Rehmannia glutinosa polysaccharide, and preparation method and application thereof

Abstract

The present application relates to a kind of fresh rehmannia polysaccharide and its preparation method and application.The fresh rehmannia polysaccharide is composed of glucose and galactose, obtained from the specific extract part of fresh rehmannia, and has better anxiolytic effect relative to other extract parts of fresh rehmannia.

Description

Technical Field

[0001] This invention relates to the field of Rehmannia glutinosa polysaccharide technology, specifically to a fresh Rehmannia glutinosa polysaccharide, its preparation method, and its application. Background Technology

[0002] Anxiety disorder (AD), also known as anxiety disorder, is a common mental illness characterized by extreme anxiety across various indicators. According to the Diagnostic and Statistical Manual of Mental Disorders (DSM) and the International Classification of Diseases (ICD), anxiety disorders can be classified into several types, including separation anxiety disorder, selective mutism, specific phobias, social anxiety disorder, agoraphobia, panic disorder, and generalized anxiety disorder (GAD).

[0003] Rehmannia glutinosa is the fresh or dried tuberous root of the plant Rehmannia glutinosa Libosch., belonging to the Scrophulariaceae family. Its chemical composition is complex, containing various compounds with different structures, mainly iridoids, ionones, phenylethyl glycosides, lignans, triterpenoids, polysaccharides, and amino acids. Rehmannia includes fresh, raw, and processed forms, with raw and processed forms being commonly used in traditional Chinese medicine, while fresh rehmannia is used relatively less frequently.

[0004] CN113896807A relates to a fresh Rehmannia glutinosa polysaccharide, its preparation method, and its application. The fresh Rehmannia glutinosa polysaccharide is obtained by enzymatic hydrolysis of fresh Rehmannia glutinosa, deproteinization, separation by ion exchange chromatography and gel column tandem chromatography. The monosaccharide units include rhamnose (Rha), arabinose (Ara), galactose (Gal), and glucose (Glc) in a molar ratio of 25.1:26.2:28.7:20.0; the molecular weight is 17.1 kDa, and it has immunomodulatory and anti-inflammatory effects.

[0005] CN110051684A relates to the application of a fresh Rehmannia glutinosa polysaccharide in the preparation of a drug for treating anxiety disorders. The polysaccharide is obtained by water extraction of fresh Rehmannia glutinosa, precipitation with 80% ethanol, washing with 95% ethanol, adsorption with anhydrous calcium chloride, centrifugation, and drying of the supernatant, and is said to have anti-anxiety effects. However, CN110051684A directly extracts fresh Rehmannia glutinosa with water and precipitates it with 80% ethanol, without organic solvent extraction. Therefore, the resulting product typically includes glycopolymers, starch, proteins, mucilage, tannins, pigments, and inorganic salts. Furthermore, because only 80% ethanol is used for precipitation, the resulting polysaccharide is a mixture of polysaccharides with various molecular weights. Therefore, the anti-anxiety active ingredient in CN110051684A cannot be clearly identified.

[0006] To date, research on the isolation and purification of carbohydrate components in Rehmannia glutinosa, especially the structural characterization of polysaccharides, is scarce, and there are no reports on the structural characterization of polysaccharides from raw and fresh Rehmannia glutinosa. Therefore, the anti-anxiety material basis of Rehmannia glutinosa polysaccharides remains unclear. Summary of the Invention

[0007] In the process of screening effective anti-anxiety components from fresh Rehmannia glutinosa, the inventors extracted petroleum ether, dichloromethane, ethyl acetate, n-butanol, and water fractions, respectively. After polysaccharide extraction from the residue, ethanol precipitation with different concentrations yielded 50%, 70%, and 90% ethanol-precipitated polysaccharides (because high molecular weight polysaccharides precipitate first in low-concentration ethanol, the 50%, 70%, and 90% ethanol-precipitated polysaccharides correspond to high, medium, and low molecular weight polysaccharide components, respectively). Finally, alkali extraction was used to extract the residue, yielding alkali-extracted polysaccharides. Through activity screening of the above fractions, the inventors unexpectedly discovered that the 70% ethanol-precipitated polysaccharide isolated from a specific extract fraction of fresh Rehmannia glutinosa exhibited better anti-anxiety effects compared to other extract fractions, thus completing this invention.

[0008] One aspect of this invention relates to a fresh Rehmannia glutinosa polysaccharide, which is composed of glucose and galactose, particularly D-glucose and D-galactose.

[0009] In some embodiments, the weight-average molecular weight (Mw) of the fresh Rehmannia glutinosa polysaccharide may be more than about 1000, more than about 1050, or more than about 1100, and less than about 1300, less than about 1250, or less than about 1200, for example, from about 1050 to about 1200; the polydispersity index (PDI) (Mw / Mn) may be more than about 1.001, more than about 1.002, and less than about 2, less than about 1.5, less than about 1.2, or less than about 1.1, for example, from about 1.001 to about 1.50. In particular, the weight-average molecular weight (Mw) of the fresh Rehmannia glutinosa polysaccharide may be about 1132 Da, the number-average molecular weight (Mn) may be about 1130 Da, and the polydispersity index (PDI) (Mw / Mn) may be about 1.002.

[0010] In some embodiments, the fresh Rehmannia glutinosa polysaccharide contains an α-pyranose ring.

[0011] In some embodiments, the fresh Rehmannia polysaccharide contains four linkage modes: Glcp-(1→,→6)-Galp-(1→,→6)-Glcp-(1→,Galp-(1→), with a molar ratio of 1:3:2:1.

[0012] In some embodiments, the fresh Rehmannia glutinosa polysaccharide has one or more of the following characteristics:

[0013] (1) Generally speaking Figure 7 The ultraviolet spectrum shown;

[0014] (2) Generally speaking Figure 8 The HPGPC spectrum shown;

[0015] (3) Generally speaking Figure 11 The infrared spectrum shown;

[0016] (4) Generally speaking Figure 13 shown 1 H-NMR spectrum;

[0017] (5) Generally speaking Figure 14 shown 13 C NMR spectrum.

[0018] In some embodiments, the fresh Rehmannia glutinosa polysaccharide has the following structure:

[0019]

[0020] Another aspect of the present invention is to provide a method for preparing fresh Rehmannia glutinosa polysaccharides, the method comprising:

[0021] S1: Fresh Rehmannia glutinosa was extracted with acetone and then subjected to solid-liquid separation to obtain the residue;

[0022] S2: Extract the medicinal residue with water and then perform solid-liquid separation to obtain an aqueous extract;

[0023] S3: Concentrate the aqueous extract to a specific gravity of about 1.06-1.22, preferably about 1.15, and centrifuge to obtain supernatant 1;

[0024] S4: Add ethanol to the supernatant 1 until the ethanol volume concentration is about 50% to precipitate, and separate the solid and liquid to obtain supernatant 2;

[0025] S5: Concentrate the supernatant 2 to a specific gravity of about 1.06-1.22, add ethanol to a volume concentration of about 70% to precipitate, and collect the precipitate by solid-liquid separation to obtain fresh Rehmannia polysaccharide.

[0026] The method for preparing fresh Rehmannia glutinosa polysaccharides according to the present invention may further include the steps of purifying and / or drying the obtained fresh Rehmannia glutinosa polysaccharides. The purification may be carried out, for example, by eluting the obtained fresh Rehmannia glutinosa polysaccharides through a cellulose anion exchange column or a gel chromatography column. The drying is not specifically limited; for example, vacuum drying, freeze drying, etc., may be performed.

[0027] The method for preparing fresh Rehmannia glutinosa polysaccharides according to the present invention may further include pretreatment of fresh Rehmannia glutinosa. The pretreatment of fresh Rehmannia glutinosa is not specifically limited; for example, it may involve cleaning the fresh Rehmannia glutinosa raw material and then crushing or chopping it, but is not limited to this.

[0028] In step S1 above, the acetone can be aqueous acetone with a volume concentration of 65%-81% (e.g., 68%, 69%, 70%, 72%, 75%, 80%, etc.). The amount of acetone used can be 7-11 times (e.g., 8, 9, 10 times) the mass of fresh Rehmannia glutinosa. There are no particular limitations on the extraction method, but tissue disruption extraction is preferred. Extraction can be performed more than once, for example, more than twice. Extraction can be carried out at 0-35°C, preferably 10-30°C.

[0029] Specifically, step S1 is performed as follows: Fresh Rehmannia glutinosa is extracted twice with 70% aqueous acetone at 8 times its mass concentration, each time for 5 minutes, and then filtered to obtain the residue.

[0030] In step S2, the extraction is carried out at 40-60℃, preferably 50-55℃. Based on 1 kg of residue, the amount of water used can be 13-31 L, that is, the material-to-liquid ratio of the extraction can be 1:13-31 (w / v, kg / L).

