Identification of AChE-Targeting Anti-Cerebral Palsy Constituents From Coptis chinensis Using Affinity Ultrafiltration And Experimental Validation

Summary

The purpose of this study was to identify the key active components of Coptis chinensis and elucidate its action in the intervention of cerebral palsy (CP) by means of affinity ultrafiltration and experimental verification.

Abstract

The purpose of this study was to identify the key active components of Coptis chinensis and elucidate its mechanisms of action in the intervention of cerebral palsy (CP) by means of affinity ultrafiltration. Using acetylcholinesterase (AChE) as the target, coptisine was screened out from the extract of C. chinensis as the component with the strongest binding capacity, which exhibited a binding rate of 21.9% and significant in vitro inhibitory activity (IC₅₀ = 3.24 µg/mL). Molecular docking and molecular dynamics simulations demonstrated that coptisine could bind stably to the active pocket of AChE. Subsequently, verification experiments were carried out on a neonatal rat model of hypoxic-ischemic brain damage (HIBD). The results showed that intervention with coptisine (10 mg/kg) significantly improved spatial learning and memory deficits in the model animals, increased cerebral blood flow in the injured hemisphere, alleviated pathological damage of brain tissues, and inhibited the abnormal activation of AChE in the brain. In summary, this study combined affinity ultrafiltration technology and confirmed that coptisine, identified from C. chinensis extract, can inhibit AChE and exert neuroprotective effects and cerebral blood flow-improving effects. It serves as the key material basis for C. chinensis to ameliorate CP-like neurological deficits, thereby providing candidate molecules and scientific evidence for the development of related drugs.

Introduction

Cerebral palsy (CP) is a permanent syndrome caused by non-progressive damage to the developing brain of fetuses or infants, characterized primarily by motor dysfunction and postural abnormalities. Although multiple clinical treatment approaches are currently available for CP, there remains a lack of ideal drugs that can fundamentally repair neurological deficits with minimal side effects. In contrast to chemically synthesized drugs, which usually act on single targets and carry relatively high potential toxicity, traditional Chinese medicines (TCMs), with their advantages of multi-component, multi-target, and holistic regulation as well as the favorable safety profiles demonstrated in long-term clinical practice, provide new insights and a reservoir of drug candidates for the treatment of complex brain disorders^1,2,3,4,5,6. Therefore, exploring highly effective and low-toxicity chemical components from TCMs holds significant scientific significance and clinical value⁷.

Among the complex pathological mechanisms of cerebral palsy, cholinergic nervous system dysfunction plays a pivotal role^8,9. Acetylcholinesterase (AChE) is a key enzyme that hydrolyzes the neurotransmitter acetylcholine (ACh). Excessively high activity of this enzyme leads to ACh depletion, impairs cholinergic signal transmission, and consequently exacerbates motor control and cognitive dysfunctions. Therefore, AChE has emerged as an important and well-established drug target for the development of neuroprotective agents. Inhibiting AChE activity to elevate ACh levels in the synaptic cleft is recognized as one of the effective strategies for ameliorating CP-related neurological deficits. In the context of hypoxic-ischemic brain injury, disruption of cholinergic neurotransmission may further aggravate neuronal dysfunction, thereby contributing to motor and cognitive impairments relevant to CP. Therefore, AChE was selected in this study not only as a classical neurofunctional target, but also as a mechanistically relevant entry point for screening bioactive compounds with potential anti-CP effects.

Modern research has shown that medicinal plants have a wide range of pharmacological activity values. As a classic traditional Chinese medicine for clearing heat and drying dampness, Coptis chinensis constitutes a complex system abundant in various isoquinoline alkaloids. In addition to the well-known berberine, it also contains a series of components with similar structures and diverse pharmacological activities, such as coptisine, epiberberine, palmatine, and jatrorrhizine¹⁰. These alkaloids collectively form the material basis for the pharmacological effects of C. chinensis. Previous studies have demonstrated that C. chinensis and its alkaloids possess anti-inflammatory, antioxidant, and potential neuroprotective properties. However, given such a complex component system, traditional research methods are difficult to rapidly and accurately identify the key active components that directly target AChE, which greatly limits the in-depth elucidation of the mechanisms underlying the effects of C. chinensis on brain injury¹¹.

