An effective part of radix cyathulae and a preparation method and application thereof

Abstract

This invention discloses an effective fraction of Achyranthes bidentata, its preparation method, and its applications, belonging to the field of traditional Chinese medicine extraction and biomedical technology. This invention discloses a composition containing 16 sterone compounds. The effective fraction of Achyranthes bidentata comprises the aforementioned composition. This invention also discloses the application of the effective fraction of Achyranthes bidentata in the preparation of drugs for the treatment and / or prevention of Parkinson's disease. The extraction and separation method provided by this invention is a simple, efficient, environmentally friendly method for extracting and separating the effective fraction of Achyranthes bidentata with high product purity. It clarifies its chemical composition and structural characteristics, and confirms its role and mechanism in treating Parkinson's disease through the TREM2 pathway. This fills a gap in the research field of the effective fraction of Achyranthes bidentata, expands its medicinal and edible value, and is suitable for widespread application.

Description

Technical Field

[0001] This invention belongs to the technical field of traditional Chinese medicine extraction and biomedicine, and relates to an effective part of Achyranthes bidentata, its preparation method and application. Background Technology

[0002] Citri Exocarpium Rubrum (CER) is the dried outer pericarp of Citrus reticulata Blanco and its cultivated varieties, belonging to the Rutaceae family. It is typically harvested in late autumn or early winter after the fruit has ripened, and the outer pericarp is peeled off and dried in the sun or shade. It has been used in traditional Chinese medicine and food for over 2000 years, possessing various pharmacological activities such as antioxidant, anti-inflammatory, and anti-cancer properties. Current research largely focuses on its saponin compounds, while systematic studies on the structural characteristics of its sterone components, efficient extraction processes, and anti-PD activity remain relatively scarce, resulting in a significant gap in the development and utilization of Citri Exocarpium Rubrum resources.

[0003] Based on the National 973 Program project "Pharmacological Study of Diuretic and Edema-Reducing Traditional Chinese Medicines" (Project No.: 2013CB531801), research has found that the "bitter" component of Achyranthes bidentata has become a hot topic in natural drug development due to its high efficacy and low toxicity. However, traditional methods for extracting polysaccharides from Achyranthes bidentata (such as high-temperature water extraction) suffer from low yields (<10%) and difficulties in subsequent purification. Although ultrasound-assisted extraction (UAE) technology is used for the separation of plant polysaccharides, strict parameter control is required when used alone to avoid polysaccharide degradation. The MCI column, through the synergistic effect of "macromolecule + macroporous resin", can improve the yield of the "bitter" component while preserving its structural integrity and bioactivity. Therefore, developing this technology for the "bitter" component of Achyranthes bidentata and clarifying its anti-PD activity and mechanism is of great significance for the development of new drugs for PD treatment and functional foods. Summary of the Invention

[0004] In order to overcome the defects in the existing technology, the present invention provides an effective part of Achyranthes bidentata, its preparation method and application, which fills the gap in the development of Achyranthes bidentata and expands its medicinal value.

[0005] The technical solution is as follows:

[0006] First, an embodiment of the present invention provides a composition comprising 16 sterone compounds, the chemical structures of which are shown in the following formulas (1)-(16):

[0007] .

[0008] Furthermore, the 16 steroidal compounds are a combination of naturally occurring forms extracted from Achyranthes bidentata.

[0009] Furthermore, the composition has a bitter taste.

[0010] Secondly, the present invention also provides an effective fraction of Achyranthes bidentata, comprising the composition described in the present invention.

[0011] Third, the present invention also provides a method for preparing the composition or the effective fraction of Achyranthes bidentata, comprising the following steps:

[0012] Step 1: Take 10 kg of Achyranthes bidentata slices, add 9 times the amount of methanol to extract by reflux for 3 hours, repeat 3 times, recover the extract under reduced pressure, concentrate, and obtain a total of 3.1 kg of extract.

[0013] Step 2: Suspend 3.1 kg of the extract in distilled water, stir evenly, and extract three times in sequence with petroleum ether, ethyl acetate, and water-saturated n-butanol. Recover the n-butanol extract under reduced pressure to obtain a total of 714 g of n-butanol fraction.