[0031] In step S2, extraction is preferably carried out in a traditional Chinese medicine hot reflux low-temperature extraction tank. Generally, the extraction temperature of the traditional Chinese medicine hot reflux low-temperature extraction tank can be 40-60℃, preferably 50-55℃, and the vacuum degree is -0.1 to -0.04MPa, preferably -0.1 to -0.08MPa.

[0032] Specifically, step S2 is performed as follows: the dregs are soaked in water at a material-to-liquid ratio of 1:20 (w / v, kg / L) in a traditional Chinese medicine hot reflux low temperature extraction tank for 12 hours, and then extracted under reduced pressure at 52℃. After 6 hours, the extraction tank and the low temperature concentration unit start the extraction-concentration cycle. The polysaccharide content of the extract in the extraction tank is monitored in real time using the phenol-sulfuric acid method until the absorbance of the extract in the extraction tank is less than 0.25.

[0033] In step S3, the concentration is preferably carried out under reduced pressure, and the concentration temperature is below 60°C or below 55°C.

[0034] In step S4, precipitation is carried out for more than 12 hours, or more than 18 hours, especially 24 hours.

[0035] In step S5, precipitation is carried out for more than 12 hours, or more than 18 hours, especially 24 hours.

[0036] Another aspect of the present invention is to provide a composition made using fresh Rehmannia glutinosa polysaccharide according to the present invention.

[0037] The composition may be a pharmaceutical composition, a food, or a beverage. The composition may be formulated into different dosage forms such as solid beverages, oral liquids, and oral tablets.

[0038] The composition may also contain one or more excipients, such as solvents, sweeteners, preservatives, flavorings, fillers, diluents, binders, lubricants, flavorings, colorings, etc., but is not limited thereto.

[0039] Another aspect of the present invention relates to the use of fresh Rehmannia glutinosa polysaccharide according to the present invention in the preparation of medicaments for the treatment or prevention of anxiety disorders.

[0040] The anxiety disorders mentioned include separation anxiety disorder, selective mutism, specific phobia, social anxiety disorder, agoraphobia, panic disorder, and generalized anxiety disorder (GAD). Attached Figure Description

[0041] Figure 1 This shows a flowchart illustrating the extraction process of different parts of fresh Rehmannia glutinosa.

[0042] Figure 2 This study demonstrates the effects of different extracts from fresh Rehmannia glutinosa on the proliferation activity of PC12 cells. The data are presented in the following format: *P<0.05, **P<0.01, n=6.

[0043] Figure 3 This study demonstrates the effects of different extracts from fresh Rehmannia glutinosa on NE levels in the extracellular fluid of PC12 cells induced by corticosterone. The data are presented in the following format: ## P < 0.01 compared with the blank group, *P < 0.05 and **P < 0.01 compared with the model group, n = 6.

[0044] Figure 4 This study demonstrates the effects of different extracts from fresh Rehmannia glutinosa on DA levels in the extracellular fluid of PC12 cells induced by corticosterone. The data are presented in the following format: ## P < 0.01 compared with the blank group, *P < 0.05 and **P < 0.01 compared with the model group, n = 6.

[0045] Figure 5 The elution curve of fresh Rehmannia glutinosa polysaccharide RG70 on a DE-52 anion exchange chromatography column is shown.

[0046] Figure 6 The elution curve of fresh Rehmannia glutinosa polysaccharide RG70 on a Sephadex G-75 gel chromatography column is shown.

[0047] Figure 7 The results of the ultraviolet spectral scan of fresh Rehmannia glutinosa polysaccharide RG70-I are shown.

[0048] Figure 8 The HPGPC chromatogram of fresh Rehmannia glutinosa polysaccharide RG70-I is shown.

[0049] Figure 9The HPLC chromatograms of the PMP pre-column derivatization products of the mixed monosaccharide standard (A) and RG70-I hydrolysate (B) are shown, where Man: mannose; Rha: rhamnose; Glc-A: glucuronic acid; Glc: glucose; Gal: galactose; Rib: ribose; Xyl: xylose; Ara: arabinose.

[0050] Figure 10 The chiral high-performance liquid chromatograms of standard monosaccharide (A) and RG70-I complete acid hydrolysate (B) are shown.

[0051] Figure 11 The infrared spectrum of fresh Rehmannia glutinosa glycoside polymer RG70-I is shown.

[0052] Figure 12 The mass spectrum shows the partially methylated alditol acetates (PMAAs) of the fresh Rehmannia glutinosa polypolymer RG70-I.

[0053] Figure 13-17 They are respectively RG70-I 1 H-NMR spectrum, 13 C-NMR spectrum, 1 H- 1 H COSY spectrum, HSQC spectrum, HMBC spectrum.

[0054] Figure 18 The primary structure prediction of the fresh Rehmannia glutinosa polypolymer RG70-I is shown.

[0055] Figure 19 The effects of RG70-I on the percentage of arm-opening distance (A), percentage of arm-opening time (B), and percentage of arm-opening times (C) in rats are shown. ## Compared with the control group, P < 0.01. ** Compared with the model group, P < 0.01.

[0056] Figure 20 The effect of RG70-I on the movement trajectory in the open field test of rats with anxiety disorder is shown. The red box represents the closed arm and the cyan box represents the open arm. A: blank group; B: model group; C: diazepam group; D: low-dose group; E: medium-dose group; F: high-dose group.

[0057] Figure 21 The effects of RG70-I on the percentage of distance traveled in the open box (A), percentage of time spent in the open box (B), and percentage of number of times the open box was used (C) in rats were shown. ## Compared with the control group, P < 0.01, ** compared with the model group, P < 0.01.

[0058] Figure 22The effect of RG70-I on the movement trajectory in the light-dark box experiment of a rat model of anxiety disorder is shown. The red box represents the dark box and the cyan box represents the light box. A: blank group; B: model group; C: diazepam group; D: low-dose group; E: medium-dose group; F: high-dose group.

[0059] Figure 23 The effects of RG70-I on resting time (A), central region movement time (B), and total distance traveled (C) in rats are shown. ## Compared with the control group, P < 0.01. ** Compared with the model group, P < 0.01.

[0060] Figure 24 The effect of RG70-I on the movement trajectory in the open field experiment of rats with anxiety disorder is shown. The red box represents the edge and the cyan box represents the center area. A: blank group; B: model group; C: diazepam group; D: low-dose group; E: medium-dose group; F: high-dose group.

[0061] Figure 25 The effects of RG70-I on serum 5-HT(A), GABA(B), and NE(C) in rats with anxiety disorder were shown. The results showed that ## compared with the control group, P < 0.01, and ** compared with the model group, P < 0.01. Detailed Implementation

[0062] Specific embodiments of the present invention will be further described below with reference to the accompanying drawings. However, the following embodiments are provided only to enable those skilled in the art to understand the present invention more clearly. The present invention is not limited to the following embodiments.

[0063] 1. Experimental Materials and Instruments

[0064] 1.1 Test medicinal materials

[0065] The fresh Rehmannia root was collected from Wen County, Jiaozuo, Henan Province. After identification by Associate Professor Xie Xiaolong of the School of Pharmacy, Henan University of Traditional Chinese Medicine, it was confirmed to be the fresh tuberous root of Rehmannia glutinosa Libosch., a perennial herb of the Scrophulariaceae family. The specimen is preserved in the School of Pharmacy, Henan University of Traditional Chinese Medicine.

[0066] 1.2 Main Reagents and Instruments

[0067] TN-type traditional Chinese medicine hot reflux low-temperature extraction and concentration unit (Zhejiang Kaidi Pharmaceutical Machinery Co., Ltd.); TDZ5-WS-type medical centrifuge (Hunan Xiangyi Laboratory Instrument Development Co., Ltd.); EYELAN-1100-type rotary evaporator (Tokyo Rika K.K., Ltd., Japan); LGJ-18 vacuum freeze dryer (Beijing Songyuan Huaxing Technology Development Co., Ltd.); CO2 cell culture incubator (Thermo Fisher Scientific); Milli-QPOD ultrapure water preparation system (Millipore GmbH, Germany); Heraeus Multifuge X1R-type high-speed benchtop refrigerated centrifuge (Thermo Fisher Scientific); multifunctional microplate reader (Berten Instruments, USA); Rat·DA / Dopamine·ELISA kit and Rat·NA / NE·ELISA kit (Wuhan Yilairui Biotechnology Co., Ltd.); corticosteroids (Cayman Chemical Inc., USA); diazepam tablets (Shandong Xinyi Pharmaceutical Co., Ltd.); SAVANT SPD2010 centrifugal concentration system (Thermo Fisher Scientific). Fisher Scientific); acetone, ethanol, dichloromethane, n-butanol, and petroleum ether (60-90℃) were all analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd.

