Rambutan polysaccharide, preparation method thereof and mood regulation application

Abstract

The present application relates to a kind of radermachera sinensis polysaccharide and its preparation method and mood regulation application, the radermachera sinensis polysaccharide NLP-II is composed of galactose, arabinose, glucose, rhamnose, glucuronic acid, mannose and xylan, and the molar ratio is 42.72%, 42.35%, 6.46%, 4.54%, 2.00%, 1.17%, 0.76% respectively.The present application carries out extraction and purification to radermachera sinensis, obtains a kind of novel structure radermachera sinensis uniform polysaccharide NLP-II, this achievement fills the blank of radermachera sinensis polysaccharide fine structure research, lays key material foundation for its structure-activity relationship research and high-value development, and the radermachera sinensis polysaccharide NLP-II obtained can improve the behavior performance of mouse, relieve CUMS induced pathological damage, and shows effective mood regulation function.

Description

Technical Field

[0001] This invention belongs to the field of polysaccharides, and more specifically relates to a rambutan polysaccharide, its preparation method, and its mood-regulating applications. Background Technology

[0002] Depression is a mental illness characterized by fatigue, lethargy, and anxiety, accompanied by psychomotor retardation and physical signs such as fatigue, appetite disturbances, and sleep disturbances.

[0003] The pathogenesis of depression is very complex and is often considered to be related to neuroendocrine, biochemical, immune, genetic and environmental factors.

[0004] In recent years, its incidence rate has been rising year by year. Although traditional drug treatment is effective, it is often accompanied by side effects and the risk of addiction.

[0005] Therefore, the search for safe, low-toxicity natural substances that can significantly improve mood has become a research hotspot.

[0006] Rambutan ( Nephelium lappaceum L. It is a tropical evergreen tree of the Sapindaceae family, native to the Malay Archipelago, and is cultivated on a large scale in places such as Baoting, Hainan, my country.

[0007] Its fruit is spherical or oval, with a skin covered with soft, fleshy spines, and turns bright red or yellow when ripe.

[0008] Rambutan is rich in carbohydrates, making it an excellent raw material for extracting natural plant polysaccharides.

[0009] Current research has found that rambutan extract has rich bioactivities such as anti-inflammatory, antioxidant, antibacterial, and anti-photoaging properties.

[0010] However, no research has been reported on the role of rambutan polysaccharides in regulating mood. Summary of the Invention

[0011] To address the aforementioned technical problems, this invention uses a specific extraction process to isolate a novel homogeneous polysaccharide, NLP-II, from rambutan and discovers that this polysaccharide has mood-regulating functions.

[0012] The above-mentioned objective of this invention is achieved through the following technical solution: A first aspect of the present invention is to provide a rambutan polysaccharide NLP-II, the structural formula of which is shown in Formula I:

[0013] In one optional embodiment, the rambutan polysaccharide NLP-II is composed of galactose, arabinose, glucose, rhamnose, glucuronic acid, mannose and xylan, with molar proportions of 42.72%, 42.35%, 6.46%, 4.54%, 2.00%, 1.17% and 0.76%, respectively.

[0014] In one optional embodiment, the rambutan polysaccharide NLP-II has an average molecular weight Mn and a weight-average molecular weight Mw of 43.00 kDa and 63.38 kDa, respectively, and a polydispersity index of 1.47.

[0015] In one optional embodiment, the main chain structure of the rambutan polysaccharide NLP-II is a repeating →6)-α-Gal-(1→), and the side chains consist of α-t-Araf, →3)-β-Glcp(1→, →5)-α-Araf(1→, →4)-β-Galp(1→ and β-t-Galp.

[0016] A second aspect of the present invention is to provide a method for preparing rambutan polysaccharide NLP-II, comprising the following steps: S1. Preparation of rambutan crude polysaccharide NLP: Remove the shells and seeds from the rambutans, crush them, add buffer solution to the resulting juice, and then add a compound enzyme for enzymatic hydrolysis. The hydrolysis temperature is 60℃ and the time is 2 hours. After enzymatic hydrolysis, an extract was obtained. The extract was then concentrated and precipitated to obtain rambutan crude polysaccharide NLP. The buffer solution is a citrate-disodium hydrogen phosphate buffer, wherein the volume ratio of citrate to disodium hydrogen phosphate is 1:10. The complex enzyme is pectinase, cellulase and protease, and the mass ratio of pectinase, cellulase and protease is 1:1:1. S2, Preparation of Rambutan Polysaccharide NLP-II: The crude polysaccharide NLP of rambutan was reconstituted, and an equal volume of TCA solution was added to remove the protein. After centrifugation, the supernatant was collected, dialyzed, and concentrated under vacuum. The obtained dialysate was separated, purified, and the eluent was collected. The eluent was concentrated and dried to obtain rambutan polysaccharide NLP-II. The molecular weight cutoff during dialysis is 3500 Da.

