New biphenanthrenes with butyrylcholinesterase inhibitory activitiy from Cremastra appendiculata

Abstract Encouraged by the in vitro potent inhibitory activity on butyrylcholinesterase (BChE) of 95% ethanol extract of Cremastra appendiculata (D. Don) Makino tubers, a further phytochemical investigation on C. appendiculata tubers was conducted, which led to the isolation of a pair of new biphenanthrene atropisomers, namely cremaphenanthrene F-G (1–2). Their structures were elucidated on the basis of extensive spectroscopic analyses and chemical method. It is the first time that biphenanthrene atropisomers have been isolated from the plant kingdom. Compound 1 showed potent BChE inhibitory effect with IC50 value of 14.62 ± 2.15 μM. Compound 2 exhibited weak BChE inhibitory effect with IC50 value of 79.56 ± 0.78 μM. Meanwhile, 1 and 2 were found to be inactive for acetylcholinesterase (AChE) inhibition. These findings suggested that compound 1 was a promising selective BChE inhibitor for AD prevention and treatment. Graphical Abstract


Introduction
The tuber of Cremastra appendiculata (D. Don) Makino (Orchidaceae) is one of main sources of the famous traditional Chinese medicine "Shancigu", which is clinically used for treating cancers with a long history (State Administration of Traditional Chinese Medicine "Chinese Materia Medica" Editorial Board 1999). Previous phytochemical investigations have shown that phenanthrenes and bibenzyls were the main antitumour active constituents (Xue et al. 2006;Liu et al. 2013;Wang et al. 2013;Liu et al. 2015Liu et al. , 2016. Recently, 95% ethanol extract of C. appendiculata tubers and three phenanthrenes isolated from this plant were reported to have potent inhibitory activities on butyrylcholinesterase (BChE) (Li et al. 2017;Tu et al. 2018), which have cast a new light on the usage of C. appendiculata tubers.
Alzheimer 0 s disease (AD) is the most common form of dementia regarded as comprising memory loss, cognitive impairment, and difficulty in thinking and problemsolving (Mantoani et al. 2016). One of its most characteristic features is decreased level of the neurotransmitter acetylcholine (ACh) (Craig et al. 2011). Cholinergic neurotransmission is terminated by two cholinesterases acetylcholinesterase (AChE) and BChE, which play essential role in the hydrolysis of ACh (Giacobini 2003). Both AChE and BChE can hydrolyse Ach, but BChE is less substrate specific than AChE (Yiannopoulou and Papageorgiou 2013). Many recent studies reveals that BChE played more important role in the AD brain and selective inhibitors of BChE could be promising drug candidates (Nordberg et al. 2013).
The potent inhibitory activities on BChE by crude extract of C. appendiculata tubers and its main constituent phenanthrenes attracted our attention. In our previous research, several benzylphenanthrenes and biphenanthrenes were reported from this herb (Liu et al. 2013(Liu et al. , 2014(Liu et al. , 2015(Liu et al. , 2016. Our continuing investigations on the constituents of ethyl acetate extract of this plant were carried out to search for more phenanthrenes possessing BChE inhibitory activities.

Results and discussion
Compound 1 was obtained as a brown amorphous powder. Its HR-ESI-MS showed an ion at m/z 679.1594 [M þ K] þ , giving the molecular formula of C 36 H 32 O 11 . The IR spectrum showed absorption bands for hydroxy and aromatic functional groups. The UV spectrum showed absorption maximum at 211, 263, and 309 nm. Twelve aromatic proton signals appeared in the 1 H NMR spectrum of 1. Twenty eight aromatic (including six oxygenated quaternary aromatic carbons) and two methoxy carbon signals were showed in the 13 C NMR spectrum of 1. These data, especially the presence of the deshielded proton signals at d H 9.53 (1H, d, J ¼ 9.0 Hz, H-5) and 9.47 (1H, d, J ¼ 9.0 Hz, H-5 0 ) indicated that 1 possessed two phenanthrene moieties with four hydroxy groups and two methoxy groups as substituents. Besides, in the 13 C NMR spectrum of 1, six carbon signals (d C 103.9, 75.1, 78.0, 71.6, 78.6, and 62.9) assigned to a glucopyranosyl moiety were observed. Therefore, 1 was inferred to be a biphenanthrene glycoside. The substituent positions of 1 were further confirmed by 2 D NMR experiments. The HMBC correlations from H-3 (d H 7.44) to C-1 (d C 116.0), C-2 (d C 155.2), C-4 (d C 160.6) and C-4a (d C 119.0); H-9 (d H 7.34) to C-4b (d C 125.5), C-8 (d C 112.4) and C-10a (d C 135.1); H-10 (d H 7.04) to C-1 (d C 116.0), C-4a (d C 119.0) and C-8a (d C 135.3); H-11 (d H 4.23) to C-4 (d C 160.6), together with the NOESY correlations between H-11 (d H 4.23) and H-3 (d H 7.44) and H-5 (d H 9.53) revealed one phenanthrene unit was a 2,7-dihydroxy-4-methoxyphenanthrene. The HMBC correlations from H- , along with the NOESY correlations between H-11 0 (d H 4.18) and H-3 0 (d H 7.00) and H-5 0 (d H 9.47) declared the existence of another 2 0 ,7 0 -dihydroxy-4 0 -methoxyphenanthrene moiety. The two phenanthrene moieties were connected by C-1 (d C 116.0) and C-1 0 (d C 113.0) through the comprehensive analyses of quaternary aromatic carbons nature, chemical shifts, and the molecular composition. In the HMBC spectrum of 1, the cross peak between H-1 00 (d H 5.03) and C-2 (d C 155.2) showed the sugar moiety was connected at C-2 (d C 155.2). The coupling constant of the anomeric proton indicated it to be a b-glucose, whose absolute configuration was confirmed to be D through GC analysis of the derivation of acid hydrolysis of 1. Due to the presence of the sugar group at C-2 (d C 155.2) and the hydroxy group at C-2 0 (d C 153.8), the two phenanthrenes moieties could not revolve freely. The CD spectrum of 1 had a positive Cotton effect at 251 nm and a negative Cotton effect at 208 nm, indicating that 1 has an R-biphenyl configuration (Liu and Li 1993). Therefore, the structure of 1 was determined as shown in Figure 1, and named cremaphenanthrene F.
Compound 2 was obtained as a brown amorphous powder. An ion at m/z 679.1597 [M þ K] þ was found in its HR-ESI-MS spectrum, indicating the molecular formula of C 36 H 32 O 11 , which was identical to that of 1. The IR, UV, and 1D NMR spectra were similar to those of 1. A further comparison of 1 H NMR, 13 C NMR, HSQC, HMBC, and NOESY spectra of 1 with those of 2 suggested that they shared the same planar structure. The CD spectrum of 2 had a negative Cotton effect at 249 nm and a positive Cotton effect at 209 nm, which was contrary with that of 1, indicating the S-biphenyl configuration for 2 (Chen et al. 2006), 2 was then named cremaphenanthrene G.
The AChE and BChE inhibitory activities of two new phenanthrenes were then tested, and the results were shown in  value of 79.56 ± 0.78 lM. Meanwhile, Both 1 and 2 were inactive for AChE inhibition, indicating that 1 could serve as a selective BChE inhibitor candidate for AD prevention and treatment.

