Tsaokoic Acid: A New Bicyclic Nonene from the Fruits of Amomum tsao-ko with Acetylcholinesterase Inhibitory Activity

A new bicyclic nonene, tsaokoic acid (1), was isolated from the fruits of Amomum tsao-ko, together with three known compounds (2–4). The structure of 1 was elucidated by analyzing spectroscopic data including 1D and 2D NMR spectra and compounds 2–4 were identified as tsaokoin, vanillin, and tsaokoarylone, respectively, by comparing their NMR spectra with previously reported data. Compounds 1–4 showed possible inhibitory activity against acetylcholinesterase (AChE) in silico molecular docking simulations. They were submitted to in vitro assay system and exhibited moderate inhibitory activity with IC50 values of 32.78, 41.70, 39.25, and 31.13 μM, respectively.


Introduction
Amomum tsao-ko Crevost et Lemair (Zingiberaceae) is a medicinal food known as "Cho-Gwa" in Korea and "Caoguo" or "Tsao-ko" in China [1]. The genus Amomum is distributed in tropical and subtropical regions of Asia and Oceania, and A. tsao-ko grows mainly in Southwestern China and Northern Vietnam [2,3]. The fruit of A. tsao-ko has been used as a spice and a traditional medicine in Asia to treat stomach disorders, dyspepsia, nausea, vomiting, diarrhea, malaria, throat infections, and abdominal pain [4][5][6]. It exhibits pharmacological activities such as anti-inflammatory, anti-tumor, anti-oxidant, antibiotic, anti-diabetic, and neuroprotective effects [3,5,7]. Previous phytochemical studies on A. tsao-ko fruit isolated various natural compounds including flavonoids, terpenoids, and diarylheptanoids [2].
Alzheimer's disease (AD) is a neurodegenerative disorder accompanied by clinical symptoms such as cognitive and language impairment, mental and behavioral difficulties, and problems with daily living activities. Although AD pathogenesis has not been clearly elucidated, one cause may be early loss of basal forebrain cholinergic neurons [8]. This explains the essential role of acetylcholine (ACh) in cognitive decline (including memory, attention, sensory, and learning) in AD [9]. Since cholinergic neurotransmission plays an important role in cognitive function, improving the acetylcholinesterase (AChE) brain level is crucial to treat AD. AChE is a cholinergic enzyme found primarily at neuromuscular junctions and chemical synapses of the cholinergic type responsible for terminating a synaptic transmission. Therefore, AChE inhibitors enhance cholinergic neurotransmission by increasing Ach levels [8,9].
In this study, a new compound, tsaokoic acid (1), was isolated with three known compounds, tsaokoin (2), vanillin (3), and tsaokoarylone (4), from the A. tsao-ko fruits ( Figure 1). 1-4 were tested for their AChE-inhibitory activity using an in silico molecular docking and an in vitro enzyme assay. This study describes their isolation, structure identification, and AChE-inhibitory activity.
Molecules 2023, 28, x FOR PEER REVIEW 2 of 9 In this study, a new compound, tsaokoic acid (1), was isolated with three known compounds, tsaokoin (2), vanillin (3), and tsaokoarylone (4), from the A. tsao-ko fruits (Figure 1). 1-4 were tested for their AChE-inhibitory activity using an in silico molecular docking and an in vitro enzyme assay. This study describes their isolation, structure identification, and AChE-inhibitory activity.
Relative configurations for 1 and 2, were determined according to their NOESY data as described in the results. , respectively, which indicates that they were not perfectly racemic. However, we were unable to find any differences in chemical shifts in the 1 H NMR spectra of (R)-and (S)-MTPA esters of compounds 1 and 2. Thus, we think that 1 and 2 are racemic mixtures. All figures, including 1D and 2D NMR spectra of compounds 1 and 2, and the 1 H NMR data for MPTA esters of compounds 1 and 2, were provided in the supplementary materials ( Figures S1-S24).

AChE-Inhibitory Activities of 1-4
In the present study, compounds 1-4 showed possible inhibitory activity against AChE in molecular docking simulations, and thus, in vitro assays have been performed against AChE and more details are as follows.