Compared with conventional activity-guided isolation, which usually requires repeated fractionation, bioactivity testing, and purification and may lead to the loss or neglect of low-abundance but high-affinity compounds, affinity ultrafiltration enables rapid screening of target-binding ligands directly from complex mixtures. This technique enables efficient and rapid "fishing" of active ligands that bind to specific targets (e.g., AChE) directly from the multi-component crude extracts of TCMs. It is particularly suitable for systems with multiple alkaloid components, such as C. chinensis, allowing for the accurate identification of effective components that directly interact with the target from the complex mixture¹². Combined with computational approaches including molecular docking and molecular dynamics simulations, it can intuitively predict the binding mode, interaction types, and complex stability between components and target proteins at the atomic level, thereby providing robust theoretical evidence and mechanistic explanations for the results of experimental screening¹³.

Based on the above background, this study proposes the following scientific hypothesis: among the various alkaloids present in C. chinensis, there exist active components that can specifically inhibit AChE, and these components are the material basis for its potential anti-cerebral palsy effects. To verify this hypothesis, we integrated modern screening techniques with pharmacological validation methods: first, using AChE as the target, affinity ultrafiltration technology was employed to rapidly screen potential inhibitors from the multi-alkaloid extract of C. chinensis; further, molecular docking and molecular dynamics simulations were utilized to clarify the binding mechanism at the atomic level; finally, in vivo animal experiments were performed to verify the improving effects of the screened components on the behavior, brain tissue pathology, and AChE activity of rats with CP.

Protocol

All animal welfare and experimental procedures were approved by the Laboratory Animal Welfare Ethics Committee of The First People's Hospital of Zunyi (Ethics No. 2025-2-362). During the modeling and treatment period, animal body condition and survival were recorded daily.

Preparation of C. chinensis extract
In this study, an Accelerated Solvent Extractor was used for the accelerated extraction of the n-butanol fraction from C. chinensis medicinal materials. A total of 1 g of dried powder of C. chinensis was accurately weighed and placed into 40 mL extraction cells, respectively. Metal gaskets and glass fiber filter membranes were sequentially placed above and below the extraction cell to filter the sample, and the sample was wrapped with quartz sand. During the extraction process, the temperature and pressure were set to 90 °C and 100 bar, respectively. The accelerated extract was collected, and the extraction solvent was evaporated to dryness using a rotary evaporator to obtain dried extracts of different polar fractions. Methanol:water (50:50, v/v) was selected as the reconstitution solvent. The extract was filtered through a 0.45 µm microporous membrane and stored at 4 °C. Before sample detection, the inhibitory activity of the blank reconstitution solvent on AChE was investigated. The results showed that its inhibitory effect was negligible, indicating that the selection of the reconstitution solvent in this experiment would not affect the experimental results.

Acetylcholinesterase inhibition assay
The experiment was performed in a 96-well plate. First, PBS was added, followed by AChE (0.1 U/mL) and C. chinensis samples sequentially. After incubation at 37 °C for 10 min, 5,5′-Dithiobis (2-nitrobenzoic acid) (DTNB) chromogenic solution (2.5 mmol/mL) and ATChI substrate (10 mmol/mL) were added. The mixture was incubated at 37 °C for another 10 min, and the reaction was terminated by adding 60 µL of 1% sodium dodecyl sulfate (SDS). The absorbance was measured at 405 nm. Each C. chinensis sample was set up with 4 groups: Control group a: PBS (120 µL), AChE solution (20 µL), DTNB solution (40 µL), ATChI solution (40 µL), and SDS solution (60 µL) were added sequentially; Blank control group b: PBS (120 µL), DTNB solution (40 µL), ATChI solution (40 µL), and SDS solution (80 µL); Sample group c: PBS (80 µL), C. chinensis extract (20 µL), AChE solution (20 µL), DTNB solution (40 µL), ATChI solution (40 µL), and SDS solution (60 µL); Sample blank group d: PBS (100 µL), C. chinensis extract (20 µL), DTNB solution (40 µL), ATChI solution (40 µL), and SDS solution (60 µL). Huperzine A was used as the positive control with an initial concentration of approximately 2.5 mg/mL. The initial concentration of the n-butanol fraction extract was about 1 mg/mL. Both huperzine A and the samples were diluted with a 60% gradient. The inhibition rate was calculated using the following formula: Inhibition rate (%) = [1 - (c - d)/(a - b)] × 100%, where a, b, c, and d represent the absorbance values of control group a, blank control group b, sample group c, and sample blank group d, respectively.

