Antiacetylcholinesterase triterpenes from the fruits of Cimicifuga yunnanensis

Two new cycloartane triterpenes, cimyunnin E (1), containing a unique oxaspiro[4.4]nonanedione moiety based on rings D and E, together with cimicifine B (2), a 25,26,27-trinortriterpene featuring a pyridine ring E, were purified from the fruits of Cimicifuga yunnanensis. Their structures were elucidated by spectroscopic methods and ECD (electronic circular dichroism calculations). Compounds 1 and 2 showed significant acetylcholinesterase (AChE) inhibition with IC50 values of 1.58 and 3.87 μM, respectively. In addition, they noticeably enhanced the neurite outgrowth of nerve growth factor (NGF) mediated PC12 cells at a concentration of 10 μM.


Introduction
Plants of the Cimicifuga genus, such as C. racemosa, C. foetida, C. dahurica, C. heracleifolia, C. simplex, and C. japonica, are wellknown herbal medicines in Europe, the United States, and East Asia. [1][2][3][4] In the past few decades, more than 300 9,19cycloartane triterpenes (CTs), which are considered to be the main active chemical constituents of this genus, have been reported.  Moreover, biological evaluations revealed these CTs possessed a variety of activities, such as cytotoxicity, 5,[12][13][14] antiosteoporotic, 31 anti-AIDS, 32 immunosuppression, 33 anti-angiogenic, 6 and anti-Alzheimer. 34 It is worth mentioning that previous studies had mainly focused on the roots of aforementioned Cimicifuga spp.  In an attempt to further explore structurally and biologically interesting CTs from this genus, we started to study some rare species, such as C. yunnanensis and C. frigida, particularly, on the fruits and owers of these plants. As a result, a number of novel bioactive CTs were discovered. Cimyunnin A, a CTs possessed an unique cyclopentenone ring G from the fruits of C. yunnanensis, was considered as an anti-angiogenic leading structure. 6 In addition, cimifrigines A-G, a series of cytotoxic CTs featured by an unusual oxime group at C-15, were identied from the owers of C. frigida. 35 Be motivated by these ndings, we continually carried out phytochemical and pharmaceutical studies on the fruits of C. yunnanensis. Consequently, another two novel triterpenes, cimyunnin E (1) and cimicine B (2), were isolated and identied (Fig. 1). Compound 1 represents the rst example of CTs with an unprecedented oxaspiro [4.4]nonanedione unite formed in ring D and E. While, compounds 2 is a trinortriterpene containing a pyridine ring E. Signicantly, biological evaluations revealed that compounds 1 and 2 were strong AChE inhibitors with IC 50 values of 1.58 and 3.87 mM, respectively (the IC 50 value of positive control tacrine is 0.17 mM, see Table S1 and Fig. S22 †). Moreover, the neuronal differentiation of NGF-mediated PC12 cells were also enhanced by compounds 1 and 2 at the concentration of 10 mM (Table S2 and Fig. S23 †). Described herein are the isolation, structure elucidation, and biological activities of these compounds.  (Table 1) and HMQC spectra revealed the existence of two olenic carbons at d C 117.0 (C-7, d) and 140.5 (C-8, s), an ester carbonyl group at d C 170.1 (s), two carbonyl carbons at d C 199.9 (C-15, s) and 204.6 (C-16, s), and six oxygenated carbon atoms at d C 87.6 (C-3, d), 67.2 (C-12, d), 87.4 (C-17, s), 78.3 (C-23, d), 79.4 (C-24, d), and 72.1 (C-25, s), respectively. These data suggested that 1 was a highly oxygenated CTs glycoside with a six-ring skeleton.