[0017] A third aspect of the present invention is to provide the application of rambutan polysaccharide NLP-II in the preparation of a mouse mood regulator.

[0018] The present invention has the following beneficial effects: (1) The rambutan polysaccharide NLP-II prepared in this invention can improve the behavioral performance of mice, alleviate the pathological damage induced by CUMS, and show effective mood regulation function.

[0019] (2) The present invention extracts and purifies rambutan to obtain a novel rambutan homogeneous polysaccharide NLP-II. This achievement fills the gap in the study of the fine structure of rambutan polysaccharides and lays a key material basis for its structure-activity relationship study and high-value development.

[0020] (3) The preparation method of rambutan polysaccharide of the present invention is simple, green, safe and economical, and the rambutan polysaccharide obtained has high purity. Attached Figure Description

[0021] Figure 1 The elution curve of Sephacryl S-100 HR for rambutan polysaccharide NLP-II of this invention is shown.

[0022] Figure 2 This is the FTIR spectrum of the rambutan polysaccharide NLP-II of the present invention.

[0023] Figure 3 This is a high-performance ion chromatogram of rambutan polysaccharide NLP-II of the present invention.

[0024] Figure 4 This is a high-performance gel permeation chromatogram of rambutan polysaccharide NLP-II of the present invention.

[0025] Figure 5 This is the GC-MS total ion chromatogram of rambutan polysaccharide NLP-II of the present invention.

[0026] Figure 6 This is the 1H NMR spectrum of rambutan polysaccharide NLP-II of the present invention.

[0027] Figure 7 This is the 13C NMR spectrum of rambutan polysaccharide NLP-II of the present invention.

[0028] Figure 8 This is the COSY spectrum of rambutan polysaccharide NLP-II of the present invention.

[0029] Figure 9 This is the HSQC spectrum of rambutan polysaccharide NLP-II of the present invention.

[0030] Figure 10 This is the HMBC spectrum of rambutan polysaccharide NLP-II of the present invention.

[0031] Figure 11 This is the NOESY spectrum of rambutan polysaccharide NLP-II of the present invention.

[0032] Figure 12 The results of the saccharide preference test in CUMS mice.

[0033] Figure 13 The results are from the open field test in CUMS mice.

[0034] Figure 14 The results are from the forced swimming test in CUMS mice.

[0035] Figure 15 The results are from the tail suspension test in CUMS mice.

[0036] Figure 16 HE staining of the DG and CA3 regions of the hippocampus. Detailed Implementation

[0037] The present invention will be further illustrated below with reference to embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0038] Example 1: Extraction, separation and purification of homogeneous polysaccharide NLP-II from rambutan

[0039] 1. Extraction of crude polysaccharide NLP from rambutan Peel and pit fresh Baoting No. 7 rambutan, crush it with a juicer, and enzymatically hydrolyze the resulting juice. Add buffer (disodium citrate phosphate buffer, pH = 6) at a ratio of 1:10 (v / v), and add compound enzymes (pectinase, cellulase, protease) at a ratio of 1:1:1 (w / w). Enzymatically hydrolyze at 60 °C for 2 h to obtain the extract. The extract was concentrated, and the residue was reconstituted and enzymatically hydrolyzed 2-3 times. The extracts were combined, and then 4 times the volume of ethanol was added for precipitation. The precipitate obtained was rambutan crude polysaccharide NLP.

[0040] 2. Isolation and purification of crude rambutan polysaccharide NLP

[0041] 2.1 Preparation of Rambutan Polysaccharide NLP-I by Ion Exchange Column Chromatography Ion exchange chromatography packing material pretreatment: Add DEAE Sepharose Fast Flow (DEAE-52) to distilled water and stir to form a suspension. Then pour the suspension into a Buchner funnel to remove the liquid and wash it repeatedly with distilled water. Repeat the above operation several times until the packing material has no ethanol odor. Add distilled water and stir well to prepare for column packing.