[0014] Step 3: Dissolve 500g of n-butanol fraction in 4000mL of distilled water and perform SP825-L macroporous resin column chromatography with gradient elution of five solvents: water, 30% ethanol, 50% ethanol, 70% ethanol, and 90% ethanol. The solvents are recovered under reduced pressure. The total sterone fraction extract of 48.62g is obtained by recovering the 50% ethanol eluent.

[0015] Step 4: Take 30.84g of the 50% ethanol eluent and perform MCI and ODS column chromatography separation. Perform gradient elution with methanol of different concentrations from 10% to 90% MeOH, and recover the solvent under reduced pressure. A total of 16 sterone compounds were separated.

[0016] Fourth, the present invention also provides the use of the composition described herein or the effective fraction of Achyranthes bidentata as described in the claims in the preparation of a medicament for the treatment and / or prevention of Parkinson's disease.

[0017] Fifth, the present invention also provides a pharmaceutical preparation comprising the composition described in any one of the present invention or the effective fraction of Achyranthes bidentata, and a pharmaceutically acceptable carrier.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] 1. Advantages of the extraction and separation process: This invention uses methanol (100%, liquid-to-solid ratio 10:1 mL / g) technology, and separates water using an MCI column; the separation process requires no chemical reagents, the reaction is mild (constant temperature 70℃, pH 6.0-6.5), which can avoid the degradation of ecdysterone compounds and preserve their biological activity;

[0020] 2. Clear Anti-PD Mechanism: Using MPTP-induced PD model mice as the research subjects, after 10 consecutive days of administration, RCS improved PD through multiple pathways: ① alleviating motor dysfunction (2.2-fold increase in dwell time at 22 r / min rotarod test, and 42% reduction in dwell time at 50 cm height / 1 cm diameter climbing pole test); ② protecting dopaminergic neurons (1.8-fold increase in the number of TH-positive neurons in the substantia nigra pars compacta, and 60% reduction in Nissl body loss); ③ inhibiting abnormal aggregation of α-synuclein (37% downregulation of relative α-Syn expression in the substantia nigra region detected by Western blot); ④ anti-inflammatory (reducing serum and substantia nigra levels of TNF-α, IL-6, and IL-1β by 41%, 44%, and 46%, respectively); ⑤ regulating the HDAC6 / NLRP3 pathway (upregulating the relative expression of TH and TREM2 proteins in the substantia nigra region by 55% and 60%, respectively); ⑥ virtual screening of effective components of Achyranthes bidentata by artificial intelligence (AI), identifying Cyasterone and Cyathsterone. Components such as C and 24,28-diepi-cyasterone can improve Parkinson's disease by targeting and regulating lipid metabolism pathways; The isolated Cyasterone (CYA) inhibited LPS-induced inflammatory factors in BV2 cells and protected Cholesterol-induced damage to SH-SY5Y cells.

[0021] 4. High resource utilization rate: This invention uses Achyranthes bidentata as raw material. After separation by MCI column, the raw material utilization rate reaches more than 71%, realizing the resource utilization of waste. Compared with the traditional production method using Achyranthes bidentata as raw material, it reduces the production cost by 23%-41%, which is in line with the concept of green production. Attached Figure Description

[0022] Figure 1 RCS alleviated MPTP-induced pathological phenotype in mice (n=3); (A) Nissl-positive neurons; (B) Immunohistochemistry; **P<0.01, *P<0.05 vs MPTP group;

[0023] Figure 2RCS improved MPTP-induced dyskinesia in mice (n=6); (A) monitoring behavioral time; (B) time on the bar during the rotating bar test; (C) travel distance; (D) total time measured during the standing bar test. **P<0.01, *P<0.05 vs MPTP group;

[0024] Figure 3 RCS inhibited the levels of TNF-α, IL-6, and IL-1β in the substantia nigra of mouse brain (n=6); all data are expressed as mean ± standard deviation; **P<0.01, *P<0.05 vs MPTP group;

[0025] Figure 4 Effects of RCS on serum TC, TG, LDL-C, and HDL-C levels in PD mice (n=6); (A) TC level; (B) TG level; (C) LDL-C level; (D) HDL-C level; All data are expressed as mean ± standard deviation; **P<0.01, *P<0.05 vs MPTP group;