General
UV spectra were run on a Shimadzu UV-2450 spectrometer. IR spectra were recorded on a Nicolet NEXUS-470 FTIR spectrometer. HR-ESI-MS were determined by a Bruker APEX IV FT-MS (7.0 T). NMR spectra were recorded on a Varian Inova-500 NMR spectrometers (25 C). A JASCO (Tokyo, Japan) J-810 circular dichroism (CD) spectrometer, connected to a Peltier temperature controller, was used to acquire CD spectra. Semi-preparative HPLC was run on a Dionex Ultimate 3000 instrument equipped with a 170 U UV detector (254 nm) and an ODS column (Thermo Sientific BDS-C 18 column, 250 mm Â 10 mm, 5 lm). Column chromatography (CC) was performed using silica gel (200-300 mesh, Qingdao Marine Chemistry Ltd., China), Sephadex LH-20 (Amersham Biosciences, Sweden) and ODS C 18 (40-63 lm, Merck, Germany). TLC was performed on glass precoated silica gel (GF254) plates. All purified compounds submitted for bioassay were at least 95% pure as judged by HPLC analyses.

Plant material
The tubers of C. appendiculata were collected in Yunnan province in June 2011, which were identified by one of the authors (Prof. P.F. Tu). The voucher specimen (No. DJL20110628) was deposited at the Herbarium of Peking University Modern Research Center for Traditional Chinese Medicine.

Complete acid hydrolysis and GC analysis of compounds 1-2
The complete acid hydrolysis of compounds 1-2 was performed in accordance with aforementioned method (Liu et al. 2015). Peaks of the sugar derivatives were identified by comparison with retention times to authentic sample of D-glucose (19.28 min) treated in the same way.

AChE/BChE inhibitory activity
The AChE/BChE inhibitory activities of the isolated compounds were investigated by applying Ellman's method (Ellman et al. 1961). 100 lL PBS (pH 7.2), 20 lL tested compounds (1 and 2, final concentrations: 100, 20, 4, 0.8, 0.16 lM, respectively), 20 lL acetylthiocholine iodide or butyrylthiocholine iodide (concentration: 1.2 mM), and 20 lL AChE or BChE (concentration: 0.05 U/mL) were added in turn to 96-well plates. After incubation for 30 min at 37 C, 20 lL SDS (concentration: 4%) and 20 lL DTNB (concentration: 0.6 mM) were added, and then followed by measuring the absorbance at 405 nm immediately. Galanthamine was used as a positive control. Inhibitory activity was calculated with the following formula: inhibition(%) where A s is the absorbance for the sample, A c is the absorbance for the control (without AChE or BChE), and A b is the absorbance for the blank (without tested compound).

Statistical analysis
All results are presented as means ± SD for three independent experiments. IC 50 values were calculated by SPSS 22.0 statistical software.

Conclusions
Our phytochemical investigations have led to the isolation of a pair of new biphenanthrene atropisomers. To the best of our knowledge, this is the first time that biphenanthrene atropisomers have been isolated from the plant kingdom. Meanwhile, 1 was found to be a promising selective BChE inhibitor for AD prevention and treatment. Our findings have confirmed the potentiality of phenanthrenes for developing selective BChE inhibitors. Our work has also given a chance to understand the traditional TCM "Shancigu" from a new sight.

Disclosure statement
No potential conflict of interest was reported by the authors.