[0042] Column mounting and balancing: After the pretreatment, the packing material obtained above is mixed with water and slowly added to the chromatography column (φ2.5 cm × 60 cm). After complete sedimentation, the peristaltic pump and the automatic collector are connected, and the pump is started to flush the column with distilled water until the gel surface is stable, thus completing the column packing.

[0043] Sample loading and elution: The crude polysaccharide NLP of rambutan was dissolved in distilled water to prepare a 10 mg / mL aqueous solution. An equal volume of TCA was added, and the mixture was centrifuged at 8000 rpm for 10 min. The supernatant was then collected and dialyzed using a dialysis bag with a molecular weight cutoff of 3500 Da. After vacuum concentration, the solution was loaded onto an anion exchange chromatography column for separation. The eluent consisted of distilled water, 0.2 M NaCl solution, and 0.5 M NaCl solution. The flow rate was set to 1 mL / min, and the solution was collected using an automatic fraction collector at a rate of 5 min / tube.

[0044] Collect 20 tubes for each eluent gradient, with 5 ml collected from each tube. Collect 26-36 tubes of eluent to obtain rambutan polysaccharide NLP-I.

[0045] 2.2 Preparation of Rambutan Polysaccharide NLP-II by Gel Filtration Column Chromatography Pretreatment of gel filtration chromatography packing materials: Add Sephacryl S-100 HR gel filtration chromatography packing material to distilled water, stir to form a suspension, then pour into a Buchner funnel to remove the liquid, and wash repeatedly with distilled water. Repeat the above operation several times until the packing material has no ethanol odor, add distilled water, stir well, and prepare for column packing.

[0046] Column mounting and balancing: After the pretreatment, the packing material obtained above is mixed with water and slowly added to the chromatography column (φ 2.5 cm × 60 cm). After complete sedimentation, the peristaltic pump and the automatic collector are connected, and the pump is started to flush the column with distilled water until the gel surface is stable, thus completing the column packing.

[0047] Sample loading and elution: The collected 26-36 tubes of rambutan polysaccharide NLP-I eluent were centrifuged at 8000 rpm for 10 min, then filtered through a 0.45 μm microporous membrane, and the filtrate was loaded onto a pre-equilibrated gel filtration chromatography column.

[0048] The sample loading volume is 1% of the column volume. After all the sample solution has entered the chromatography column, the eluent is distilled water and collected using an automatic fraction collector, with 5 ml collected per tube.

[0049] The polysaccharide content (hereinafter referred to as polysaccharide content) in each eluent tube was detected at 490 nm using the phenol-sulfuric acid method. The polysaccharide elution curve was then obtained by plotting the number of tubes on the x-axis and absorbance on the y-axis, as shown below. Figure 1 As shown.

[0050] 3. Preparation of Rambutan Polysaccharide NLP-II The eluents from tubes 3 to 8 were combined and collected, concentrated under reduced pressure, and freeze-dried under vacuum to obtain homogeneous rambutan polysaccharide NLP-II, with a total sugar content of 90.28 ± 0.66%.

[0051] The polysaccharide is light brown in color and has good resolubility.

[0052] Example 2: Structural Identification of Rambutan Polysaccharide NLP-II

[0053] I. Characteristic Functional Groups in Infrared Spectroscopy The determination and analysis were performed using a Fourier transform infrared spectrometer, employing the potassium bromide (KBr) pellet method.

[0054] The lyophilized sample and KBr were thoroughly ground at a ratio of 1:100 (w / w) and then compressed into tablets. The tablets were prepared at wavenumbers in the range of 400-4000 cm⁻¹. -1 ) 64 scans were performed inside.

[0055] The results are as follows Figure 2 As shown, rambutan polysaccharide NLP-II at 4000-400 cm⁻¹ -1 The absorption peaks exhibited typical characteristics of polysaccharides within the range.

[0056] 3420.7 cm -1 There is a broad, strong absorption peak nearby, which is the result of the OH stretching vibration in the sugar residue, at 2928.2 cm⁻¹. -1 The presence of a strong absorption peak nearby indicates the stretching vibration of the asymmetric CH in sugars, which may contain -CH2 or -CH3.

[0057] At 1406.1 cm -1 The weaker absorption peaks appearing at this point are attributed to the bending vibrations of OH or CH2.