[0026] Figure 5 Effects of RCS on α-Syn, TH, and TREM2 proteins in the substantia nigra of PD mice (n=3); (A) α-Syn protein; (B) TH protein; (C) TREM2 protein; All data are expressed as mean ± standard deviation; **P<0.01, *P<0.05 vs MPTP group;

[0027] Figure 6 AI technology was used to virtually screen effective components of RCS targeting lipid metabolism disorders in PD; (A) Drug screening results; (B) Dynamic changes in lipid metabolism and therapeutic effects; (C) Changes in the activity of key lipid metabolism enzymes in the PD model; (D) Lipidomics differential expression heatmap; (E) Lipid metabolism pathway enrichment analysis; (F) Lipidomics multivariate statistical analysis;

[0028] Figure 7 Steroidal ketones and triterpenoid saponins isolated from Achyranthes bidentata 1-21;

[0029] Figure 8 Molecular docking of CYA component with TREM2 target protein: overall docking diagram (left), partial docking diagram (middle), and 2D interaction diagram (right);

[0030] Figure 9Effects of Cyasterone (CYA) on BV2 cells (n=3), all data are expressed as mean ± standard deviation; (A) Cell viability as determined by CCK8 assay; (B) LPS+CYA cell viability; (C) Levels of inflammatory factors (TNF-α, IL-6, and IL-1β); **P<0.01, *P<0.05 vs NC; **P<0.01, *P<0.05 vs LPS group;

[0031] Figure 10 Effects of Cyasterone on Cholesterol-induced SH-SY5Y cells (n=3); (A) Cell viability as determined by CCK8 assay; (B) Cholesterol+CYA cell viability; (C) LDH level; (D) TH protein; (E) TREM2 protein; All data are expressed as mean ± standard deviation, *P<0.05, **P<0.01 vs Cholesterol group. Detailed Implementation

[0032] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0033] 1. Pathological effects of bitter components of Achyranthes bidentata on PD mice

[0034] In the high- and low-dose groups of the bitter component of Achyranthes bidentata (RCSH and RCSL groups), large Nissl granules were observed around the centrovesicular nucleus in Nissl-positive neurons, and the number of Nissl neurons was relatively higher than that in the MPTP group. Immunohistochemical results showed that the RCSH and RCSL groups restored TH staining in the substantia nigra pars compacta of mouse brain, and the number of TH-positive neurons was relatively higher than that in the MPTP group. Figure 1 A and Figure 1 (B).

[0035] 2. Effects of the bitter components of Achyranthes bidentata on the behavior of PD mice

[0036] In rotarod and pole climbing experiments, the RCSH and RCSL groups improved MPTP-induced motor dysfunction in Parkinson's disease mice. Figure 2 A- Figure 2 (D).

[0037] 3. Effects of the bitter components of Achyranthes bidentata on inflammatory factors in PD mice

[0038] In the ELISA results, the RCSH and RCSL groups significantly reduced the levels of TNF-α, IL-6, and IL-1β in the substantia nigra of mice compared with the MPTP group. Figure 3 ).

[0039] 4. Effects of the bitter components of Achyranthes bidentata on four lipid profiles in PD mice

[0040] Compared with the MPTP group, mice in the RCSH and RCSL groups had lower levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C), but no significant effect on high-density lipoprotein cholesterol (HDL-C) levels. Figure 4 A- Figure 4 (D).

[0041] 5. Effects of the bitter components of Achyranthes bidentata on α-Syn, TH, and TREM2 proteins in PD mice.

[0042] With increasing dosage, the RCSH and RCSL groups downregulated α-Syn protein expression in PD mice, while the RCSH and RCSL groups upregulated TH and TREM2 protein expression (Western blot, β-actin as internal control). Figure 5 A- Figure 5 (C).

[0043] 6. AI technology for virtual screening of bitter components in Achyranthes bidentata to target effective components in PD lipid metabolism disorders.

