the School of Chinese Medicine, the Chinese University of Hong Kong. Voucher specimens were deposited at the same institute. Fresh twigs were cut into small pieces, dried under sun and milled into fine powder.

4.3. Extraction and isolation

The pulverized twigs (1 kg) were exhaustively extracted by percolation with 90% EtOH at room temperature and dried under reduced pressure. The dried extract (232 g) was partitioned into petroleum ether-soluble (39 g), ethyl acetate-soluble (50 g), butanol-soluble (63 g) and water-soluble (78 g) fractions. The ethyl acetate-soluble part was separated into 47 fractions by a flash column of SiO₂ eluted by mixtures of petroleum ether and EtOAc (100:0 to 0:100). Sixteen compounds (1—16) were purified following repeated column chromatography and semi-preparative HPLC.

4.3.1. 3-O-Benzoyl-myrcenediol (1)

Colorless amorphous powder, 2 mg; ${[\alpha ]}_{\mathrm{D}}^{25}$ + 1.4° (c 0.07, MeOH); IR (film) ν_max 3429, 2971, 2936, 2366, 2342, 1713, 1275, 1119, 716 cm^—1; ¹H NMR and ¹³C NMR data, see Table 1; HR-ESI-MS m/z 275.1569 ([M+H]⁺, Calcd. for C₁₇H₂₃O₃, 275.1647).

4.3.2. 3,6-Dimethyl-3-hydroxy-tetrahydro-2H-pyran-2-one (2)

Yellow oil, 1.5 mg; ${[\alpha ]}_{\mathrm{D}}^{25}$ —2.2° (c 0.28, MeOH); IR (film) ν_max 3366, 2970, 2933, 1779, 1705, 1378, 1247, 1122 cm^—1; ¹H NMR and ¹³C NMR data, see Table 2; HR-ESI-MS m/z 145.0866 ([M+H]⁺, Calcd. for C₇H₁₃O₃, 145.0861).

4.3.3. Majusanic acid B (3)

Colorless amorphous powder, 8 mg; ${[\alpha ]}_{\mathrm{D}}^{25}$ + 75.75° (c 0.8, MeOH); IR (film) ν_max 3400, 2958, 2934, 1701, 1046, 1025, 1000 cm^—1; HR-ESI-MS m/z 332.1988 ([M+H]⁺, Calcd. for C₂₀H₂₈O₄, 332.1980).

4.3.4. Majusanic acid C (4)

Colorless amorphous powder, 11 mg; ${[\alpha ]}_{\mathrm{D}}^{25}$ + 88.0° (c 0.1, MeOH); IR ν_max 2964, 2931, 1692, 1471, 1256, 1171, 1147, 1071, 755 cm^—1; HR-ESI-MS m/z 330.2195 ([M+H]⁺; Calcd. for C₂₁H₃₀O₃, 330.2187).

4.3.5. Angustanoic acid E (5)

Colorless amorphous powder, 9 mg; ${[\alpha ]}_{\mathrm{D}}^{25}$ + 22.1° (c 0.19, MeOH); IR (film) ν_max 2956, 2931, 2850, 1695, 1437, 1231, 108, 950, 888 cm^—1; HR-ESI-MS m/z 299.2004 ([M+H]⁺, Calcd. for C₂₀H₂₇O₂, 299.2004).

4.3.6. Angustanoic acid F (6)

Colorless crystal, 10 mg; ${[\alpha ]}_{\mathrm{D}}^{25}$ + 88.0° (c 0.1, MeOH); IR (film) ν_max 3400, 2962, 2930, 1706, 1436, 1230, 1155, 1017, 951 cm^—1; HR-ESI-MS m/z 315.1966 ([M—H]^—, Calcd. for C₂₀H₂₇O₃, 315.1953).

4.3.7. Angustanol (7)

Colorless amorphous powder, 24 mg; ${[\alpha ]}_{\mathrm{D}}^{25}$ + 9.0° (c 0.1, acetone); IR (film) ν_max 3357, 2960, 2931, 2906, 2871, 1697, 1455, 1375, 1139, 1041 cm^—1; HR-ESI-MS m/z 302.2244 ([M+H]⁺, Calcd. for C₂₀H₃₀O₂, 302.2238).

4.3.8. Angustanoic acid G (8)

Colorless crystal, 27 mg; ${[\alpha ]}_{\mathrm{D}}^{25}$ + 135.3° (c 0.17, MeOH); IR (film) ν_max 2932, 1706, 1677, 1271, 1017, 951 cm^—1; HR-ESI-MS m/z 301.1786 ([M+H]⁺, Calcd. for C₁₉H₂₅O₃, 301.1797).

4.3.9. (—)-τ-Muurolol (9)

Yellow oil, 6 mg; ${[\alpha ]}_{\mathrm{D}}^{25}$ —70.0° (c 0.1, DCM); IR (film) ν_max 3345, 2957, 2932, 2907, 2870, 1713, 1668, 1454, 1369, 1019, 952 cm^—1; HR-ESI-MS m/z 205.1948 ([M—H₂O+H]⁺, Calcd. for C₁₅H₂₅, 205.1950).