[0058] At 1098.9 cm -1 The absorption peaks that appear between [the two points] are due to the asymmetric stretching vibration of the COC on the pyranose ring, indicating the presence of pyranose glycosidic bonds. (899.5 cm⁻¹) -1 The presence of a weak absorption peak indicates that NLP-II contains both β- and α-glycosidic bonds.

[0059] II. High-performance anion chromatography for the analysis of monosaccharide composition The monosaccharide composition of the purified fraction NLP-II was determined by high performance anion chromatography.

[0060] Weigh 5 mg of polysaccharide sample, add 1 ml of 2M TFA acid solution, and heat at 121 °C for 2 hours to allow for complete acid hydrolysis.

[0061] Blow dry with nitrogen. Add methanol to clean and blow dry again. Repeat the methanol cleaning 2-3 times to remove TFA completely.

[0062] Dissolve in sterile water, filter through a 0.22 μm aqueous filter membrane, and transfer to a chromatographic vial for analysis.

[0063] Chromatographic conditions: An ion chromatography system (ICS 5000+, Thermo Fisher Scientific, USA) is used to analyze and detect monosaccharide components using an electrochemical detector.

[0064] A Dionex™ CarboPac™ PA20 (150*3.0 mm, 10 μm) liquid chromatography column was used. The mobile phase A was H2O, the mobile phase B was 0.1 M NaOH, and the mobile phase C was 0.1 M NaOH and 0.2 M NaAc. The injection volume was 5 μl, the flow rate was 0.5 ml / min, and the column temperature was 30 ℃.

[0065] The results are as follows Figure 3 The ion chromatograms of NLP-II and monosaccharide standards are shown.

[0066] The monosaccharide composition and molar percentage of NLP-II are shown in Table 1.

[0067] Table 1 Monosaccharide composition and molar percentage of NLP-II

[0068] III. Determination of Molecular Weight by High-Performance Gel Permeation Chromatography The determination was performed using high-performance gel permeation chromatography.

[0069] The chromatographic conditions are as follows: The Shimadzu RID-20 differential refractive index detector is equipped with a TOSOH (TSK) TSKgel GMPWXL (7.8 mm × 300 mm) aqueous gel chromatography column, and the mobile phase is 0.1 M NaNO3. 3+ The column was prepared using 0.05% NaN3 in pure water at a flow rate of 0.6 mL / min and a column temperature of 35 ℃. 5 mg of rambutan polysaccharide NLP-II sample was weighed and dissolved thoroughly in ultrapure water, filtered through a 0.22 μm filter membrane, and then analyzed by the instrument.

[0070] The results are as follows Figure 4As shown, the purified rambutan polysaccharide NLP-II exhibited a single symmetrical peak, indicating that the purified fraction of rambutan polysaccharide NLP-II has high purity. This result is consistent with the purification results of Sephacryl S-100 HR column chromatography, indicating that rambutan polysaccharide NLP-II with high purity and good homogeneity was isolated and purified. Based on the molecular weight standard curve, comparing the elution times of rambutan polysaccharide NLP-II, the average molecular weight (Mn) of rambutan polysaccharide NLP-II was calculated to be approximately 43.00 kDa, the weight-average molecular weight (Mw) was approximately 63.38 kDa, and the polydispersity index was 1.47.

[0071] IV. Methylation Analysis of Polysaccharide Linkages Accurately weigh 3 mg of the NLP-II sample to be tested, dissolve it in 500 μL of dimethyl sulfoxide, then add 1.0 mg of NaOH, stir magnetically for 3 h at room temperature, add 50 μL of iodomethane solution and react for 3 h, then add a few drops of distilled water to terminate the reaction.

[0072] Add 2 mL of dichloromethane, vortex centrifuge, discard the aqueous phase, repeat the washing with water 3 times, evaporate the dichloromethane layer to dryness, add 100 μL of 2.0 M TFA solution, react at 121 °C for 90 min, blow dry with nitrogen, add 50 μL of 2.0 M ammonia and 50 μL of 1.0 M sodium borodeuteride solution, mix well, react at room temperature, add 20 μL of acetic acid to terminate the reaction, blow dry with nitrogen, wash twice with 250 μL of methanol, blow dry with nitrogen, add 250 μL of acetic anhydride, vortex mix well, react at 100 °C for 2.5 h, add 1 mL of water and let stand for 10 min, then add 500 μL of dichloromethane, vortex centrifuge, discard the aqueous phase, repeat the washing with water 3 times.