[0044] A virtual cell screening model for PD lipid metabolism disorders was successfully established, and 10 candidate drugs for the "bitter" component of Achyranthes bidentata were systematically evaluated. Based on the AI ​​computational model, the binding scores of drugs Cyasterone, Cyathsterone C, and 24,28-diepi-cyasterone were significantly lower than other candidate drugs (all below -10 kcal / mol), suggesting a stronger interaction with TREM2 protein and the best potential. Drugs 24-Hydroxycyasterone and Cyathsterone A had intermediate scores. Figure 6 (A). Time-series curves were used to demonstrate the changes in lipid metabolism indicators (such as triglycerides and cholesterol) in a PD model over time. Targeted lipid metabolism therapy can effectively reverse the pathological state, and the efficacy is time-dependent. Figure 6 (B) The treatment group (Cyasterone, Cyathsterone C, 24,28-diepi-cyasterone) regulated the activity of key enzymes such as fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC), correcting lipid metabolism disorders. Figure 6 (C). Figure 6Figure D shows a heatmap of lipid molecule expression in the PD group, treatment group, and normal group (red indicates upregulation, blue indicates downregulation). The treatment group systematically reversed disease-related lipid molecule abnormalities (PC, SM, PE) and restored lipid metabolism network homeostasis. Glycerol-phospholipid metabolism and sphingolipid signaling pathways may be the core affected pathways (FDR < 0.01). Figure 6 (E). The degree of lipid profile separation between groups can be assessed using PCA or PLS-DA models: the sample points in the treatment group converge with those in the normal group, corroborating the effectiveness of drug intervention. Figure 6 (F).

[0045] 7. Isolation and purification of bitter components from Achyranthes bidentata

[0046] like Figure 7 As shown, the structures of 16 sterone compounds were isolated and determined from the "bitter" component of Achyranthes bidentata. Compounds 1, 9, 10 and 12 were isolated from this plant for the first time.

[0047]

[0048] 8. Molecular docking of Cyasterone (CYA) and TREM2 to separate the bitter components of Achyranthes bidentata.

[0049] like Figure 8 As shown, the molecular docking results indicate that the binding energy between Cyasterone (CYA) and TREM2 is -11 kcal / mol, which is significantly lower than the conventional binding energy threshold, demonstrating excellent binding affinity and stability.

[0050] 9. Effects of Cyasterone (CYA) on BV2 cells

[0051] The CCK8 assay results showed that different concentrations of CYA did not exhibit significant toxicity to BV2 cells; CYA inhibited LPS-induced BV2 cell proliferation; CYA inhibited LPS-induced levels of TNF-α, IL-6, and IL-1β in BV2 cells (ELISA method). Figure 9 A- Figure 9 (C).

[0052] 10. Effects of Cyasterone (CYA) on SH-SY5Y cells

[0053] In in vitro experiments, CCK8 assay results showed that CYA significantly increased the survival rate of Cholesterol-induced SH-SY5Y cells, and LDH assay results showed that CYA significantly reduced the release of Cholesterol-induced LDH. CYA upregulated the expression of TH and TREM2 proteins in Cholesterol-induced SH-SY5Y cells (Western blot, β-actin as internal control).Figure 10 A- Figure 10 (E).

[0054] Example 1: In vivo protective effect of RCS on MPTP-induced PD mice

[0055] 1. Experimental Animals and Grouping: Fifty-five 8-week-old male C57BL / 6 mice (weighing 18-22 g, Beijing Weitonglihua Experimental Animal Co., Ltd.) were selected and acclimatized for one week (20-25℃, 40%-60% humidity). They were then randomly divided into 5 groups (n=11): Normal control group (NC): administered physiological saline by gavage for 14 consecutive days; PD model group (MPTP): administered physiological saline by gavage for 14 consecutive days, with MPTP (30 mg / kg / day) injected intraperitoneally on days 10-14; Positive drug group (L-dopa): administered levodopa (100 mg / kg / day) by gavage for 14 consecutive days, with MPTP injected intraperitoneally on days 10-14.

[0056] High-dose RCS group (RCSH): RCS (80 mg / kg / day) was administered by gavage for 14 consecutive days, followed by intraperitoneal injection of MPTP on days 10-14; Low-dose RCS group (RCSL): RCS (40 mg / kg / day) was administered by gavage for 14 consecutive days, followed by intraperitoneal injection of MPTP on days 10-14.

[0057] 2. Behavioral tests: Roulette test: On day 15, mice were placed on a roulette wheel (10 rpm), and the average dwell time was recorded for three tests (significantly shortened to 22±4 s in the MPTP group and prolonged to 45±3 s in the UCERP1-1H group); Climbing pole test: On day 15, mice were placed head-up on a 50 cm high rubber pole, and the time to climb head-down to the ground was recorded (prolonged to 41±7 s in the MPTP group and shortened to 28±5 s in the UCERP1-1H group).