4.3.10. Clovane-2,9-diol (10)

Colorless amorphous powder, 58 mg; ${[\alpha ]}_{\mathrm{D}}^{25}$ —5.0° (c 0.08, CHCl₃); IR (film) ν_max 3366, 2920, 2851, 1744, 1718, 1463, 1367, 1245, 1073 cm^—1; HR-ESI-MS m/z 238.1933 ([M+H]⁺, Calcd. for C₁₅H₂₆O₂, 238.1926).

4.3.11. Angustisepalin (11)

Colorless amorphous powder, 30 mg; ${[\alpha ]}_{\mathrm{D}}^{25}$ —33.3° (c 0.06, CHCl₃); IR (film) ν_max 3480, 2955, 1785, 1754, 1731, 1451, 1372, 1250, 1178, 1091, 711 cm^—1; HR-ESI-MS m/z 417.1532 ([M+H]⁺, Calcd. for C₂₂H₂₅O₈, 417.1542).

4.3.12. Majucin (12)

Colorless crystal, 9 mg; ${[\alpha ]}_{\mathrm{D}}^{25}$ —71.4° (c 0.22, MeOH); IR (film) ν_max 3453, 2939, 2874, 1770, 1731, 1510, 1453, 1372, 1211, 1123, 1008 cm^—1; HR-ESI-MS m/z 328.1158 ([M+H]⁺, Calcd. for C₁₅H₂₀O₈, 328.1152).

4.3.13. 6β-Hydroxy-4-stigmasten-3-one (13)

Colorless crystal, 3 mg; ${[\alpha ]}_{\mathrm{D}}^{25}$ + 1.42° (c 0.07, MeOH); IR (film) ν_max 3400, 2918, 2849, 2359, 1341 cm^—1; HR-ESI-MS m/z 429.3586 ([M+H]⁺, Calcd. for C₂₉H₄₉O₂, 429.3720).

4.3.14. Thymol (14)

Colorless powder, 9 mg. The identity of thymol was confirmed by means of co-TLC with a reference standard (Sigma—Aldrich) and also by analysis of GC—MS data. In the TLC analysis, the R_f values of 14 and the reference standard were both 0.9 after elution with petroleum ether–ethyl acetate (6:4). The GC chromatogram displayed signal at 12.67 and 12.66 min, respectively, for 14 and the reference standard. GC—MS revealed the presence of [M+H]⁺ at m/z 150.00 for both compounds.

4.3.15. 2,6-Dimethoxychavicol (15)

Yellow oil, 30 mg; ${[\alpha ]}_{\mathrm{D}}^{25}$ —0.3° (c 0.69, MeOH); IR (film) ν_max 3443, 2937, 1613, 1515, 1459, 1428, 1328, 1239, 1213, 1119 cm^—1; HR-ESI-MS m/z 194.0943 ([M+H]⁺, Calcd. for C₁₁H₁₄O₃, 194.0939).

4.3.16. (+)-Taxifolin (16)

Yellow amorphous powder, 57 mg; ${[\alpha ]}_{\mathrm{D}}^{25}$ + 18.0° (c 0.5, acetone); IR (film) ν_max 3344, 1638, 1469, 1283, 1162, 1086 cm^—1; HR-ESI-MS m/z 303.0502 ([M—H]^—, Calcd. for C₁₅H₁₁O₇, 303.0501).

4.4. Cytotoxicity assay

Cancer cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA), and the assay procedures previously described were adopted²⁶.

4.5. Antibacterial assay

The antibacterial activity against strains of A. calcoaceticus, B. anthracis sterne, B. cereus 14579, Enterococcus faecalis V583, E. coli MG1655, E. coli BW25113 ∆TolC, S. aureus USA 300, and S. aureus MSSA 476, were performed according to established procedures²⁷.

4.6. Neuroprotection assay

PC12 cells were obtained from the American Type Culture Collection (Rockville, MD, USA) and treated as previously described²⁸. The cells were seeded onto 96-well culture plates at a density of 2 × 10⁴ cells/well, cultured in serum-free medium and incubated for 24 h in the presence or absence of MPP⁺ (500 µmol/L) and test compounds. Cell viability was measured using a CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA).

4.7. Acetylcholinesterase inhibition assays

Two assays were performed to test for anti-acetylcholinesterase activity. The TLC-autobiographic assay was performed according to the modified Marston׳s method29, 30. For detection, Fast Blue B salt solution was sprayed onto the TLC plate to give a purple coloration. Acetylcholinesterase inhibitors produced white spots on the purple background. A microplate assay was also adopted from the Ellman׳s method³¹. Enzyme inhibition was expressed as pIC₅₀ (negative logarithms of molar concentrations of inhibitor required to decrease acetylcholinesterase activity by 50%). The percent of inhibition was calculated relative to a blank.