[0073] The organic phase was purged with nitrogen to about 1 mL, passed through a 0.22 μm membrane, and analyzed by GC-MS.

[0074] Chromatographic conditions: The chromatographic system used was an Agilent gas chromatographic system (Agilent 6890A; Agilent Technologies, USA), with a BPX70 column (30 m × 0.25 mm × 0.25 µm, SGE, Australia).

[0075] The injection volume was 1 μl, the split ratio was 10:1, the carrier gas was high-purity helium, and the flow rate was 1.5 ml / min; The initial temperature of the column oven was 140℃ and held for 2.0 min. The temperature was then increased to 230℃ at a rate of 3℃ / min and held for 3 min.

[0076] Mass spectrometry detection conditions: The mass spectrometry system used is a quadrupole mass spectrometer detection system (Agilent 5977B; Agilent Technologies, USA) from Agilent Technologies, equipped with an electron impact ion source (EI) and a MassHunter workstation.

[0077] Electron impact ionization (EI) was used, and the analytes were detected in full scan (SCAN) mode. The ion source temperature was 200℃, the MS quadrupole temperature was 110℃, the ionization energy was 50 EV, the transfer line temperature was 210℃, and the mass scan range (m / z) was 50-350.

[0078] The results are as follows Figure 5 As shown, NLP-II has 7 different glycosidic bond linkages, mainly 3,6-Galp, t-Araf and 3-Glcp.

[0079] The specific data for glycosidic bonds are shown in Table 2.

[0080] Table 2. Analysis of NLP-II methylated sugar alcohol acetyl ester (PMAA) results

[0081] V. Nuclear Magnetic Resonance Spectroscopy for Molecular Structure Analysis Weigh 60 mg of NLP-II, add 10 mL of heavy water to dissolve it completely, filter it through a 0.22 μm filter membrane, freeze-dry it, and repeat the exchange with heavy water 3 times. The freeze-dried sample is then vacuum-dried overnight at 55 °C.

[0082] Finally, it was dissolved in 0.7 mL of heavy water, and after it was fully dissolved, it was transferred to an NMR tube and allowed to stand at room temperature for 3 h before testing.

[0083] The tests included one-dimensional 1H NMR and 13C NMR, and two-dimensional COSY, HSQC, HMBC, and NOESY.

[0084] The experiment was conducted on a 600 MHz Bruker AVANCE spectrometer, and the NMR spectral data were analyzed and processed using Mest ReNova software.

[0085] The structure of NLP-II was further investigated using one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy.

[0086] This section mainly focuses on a detailed analysis of the one-dimensional and two-dimensional NMR spectra of NLP-II. The results for 1H NMR, 13C NMR, COSY, HSQC, HMBC, and NOESY are as follows: Figures 6-11 As shown, the summary is shown in Tables 3 and 4.

[0087] Figure 6 , Figure 7 The 1H NMR and 13C NMR spectra of NLP-II are shown, respectively.

[0088] according to Figure 6 The 1H NMR spectra show that the anomeric hydrogen signals at δ 5.02, 5.18, 4.44, 5.14, 5.02, 4.44 and 4.62 are labeled as residues A, B, C, D, E, F and G.

[0089] Polysaccharides have large molecular weights and complex structures, and the peaks in their one-dimensional spectra often overlap significantly. Therefore, nuclear magnetic resonance spectroscopy examinations are usually a combination of one-dimensional and two-dimensional analyses.

[0090] The corresponding anodic carbon signal is based on Figure 7 13C NMR spectrum and Figure 9 The HSQC spectra were labeled, and the corresponding anomaly carbon signals were δ107.40, 109.24, 103.72, 107.40, 101.74, 103.72 and 103.72, respectively.

[0091] Combination Figure 8 COSY, Figure 9 HSQC Figure 10 HMBC and Figure 11 The NOESY spectra were used to assign values ​​to the experimental data, and the results are summarized in Tables 3 and 4.

[0092] Among them, Figure 8-11 In the table, the first capital letter (AG) represents different sugar residues (corresponding to the serial numbers in Tables 3 and 4). H represents the hydrogen atom on the sugar residue, and C represents the carbon atom on the sugar residue; The number represents the number of hydrogen atoms / carbon atoms bonded to the carbon atom of the sugar residue. For example, AH1 represents the hydrogen atom bonded to the carbon atom of sugar residue A; AC1 represents the carbon atom of sugar residue A.