[0068] 1.3 Cell lines

[0069] Pheochromocytoma cells (PC12 cells) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0070] 2. Preparation and activity detection of different extract fractions from fresh Rehmannia glutinosa

[0071] 2.1 Preparation of different parts of fresh Rehmannia glutinosa

[0072] Figure 1 The flowchart shows the extraction process of different parts of fresh Rehmannia glutinosa. The specific operation is as follows.

[0073] 50 kg of fresh Rehmannia glutinosa was cut into small pieces and extracted twice with 8 times the amount of 70% (v / v) aqueous acetone in a flash extractor for 5 minutes each time. The extracts were filtered to obtain the filtrate and residue. The filtrates were combined and concentrated under reduced pressure to obtain 7 kg of extract.

[0074] After dispersing the extract with an appropriate amount of water, it was successively extracted with petroleum ether, dichloromethane, ethyl acetate, and water-saturated n-butanol. Each organic solvent was used for extraction three times consecutively, with each extraction being 1.5 times the volume of the extract solution. The resulting solutions were obtained from the petroleum ether, dichloromethane, ethyl acetate, and water-saturated n-butanol extracts. The remaining fraction was used as the water fraction.

[0075] The solutions from each fraction were concentrated under reduced pressure using a rotary evaporator and freeze-dried to obtain extracts of petroleum ether, dichloromethane, ethyl acetate, n-butanol, and water fractions, which are hereinafter referred to as the petroleum ether fraction, dichloromethane fraction, ethyl acetate fraction, n-butanol fraction, and water fraction, respectively.

[0076] After acetone extraction, the residue was dried and placed in a non-woven filter bag. It was then placed in a TN-type traditional Chinese medicine hot reflux low-temperature extraction tank. Water was added at a material-to-liquid ratio of 1:20 (w / v, kg / L) and soaked for 12 hours. Extraction was then performed under reduced pressure at 52℃. After 6 hours, the extraction tank and low-temperature concentrator were connected to the extraction-concentration cycle. The polysaccharide content of the extract was monitored in real-time using the phenol-sulfuric acid method until the absorbance of the extract was less than 0.25, indicating complete polysaccharide extraction. After concentration by the low-temperature concentrator, the extract was further concentrated under reduced pressure at 50℃ using a rotary evaporator to a specific gravity of 1.15. The extract was then centrifuged (4000 rpm, 5 min) to obtain the supernatant.

[0077] While stirring, ethanol was slowly added to the supernatant 1 until the ethanol concentration reached 50% (v / v). The mixture was then allowed to stand for 24 hours. After centrifugation, the precipitate was collected and named 50% crude sugar polymer (RG 50). Supernatant 2 was obtained at the same time.

[0078] Supernatant 2 was concentrated under reduced pressure to a specific gravity of 1.06-1.21. Ethanol was slowly added to the concentrate until the ethanol concentration reached 70% (v / v). The mixture was allowed to stand for 24 hours and then centrifuged. The precipitate was collected and named 70% crude sugar polymer (RG 70). Supernatant 3 was obtained at the same time.

[0079] The supernatant 3 was concentrated under reduced pressure until the specific gravity of the extract was 1.06-1.21. Ethanol was slowly added to the concentrate until the ethanol concentration reached 90% (v / v). After standing for 24 hours, the extract was centrifuged, and the precipitate was collected and named 90% crude sugar polymer (RG 90).

[0080] Because high molecular weight polysaccharides precipitate first in low concentrations of ethanol, the 50% crude sugar polymer (RG 50), 70% crude sugar polymer (RG 70), and 90% crude sugar polymer (RG 90) obtained above correspond to a high molecular weight, medium molecular weight, or low molecular weight polysaccharide component, respectively.

[0081] After drying the residue following water extraction, 0.3M sodium hydroxide solution was added at a material-to-liquid ratio of 1:10 (w / v, kg / L). The mixture was soaked at room temperature for 24 hours and then filtered to obtain the extract. This extraction was repeated several times until the polysaccharides were completely extracted. The extracts were combined and neutralized to pH 7.0 with 0.5M hydrochloric acid solution. The neutralized extract was concentrated under reduced pressure and centrifuged. The supernatant was precipitated with 75% ethanol (v / v), allowed to stand for 24 hours, centrifuged, and the precipitate was collected. The precipitate was dissolved in 10 times its volume of distilled water and dialyzed against a 1000 Da dialysis bag for 72 hours. The dialysate was concentrated under reduced pressure at 50°C, precipitated with 75% ethanol, allowed to stand for 24 hours, centrifuged, and the precipitate was collected. This precipitate was named alkaline-extracted crude polysaccharide polymer (RGB).

[0082] 2.2 Determination of the content of crude saccharide polymers in fresh Rehmannia glutinosa

[0083] The sugar content of the above crude saccharide polymers RG 50, RG 70, RG 90 and RGB was determined by the phenol-sulfuric acid method.

[0084] The results showed that the sugar content was 62.7% in RG 50, 68.5% in RG 70, 71.2% in RG 90, and 58.6% in RGB. The content of crude glycopolymers in each part of the fresh Rehmannia glutinosa was all above 50%, indicating that glycopolymers were the main component of the crude glycopolymers obtained through water extraction / alkali extraction, fractionation, and alcohol precipitation.

[0085] 2.3 Preparation of Activity Screening Sample Solutions

[0086] Accurately weigh 10.0 mg of each fraction (petroleum ether, dichloromethane, ethyl acetate, n-butanol, water, 50% crude saccharide polymer, 70% crude saccharide polymer, 90% crude saccharide polymer, and alkali-extracted crude saccharide polymer) and place them in 5 mL volumetric flasks. Dissolve the petroleum ether, dichloromethane, ethyl acetate, n-butanol, and water fractions separately in an appropriate amount of DMSO by vortexing, allow to stand at room temperature, dilute to the mark with DMSO, and mix well. Dissolve the 50%, 70%, 90%, and alkali-extracted crude saccharide polymer fractions separately in an appropriate amount of ultrapure water by vortexing, allow to stand at room temperature, dilute to the mark with ultrapure water, and mix well. Filter each solution through a 0.22 μm microporous membrane, and use the filtrate as a stock solution for testing, diluted before use.

[0087] 2.4 Toxic side effects of different parts of fresh Rehmannia glutinosa on PC12 cells

[0088] The viability of PC12 cells was detected by CCK-8 assay to evaluate the toxic effects of different parts of fresh Rehmannia glutinosa on PC12 cells.

[0089] PC12 cells in the logarithmic growth phase were collected and the cell suspension density was adjusted to 2.5 × 10⁻⁶ cells / mL using DMEM medium containing 5% fetal bovine serum and 10% horse serum (containing 200 kU / L penicillin sodium, 10 mg / L streptomycin, pH 7.4). 6 Cells were seeded at a rate of 100 μL per well into a 96-well culture plate and incubated for 24 h. After the cells had adhered well, the original culture medium was discarded, and the cells were randomly divided into a control group, a group receiving different concentrations of petroleum ether, a group receiving different concentrations of dichloromethane, a group receiving different concentrations of ethyl acetate, a group receiving different concentrations of n-butanol, a group receiving different concentrations of water, a group receiving different concentrations of 50% crude saccharide polymer, a group receiving different concentrations of 70% crude saccharide polymer, a group receiving different concentrations of 90% crude saccharide polymer, and a group receiving different concentrations of alkali-extracted crude saccharide polymer. The concentration design of the drug groups is shown in Table 2.

[0090] The preparation method of the test solution is as follows: Take the test solution stock solution under "2.3", add DMEM medium containing 5% fetal bovine serum and 10% horse serum and dilute to the predetermined concentration, vortex and mix well to obtain the solution.

[0091] The control group was given a culture medium containing 5% fetal bovine serum and 10% horse serum, while the other drug-treated groups were given relative concentrations of the drug. Each experimental group was configured with 6 replicates. After drug administration, the cells were cultured for 24 hours in a cell culture incubator at 37°C, 5% CO2, and saturated humidity. After 24 hours, CCK-8 reagent solution was added, and the cells were cultured for another 3 hours. The absorbance of each group was measured at 450 nm using a microplate reader, and the cell viability of each group was calculated.

[0092] Cell viability = (Absorbance value of drug-treated group / Absorbance value of control group) × 100%

[0093] Table 2. Dosage group concentration design

[0094]

[0095] Figure 2 This study investigated the effects of different extracts of fresh Rehmannia glutinosa on the proliferation activity of PC12 cells. The results showed that a significant effect on PC12 cell activity only began to occur when the concentrations of the petroleum ether fraction (50 μg / mL), the dichloromethane fraction (400 μg / mL), the ethyl acetate fraction (200 μg / mL), the n-butanol fraction (400 μg / mL), and the water, 50%, 70%, 90%, and alkali-extracted crude saccharide polymer fractions (1000 μg / mL) were reached. This indicates that none of the crude saccharide polymer fractions had significant toxic side effects on PC12 cells.