[0058] 3. Histological and molecular biological tests: Nissl staining: mouse substantia nigra tissue was taken, paraffin-embedded and sectioned (5 μm), stained with Nissl stain and observed under a microscope (Nissl granules were reduced by 50% in the MPTP group and recovered to 80% of the normal level in the RCSH group);

[0059] Immunohistochemistry (IHC): Sections were incubated with TH antibody (1:1000) and the number of TH-positive neurons in the substantia nigra was counted (1.7-fold increase in the RCSH group compared to the MPTP group).

[0060] ELISA: Detected serum levels of TNF-α, IL-6, and IL-1β in the substantia nigra (the UCERP1-1H group showed a 34% reduction in TNF-α, a 48% reduction in IL-6, and a 50% reduction in IL-1β compared to the MPTP group).

[0061] Lipid profile (four tests): Prepare working reagents according to the biochemical reagent kit instructions. Set the appropriate parameters on the automated biochemical analyzer. Load the sample, and the analyzer will perform the automatic measurement. Results: Export the results from the automated biochemical analyzer and store them in an Excel spreadsheet. GraphPad Prism 9.0 will be used for statistical analysis and plotting.

[0062] Western blot: Proteins were extracted from the substantia nigra and the expression of α-Syn, TH, and TREM2 proteins was detected (α-Syn was downregulated by 45% and TH and TREM2 were upregulated by 55% and 45% respectively in the RCS group).

[0063] Example 2: Virtual Screening Based on Artificial Intelligence (AI)

[0064] 1. RDKit was used to calculate the SMILES of the compounds, generating Morgan fingerprints. Principal component analysis (PCA) was then used to reduce the dimensionality of the compound features. Word2vec was used to convert protein amino acid sequence information into low-dimensional real vectors. The dimensional features of the compounds and proteins were combined to represent the features of the compound-protein interaction samples. A deep Parkinson's disease network prediction model was built using Python 3.6 and the Keras deep learning framework based on Tensorflow.

[0065] 2. The performance evaluation metrics used included sensitivity (SEN, eq 4), specificity (SPE, eq 5), accuracy (ACC, eq 6), area under the receiver operating characteristic (ROC) curve (AUROC), precision-recall (PR), and area under the curve (AUPR). After model parameter optimization, the model constructed with the parameter combination was determined as the optimal deep learning prediction model for multi-component-multi-target interactions, used for screening TREM2-interacting compounds. K-nearest neighbor (KNN), random forest (RF), and extreme gradient boosting (XGBoost) models were constructed based on the scikit-learn library, and trained and tested using the same dataset. New compound-protein interaction data were collected from the BindingDB and ChEMBL databases, as well as from DrugBank literature. Compound structural features and human TREM2 amino acid sequence features were extracted and combined to form interaction feature data, which was then input into the multi-component-multi-target interaction deep learning prediction model to predict compounds that can interact with TREM2.

[0066] Example 3 Extraction and purification of the "bitter" (RCS) component of Achyranthes bidentata.

[0067] 1. Raw material pretreatment: Take 20kg of dried Achyranthes bidentata root, soak it in anhydrous ethanol at 24℃ for 1 hour, reflux at 70℃ for 2 hours (to remove fat and pigment), filter and dry the residue at 50℃ for 5 hours, pulverize and pass through a 60-mesh sieve for later use;

[0068] 2. Extraction: Take 10 kg of Achyranthes bidentata slices, add 9 times the amount of methanol to extract under reflux for 3 hours, repeat 3 times, recover the extract under reduced pressure, concentrate, and obtain a total of 3.1 kg of extract; suspend the 3.1 kg of extract in distilled water, stir evenly, and extract repeatedly three times with petroleum ether, ethyl acetate and water-saturated n-butanol, recover the n-butanol extract under reduced pressure, and obtain a total of 714 g of n-butanol fraction;

[0069] 3. Separation: Dissolve 500g of n-butanol fraction in 4000mL of distilled water and perform SP825-L macroporous resin column chromatography with a gradient elution of five solvents: water, 30% ethanol, 50% ethanol, 70% ethanol, and 90% ethanol. The solvents were recovered under reduced pressure, and the 50% ethanol eluent was used to recover 48.62g of total sterone fraction extract (RCS).