[0093] Table 3 C and H chemical shifts of NLP-II sugar residues (D2O, 25℃, δ)

[0094] Table 4. Information on the correlation between C and H residues on adjacent residues in NLP-II

[0095] Combining the information in Tables 3 and 4 above, we can see that the δ signal H5.32 is derived from the chemical shift of the anomeric proton of residue A. The corresponding δ values ​​for H1-H6 and C1-C6 of residue A are δ5.02 / 107.40, δ4.06 / 68.62, δ3.86 / 83.86, δ3.94 / 76.79, δ4.18 / 68.60, and δ3.84 / 73.85, respectively. Similarly, the chemical shifts of the anomeric protons and carbon atoms of residues B, C, D, E, F, and G can also be deduced. Based on these NMR data, residue A was designated as →3,6)-α-Galp(1→), residue B as α-t-Araf, residue C as →3)-β-Glcp(1→), residue D as →5)-α-Araf(1→), residue E as →6)-α-Galp(1→), residue F as →4)-β-Galp(1→), and residue G as β-t-Galp.

[0096] Based on the above data, the structural formula of NLP-II is deduced as follows.

[0097]

[0098] Example 3: Application of Rambutan Polysaccharide NLP-II in Mood Regulation I. Animal Experiment Design Sixty adult (5-7 weeks old) male C57BL / 6 mice were housed under standardized environmental conditions: temperature (23±2 ℃), humidity (60%±5%), and a 12:12 h light / dark cycle. The mice had free access to food and water during the rearing period. All animal experimental procedures were approved by the Animal Ethics Committee of Hainan University (NUAUCC-2025-00218).

[0099] After one week of acclimatization, the experimental mice were randomly divided into four groups (n = 10 per group): blank control group (NC), model group (MC), and high- and low-dose NLP-II groups (NLP-H: 200 mg / kg, NLP-L: 100 mg / kg) (the dosage was converted according to the equivalent dose ratio table based on body surface area between humans and animals). A mouse model of depression was established using chronic unpredictable mild stimulation (CUMS), which lasted for four weeks. Behavioral tests were used to determine the success of the model establishment. After four weeks, gavage was initiated. To prevent spontaneous remission, stimulation was administered simultaneously with gavage. The control and model groups were administered 0.2 mL of physiological saline, while the NLP-II group received the same volume of polysaccharide solution via gavage. Gavage was continued for four weeks. Weight changes were recorded every other day during the experiment. Finally, the brains, intestines, and feces of the mice were collected and stored at -80 °C. The specific CUMS procedure is shown in Table 5, which outlines the modeling procedures for the first four weeks, with subsequent procedures repeated every four weeks until the end of the experiment.

[0100] Table 5 CUMS Operation Schedule

[0101] II. Behavioral Testing Sugar water preference experiment: In the eighth week of modeling, mice were given free access to a 1% sucrose solution for 24 hours, followed by free access to pure water for 24 hours. They were then deprived of food and water for 12 hours before the test. All mice were housed individually and had free access to the 1% sucrose solution and pure water for 12 hours. The volumes of sucrose solution and pure water consumed were calculated. Sucrose preference was calculated as follows: Sucrose preference = (Sucrose solution consumption / Total consumption) × 100%.

[0102] (a) Open field experiment Mice were placed individually in a 40 cm × 40 cm × 30 cm apparatus with 16 equal squares drawn on the bottom, allowing them to adapt to the new environment in a quiet setting. The mice were then placed in the center of the apparatus, and the total time spent in the central area of ​​the open field over 6 minutes was observed and recorded.

[0103] (ii) Forced swimming experiment Mice were placed individually in a cylindrical container 30 cm high and 20 cm in diameter, containing 20 cm of water, and tested at 25 °C. The cumulative time the mice remained still during the last 4 minutes of the 6-minute test was recorded. A mouse was considered still when it stopped struggling or floated.

[0104] (III) Tail Suspension Test The animal was suspended 50 cm above a table using pressure-sensitive adhesive tape, with the tape approximately 1 cm from the tip of its tail. The test lasted 6 minutes. The time the mouse remained motionless was recorded during the last 4 minutes of the test. Mice were considered motionless when passively suspended and completely still.