In Silico Docking Simulation
Before the in vitro anti-AChE activity test, an in silico docking simulation was performed to predict the specific pharmacological effects of the four compounds (1-4) against AChE. The binding sites in the enzyme and binding energies of each compound were predicted through a docking simulation utilizing three systems (Autodock vina, Autodock 4, LeDock). Figure 3 shows the binding sites of the AChE receptor and ligands. FP1, the positive control, had two hydrogen bonds and two hydrophobic interactions with AChE ( Figure 3a). The binding affinity of the tested compounds was compared to those of the control (FP1) for hydrogen bonding and hydrophobic interaction. Tsaokoic acid (1) and tsaokoin (2) did not have hydrophobic interactions with the enzyme and thus showed lower binding affinities than the control (FP1) (Figure 3b,c). Vanillin (3) possessed one hydrogen bond and one hydrophobic interaction with AChE, showing a lower binding force than the control (Figure 3d). On the other hand, tsaokoarylone (4) showed a stronger binding affinity than the control (FP1) as four hydrophobic interactions and two hydrogen bonds were observed (Figure 3e).

In Vitro Assay for AChE-Inhibitory Activity
On the basis of the results from the in silico docking simulation for AChE-inhibitory activity, 1-4 were tested in vitro screening system at total concentrations of 2, 10, and 50 µM and berberine was used as a positive control (Figure 4 and Table S1). The halfmaximal inhibitory concentrations (IC 50 ) of 1-4 were 32.78, 41.70, 39.25, and 31.13 µM, respectively, as shown in Figure 4. These results can be considered as moderate AChEinhibitory activity compared to the positive control, berberine (IC 50 0.19 µM). Isolates 1-4 showed consistent activity between in silico and in vitro experiments. Therefore, we can utilize this in silico docking simulation system to find any possible AChE inhibitors before we perform the in vitro or in vivo experiments to save our time and expenses. This is the first report on the AChE-inhibitory activities of compounds 1, 2, and 4. Compound 2, tsaokoin, was reported to have weak antifungal activity in previous studies, but its AChEinhibitory activity has not been reported [10]. Previous in vitro and in vivo reports indicate that compound 3 has AChE-inhibitory activities [12][13][14]. In this study, in silico docking experiments on AChE for vanillin (3) were performed for the first time as well as for compounds 1, 2, and 4. This study is considered a good example of in silico research that aligns with in vitro results. Their AChE-inhibitory activity indicates that compounds 1-4 have some possibilities to treat mild Alzheimer's disease, by increasing the level of ACh.

In Vitro Assay for AChE-Inhibitory Activity
On the basis of the results from the in silico docking simulation for AChE-inhibitory activity, 1-4 were tested in vitro screening system at total concentrations of 2, 10, and 50 μM and berberine was used as a positive control (Figure 4 and Table S1). The half-maximal inhibitory concentrations (IC50) of 1-4 were 32.78, 41.70, 39.25, and 31.13 μM, respectively, as shown in Figure 4. These results can be considered as moderate AChE-inhibitory activity compared to the positive control, berberine (IC50 0.19 μM). Isolates 1-4 showed consistent activity between in silico and in vitro experiments. Therefore, we can utilize this in silico docking simulation system to find any possible AChE inhibitors before we perform the in vitro or in vivo experiments to save our time and expenses. This is the first report on the AChE-inhibitory activities of compounds 1, 2, and 4. Compound 2, tsaokoin, was reported to have weak antifungal activity in previous studies, but its AChE-inhibitory activity has not been reported [10]. Previous in vitro and in vivo reports indicate that compound 3 has AChE-inhibitory activities [12][13][14]. In this study, in silico docking experiments on AChE for vanillin (3) were performed for the first time as well as for compounds 1, 2, and 4. This study is considered a good example of in silico research that aligns with in vitro results. Their AChE-inhibitory activity indicates that compounds 1-4 have some possibilities to treat mild Alzheimer's disease, by increasing the level of ACh.

General Experimental Procedures
Optical rotation data were obtained on a JASCO P-2000 polarimeter (Tokyo, Japan). UV spectra were measured on a Hitachi U-3000 UV/Vis spectrophotometer (Tokyo, Japan). IR spectra were recorded on a Thermo Fisher Nicolet iS 10 FT-IR spectrometer (Waltham, MA, USA). NMR spectra were acquired on an Agilent DD2 400 MHz FT-NMR instrument (Agilent Technologies, Santa Clara, CA, USA) using tetramethylsilane as an internal standard and analyzed with MestreNova 9.