The inhibitory rate at each tested concentration was calculated using the formula described above. Concentration–inhibition curves were then constructed by plotting the inhibitory rate against the logarithm of compound concentration. IC20, IC50, and IC80 values were calculated by nonlinear regression fitting using GraphPad Prism software based on the fitted concentration–response curves, and were defined as the concentrations required to produce 20%, 50%, and 80% inhibition of AChE activity, respectively.

Affinity ultrafiltration assay
Affinity ultrafiltration was performed to screen AChE-binding compounds from the C. chinensis extract. Briefly, AChE solution (0.1 U/mL in PBS, pH 7.4) was used as the active enzyme. The inactivated enzyme control was prepared by heating the same AChE solution in a boiling water bath for 10 min. For each reaction, 200 µL of C. chinensis extract solution was mixed with 200 µL of active or inactivated AChE solution and incubated at 37 °C for 30 min. After incubation, the mixture was transferred to a 30 kDa ultrafiltration centrifuge tube and centrifuged at 12,000 × g for 10 min at 4 °C. The retained enzyme-ligand complex was washed three times with 200 µL of PBS under the same centrifugation conditions to remove unbound components. Subsequently, 200 µL of 90% methanol was added to dissociate and elute the ligands bound to AChE, followed by incubation for 10 min and centrifugation at 12,000 × g for 10 min. The eluate was collected, filtered, and analyzed by high-performance liquid chromatography (HPLC). Compounds enriched in the active enzyme group relative to the inactivated enzyme control were considered potential AChE-binding components¹⁴.

HPLC analysis of major alkaloids metabolites
Chromatographic analysis was performed using an HPLC system equipped with a quaternary pump, an autosampler, a column oven, and a ultraviolet–visible (UV-Vis) detector. Separation was achieved on a C18 chromatographic column (250 × 4.6 mm, 5 µm) maintained at 25 °C. The mobile phase consisted of acetonitrile (A) and 30 mmol/L ammonium bicarbonate solution containing 0.7% ammonia and 0.25% triethylamine (B), delivered at a flow rate of 1.0 mL/min. The gradient elution program was as follows: 0–15 min, 90%–75% B; 15–25 min, 75%–70% B; 25–50 min, 70%–55% B. The injection volume was 5 µL, and UV detection was performed at 270 nm.

For quantitative analysis of the major alkaloids in C. chinensis, each sample (0.02 g) was accurately weighed and ultrasonically extracted with 10 mL of hydrochloric acid–methanol solution (1:100, v/v) for 30 min. After filtration, the weight loss was compensated with the same extraction solvent to the original weight, and the final solution was filtered through a 0.45 µm membrane before HPLC analysis. Eight authentic reference standards, including magnoflorine, glaucine, jatrorrhizine, columbamine, epiberberine, coptisine, palmatine, and berberine, were analyzed under the same chromatographic conditions. Calibration curves were constructed for these analytes using reference solutions. Because the abundance levels and detector responses of the eight alkaloids differed considerably, the calibration range for each analyte was individually optimized based on literature reports and preliminary experiments, rather than applying an identical fold-dilution scheme to all compounds. Therefore, the high-to-low concentration ratios of the linear ranges were not expected to be identical among the eight analytes. The limits of detection (LOD) and limits of quantification (LOQ) were estimated at signal-to-noise ratios of approximately 3 and 10, respectively.

For analysis of affinity ultrafiltration eluates, the ligands dissociated from AChE after ultrafiltration were collected from repeated experiments and concentrated prior to HPLC analysis to ensure adequate signal intensity for detection. The concentrated eluates were then analyzed using the same HPLC conditions as described above. The chromatographic peaks in the eluates were assigned by targeted comparison with the eight authentic reference standards and with the chromatographic profile of the C. chinensis extract obtained under identical analytical conditions. Therefore, the present analysis was not intended for de novo structural elucidation of unknown compounds, but for targeted determination of whether the known major alkaloids in C. chinensis were enriched in the active-enzyme eluate relative to the inactivated-enzyme control.