[0105] The results are as follows: Figure 12-15 As shown, behavioral test results of CUMS mice indicate that the sucrose preference rate in the MC group was significantly lower than that in the NC group ( Figure 12 The total time spent in the central area of ​​the open field was significantly reduced. Figure 13 Forced swimming and suspended tail stillness time were significantly increased (p < 0.05). Figure 14 and 15 The results showed that the mice exhibited a decreased preference for sweet water, lost their curiosity to explore new things, and gave up struggling with adverse environments, indicating the success of the CUMS model. Gavage administration of NLP-II significantly improved this condition, bringing the mice's behavioral performance closer to that of the NC group. The results demonstrate that NLP-II can improve CUMS-induced behavior and possesses a certain degree of emotion regulation ability.

[0106] III. Histopathological Observation Brain tissue was harvested, washed with physiological saline to remove blood, and placed in 4% tissue fixative. After 24 hours, the tissue was dehydrated, embedded in paraffin, sectioned, stained with eosin-hematoxylin, and observed and photographed using an optical microscope.

[0107] The results are as follows Figure 16 As shown in the optical microscope, the NC group mice exhibited a large number of pyramidal cells in the CA3 region (angle 3 of the hippocampus) and granule cells in the DG region (dentate gyrus of the hippocampus). These cells were plump, structurally intact, and arranged in a regular and dense manner. In contrast, the MC group showed unclear CA3 region structure, disordered cell arrangement, and extensive pyknosis of granule cell nuclei in the DG region (indicated by the arrows). This indicates that the MC group showed varying degrees of pathological damage to both the CA3 and DG regions of the hippocampus. Compared to the MC group, NLP-H and NLP-L improved the disordered arrangement of pyramidal cells in the CA3 region and granule cells in the DG region of the hippocampus in the MC group (indicated by the arrows). This suggests that NLP-II can improve CUMS-induced pathological damage and has a certain mood-regulating ability.

[0108] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A rambutan polysaccharide NLP-II, characterized in that, The structural formula of the rambutan polysaccharide NLP-II is shown in Formula I: 。 2. The rambutan polysaccharide NLP-II according to claim 1, characterized in that, The rambutan polysaccharide NLP-II is composed of galactose, arabinose, glucose, rhamnose, glucuronic acid, mannose and xylan, with molar proportions of 42.72%, 42.35%, 6.46%, 4.54%, 2.00%, 1.17% and 0.76%, respectively.

3. The rambutan polysaccharide NLP-II according to claim 1, characterized in that, The average molecular weight (Mn) and weight-average molecular weight (Mw) of the rambutan polysaccharide NLP-II were 43.00 kDa and 63.38 kDa, respectively, and the polydispersity index was 1.

47.

4. The rambutan polysaccharide NLP-II according to claim 1, characterized in that, The main chain structure of the rambutan polysaccharide NLP-II is a repeating →6)-α-Gal-(1→), and the side chains are composed of α-t-Araf, →3)-β-Glcp(1→, →5)-α-Araf(1→, →4)-β-Galp(1→ and β-t-Galp.

5. The method for preparing rambutan polysaccharide NLP-II according to any one of claims 1-4, characterized in that, Includes the following steps: S1. Preparation of crude polysaccharide NLP from rambutan: The rambutan was shelled and pitted, crushed, and then buffer was added to the resulting juice. Then, a compound enzyme was added for enzymatic hydrolysis. The hydrolysis temperature was 60℃ and the time was 2 h. After enzymatic hydrolysis, an extract was obtained. The extract was concentrated and precipitated to obtain rambutan crude polysaccharide NLP. The buffer solution was citrate-disodium hydrogen phosphate buffer, wherein the volume ratio of citrate to disodium hydrogen phosphate was 1:

10. The complex enzyme consisted of pectinase, cellulase and protease, and the mass ratio of pectinase, cellulase and protease was 1:1:

1. S2. Preparation of rambutan polysaccharide NLP-II: The crude rambutan polysaccharide NLP was reconstituted, and an equal volume of TCA solution was added to remove the protein. After centrifugation, the supernatant was collected, dialyzed, and concentrated under vacuum. The obtained dialysate was separated, purified, and the eluent was collected. The eluent was concentrated and dried to obtain rambutan polysaccharide NLP-II. The molecular weight cutoff during dialysis was 3500 Da.

6. The application of the rambutan polysaccharide NLP-II according to claim 1 in the preparation of a mouse mood regulator.

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