[0096] 2.5 ELISA method for detecting neurotransmitter levels in PC12 cell supernatant

[0097] Norepinephrine (NE) and dopamine (DA) are the main neurotransmitters secreted by PC12 cells. This experiment used NE and DA as indicators, and employed a corticosterone (CORT)-induced PC12 cell damage model as a cell model of anxiety, to evaluate the ability of different parts of Rehmannia glutinosa to regulate neurotransmitter levels.

[0098] PC12 cells in the logarithmic growth phase were digested, and the density of the cell suspension was adjusted to 1×10⁻⁶. 5 Cells were seeded at a rate of 1 mL / well in 12-well plates and cultured for 24 h in a cell culture incubator with 37°C, 5% CO2, and saturated humidity. After cell adhesion and growth, cells were randomly divided into control group, model group, diazepam group, petroleum ether fraction group, dichloromethane fraction group, ethyl acetate fraction group, n-butanol fraction group, water fraction group, 50% crude saccharide polymer fraction group, 70% crude saccharide polymer fraction group, 90% crude saccharide polymer fraction group, and alkali-extracted crude saccharide polymer fraction group. The model group and each drug-treated group were treated with 250 μmol / L corticosterone, and each drug-treated group was further treated with the corresponding drug. The control group was treated with drug-free culture medium, and the positive control group was treated with diazepam (the toxic blood concentration of diazepam is 30 μg / mL, so the dosage was set to 1 / 10 of the dosage, i.e., 3.08 μg / mL). After 24 h of treatment, the cell culture supernatant was collected and analyzed according to the instructions of the NE and DA detection kits.

[0099] Figure 3 This study investigated the effects of different fractions of fresh Rehmannia glutinosa on the extracellular NE (norepinephrine) levels in PC12 cells induced by corticosterone. The results showed that, compared with the control group, the NE level in the model group was significantly reduced (P < 0.01), indicating the successful establishment of the corticosterone-induced PC12 cell damage model. Compared with the model group, the diazepam group, as well as the dichloromethane, ethyl acetate, and water fractions of fresh Rehmannia glutinosa, and all crude saccharide polymer fractions, significantly increased NE levels (P < 0.01), while the petroleum ether and n-butanol fractions had no significant effect on NE levels (P > 0.05). The crude saccharide polymer fractions were significantly superior to the other fractions, with the 70% crude saccharide polymer fraction showing the best results.

[0100] Figure 4This study investigated the effects of different extracts from fresh Rehmannia glutinosa on the extracellular dopamine (DA) levels in PC12 cells induced by corticosterone. The results showed that the DA level in the model group was significantly lower than that in the control group (P < 0.01), indicating the successful establishment of the corticosterone-induced PC12 cell damage model. Compared with the model group, the diazepam group, the ethyl acetate fraction group, the water fraction group, and all crude saccharide polymer fraction groups significantly increased DA levels (P < 0.05, P < 0.01), while the petroleum ether fraction, dichloromethane fraction, and n-butanol fraction had no significant effect on DA levels (P > 0.05). The crude saccharide polymer fraction groups were significantly better than other fraction groups, with the 70% crude saccharide polymer fraction showing the best results.

[0101] The results showed that the dichloromethane, ethyl acetate, and water extracts of fresh Rehmannia glutinosa, as well as various crude saccharide polymers, significantly increased the levels of NE and DA in a corticosterone-induced PC12 cell injury model (P < 0.05, P < 0.01). Among them, the effects of each crude saccharide polymer were significantly better than those of other extracts, especially the 70% crude saccharide polymer, which showed the best effect.

[0102] In summary, the 70% crude saccharide polymer had no obvious toxic side effects on PC12 cells, and it was effective in regulating the extracellular neurotransmitter NE and DA levels in the CORT-induced PC12 cell injury model. Therefore, further research will be conducted on the 70% crude saccharide polymer of fresh Rehmannia glutinosa.

[0103] Purification and compositional identification of 70% crude saccharide polymer from fresh Rehmannia glutinosa

[0104] 3.1 Main Reagents, Materials and Instruments

[0105] Dextran 1000, 5000, 12000, 25000, 50000, 80000, 150000, 270000, 410000, and 670000 were purchased from Sigma-Aldrich; Cellulose DE-52 (Whatman, UK); Sephadex G-75 (Beijing Solarbio Science & Technology Co., Ltd.); HL-2B digital constant flow pump and BS-100A automatic fraction collector were purchased from Shanghai Huxi Analytical Instrument Co., Ltd.; Rudolph AP-IV Polarimeter was purchased from Rudolph, USA; 2410 differential detector, 1515 high performance liquid chromatograph, and 2707 autosampler were purchased from Waters, USA; OHpak SB-803HQ, OHpak SB-804HQ, and OHpak SB-805HQ polymer matrix water-soluble SEC (GFC) columns.

[0106] 3.2 Separation and purification of 70% crude saccharide polymer from fresh Rehmannia glutinosa

[0107] The crude saccharide polymer (RG70) of 70% fraction of fresh Rehmannia glutinosa was initially separated using a DE-52 anion exchange column. Accurately weigh the 70% fraction crude saccharide polymer sample, add ultrapure water and heat to dissolve to 1 g / mL. After centrifugation, the supernatant was loaded onto the sample. Gradient elution was performed using deionized water and 0.05, 0.10, 0.20, and 0.30 mol / L NaCl solutions as eluents at a flow rate of 1 mL / min. Elutions were collected in individual tubes every 10 min. The eluent was developed using the phenol-sulfuric acid method, and the absorbance was measured at 485 nm using a UV-Vis spectrophotometer. An elution curve was plotted with the tube number on the x-axis and the absorbance value on the y-axis. The eluents at each gradient peak were collected.

[0108] Figure 5 The elution curve of fresh Rehmannia glutinosa glycopolymer RG70 is shown on a DE-52 anion exchange chromatography column. As can be seen from the figure, a main peak is obtained from RG70, which is present in the deionized water eluent.

[0109] The main peak eluents were combined, concentrated under reduced pressure, dialyzed through a 1000 Da dialysis bag, and freeze-dried to obtain the preliminarily purified fresh rehmannia glutinosa polymer RG70 sample.

[0110] The preliminarily purified product was further purified using a Sephadex G-75 gel chromatography column. The preliminarily purified glycopolymer RG70 sample was weighed, dissolved in ultrapure water by heating to a concentration of 10 mg / mL, filtered through a 0.45 μm microporous membrane, and loaded onto the filter. Elution was performed using ultrapure water at a flow rate of 4.8 mL / 15 min. Elution was collected in separate tubes every 15 min, and the absorbance of the eluent was measured to plot the elution curve.

[0111] Figure 6 The figure shows the elution curve of fresh Rehmannia glutinosa glycopolymer RG70 on a Sephadex G-75 gel chromatography column. As can be seen from the figure, the elution curve on the Sephadex G-75 gel column has a sharp and concentrated peak, while no obvious peaks are observed in other parts.

[0112] The eluent from the main peak was collected, concentrated under reduced pressure, and freeze-dried to obtain the purified glycopolymer RG70-I from fresh Rehmannia glutinosa.

[0113] 3.3 Purity verification and molecular weight determination of fresh Rehmannia glutinosa polysaccharide RG70-I

[0114] 3.3.1 Ultraviolet Spectroscopy Scan

[0115] Accurately weigh 2 mg of RG70-I sample and add 2 mL of ultrapure water to prepare a 1 mg / mL glycopolymer sample solution. Use a UV-Vis spectrophotometer to perform UV scanning in the wavelength range of 200-400 nm, with ultrapure water as a blank control, to determine whether the sample contains nucleic acids and proteins.

[0116] Figure 7 Display the results of the ultraviolet spectral scan. For example... Figure 7 As shown, the fresh Rehmannia glutinosa glycopolymer RG70-I did not show ultraviolet absorption peaks at two wavelengths of 260nm and 280nm, indicating that RG70-I is a homogeneous glycopolymer and does not contain nucleic acids or proteins.

[0117] 3.3.2 Determination of specific rotation

[0118] Accurately weigh 1g of fresh Rehmannia glutinosa polysaccharide RG70-I sample, dissolve it in 10mL of ultrapure water, and add anhydrous ethanol until a large amount of precipitate appears in the solution. Record the ethanol concentration at this point. After standing for 24 hours, centrifuge to obtain precipitate I. Continue adding anhydrous ethanol to the supernatant until another precipitate forms, and record the ethanol concentration at this point. After standing for 24 hours, centrifuge again to obtain precipitate II. Perform lyophilization on precipitates I and II respectively. Dissolve 347mg of the precipitate in 5mL of ultrapure water to prepare a solution with a concentration of 69.4mg / mL, and determine its specific rotation. The measurement conditions were: temperature 20±0.3℃, detection wavelength 589nm, and polarimeter tube length 100mm. The specific rotation analysis results are shown in Table 5.