Molecular docking assay
In the present study, docking analysis was focused on coptisine because it was identified as the top-ranked AChE-binding constituent by affinity ultrafiltration and also showed the strongest in vitro inhibitory activity among the major alkaloids. Protein crystals were retrieved from the Protein Data Bank (PDB). Protein crystals with co-crystallized ligands were selected as the final docking receptors, and all target protein crystals were derived from humans. The receptor file was opened using PyMOL software, and the receptor and ligand files were separated and saved; the receptor was opened with MGLTools software, followed by removing water molecules, adding hydrogen atoms and charges, merging non-polar hydrogen atoms, calculating local atomic charges, etc., and then saved in pdbqt format; the ligand file was opened with MGLTools software, hydrogen atoms and charges were added, and saved as a pdbqt format file; grid box was opened, spacing was set to 1.000, the number of grid points was set, the central point coordinates of the box were selected according to the co-crystallized ligand, and saved as a GPF file^15,16.

Chemical component structures were first collected from the SciFinder database and saved in cdx format; each molecular three-dimensional structure was displayed using the ChemBio3D Ultra module, energy optimization was performed with the MM2 force field, saved in mol2 format, and charge parameters were removed. Using AutoDock, the mol2 format file was opened with MGLTools software, hydrogen atoms and charges were added to the ligand, rotatable chemical bonds were observed, and saved as a pdbqt format file. The AutoDock Vina (version 1.2.5) model was applied to perform molecular docking evaluation.

Molecular dynamics simulation assay
Gaussian 16 and GaussView 6 software were used for ligand structure optimization. After importing the mol2 file of the ligand structure into GaussView 6, the task type was set to Optimization, the state of the target molecule was selected as Ground State, the calculation method was density functional theory (DFT), the spin multiplicity was defaulted to Default Spin, the functional used was B3LYP, the solvent model was IEFPCM, and the solvent was water. After the completion of molecular structure optimization, Gaussian 16 was used to convert the generated chk file into an fch file, which was then imported into Multiwfn software to calculate the RESP charge of the ligand.

After the calculation of RESP charge, the generated chg file was imported into sobtop_1.0 (dev5) software to generate the ligand topology file required for molecular dynamics simulation. The molecular dynamics simulation was performed using GROMACS 2019.6 software with the AMBER99 force field. The structure files of the protein molecule and small molecule were imported into the software, a triclinic box with a periodic boundary of 1.0 nm was established, and the TIP3P water model was used for filling. The Particle Mesh Ewald (PME) method was employed to calculate long-range electrostatic interactions, and the gmx geion command was used to introduce an appropriate number of sodium ions and chloride ions to neutralize the charge of the entire system. The simulation was carried out at a constant temperature of 298 K and a standard atmospheric pressure of 100 kPa. For energy minimization, the steepest descent method (steep) was implemented. The gmx grompp and gmx mdrun commands were used to perform 10,000 steps of NVT (isothermal-isochoric ensemble) equilibrium and NPT (isothermal-isobaric ensemble) equilibrium, with the temperature coupling constant and pressure coupling constant set to 0.1 ps and 0.5 ps, respectively, and the duration of the equilibrium simulation was 100 ps. After the system reached equilibrium, the time step was set to 2 fs, and a 10 ns molecular dynamics simulation was performed on the complex, with trajectory data saved every 2 ps. The 10 ns production run was selected as an exploratory simulation length to provide a short-timescale dynamic assessment of the docked coptisine–AChE complex, rather than exhaustive conformational sampling. This setting was used to evaluate whether the docked complex could maintain a relatively stable binding mode over time. The gmx rms, gmx rmsf, gmx gyrate, and gmx hbonds commands were further used to calculate the root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration of the protein, hydrogen bonds, and solvent-accessible surface area. QTgrace was used for result visualization.

