Sesquilignans and sesquiterpenoid from the stem barks of Illicium simonsii and their anti-AChE activity

Three new sesquilignans, 1–3, a new sesquiterpenoid, 4, and three known compounds were isolated from the stem barks of Illicium simonsii. The structures of new compounds (1–4) were elucidated by spectroscopic methods. A biosynthetic pathway was proposed for simonsienols A-C (1–3). Anti-AChE activity and anti-BuChE activity were evaluated for all compounds except for α-cadinol ethyl ether (4). As a result, isodunnianol (7) exhibited anti-AChE activity with an IC50 value of 13.0 µM. Electronic Supplementary Material Supplementary material is available for this article at 10.1007/s13659-012-0026-z and is accessible for authorized users.


Introduction
The genus Illicium is the only member of the family Illiciaceae and is an evergreen shrub or tree. About 40 species have been found disjunctively in eastern North America, Mexico, the West Indies and the eastern Asia. The highest concentration of species is in the northern Myanmar and the southern China where nearly 35 species have been described. 1,2 seco-Prezizaane-type sesquiterpenes, prenylated C 6 -C 3 compounds and sesqui-neolignans are the secondary metabolites characteristic of the Illicium plants. 3 4-epi-illicinone E-12shikimate and 3-hydroxyillifunone B were isolated from the fruits of I. simonsii. 4 Simonin A and 1-hydroxyl-2-O-β-D-6′acetyl-glucopyranosyl-4-allybenzene were isolated from the stems of I. simonsii and simonin A showed activity against oral microbial organisms. 5 The unique structures and interesting biological activities have lured us to extensively investigate on the bioactive compounds of I. species. As a result, three new sesquilignans (1-3) and one new sesquiterpenoid (4), together with three known compounds macranthol (5), 6 dunnianol (6), 6 and isodunnianol (7) 7 were isolated from the stem barks of I. simonsii. Herein we report the isolation, structure elucidation and anti-AChE activity of these compounds.

Results and Discussion
The aired-dried stem barks of I. simonsii were extracted with EtOH for three times at room temperature. The extract was partitioned successively with petroleum ether, CHCl 3 and n-BuOH. The CHCl 3 fraction was subjected to a multistep chromatographic separation and purification procedures to afford pure compounds 1-7.
The   Table 2) of 1 showed the presence of 27 carbons, which were categorized into nine quaternary, twelve methine and six methylene. HMBC correlations (Figure 1) of H-3″/C-5″, H-5″/C-1″, 3″, H-6″/C-1″, 2″, 4″, 5″, H-7″/C-3″, 4″, 5″ and 1 H-1 H COSY correlation between H-5″ and H-6″ indicated that the position of the hydroxy group and allyl group on ring C should be at C-1″ and C-4″, respectively. Furthermore, HMBC correlations of H-3″/C-2 and H-3/C-2″ showed that ring C was connected to C-2 of ring A. Ring B was similarly confirmed to be connected to C-4 of ring A, indicating by the analyses of 1 H-1 H COSY and HMBC spectras. HMBC correlations of H-3, 5, 6/C-1 (δ C 154.0) and 1 H-1 H COSY correlation between H-5 and H-6 indicated that an oxygenated group might be at C-1 of ring A. According to the above NMR analysis, 1 was indicated to be a sesquilignan, which was similar to macranthol (5). 6 The significant difference was that an allyl unit was connected to C-1 through an oxygen atom, which was confirmed by HMBC correlations of H-7 (δ H 4.68)/C-1 (δ C 154.0), 8, 9 and 1 H-1 H COSY correlations of H-8/H-7, 9 ( Figure 1). At last, the structure of 1 was established as shown and named simonsienol A.
The molecular formula of 3 was determined as C 24 H 22 O 4 by HRESIMS. Analysis of its NMR (Tables 1 and 2) and MS data showed that 3 was similar to 2 except for the absence of one allyl group. Comparing with 2, a significant upfield chemical shift of C-4′ (δ C 118.0) was observed in 3. HMBC correlations ( Figure 1) of H-7/C-3, 4, 5, 8, 9, H-7″/C-3″, 4″, 5″, 8″, 9″, together with 1 H-1 H COSY correlation between H-4′ and H-5′ on ring C further supported the above assignment. Thus, the structure of 3 was established as shown and named simonsienol C. A possible mechanism for the formation of 1, 2 and 3 is shown in Scheme 1. . The 13 C NMR data (Experimental Section) showed that 4 was a sesquiterpenoid, which was very similar to α-cadinol methyl ether 9 except for the presence of an ethyl group. Diagnostic HMBC correlations were observed between C-10 (δ C 76.0), 17 and H-16, together with 1 H-1 H COSY correlatons (Figure 2) of H-16/H-17, implying the connection through oxygen bridge between C-16 and C-10. 4 might be an artificial product which was produced through the reaction with EtOH during the extraction process. All the groups had the same orientations as those in α-cadinol  All compounds except for compound 4 were evaluated for acetylcholinesterase (AChE) inhibitory activity by using 96-well microplate reader (680 XR, USA). Tacrine was used as a positive control with an IC 50 value of 0.33 µM. As a result, compound 7 was found to exhibit anti-AChE activity with an IC 50 value of 13.0 µM. However, all the tested compounds were inactive when BuChE inhibitory activity and neurotrophic effect were assayed.
Anti-BuChE assay. The Anti-BuChE assay was performed as previously described.
Neurotrophic Bioassay. 11 PC12 cells are bought from Kunming Institute of Zoology, Chinese Academy of Scienses and suspended in 12.5% HS + 2.5% FBS, then seeded at 50,000 cells/mL into poly-L-lysine-coated 48 well culture plates. After 48 h, the medium is changed to a serum-free medium F12 (10% HS + 5% FBS + 10 mg/mL NGF). 12 Samples at 50 µM and 5 µM are added into F12. The length of axon was measured and calculated by using microscope.

Electronic Supplementary Material
Supplementary material is available in the online version of this article at http://dx.doi.org/ 10.1007/s13659-012-0026-z and is accessible for authorized users.