Results and discussion
The detailed 1D and 2D NMR analyses established the planar structure of 1. The 1 H-1 H COSY (Fig. 2) spectrum disclosed that 1 has partial structures -CH 2 CH 2 CH-(due to C-1 to C-3), -CHCH 2 CH-(for C-5 to C-7), and -CH 2 CH-(for C-11 to C-12), that were compatible for rings A, B, and C of the CTs with a pair of double bond at C-7 and C-8. 27,29 The existence of the downeld shied cyclopropane methylene (d H 0.69 and 1.04) further supported this deduction. The acetoxy group was located to C-12 on the basis of the HMBC correlation of H-12 (d H 6.20) and the ester carbonyl group (d C 170.1). Similarly, the sugar unit was attached to C-3 by the HMBC coupling between  the anomeric proton (d H 4.76) and C-3 (d C 87.6). In addition, the sugar obtained aer acid hydrolysis was identied as L-arabinose by comparing its TLC and specic rotation with a standard. Thus, ring A, B, C, F, and the sugar unit of 1 was constructed as shown (Fig. 2).
The spin system -CH 3 CHCH 2 CHCH-due to Me-21, C-20, C-22, C-23 and C-24 was also deduced from 1 H-1 H COSY correlations (Fig. 2). In addition, HMBC correlations from CH 3 -21 (d H 1.29) and H-22 (2.92, 1H) to the oxygenated quaternary carbon at d C 87.4 (C-17) indicated the linkage of C-20 and C-17 (Fig. 2). Similarly, the connection of C-24 to the isopropanol group (C-25, C-26, and C-27) was elucidated by the HMBC couplings of H-24 (d H 3.74) CH 3 -26 (d H 1.58) and CH 3 -27 (d H 1.50) to the hydroxyl substituted quaternary carbon at d C 72.1 (C-25). By now, there were still one oxygen atom and two carbonyl carbons (C-15, d C 199.9; C-16, d C 204.6) need to be assigned and four degree of unsaturation unaccounted for, requiring another two rings in the nal structure. In the HMBC spectrum, correlations of CH 3 -28 (d H 1.67) to C-14 (d C 54.9) and C-15 (d C 199.9) were observed, indicating the connection between C-14 and C-15. Similarly, the connection of C-13 and C-17 was deduced from HMBC correlations of CH 3 -18 (d H 1.37) to C-13 (d C 46.8), and C-17 (d C 87.4), respectively. Thus, to fulll the unsaturation requirement, the molecular weight, and the chemical shi of C-15 and C-16, C-17 was connected to C-16 and C-23 was linked to C-17 by an oxygen atom (based on HMBC correlations, C-17 and C-23 could be connected by the carbonyl carbon C-16. Thus, ring D would be a ve-membered lactone ring. In that case, the chemical shi for C-15 should around d C 170.0). Therefore, an unique 15,16-cyclopentanedione-17-spiro-17,23-oxolane moiety was established in 1. Finally, the planar structure of 1 was constructed as shown (Fig. 2).
In the ROESY spectrum ( Fig. 2), correlations of H-5 (biogenetically a-oriented)/H-3, H-3/H-1 0 , and Me-28 (biogenetically aoriented)/H-12 indicated the a-orientation of H-1 0 , H-3, and H-12. Our efforts to make ne crystals from 1 failed which precluded the possibility to determine the absolute conguration directly by X-ray crystallography. However, the diagnostic ROESY couplings of Me-18 (biogenetically b-oriented) to H-20, H-20 to H-23, and acetoxy methyl to Me-21 (biogenetically aoriented) were observed, which help to determine b-orientation of H-20 and H-23 and the conformation of ring E as shown (Fig. 2). Therefore, the relative conguration of C-17, C-20, and C-23 of 1 was assigned as S*, R*, and R*, respectively. For the 17R*, 20S*, and 23S* stereoisomer of 1, those correlations would not be observed (except H-20 to H-23, Fig. S21 †), which further conrmed this deduction. On the basis of ROESY correlations of H-23/H-24, H-24/H-22, and H-23/Me-27 and the 3 J H,H value of H-23 and H-24 (6.3 Hz), the Newman projection of C-23/C-24 coupling system was established as shown (Fig. 3), which help to establish the S* conguration of C-24. Finally, the ECD calculation was applied to determine the absolute conguration of 1. As shown in Fig. 4, spectrum calculated for the 17S, 20R, 23R, and 24S one was nearly identical with the experimental data of 1 over the whole range of wavelengths under investigation, whereas the stereoisomer exhibited very different ECD behaviour between 250-300 nm. Therefore, the absolute congurations of C-17, C-20, C-23, and C-24 of 1 were determined as S, R, R, and S, respectively.
The molecular composition of cimicine B (2) . Therefore, aforementioned data suggested that 2 was a trinortriterpene Fig. 3 The Newman projection of C-23/C-24 coupling system of 1. glycoside and a six-ring structure, which included an unsaturated azacycle, was required to fulll the unsaturation requirement.
Extensive analyses of 2D NMR spectra revealed the conformation of ring A, B, C, D, and F of 2 as shown (Fig. 5), which was similar to those of known compounds.  In the 1 H-1 H COSY spectrum (Fig. 5) . Thus, to fulll the double-bond equivalents, a pyridine ring E should be fused to ring D through C-16 and C-17. Similarly, to fulll the molecular weight of 2 an aldehyde group was assigned to C-24, which was further supported by the HMBC correlation of an active hydrogen (d H 10.22) and C-23 (d C 151.4). Finally, by the same way as that of 1, the sugar was identied as L-arabinose and located at C-3. The orientations of H-3 and H-11 were ascribed by the ROESY correlations (Fig. 5) between H-5 (biogenetically a-oriented) and H-3, and H-11. Thus, the structure of 2 was determined as shown.
Hypothetically, prevention of acetylcholine (Ach) hydrolysis could increase the efficiency of cholinergic transmissions, which has been reported to be associated with the onset of Alzheimer's disease (AD). 36 Thus, enhancement of ACh levels by potent AChE inhibitors in the brain has been considered to be an effective approach for treating AD. [37][38][39] Although prominent biological activities of CTs from Cimicifuga spp. have been reported, to date, however, anti-AChE knowledge of those chemical constituents is mainly not yet involved. Thus, the AChE inhibitory activities of compounds 1 and 2 were evaluated using the Ellman method. 40 Unexpectedly, compound 1 exhibited signicant inhibition on AChE with an IC 50 value of 1.58 mM (Table S1 and Fig. S22 †). Similarly, compound 2 showed noticeable inhibitory effect on AChE, having an IC 50 value of 3.87 mM. Tacrine was used as the positive control and had an IC 50 value of 0.17 mM.
AD is a type of neurodegenerative diseases. Any agent with neurotrophic activity may benet AD. Therefore, the effects of 1 and 2 to stimulate NGF-mediated neurite outgrowth on PC12 cells were further evaluated. As a result, 1 and 2 obviously increased the neuronal differentiation at a concentration of 10 mM. The differentiation rates are 15.34% and 11.72% for 1 and 2, respectively, compared with 4.17% of the negative control and 19.18% of the positive control (Table S2 and Fig. S23 †).