Animal experiment verification
Eighteen healthy neonatal Sprague-Dawley (SD) rat pups (7 days postnatal) of either sex were selected. All experimental animals were housed in a specific pathogen-free (SPF) animal experimental facility. This facility was equipped with a strict barrier system, which could effectively prevent the invasion of microorganisms and parasites. The environmental parameters of the housing room were automatically monitored and regulated by a central control system: temperature 22 ± 2 °C, relative humidity 55% ± 10%, and light cycle 12-h light/12-h dark cycle. A neonatal rat model of hypoxic-ischemic brain damage (HIBD) modified by the classic Rice-Vannucci method was used to simulate the pathological and behavioral characteristics of CP.

Briefly, postnatal day 7 rat pups were anesthetized with 2–3% isoflurane for induction and 1–2% for maintenance. After adequate anesthesia, a midline cervical incision was made, and the left common carotid artery was carefully isolated and permanently ligated. The incision was then sutured, and the pups were allowed to recover on a warming pad for 1 h. Subsequently, the pups in the model and coptisine groups were exposed to hypoxia in a chamber containing 8% O₂ balanced with 92% N₂ for 2 h at 37 °C to induce hypoxic-ischemic brain injury. Sham-operated animals underwent the same anesthesia and surgical exposure without carotid ligation or hypoxic treatment. After hypoxia, the pups were returned to their dams and monitored daily for general condition, survival, and feeding behavior throughout the experimental period.

The experimental rats were randomly divided into 3 groups: Sham group: only the left common carotid artery was separated without ligation or hypoxia treatment, and an equal volume of solvent was administered postoperatively; Model group: hypoxic-ischemic brain damage modeling surgery was performed, and an equal volume of solvent was administered postoperatively; Coptisine group: after modeling, coptisine (10 mg/kg) was administered by gavage. The dose of coptisine (10 mg/kg) was selected as an in vivo dose based on previously reported in vivo/pharmacokinetic studies of coptisine¹⁷. Drugs were administered once a day at a fixed time for 14 consecutive days of intervention.

Detection of animal indicators
The Morris water maze (MWM) test equipment consisted of a large circular water pool (diameter approximately 150 cm) with obvious visual cues on the pool wall. The pool water was stratified into opaque milky white, and the water temperature was controlled at 23 ± 1 °C. A transparent platform was placed in the target quadrant, submerged 1–2 cm below the water surface. Training was conducted 4 times a day: the animals were placed into the water facing the pool wall from four different entry points on the pool wall, and the time required for the animals to find and climb onto the hidden platform from entry into the water, namely the "escape latency", was recorded. Each training session was limited to 60 s. If the animal found the platform within 60 s, it was allowed to stay on it for 15 s to consolidate memory. If not found, the animal was gently guided to the platform and allowed to stay for 15 s. After 7 days of training, the hidden platform was removed. The animals were placed into the water from the entry point opposite the original platform and allowed to swim freely for 60 s, and the swimming trajectory of the animals was recorded by a video system. Through the MWM test, indicators such as average escape latency, time spent in the target quadrant, and the number of crossings over the original platform position were recorded.

For laser speckle contrast imaging (LSCI) detection: After anesthesia, the rats were fixed on a stereotaxic instrument, and the skull was exposed and kept moist. A laser speckle blood flow imaging system was used to continuously collect raw speckle images for 30 s directly above the skull. Professional software was used to convert the spatiotemporally varying speckle contrast into a cerebral blood flow perfusion unit (PU) distribution map. For quantitative analysis, a manually delineated cortical ROI was selected within the exposed ipsilateral hemisphere in each image according to the lesion-relevant cortical perfusion area and visible anatomical landmarks, and the average perfusion value within this ROI was used for intergroup comparison.

Brain tissues were collected, and pre-cooled PBS was added according to the weight-volume ratio. Mechanical homogenization was performed on ice, followed by centrifugation at 8000 × g at 4 °C for 10–15 min, and the supernatant was collected for testing. Brain tissues were quickly put into 4% paraformaldehyde for fixation for more than 24 h to maintain tissue morphology. Hematoxylin-eosin (HE) staining was performed through operations such as dehydration, clearing, and paraffin embedding, and the degree of brain injury was observed under an optical microscope.