[0119] Table 5 Specific rotation analysis of RG70-I

[0120]

[0121] Table 5 shows that the difference in specific rotation between the two fractions obtained by fractional alcohol precipitation of RG70-I is within 5, indicating that RG70-I is a homogeneous component.

[0122] 3.3 High-performance gel permeation chromatography

[0123] 3.3.3.1 Preparation of reference solution

[0124] Accurately weigh dextran standards of different molecular weights (analytical standards of molecular weights of 1000, 5000, 12000, 25000, 50000, 80000, 150000, 270000, 410000, and 670000), and add 0.05M NaCl solution to prepare standard solutions of 5 mg / mL dextran. Filter the solutions through a 0.22 μm microporous membrane to obtain a series of reference solutions.

[0125] 3.3.3.2 Preparation of the test sample solution

[0126] Accurately weigh 5 mg of RG70-I sample, add 0.05 M NaCl solution to prepare a 5 mg / mL test sample solution, centrifuge at 8000 rpm for 10 min, take the supernatant, and filter it through a 0.22 μm microporous membrane to obtain the test sample solution.

[0127] 3.3.3.3 Chromatographic conditions

[0128] Three polymer-based water-soluble SEC (GFC) columns in series were used: OHpak SB-803HQ, OHpak SB-804HQ, and OHpak SB-805HQ (8 × 300 mm). Differential detectors were used for detection. The mobile phase was 0.05 M NaCl solution at a flow rate of 0.65 mL / min, the column temperature was set at 40 °C, and the injection volume was 30 μL.

[0129] 3.3.3.4 Results of high performance gel permeation chromatography

[0130] The sample solution and blank solvent of fresh Rehmannia glutinosa polysaccharide RG70-I were analyzed by HPGPC method. The test sample solution under section "3.3.3.2" was injected and determined according to the chromatographic conditions under section "3.3.3.3". The results are shown in [Figure number missing]. Figure 8 .

[0131] Depend on Figure 8 As can be seen, RG70-I exhibits a single symmetrical peak, indicating that the molecular weight of the RG70-I glycopolymer is relatively concentrated and its homogeneity is good. Calculations show that the weight-average molecular weight (Mw) of RG70-I is approximately 1132 Da, the number-average molecular weight (Mn) is approximately 1130 Da, the polydispersity index (PDI) (Mw / Mn) is approximately 1.002, and the purity is approximately 100%.

[0132] Structural identification of RG70-I, a homogeneous saccharide polymer from fresh Rehmannia glutinosa.

[0133] 4.1 Experimental Materials and Equipment

[0134] D-glucose reference standard (Millipore Sigma); D-xylose reference standard, D-glucuronic acid reference standard, L-arabinose reference standard, D-mannose reference standard, D-galactose reference standard, D-ribose reference standard, and L-rhamnose reference standard were all purchased from Shanghai Yuanye Biotechnology Co., Ltd.; D-galacturonic acid reference standard and D-mannuronic acid reference standard were purchased from Beijing Solarbio Technology Co., Ltd.; L-fucose reference standard and D-fructose reference standard were purchased from Sigma-Aldrich, USA; D-guluronic acid reference standard was purchased from Sichuan Weikeqi Biotechnology Co., Ltd.; methanol and acetonitrile (chromatographic grade) were purchased from Thermo Fisher Scientific; 1-phenyl-3-methyl-5-pyrazolone (PMP) and iodomethane were purchased from Shanghai Mairui Chemical Technology Co., Ltd.; trifluoroacetic acid (TFA) was purchased from Aladdin Reagent (Shanghai) Co., Ltd.; heavy water was purchased from Cambridge Isotope Laboratories; Congo red was purchased from Tianjin Kemeio Chemical Reagent Development Center.

[0135] The following instruments were used: 1260 Infinity II high-performance liquid chromatograph and 1260 Infinity II DAD detector (Agilent Technologies, Inc., USA); EVO300 PC UV-Vis spectrophotometer, Thermo ICS 5000+ ion chromatography system and electrochemical detector, TRACE1300-TSQ8000EVO gas chromatograph-mass spectrometer, and Nicolet IS50 FT-IR spectrometer (Thermo Fisher Scientific); AV-500 nuclear magnetic resonance spectrometer (Bruker Technologies, Germany); and NovaNanoSEM450 field emission scanning electron microscope (FEI, Inc., USA).

[0136] 4.2 Monosaccharide composition analysis of RG70-I

[0137] 4.2.1 PMP-HPLC method

[0138] 4.2.1.1 Preparation of 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatives from mixed monosaccharide standards

[0139] Accurately weigh appropriate amounts of glucose, xylose, glucuronic acid, galacturonic acid, arabinose, mannose, galactose, ribose, and rhamnose reference standards, and add them to ultrapure water to prepare a monosaccharide standard stock solution with a concentration of 1 mg / mL. Accurately pipette 100 μL of the above reference solution, add 100 μL of 0.6 mol / L NaOH solution and 200 μL of 0.5 mol / L PMP methanol solution, mix well, heat in a 70°C water bath for 50 min, remove and cool to room temperature, add 200 μL of 0.3 mol / L HCl solution and 600 μL of chloroform solution, shake thoroughly, centrifuge and discard the chloroform layer, repeat the extraction twice, and filter the aqueous layer through a 0.45 μL microporous membrane to obtain the final product.

[0140] 4.2.1.2 Preparation of PMP derivatives of monosaccharides from RG70-I sample

[0141] Accurately weigh 4.0 mg of RG70-I sample into an ampoule, add 2 mL of 2M trifluoroacetic acid (TFA), mix well, seal, and place in an oil bath at 120℃ for 6 h for hydrolysis. Cool to room temperature, add methanol and concentrate under reduced pressure to dryness, repeating several times to remove TFA. Add 1 mL of ultrapure water to dissolve completely. Accurately pipette 100 μL of sample solution, add 100 μL of 0.6 mol / L NaOH solution and 200 μL of 0.5 mol / L PMP methanol solution, mix well, heat in a water bath at 70℃ for 50 min, remove and cool to room temperature, add 200 μL of 0.3 mol / L HCl solution and 600 μL of chloroform solution, shake thoroughly, centrifuge and discard the chloroform layer, repeat extraction twice, and filter the aqueous layer through a 0.45 μL microporous membrane to obtain the final product.

[0142] 4.2.1.3 Chromatographic conditions

[0143] The chromatographic column used was Diamonsil C10. 18 (4.6mm×250mm, 5μm); Detector: UV detector; Detection wavelength: 254nm; Isocratic elution with 0.05mol / L phosphate (pH 6.8) buffer-acetonitrile (v / v 82:18) as the mobile phase; Flow rate: 1mL / min; Column temperature: 30℃; Injection volume: 1μL.

[0144] 4.2.1.4 Results of PMP-HPLC analysis

[0145] The mixed monosaccharide standards and the PMP derivative solution of the completely acid-hydrolyzed product of fresh Rehmannia glutinosa glycopolymer RG70-I were separately analyzed by injection. The results are shown in the figure. Figure 9 .Depend on Figure 9 It is known that the fresh rehmannia glutinosa polypolymer RG70-I is composed of glucose and galactose.

[0146] 4.2.2 Chiral-HPLC Analysis

[0147] 4.2.2.1 Preparation of Standards

[0148] Weigh D-anhydrous glucose, D-galactose, and D-fructose reference standards accurately, add ultrapure water to prepare a standard mixture of approximately 1 mg / mL, filter through a 0.22 μm microporous membrane, and set aside for later use.

[0149] 4.2.2.2 Preparation of the test solution

[0150] Accurately weigh 2.0 mg of RG70-I sample into an ampoule, add 2 mL of 2M trifluoroacetic acid (TFA), mix well, seal, and place in an oil bath at 120°C for 6 h for hydrolysis. Cool to room temperature, add methanol, concentrate under reduced pressure to dryness, repeat several times to remove TFA, add a small amount of ultrapure water to dissolve completely, add ultrapure water to make up to 1 mL, filter through a 0.22 μm filter membrane, and set aside for later use.

[0151] 4.2.2.3 Chromatographic conditions

[0152] The chromatographic system was: Agilent 1260 Infinity II; detector: 1260 Infinity II evaporative light scattering detector; column: CHIRALPAK AD-H, 5μm, 4.6×250mm; evaporator temperature: 45℃, nebulization temperature: 45℃, gas flow rate: 1.60L / min; mobile phase: n-hexane (A)-anhydrous ethanol (B); gradient elution: 0–10 min (15–30% B), 10–30 min (30–34% B), 30–36 min (34–40% B), 36–45 min (40–15% B), 45–55 min (15% B); flow rate: 0.5 mL / min; column temperature: 30℃; injection volume: 3μL.