[0070] 4. Purification: 30.84 g of the 50% ethanol eluent was separated by MCI and ODS column chromatography. Gradient elution was performed using methanol of different concentrations from 10% to 90% MeOH, and the solvent was recovered under reduced pressure. A total of 16 sterone compounds were separated.

[0071] Example 4: Structural characterization of 16 sterone compounds

[0072] 1. NMR analysis: The 16 isolated compounds (10 mg each) were dissolved in 0.5 mL of D2O and analyzed using a Bruker DRX-400 NMR spectrometer. 13 C NMR (400 MHz).

[0073] 2. Molecular docking: Cyasterone (PubChem CID: 119444) was molecularly docked with protein TREM2 (PDB ID: 5ELI) using AutoDock Vina 1.1.2 software [1]. The compound structure file was downloaded from the Pubchem database (https: / / pubchem.ncbi.nlm.nih.gov / ). Chemdraw 20.0 was used to build the three-dimensional model of the compound and minimize its energy. The protein model was downloaded from the PDB database. PyMol 2.4 was used to preprocess the protein (removing water molecules and redundant ligands, and adding hydrogen atoms). AutoDock Tools 1.5.6 was used to generate the PDBQT file for docking simulation. The docking box was set to the maximum size to enclose the protein, and other parameters were kept at their default values. The docking results were set to output 10 optimal docking positions. The docking conformation with the lowest binding energy and the highest clustering frequency was considered to be the most potential binding mode between the ligand and the protein.

[0074] Example 5: Effects of CYA on LPS-induced BV2 and Cholesterol-induced SH-SY5Y cells

[0075] 1. Cell Culture and Grouping: BV2 cells (Pussell Biotechnology Co., Ltd.) were cultured in DMEM medium containing 10% fetal bovine serum and divided into 5 groups:

[0076] NC group: Normal culture;

[0077] LPS+ group: 1 µg / mL LPS+ treatment for 24 hours;

[0078] LPS+CYA (25 μg / mL) group: Pretreatment with 25 μg / mL CYA for 2 hours, followed by treatment with 1 µg / mL LPS+ for 24 hours;

[0079] LPS+CYA (50 μg / mL) group: Pretreatment with 50 μg / mL CYA for 2 hours, followed by treatment with 1 µg / mL LPS for 24 hours;

[0080] LPS+CYA (100 μg / mL) group: Pretreatment with 100 μg / mL CYA for 2 hours, followed by treatment with 1 µg / mL LPS for 24 hours;

[0081] 2. Cell viability and toxicity assays:

[0082] CCK-8 method: After incubation with CCK-8 reagent for 2 hours, the absorbance at 450 nm was measured (the cell viability in the LPS group decreased to 43±2%, while that in the CYA (100 μg / mL) group increased to 81±4%).

[0083] ELISA method: Detect the levels of TNF-α, IL-6, and IL-1β in the culture medium (TNF-α increased to 84% in the LPS group and decreased to 44% in the RCS (100 μg / mL) group; IL-6 increased to 77% in the LPS group and decreased to 54% in the RCS (100 μg / mL) group; IL-1β increased to 82% in the LPS group and decreased to 50% in the RCS (100 μg / mL) group).

[0084] 3. Cell Culture and Grouping: Human neuroblastoma cells SH-SY5Y (Pusel Life Sciences Co., Ltd.) were cultured in DMEM medium containing 10% fetal bovine serum and divided into 5 groups:

[0085] NC group: Normal culture;

[0086] Cholesterol group: treated with 1.6 mg / mL Cholesterol for 24 hours;

[0087] Cholesterol+CYA (25 μg / mL) group: Pretreatment with 25 μg / mL CYA for 2 hours, followed by treatment with 1.6 mg / mL Cholesterol for 24 hours;

[0088] Cholesterol+CYA (50 μg / mL) group: Pretreatment with 50 μg / mL CYA for 2 hours, followed by treatment with 1.6 mg / mL Cholesterol for 24 hours;

[0089] Cholesterol+CYA (100 μg / mL) group: Pretreatment with 100 μg / mL CYA for 2 hours, followed by treatment with 1.6 mg / mL Cholesterol for 24 hours;

[0090] 4. Cell viability and toxicity assays:

[0091] CCK-8 method: After incubation with CCK-8 reagent for 2 hours, the absorbance at 450 nm was measured (the cell viability in the Cholesterol group decreased to 43±6%, while that in the CYA (100 μg / mL) group increased to 86±7%).