Conclusions
In our continual investigation on the fruits of C. yunnanensis, another two unusual CTs were obtained. Cimyunnin E (1) is the rst CTs possessing an oxaspiro [4.4]nonanedione moiety in ring D and E. While, cimicine B (2) is a trinortriterpene contains a fused pyridine ring E. Signicantly, these two compounds showed potent anti-AChE effects and neurotrophic activities. Therefore, the bioactivities of cimyunnin E and cimicine B deserve further study. In summary, once again, novel active constituents were isolated from fruits of C. yunnanensis, the more sophisticated parts of this genus, such as pollen and vegetative organ are worth studying in future.

General experimental procedures
A JASCO P-1020 digital polarimeter was applied to record optical rotations, using MeOH as solvent. 1D and 2D NMR spectra were performed on Bruker DRX-500 and Avance III-600 MHz spectrometers (Bruker, Z} urich, Switzerland) with solvent signal as internal reference. ESIMS and HRESIMS were run on an Agilent G6230 TOF MS (Agilent Technologies, Palo Alto, USA). Infrared spectra were tested on a Shimadzu IR-450 instrument with KBr pellets. Column chromatography (CC) was run on Silica gel (200-300 mesh, Qingdao Marine Chemical, Inc.), and Lichroprep RP-18 (40-63 mm, Merck). Semipreparative HPLC was carried out on an Agilent 1100 liquid chromatography system using an YMC-Pack 10 mm Â 250 mm column (Pro C18 RS). Precoated TLC plates (200-250 mm thickness, silica gel 60 F 254 , Qingdao Marine Chemical, Inc.) were used for thin-layer chromatography. The spots in TLC were visualized by heating aer spraying with 10% aq. H 2 SO 4 .

Plant material
The fruits of Cimicifuga yunnanensis (1.5 kg) were collected from Bomi County, Tibet, China, in September 2013. Prof. Wang Zongyu, Kunming Institute of Botany, Chinese Academy of Sciences, identied the species. A voucher specimen (KUN no. 201309007) has been deposited at the State Key Laboratory of  Table 1 for 1 H NMR (600 MHz, C 5 D 5 N) and 13 Table 1 for 1 H NMR (600 MHz, C 5 D 5 N) and 13

Hydrolysis and identication of the sugar units in compounds 1 and 2
The MeOH solution (3 mL) of each compound (1.5 mg) was reuxed with 0.5 N HCl (2 mL) for 2 h. CHCl 3 (3 Â 6 mL) was used to extract the reaction mixture aer diluting with H 2 O. A monosaccharide was given by neutralizing each aqueous layer with Ag 2 CO 3 and ltering the precipitate. The monosaccharide from compounds 1 and 2 had an R f (EtOAc-CHCl 3 -MeOH-H 2 O, 3 : 2 : 2 : 1) and specic rotation of [a] 20 D +85.4 (c 0.08, MeOH) corresponding to those of L-arabinose (Sigma-Aldrich).