[0153] 4.2.2.4 Chiral-HPLC Analysis Results

[0154] Figure 10 This displays a chiral high-performance liquid chromatogram of the standard monosaccharide (A) and the completely acid-hydrolyzed product of RG70-I. Figure 10 It is known that the fresh rehmannia glutinosa polymer RG70-I is composed of D-glucose and D-galactose.

[0155] 4.2.3 Infrared Spectroscopic Analysis

[0156] Accurately weigh 1 mg of dried RG70-I sample and 100 mg of KBr into a mortar, grind thoroughly, compress into tablets, and analyze using FT-IR at 4000-400 cm⁻¹. -1 Infrared spectra of RG-70-I sample were obtained by range scanning.

[0157] Figure 11The infrared spectrum of fresh rehmannia glutinosa polymer RG70-I is shown. Figure 11 As shown, at a wavenumber of 3397.71 cm⁻¹ -1 A broad and strong absorption peak at 2925.03 cm⁻¹ is observed. -1 The characteristic absorption peaks at 1650.84 cm⁻¹ are for the OH stretching vibration and the CH stretching vibration, respectively, which are characteristic absorption peaks of glycopolymers. -1 and 1402.66cm -1 The absorption peak at 546.02 cm⁻¹ indicates that RG70-I may contain water of crystallization. -1 Absorption peak at 1000.83 cm⁻¹ -1 1073.31cm -1 1151.12cm -1 The three absorption peaks at 868.05 cm⁻¹ suggest the presence of a pyranose ring in RG70-I. -1 The absorption peak indicates the presence of α-configuration glycosidic bonds in RG70-I, and the presence of an α-type pyranose ring in RG70-I.

[0158] 4.2.4 Methylation reaction of RG70-I and GC-MS analysis

[0159] 4.2.4.1 Glucuronate reduction and methylation reaction of RG70-I

[0160] Accurately weigh 8.0 mg of dried RG70-I sample into a reaction flask, add 3 mL of anhydrous methanol, mix well, and concentrate under reduced pressure to dryness. Repeat 3 times, then add 7 mL of anhydrous DMSO and sonicate to completely dissolve the sample.

[0161] Weigh 400 mg of dry NaOH, quickly add 7 mL of anhydrous DMSO, sonicate to dissolve, then mix with the glycopolymer sample solution and sonicate for 1 h. After protecting the reaction flask from light, add 300 μL of CH3I and sonicate in an ice-water bath for 10 min. Remove the reaction flask, add another 300 μL of CH3I, and continue sonicating in an ice-water bath for 10 min; finally, add another 300 μL of CH3I and sonicate in an ice-water bath for 1 hour. After the reaction is complete, add 6 mL of ultrapure water and 4 mL of CHCl3, shake thoroughly, centrifuge, separate the chloroform layer, wash the CHCl3 layer three times, and add ultrapure water. Finally, aspirate the CHCl3 layer and spot it onto a KBr slide for FT-IR analysis. If the temperature is between 3600-3200 cm⁻¹... -1 The hydroxyl peaks within the range almost disappeared, indicating that RG70-I was completely methylated.

[0162] 4.2.4.2 Complete acid hydrolysis, reduction, and acetylation of methylated glycopolymers

[0163] The fully methylated RG70-I sample was placed in an ampoule, 2 mL of 2 mol / L TFA was added, and the ampoule was sealed and hydrolyzed in a constant temperature oil bath at 120 °C for 6 h. After the reaction, 3 mL of anhydrous methanol was added and the mixture was concentrated to dryness under reduced pressure. This process was repeated 4 times until the sample pH was neutral. 3 mL of ultrapure water was added to dissolve the sample, and 25 mg of NaBH4 was added to reduce the hydrolysis product in a water bath at 40 °C. After the reaction was completed, 100 μL of glacial acetic acid was added to terminate the reaction. The sample was concentrated to dryness under reduced pressure, and 2 mL of acetic anhydride and pyridine were added. The mixture was kept in a 95 °C oil bath for 1 h. After the reaction, 3 mL of anhydrous methanol was added and the mixture was concentrated to dryness under reduced pressure. This process was repeated 3 times. The sample was dissolved in 2 mL of chloroform, and the chloroform layer was washed 4 times with an equal volume of ultrapure water. The aqueous layer was discarded by centrifugation, and the chloroform layer was filtered through a 0.22 μm microporous membrane for GC-MS analysis.

[0164] 4.2.4.3 Chromatographic conditions

[0165] The chromatographic column was a TG-5 SILMS column (weakly polar 5% phenyl polysiloxane, 30m × 0.25mm, 0.25μm). The temperature program was as follows: initial temperature 80℃, maintained for 5 min, increased to 200℃ at 5℃ / min, maintained at 200℃ for 10 min, decreased to 80℃ at 50℃ / min, and maintained at 80℃ for 2 min. High-purity helium was used as the carrier gas at a flow rate of 1 mL / min. The ionization method was electron impact ionization (EI), with an ion source temperature of 230℃, an injection port temperature of 250℃, and an injection volume of 1 μL.

[0166] 4.2.4.4 Methylation / GC-MS Analysis of Fresh Rehmannia Glutamate Polymer RG70-I

[0167] The RG70-I fully methylated product was subjected to complete acid hydrolysis, reduction, and acetylation treatments. The final product was analyzed by GC-MS, and the results are shown in Table 8. Figure 12 This indicates that the fresh Rehmannia glutinosa polypolymer RG70-I contains four linkage modes: Glcp-(1→,→6)-Galp-(1→,→6)-Glcp-(1→,Galp-(1→), and the molar ratio is approximately 1:3:2:1.

[0168] Table 8. Results of RG70-I methylation analysis of fresh Rehmannia glutinosa polymer.

[0169]

[0170] 4.2.5 Nuclear Magnetic Resonance Analysis of RG70-I

[0171] Accurately weigh 80 mg of RG70-I sample, add 500 μL of heavy water, heat and sonicate to completely dissolve the sample, filter through a 0.45 μm microporous membrane, transfer to an NMR tube, and obtain a one-dimensional NMR spectrum using an NMR spectrometer.1 H NMR and 13 CNMR and two-dimensional NMR spectra HSQC, HMBC and 1 H- 1 H COSY, see Figure 13-17 .

[0172] from 1 The H-NMR spectrum shows severe overlap of anomeric proton signals, making it difficult to determine the number of anomeric protons. 13 C-NMR spectroscopy revealed four anomeric carbon signals in RG70-I, named A, B, D, and E. HSQC spectroscopy analysis assigned the anomeric protons and their respective anomeric carbons, yielding the following peak signals: A 5.00 / 103.69 ppm, B 5.41 / 5.42 / 92.06 ppm, D 5.00 / 97.89 ppm, and E 5.43 / 5.44 / 91.99 ppm. This indicates that RG70-I contains four sugar residues. 1 Chemical shifts and coupling constants in the H-NMR spectra confirmed that the relative configurations of these four sugar residues were all α-terminal. Further analysis using RG70-I... 1 H- 1 HCl and HSQC spectral analysis revealed the fragments. 1 H and its associated 13 The C signal was used to assign the signals of each sugar residue in RG70-I (results are shown in Table 9). The linkage sites and sequence of each sugar residue were then inferred using HMBC spectroscopy. The HMBC spectroscopy showed that signals 5.43 / 5.44 / / 103.69 (E-H1 / A-C1) indicated that the O-1 of residue E was linked to the C-1 of residue A. Similarly, signals 3.88 / 103.69 (A-H6 / A-C1), 5.00 / 70.93ppm (D-H1 / A-C6), 5.00 / 72.60ppm (A-H1 / D-C6), and 3.68 / 1 03.69ppm (D-H6 / A-C1), 5.41 / 5.42 / / 72.60ppm (B-H1 / D-C6) indicate that O-6 of residue A is linked to C-1 of residue A, O-1 of residue D is linked to C-6 of residue A, O-1 of residue A is linked to C-6 of residue D, O-6 of residue D is linked to C-1 of residue A, and O-1 of residue B is linked to C-6 of residue D.

[0173] Table 9 Chemical shift values ​​of monosaccharide residues in fresh Rehmannia glutinosa glycoside polymer RG70-I

[0174]

[0175] The primary structure of RG70-I was obtained through comprehensive analysis of monosaccharide composition, infrared spectroscopy, methylation, and nuclear magnetic resonance spectroscopy. Figure 18 As shown.

[0176] Pharmacodynamics of 5RG70-I for Anxiety

[0177] Using serum 5-hydroxytryptamine (5-HT), γ-aminobutyric acid (GABA), and norepinephrine (NE) levels, as well as behavioral experiments such as the elevated plus maze (EPM), open field test (OFT), and light-dark box (LDB), as indicators, this study explored the anti-anxiety effect of RG70-I in an animal model of anxiety disorder.