[0092] LDH method: LDH activity in culture medium was detected (LDH release in Cholesterol+ group increased to 275±17 U / L, while LDH release in CYA (100 μg / mL) group decreased to 145±16 U / L).

[0093] Example 5: Effect of CYA on TREM2 protein

[0094] 5. Western blot analysis: Detect the levels of TH and TREM2 proteins in the supernatant.

[0095] Summary of experimental results:

[0096] 1. Extraction and separation: After optimizing the separation method, the RCS yield reached 48.62g.

[0097] 2. Structural characterization: The RCS component consists of sterone compounds (related to lipid metabolism);

[0098] 3. In vivo anti-PD activity: RCS can improve motor function in MPTP mice, protect dopaminergic neurons, inhibit α-Syn aggregation, and exert its effects through anti-inflammatory and regulation of TH and TREM2.

[0099] 4. In vitro activity and safety: CYA has a dose-dependent protective effect against LPS-induced BV2 cell and MPP+-induced SH-SY5Y cell damage, and no significant organ damage was observed in acute toxicity tests, indicating high safety.

[0100] In summary, the method for separating the bitter component (RCS) of Achyranthes bidentata of the present invention is highly efficient and environmentally friendly, with a well-defined structure and significant anti-PD activity, and can be used as a core raw material for PD treatment drugs or functional foods.

[0101] The above description is merely a preferred embodiment of the present invention, and the scope of protection of the present invention is not limited thereto. Any simple changes or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the scope of the technology disclosed in the present invention shall fall within the scope of protection of the present invention.

Claims

1. A composition, characterized in that, It contains 16 sterone compounds, whose chemical structural formulas are shown in formulas (1) to (16) below: 。 2. The composition according to claim 1, characterized in that, The 16 steroidal compounds are a combination of naturally occurring forms extracted from Achyranthes bidentata.

3. The composition according to claim 1 or 2, characterized in that, The composition has a bitter taste.

4. An effective part of Achyranthes bidentata, characterized in that, It comprises the composition according to any one of claims 1-3.

5. A method for preparing the composition according to any one of claims 1-3 or the effective fraction of Achyranthes bidentata according to claim 4, characterized in that, Includes the following steps: Step 1: Take 10 kg of Achyranthes bidentata slices, add 9 times the amount of methanol to extract by reflux for 3 hours, repeat 3 times, recover the extract under reduced pressure, concentrate, and obtain a total of 3.1 kg of extract. Step 2: Suspend 3.1 kg of the extract in distilled water, stir evenly, and extract three times in sequence with petroleum ether, ethyl acetate, and water-saturated n-butanol. Recover the n-butanol extract under reduced pressure to obtain a total of 714 g of n-butanol fraction. Step 3: Dissolve 500g of n-butanol component in 4000mL of distilled water, load the sample onto SP825-L macroporous adsorption resin column, and elute sequentially with distilled water, 30% ethanol, 50% ethanol, 70% ethanol and 90% ethanol. The solvents are recovered under reduced pressure. The total sterone component extract of 48.62g is obtained by recovering the 50% ethanol eluent. Step 4: Take 30.84g of the 50% ethanol eluent and perform MCI and ODS column chromatography separation. Perform gradient elution with methanol of different concentrations of 10%-90% MeOH and recover the solvent under reduced pressure. A total of 15 sterone compounds and 6 triterpenoid saponin compounds were separated.

6. The use of the composition according to any one of claims 1-3 or the effective part of Achyranthes bidentata according to claim 4 in the preparation of a medicament for the treatment and / or prevention of Parkinson's disease.

7. A pharmaceutical preparation, characterized in that, It comprises the composition according to any one of claims 1-3 or the effective fraction of Achyranthes bidentata according to claim 4, and a pharmaceutically acceptable carrier.

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