For AChE activity measurement, the cerebral cortex from the injured (ipsilateral) hemisphere was collected and homogenized according to the manufacturer's instructions. Tissue samples were homogenized in extraction buffer and centrifuged at 8000 × g for 10 min at 4 °C, and the supernatant was collected for analysis. AChE activity was determined using a commercial acetylcholinesterase activity assay kit (microplate/colorimetric method). This assay is based on the hydrolysis of acetylcholine by AChE, followed by reaction with DTNB to generate a chromogenic product, and the absorbance was measured at 412 nm for the calculation of enzyme activity. Because this kit is an enzyme activity-based colorimetric assay rather than a conventional antigen-quantifying enzyme-linked immunosorbent assay (ELISA) kit, a standard detection range and analytical sensitivity were not provided by the manufacturer. In the present study, the measured AChE activity values in cortical tissue were approximately within the range of 25–80 nmol/min/mg protein.

Statistical analysis
All experimental data were expressed as mean ± standard deviation (SD) and analyzed using GraphPad Prism software. Comparisons among groups were performed using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test. For the Morris water maze test, the final training result and probe trial parameters were analyzed using one-way ANOVA followed by Tukey's multiple comparisons test. A value of P < 0.05 was considered statistically significant¹⁸. The correlation between cerebral blood flow perfusion in the ROI and AChE activity in the ipsilateral cerebral cortex was analyzed using Pearson correlation analysis. A value of P < 0.05 was considered statistically significant.

Results

Analysis of chemical components in C. chinensis extract
Alkaloids are the main accumulated components and also the major active components of C. chinensis medicinal materials. In this study, chromatographic technology was used to analyze the main chemical components in C. chinensis (Figure 1A). As shown in Table 1, all eight analytes exhibited good linearity within their respective calibration ranges. Because the contents and d...

Discussion

Currently, various methods and technologies have been developed for the prevention and treatment of brain diseases^20,21,22. In this study, an integrated strategy of in vitro screening, computational simulation, and in vivo verification was adopted to systematically clarify the material basis and multi-target mechanism of C. chinensis in improving cerebral palsy-like symptoms induced by HIBD. The core f...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5,5'-dithiobis(2-nitrobenzoic acid) (DTNB)	CATO Research Chemicals Inc.	416046844	Acetylcholinesterase inhibition assay
Acetylcholinesterase (AChE)	Sigma-Aldrich Trading Co., Ltd	C1682	Acetylcholinesterase inhibition assay
Acetylthiocholine iodide (ATChI)	Shanghai Adamas Reagent Co., Ltd	1037408	Acetylcholinesterase inhibition assay
Aetonitrile	Thermo Fisher Scientific Inc	1162577	Liquid chromatography mobile phase
AutoDock Vina	AutoDock	Version 1.2.5
Berberine standard	Chengdu Keloma Reagent Co., Ltd	110713-201814	Chemical composition analysis
Columbamine standard	Chengdu Keloma Reagent Co., Ltd	CHB180712	Chemical composition analysis
Coptisine standard	Chengdu Keloma Reagent Co., Ltd	CHB180309	Chemical composition analysis
E-916 accelerated solvent extractor	Büchi Labortechnik AG, Switzerland	E916-008	Extraction of n-butanol fraction
Epiberberine standard	Chengdu Keloma Reagent Co., Ltd	CHB180309	Chemical composition analysis
Formic acid	Thermo Fisher Scientific Inc	1270563	Liquid chromatography mobile phase
GaussView 6	Gaussian	https://gaussian.com/gaussview6/
Glaucine standard	Chengdu Keloma Reagent Co., Ltd	CHB180615	Chemical composition analysis
Jatrorrhizine standard	Chengdu Keloma Reagent Co., Ltd	CHB180607	Chemical composition analysis
Magnoflorine standard	Chengdu Keloma Reagent Co., Ltd	CHB180205	Chemical composition analysis
Methanol	Thermo Fisher Scientific Inc	1162578	Liquid chromatography mobile phase
Microporous membrane	Millipore Filter Company	2139547	Filtration of extracts
Palmatine standard	Chengdu Keloma Reagent Co., Ltd	CHB180226	Chemical composition analysis
Prism	GraphPad	https://www.graphpad.com/
PyMOL	PyMOL	https://pymol.org/
Sodium carbonate	Shanghai Adamas Reagent Co., Ltd	15138805	Acetylcholinesterase inhibition assay
Sodium dodecyl sulfate (SDS)	Shanghai Adamas Reagent Co., Ltd	11019349	Acetylcholinesterase inhibition assay