[0178] 5.1 Main Test Materials and Instruments

[0179] Diazepam tablets were purchased from Shandong Xinyi Pharmaceutical Co., Ltd.; Rat·ST / 5-HT·ELISA kits, Rat·NA / NE·ELISA kits, and Rat·GABA·ELISA kits were all purchased from Wuhan Yilairuit Biotechnology Co., Ltd.; the XR-XC404 SuperFcs conditioned fear test analysis system, rat elevated cross maze, light and dark chamber, and open field chamber were purchased from Shanghai Xinruan Information Technology Co., Ltd.; the Smart3.0 small animal behavior tracking system was provided by Shenzhen Ruiwode Life Science Co., Ltd.; the E100 upright optical microscope (Nikon Eclipse, Japan); the M204E analytical balance (Mettler Instruments, Switzerland); and the multi-functional microplate reader (Berten Instruments, USA) were all purchased.

[0180] The specifications of the elevated plus maze (EPM) for rats are as follows: closed arm length × width: 500 × 100 mm, wall height: 400 mm, open arm length × width: 500 × 100 mm, edge height: 10 mm, and height from the ground: 50 cm.

[0181] Open Field Test (OFT): Dimensions are 100cm x 100cm x 40cm (length x width x height). It is open-topped with smooth acrylic sheets for both the walls and bottom.

[0182] Light / dark box (LDB) specifications: length × width × height: 45 cm × 27 cm × 27 cm, the ratio of light box to dark box is 1:1. Each box is independently covered at the top and equipped with a camera. The inner wall and bottom of the light box are both white and equipped with lighting equipment. The inner wall and bottom of the dark box are both black. There is a 7.5 cm × 7.5 cm hole in the middle of the light and dark boxes for rats to freely shuttle through.

[0183] 5.2 Experimental animals

[0184] A total of 150 healthy SPF-grade male SD rats were used in this study, with a weight range of (200 ± 20 g). They were purchased from Beijing Huafukang Biotechnology Co., Ltd. (license number: SCXK(Beijing)2019-0008). These rats were raised in the Animal Experiment Center of Henan University of Chinese Medicine, with the relevant license number SYXK(Yu)2020-0004. The feeding conditions were a temperature of 22 - 24 °C and a relative humidity controlled at 50 ± 2%. The rats were fasted before the experiment, but water intake was not restricted. All procedures related to the feeding and experiments of experimental animals followed the regulations of the "Regulations on the Management of Experimental Animals in Henan Province".

[0185] 5.3 Experimental methods

[0186] 5.3.1 Drug preparation

[0187] 5.3.1.1 Preparation of RG70-I solution

[0188] Take the RG70-I freeze-dried powder prepared in section 3.2 above and add 0.5% CMC-Na solution to make a 20 mg / mL solution.

[0189] 5.3.1.2 Preparation of diazepam tablet suspension

[0190] Take 10 diazepam tablets (2.5 mg / tablet), add physiological saline and dissolve them by ultrasonic wave to prepare a 0.2 mg / mL suspension.

[0191] 5.3.2 Grouping, drug administration and model establishment

[0192] Healthy male SD rats were acclimatized for 7 days and then randomly divided into four groups: Control, Model, Diazepam (2 mg / kg / d), Low-dose RG70-I (100 mg / kg / d), Medium-dose RG70-I (200 mg / kg / d), and High-dose RG70-I (400 mg / kg / d), with 25 rats in each group. Thirty minutes after model establishment, the low-, medium-, and high-dose RG70-I and Diazepam groups were administered the corresponding doses by gavage, while the Control and Model groups were administered the same volume of 0.5% CMC-Na solution by gavage. Administration was continued for 14 days at a volume of 10 mL / kg.

[0193] Establishment of an anxiety disorder model: This study established an anxiety disorder model using chronic unpredictable stress (CUS). Stress methods included: restraint for 6 hours, moist bedding (500 mL water per cage) for 24 hours, fasting and water deprivation for 24 hours, two daily unpredictable empty bottle stresses (10 min each), foot shocks (2 mA, 1 s / time, 10 times / min, lasting 3 min), and 90 dB noise stimulation for 10 min. Except for the control group, rats in each group received continuous CUS stimulation for 14 days during the modeling period, with one stress method randomly assigned daily, and the same stress method was not repeated. The order of stress was determined using a random number table. All animals were housed individually during the modeling period.

[0194] Table 11 Methods for establishing rat CUS model

[0195]

[0196] 5.3.3 Behavioral Evaluation Methods

[0197] The illuminance in the testing room was kept below 60 Lux, the room temperature was controlled at 22-24℃, and the environment was kept quiet. After each rat was tested, its feces and urine were cleaned up, and the equipment was disinfected with 75% alcohol. The equipment was dried before the next rat could be tested.

[0198] EPM Experiment: Each rat was placed in an empty cage for 5 minutes before the test, then placed in the central area of ​​the EPM platform with its head facing the open arm. The following parameters were recorded during the experiment, each lasting 5 minutes: number of times the rat entered the open arm (OE), number of times it entered the closed arm (CE), time spent in the open arm (OT), time spent in the closed arm (CT), distance traveled into the open arm (OD), and distance traveled into the closed arm (CD). After the experiment, the percentage of rats entering the open arm out of the total number of arm entries, the percentage of time spent in the open arm out of the total time, and the percentage of distance traveled into the open arm out of the total distance traveled were calculated. The specific calculation formulas are as follows:

[0199] Open arm time percentage: OT% = OT / (OT+CT) × 100%

[0200] Percentage of arm openings: OE% = OE / (OE+CE)×100%

[0201] Open arm travel percentage: OD% = OD / (OD+CD) × 100%

[0202] Successful modeling is indicated when the OD%, OT%, and OE% of the model group are significantly lower than those of the blank group.

[0203] LDB experiment: The number of times rats entered the open box, the percentage of time spent in the open box, and the percentage of distance traveled in the open box were used as evaluation indicators. If the number of times rats entered the open box, the percentage of time spent in the open box, and the percentage of distance traveled in the open box were significantly lower in the model group than in the control group, it indicated that the model was successfully established.

[0204] OFT experiment: Before the test, each rat was placed in an empty cage for 5 minutes of free exploration, and then quickly moved to the edge area of ​​an open field. Its activity in the open field was then recorded via video for 5 minutes. The main observation indicators included the time the rat remained still, the time spent active in the central area, and the total distance traveled. If the model group rats had significantly less time remaining still in the open field, less activity time in the central area, and a significantly shorter total distance traveled compared to the control group, the model was considered successfully established.

[0205] 5.3.4 Determination of serum neurotransmitters 5-HT, GABA, and NE in rats

[0206] Immediately after the behavioral experiments, rats were anesthetized by intraperitoneal injection of 10% chloral hydrate at a dose of 0.4 mL per kilogram of body weight. Blood was then collected via the abdominal aorta, centrifuged at 3000 rpm for 15 min, and the supernatant serum was collected for non-targeted metabolomics analysis and detection of 5-HT (5-hydroxytryptamine), GABA (gamma-aminobutyric acid), and NE neurotransmitter levels. Twelve rats underwent craniotomy, and the prefrontal cortex, bilateral hippocampus, and amygdala were extracted for targeted metabolomics analysis, RNA sequencing (RNA-seq), polymerase chain reaction (PCR), and Western blot analysis. In addition, the whole brains of nine other rats were washed with PBS solution; six of these were cryopreserved in liquid nitrogen and stored at -80°C for non-targeted metabolomics analysis; the remaining three rats were fixed in 4% paraformaldehyde for pathological examination and immunofluorescence sectioning. In addition, the cecal contents of all rats were pre-cooled with liquid nitrogen and stored at -80°C for later use.

[0207] Serum samples were tested using the ELISA method, strictly following the instructions. The levels of 5-HT, GABA, and NE in the serum were measured using a microplate reader at a wavelength of 450 nm. The results are expressed as absorbance (OD) values, and the concentrations of each indicator are calculated in ng / mL.

[0208] 5.3.5 Data Processing

[0209] Data were processed using SPSS Statistics 26.0 statistical analysis software. One-way ANOVA was used for inter-group comparisons. The least significant value (LSD) method was used for comparisons meeting the homogeneity of variance test criteria, and Dunnett's T3 method was used for comparisons not meeting the homogeneity of variance test criteria. Results are expressed as mean ± standard deviation. The description was performed, and the significance level was set at α = 0.05.