General Experimental Procedures
Optical rotation data were obtained on a JASCO P-2000 polarimeter (Tokyo, Japan). UV spectra were measured on a Hitachi U-3000 UV/Vis spectrophotometer (Tokyo, Japan). IR spectra were recorded on a Thermo Fisher Nicolet iS 10 FT-IR spectrometer (Waltham, MA, USA). NMR spectra were acquired on an Agilent DD2 400 MHz FT-NMR instrument (Agilent Technologies, Santa Clara, CA, USA) using tetramethylsilane as an internal standard and analyzed with MestreNova 9.0.0 software (Mestrelab Research S.L., Santiago de Compostela, Spain). HRESIMS was performed on an Agilent 6230 TOF LC/MS instrument (Agilent Technologies, Santa Clara, CA, USA) equipped at Ewha Drug Development Re-search Core Center. Adsorption column chromatography was conducted using silica gel (63-200 µm, Merck, Darmstadt, Germany). MPLC was run on a CombiFlash Rf-200 instrument (Teledyne Isco, Lincoln, NE, USA) and RediSep ® Silver Silica Gel Disposable Flash Columns 330.0 g and 24.0 g (Teledyne Isco, Lincoln, NE, USA) were used for separations. The Acme 9000 system (Young Lin, Anyang-si, Gyeonggi-do, Republic of Korea) with UV detection was used for analytic HPLC, equipped with an Agilent Prep-C18 Scalar column (4.6 × 250 mm, 5 µm, Santa Clara, CA, USA). Preparative HPLC was carried out on a YMC-Pack Pro C 18 column (20 × 250 mm, 5 µm, Asan-si, Chungcheongnam-do, Republic of Korea) using a Waters system equipped with a Waters 600 pump and a Waters 996 photodiode array detector (Waters, MA, USA). Thin-layer chromatography (TLC) was conducted using Kieselgel 60 F 254 aluminum sheets (Merck, Darmstadt, Germany) and RP-18 F 254s aluminum sheets (Merck, Darmstadt, Germany). TLC plates were visualized under UV (254 and 365 nm) after being dipped in a 10% (v/v) sulfuric acid solution and heated at 300 • C for 1 min. The solvents used for HPLC experiments were HPLC-grade (Daejung Chemicals & Metals, Siheung-si, Gyeonggi-do, Republic of Korea). Solvents for NMR experiments were purchased from Cambridge Isotope Laboratories (Tewksbury, MA, USA).

Plant Material
The dried fruits of Amomum tsaoko Crevost et Lemaire (Zingiberaceae) were purchased from Nonglim Saengyak Company (Agricultural and Forestry Herb Market) in Seoul, South Korea in June 2020. A voucher specimen (no. EA389) was deposited at the Natural Product Chemistry Laboratory, College of Pharmacy, Ewha Womans University.

In Vitro AChE-Inhibitory Assay
The inhibitory activities of the compounds on AChE were measured using the spectrophotometric method developed by Ellman et al. 1961 [17]. The reaction mixtures contained 140 µL of sodium phosphate buffer (pH 8.0), 20 µL of tested sample solution, and 20 µL of AChE solution, which were mixed and incubated for 15 min at room temperature. All tested compounds and positive control (berberine) were dissolved in 10% DMSO Reactions were initiated with the addition of 10 µL of dithiobisnitrobenzoic acid (DTNB) and 10 µL of ACh. The hydrolysis of ACh was monitored by tracking the formation of 5-thio-2-nitrobenzoate anion at 412 nm for 15 min, resulting from the reaction of DTNB with the thiocholine released by the enzyme. Each reaction was performed in triplicate and the results were measured in 96-well microplates using a microplate spectrophotometer (Tecan, Sunrise, Austria). Percent inhibition was calculated using the formula (1 − S/E) × 100, where E and S are enzyme activities with and without the test sample, respectively. The inhibitory activity of each compound against AChE was expressed as an IC 50 (the µM concentration required to inhibit substrate hydrolysis by 50%), as calculated using log-dose inhibition curves.

Conclusions
In this study, tsaokoic acid (1), tsaokoin (2), vanillin (3), and tsaokoarylone (4) were isolated from the EtOAc fraction of the A. tsao-ko fruits. Compound 1 was elucidated as a new compound, tsaokoic acid, using various spectroscopic data including 1D and 2D NMR techniques such as COSY, NOESY, HSQC, and HMBC NMR experiments. Relative configurations for 1 and 2 were determined according to their NOESY data as described in the results. Compounds 1-4 exhibited moderate AChE-inhibitory activities at IC 50 values of 32.78, 41.70, 39.25, and 31.13 µM, respectively, which are consistent with the results of in silico docking simulations. From these results, we can conclude that in silico docking simulation system for AChE-inhibitory activiy could be a guide for in vitro system. Compounds 1-4 isolated from the fruits of A. tsao-ko, have some possibilities to enhance cognition in humans, treating mild Alzheimer's disease.