Acetylcholinesterase inhibitory activity
Acetylcholinesterase (AChE) inhibitory activities of compounds 1 and 2 were assayed by the spectrophotometric method developed by Ellman et al. 40 with slightly modication. S-Acetylthiocholine iodide, 5,5 0 -dithio-bis-(2-nitrobenzoic) acid (DTNB, Ellman's reagent), and acetylcholinesterase derived from human erythrocytes were purchased from Sigma Chemical. Compounds 1 and 2 were dissolved in DMSO. The reaction mixture (totally 200 mL) containing phosphate buffer (pH 8.0), test compound (50 mM for preliminary screening; 100, 50, 30, 10, 3, 1, and 0.2 mM for IC 50 value assay), and acetylcholinesterase (0.02 U mL À1 ) was incubated for 20 min (30 C). Then, the reaction was initiated by the addition of 40 mL of solution containing DTNB (0.625 mM) and acetylthiocholine iodide (0.625 mM) for AChE inhibitory activity assay. The hydrolysis of acetylthiocholine was monitored at 405 nm every 30 seconds for one hour. Tacrine was used as positive control with nal concentration of 0.333 mM for preliminary screening and 2, 1, 0.5, 0.2, 0.04, 0.008, and 0.0016 mM for IC 50 value assay. All the reactions were performed in triplicate. The percentage inhibition was calculated as follows: % inhibition ¼ (NC À S)/NC Â 100 (NC (negative control) is the activity of the enzyme without test compound and with 2% DMSO and S is the activity of enzyme with test compound and the nal concentration of DMSO is 0.1%). Inhibition curves were obtained for each compound by plotting the percent inhibition versus the logarithm of inhibitor concentration in the assay solution. The linear regression parameters were determined for each curve and the IC 50 values extrapolated. The same procedure was applied for the positive control tacrine.

Neurite outgrowth-promoting activity
The neurotrophic activities of the tested compounds were examined according to an assay using PC12 cells as reported. 41 Briey, PC12 cells (purchased from Kunming institute of zoology) were maintained in F12 medium (Ham's F12K, Gibco's reagent) supplemented with 12.5% horse serum (HS, Hyclone's reagent), and 2.5% fetal bovine serum (FBS, Hyclone's reagent), and incubated at 5% CO 2 and 37 C. Tested compounds were dissolved in DMSO. For the neurite outgrowth-promoting activity bioassay, PC12 cells were seeded at a density of 5 Â 10 4 cells per mL in 48-well plate coated with poly-L-lysine (sigma's reagent). Aer 24 h, the medium was changed to that containing 10 mM of each test compounds plus 5 ng mL À1 NGF (sigma's reagent), or different concentrations of NGF (50 ng mL À1 for the positive control, 5 ng mL À1 for the negative control). The nal concentration of DMSO was 0.05%, and the same concentration of DMSO was added into the negative control. Aer 72 h incubation, the neurite outgrowth was assessed under a phase contrast microscope. Neurite processes with a length equal to or greater than the diameter of the neuron cell body were scored as neurite bearing cells. The ratio of the neurite-bearing cells to total cells (with at least 100 cells examined per view area; 5 viewing area per well) was determined and expressed as a percentage.

ECD calculation
The theoretical calculations were carried out using Gaussian 09. 42 Structures were rst optimized at PM6 using semiempirical theory method and then optimized at HF/6-31G(d) theory level. Room-temperature equilibrium populations were calculated according to Boltzmann distribution law (eqn. (1)). The conformers with Boltzmann-population of over 1% were chosen and further optimized at B3LYP/6-311G(d,p) in methanol using the IEFPCM model (Table S3 †). Vibrational frequency analysis conrmed the stable structures.
where N i is the number of conformer i with energy E i and degeneracy g i at temperature T, and k B is Boltzmann constant. Under the same condition, the ECD calculation was conducted using time-dependent density functional theory (TD-DFT). Rotatory strengths for a total of 30 excited states were calculated. The ECD spectrum was simulated in SpecDis 43 by overlapping Gaussian functions for each transition according to (eqn. (2)): where s represents the width of the band at 1/e height, and DE i and R i are the excitation energies and rotatory strengths for transition i, respectively. Parameters of s and UV-shi for enantiomers were 0.5 eV and 1 nm, respectively.

Conflicts of interest
There are no conicts of interest to declare.