[0210] 5.4 Experimental Results

[0211] 5.4.1 Behavioral Results

[0212] 5.4.1.1 EPM Experiment

[0213] The results of the anxiolytic effect of RG70-I on rats in the EPM experiment are shown in Table 12 and... Figure 19 and 20The results showed that, compared with the control group, the percentage of arm-opening distance, the percentage of arm-opening time, and the percentage of arm-opening frequency were all significantly reduced in the model group (P<0.01), indicating that the anxiety model was successfully established. Compared with the model group, the diazepam group and the low, medium, and high dose groups of RG70-I all showed significant increases in the percentage of arm-opening distance, the percentage of arm-opening time, and the percentage of arm-opening frequency (P<0.01), with the high dose group showing the most significant increase, approaching the level of the diazepam group.

[0214] Table 12 shows the anxiolytic effect of RG70-I on rats in the EPM experiment. n=12)

[0215]

[0216] Note: ## Compared with the control group, P < 0.01. ** Compared with the model group, P < 0.01

[0217] 5.4.1.2 LDB Experiment

[0218] The results of the anxiolytic effect of RG70-I on rats in the LDB experiment are shown in Table 13. Figure 21 and Figure 22 The results showed that, compared with the control group, the percentage of distance traveled in the box, the percentage of time spent in the box, and the percentage of the number of times the rats were exposed to the box were all significantly reduced in the model group (P<0.01), indicating that the rat anxiety model was successfully established. Compared with the model group, the diazepam group and the low, medium, and high dose groups of RG70-I all showed significant increases in the percentage of distance traveled in the box, the percentage of time spent in the box, and the percentage of the number of times the rats were exposed to the box (P<0.01), with the medium and high dose groups showing particularly significant increases. There was little difference between the two groups and no significant difference from the diazepam group.

[0219] Table 13. Anxiety-relieving effect of RG70-I on rats in LDB experiment ( n=12)

[0220]

[0221] Note: ## Compared with the control group, P < 0.01. ** Compared with the model group, P < 0.01

[0222] 5.4.1.3 OFT Experiment

[0223] The results of the anxiolytic effect of RG70-I on rats in the OFT experiment are shown in Table 14. Figure 23 and Figure 24The results showed that, compared with the control group, the model group rats had a significantly increased immobility time (P<0.01), while the central region movement time and total movement distance were significantly reduced (P<0.01), indicating that a rat anxiety model was successfully established. Compared with the model group, the diazepam group and the low, medium, and high dose groups of RG70-I had significantly reduced immobility time (P<0.01), while the central region movement time and total movement distance were significantly increased (P<0.01), with the medium dose group showing a particularly significant increase, which was not significantly different from the diazepam group.

[0224] Table 14. Anxiety-relieving effects of RG70-I on rats in the OFT experiment ( n=12)

[0225]

[0226] Note: ## Compared with the control group, P < 0.01. ** Compared with the model group, P < 0.01

[0227] The results showed that low, medium, and high doses of RG70-I significantly improved anxiety-like behaviors in anxiety model rats (P < 0.01), with the medium and high doses showing particularly significant effects. This indicates that all doses of the fresh rehmannia glycopolymer RG70-I can effectively improve anxiety-like behaviors, especially the medium and high doses, which are similar in effect to diazepam.

[0228] 5.4.2 Effects of RG70-I on serum neurotransmitters 5-HT, GABA, and NE in rats

[0229] like Figure 25 As shown, compared with the blank group, the levels of 5-HT, GABA, and NE in the model group were significantly reduced (P < 0.01); compared with the model group, the levels of 5-HT, GABA, and NE in the diazepam group and the low, medium, and high dose groups of RG70-I were significantly increased (P < 0.01), with the medium and high dose groups showing more significant increases, and there was no significant difference compared with the diazepam group.

[0230] Therefore, it is evident that the fresh Rehmannia glutinosa glycopolymer RG70-I can exert its anti-anxiety effect by increasing the levels of 5-HT, GABA, and NE in the serum of rats with anxiety disorders. In particular, the effects of medium and high doses of RG70-I are similar to those of diazepam. Furthermore, there was no significant difference in efficacy between the medium and high dose groups, indicating that the effect between these two groups is not dose-dependent, and the medium dose has reached the optimal dosage of RG70-I for treating CUS-type anxiety disorders.

Claims

1. A fresh Rehmannia glutinosa polysaccharide, said fresh Rehmannia glutinosa polysaccharide being composed of glucose and galactose, particularly composed of D-glucose and D-galactose.

2. The fresh Rehmannia glutinosa polysaccharide according to claim 1, wherein, The weight-average molecular weight of the fresh Rehmannia glutinosa polysaccharide is above 1000 and below 1300, or between 1050 and 1200, and the polydispersity index (PDI) is above 1.001 and below 2, for example, between 1.001 and 1.

50. Specifically, the weight-average molecular weight of the fresh Rehmannia glutinosa polysaccharide is 1132 Da, the number-average molecular weight is 1130 Da, and the polydispersity index (PDI) is 1.

002.

3. The fresh Rehmannia glutinosa polysaccharide according to claim 1, wherein, The fresh Rehmannia glutinosa polysaccharide contains an α-type pyranose ring. Specifically, the fresh Rehmannia polysaccharide contains four linkage modes: Glcp-(1→,→6)-Galp-(1→,→6)-Glcp-(1→, Galp-(1→), with a molar ratio of 1:3:2:

1.

4. The fresh Rehmannia glutinosa polysaccharide according to claim 1, wherein, The fresh Rehmannia glutinosa polysaccharide has one or more of the following characteristics: (1) The ultraviolet spectrum shown in Figure 7; (2) The HPGPC spectrum shown in Figure 8; (3) The infrared spectrum shown in Figure 11; (4) As shown in Figure 13 1 H-NMR spectrum; (5) As shown in Figure 14 13 C NMR spectrum.

5. The fresh Rehmannia glutinosa polysaccharide according to claim 1, wherein, The fresh Rehmannia glutinosa polysaccharide has the following structure:

6. A method for preparing fresh Rehmannia glutinosa polysaccharide, the method comprising: S1: Fresh Rehmannia glutinosa was extracted with acetone and then subjected to solid-liquid separation to obtain the residue; S2: Extract the medicinal residue with water and then perform solid-liquid separation to obtain an aqueous extract; S3: Concentrate the aqueous extract to a specific gravity of 1.06-1.22, preferably 1.15, and centrifuge to obtain supernatant 1; S4: Add ethanol to the supernatant 1 until the ethanol volume concentration is 50% to precipitate, and separate the solid and liquid to obtain supernatant 2; S5: Concentrate the supernatant 2 to a specific gravity of 1.06-1.22, add ethanol to a volume concentration of 70% to precipitate, and collect the precipitate by solid-liquid separation to obtain fresh Rehmannia polysaccharide.

7. The method according to claim 6, further comprising the steps of purifying and / or drying the obtained fresh Rehmannia glutinosa polysaccharide.

8. The method according to claim 6, wherein, In step S1, the acetone is aqueous acetone with a volume concentration of 65%-81%; preferably, the amount of acetone used is 7-11 times the mass of fresh Rehmannia glutinosa; preferably, tissue disruption extraction is used; preferably, the extraction is carried out at 0-35°C. Specifically, step S1 is performed as follows: Fresh Rehmannia glutinosa is extracted twice with 8 times its mass of 70% aqueous acetone for 5 minutes each time, followed by filtration to obtain the residue; and / or In step S2, the extraction is carried out at 40-60℃; preferably, the amount of water used is 13-31L based on 1kg of medicinal residue; preferably, the extraction is carried out in a traditional Chinese medicine hot reflux low temperature extraction tank, the extraction temperature is 40-60℃, preferably 50-55℃, and the vacuum degree is -0.1 to -0.04MPa, preferably -0.1 to -0.08MPa; Specifically, step S2 is performed as follows: The dregs are soaked in a traditional Chinese medicine hot reflux low-temperature extraction tank with water at a material-to-liquid ratio of 1:20 kg / L for 12 hours, followed by vacuum extraction at 52℃. After 6 hours, the extraction tank and the low-temperature concentration unit are connected to start the extraction-concentration cycle. The polysaccharide content of the extract in the extraction tank is monitored in real time using the phenol-sulfuric acid method until the absorbance of the extract in the extraction tank is less than 0.25; and / or In step S3, concentration is carried out under reduced pressure, preferably at a concentration temperature below 60°C or below 55°C; and / or In step S4, precipitation is carried out for more than 12 hours, or more than 18 hours, especially 24 hours; and / or In step S5, precipitation is carried out for more than 12 hours, or more than 18 hours, especially 24 hours.

9. A composition made using the fresh Rehmannia glutinosa polysaccharide according to any one of claims 1-5.

10. The use of the fresh Rehmannia polysaccharide according to any one of claims 1-5 in the preparation of a medicament for treating or preventing anxiety disorders, wherein the anxiety disorders include separation anxiety disorder, selective mutism, specific phobias, social anxiety disorder, agoraphobia, panic disorder, and generalized anxiety disorder.

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