Preparation method of fructus evodiae pectin polysaccharide, polysaccharide and application thereof

Abstract

This invention relates to the field of Alpinia oxyphylla pectin polysaccharide technology, specifically a method for preparing Alpinia oxyphylla pectin polysaccharide, its polysaccharide composition, and its applications. The preparation method includes the following steps: S1: Pulverize whole Alpinia oxyphylla fruit, extract using hot water reflux, then concentrate, centrifuge, and add ethanol for precipitation to obtain an alcohol precipitate; S2: Dissolve the alcohol precipitate in water, add a protein removal reagent to remove plant protein, add ethanol again for precipitation, separate the precipitate, and obtain crude Alpinia oxyphylla polysaccharide; S3: Separate the crude Alpinia oxyphylla polysaccharide using a DEAE-52 cellulose column, and use a salt gradient elution to obtain two Alpinia oxyphylla polysaccharide solutions; the whole Alpinia oxyphylla fruit includes the pericarp and kernel. This invention uses whole Alpinia oxyphylla fruit as material to separate and extract two novel polysaccharide components, which have antidepressant and intestinal flora imbalance treatment effects, and can be used to prepare antidepressant or intestinal flora imbalance treatment drugs or functional foods.

Description

Technical Field

[0001] This invention relates to the field of Alpinia oxyphylla pectin polysaccharide technology, specifically a method for preparing Alpinia oxyphylla pectin polysaccharide, the polysaccharide itself, and its applications. Background Technology

[0002] Depression is a leading cause of suicide and disability, imposing a significant burden on global social functioning and economic productivity. Currently, commonly used antidepressants include selective serotonin reuptake inhibitors (SSRIs) (representative drugs: fluoxetine, sertraline, etc.), serotonin and norepinephrine reuptake inhibitors (representative drugs: venlafaxine, duloxetine, mirtazapine, etc.), norepinephrine and specific serotonergic antidepressants (representative drugs: mirtazapine, etc.), and monoamine oxidase inhibitors (representative drugs: moclobemide, phenelzine, etc.). However, the efficacy of these chemical drugs is limited, and approximately one-third of patients with depression do not respond well to existing treatments. Furthermore, long-term use of antidepressants can lead to side effects such as emotional numbness, fatigue, poor concentration, increased anxiety, sexual dysfunction, and withdrawal syndrome. Therefore, it is necessary to find natural drug components with fewer side effects, milder effects, and good antidepressant efficacy.

[0003] Alpinia oxyphylla is a traditional Chinese medicine, derived from the plant Alpinia oxyphylla in the ginger family (Zingiberaceae). Alpinia oxyphylla Miq. The fruit of Alpinia oxyphylla is mainly produced in Hainan, Guangdong, Guangxi, and Yunnan. The dried fruit is spindle-shaped or oval, encased in a tough, reddish-brown outer shell. Although some traditional Chinese medicine practitioners believe that the outer shell of Alpinia oxyphylla has certain medicinal properties and can be used in medicine with the shell on, the Chinese Pharmacopoeia clearly stipulates that processed Alpinia oxyphylla seeds with the outer shell removed, as well as their salt-processed form, are the standard ingredients. Therefore, in modern clinical practice, the shell is usually removed before use.

[0004] The traditional efficacy of Alpinia oxyphylla is to treat spleen and stomach deficiency-cold, vomiting, diarrhea, abdominal cold pain, excessive salivation, kidney deficiency-induced urination, frequent urination, seminal emission, and leukorrhea. Modern research shows that Alpinia oxyphylla also has anti-diuretic, memory-enhancing, gastrointestinal function-improving, anti-fatigue, and sex hormone-like effects. In recent years, some scholars have proposed that Alpinia oxyphylla also has anti-depressant effects. For example, the studies by Cui Lujie (2022) and Wu Xinlei (2022) found that the traditional prescriptions containing Alpinia oxyphylla, "Yizhi Jieyu Tang" or "Yishen Jieyu Tang", have the effect of treating depression and cognitive impairment. However, they did not specifically study which Chinese medicines and chemical components in the prescription play a major role in the treatment. Deng Jinlan (2023) proposed that the alcohol extract of Alpinia oxyphylla has the effect of improving diabetic depression and conducted a preliminary identification of the alcohol extract. However, the composition of its alcohol extract is complex, including terpenes, flavonoids, proteins, sugars and other components. Not all components have therapeutic effects, and this study did not analyze which specific components play a major role.

[0005] Furthermore, numerous studies have shown that patients with depression often experience gut microbiota dysbiosis, increased intestinal permeability, and systemic inflammation. "Mental probiotics" therapy, which modulates gut microbiota through probiotics, prebiotics, or dietary microbiota, has emerged as a potential new treatment direction. Existing research indicates that pectin polysaccharides can demonstrate good therapeutic potential in the treatment of cognitive impairment and mental illness by regulating the gut microenvironment and improving microbiota metabolic disorders; however, there are currently no reports on the antidepressant effects of Alpinia oxyphylla pectin polysaccharides.

[0006] In summary, although current research indicates that Alpinia oxyphylla has antidepressant effects, its mechanism is unclear. Direct use of Alpinia oxyphylla or its crude extracts is not very effective and has low medicinal value. Therefore, it is urgent to extract and isolate the chemical components with the main therapeutic effects from Alpinia oxyphylla to support the subsequent research and development of natural drugs for antidepressant and gut microbiota regulation. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing Alpinia oxyphylla pectin polysaccharide to achieve efficient extraction and separation of Alpinia oxyphylla pectin polysaccharide, and the extracted Alpinia oxyphylla pectin polysaccharide has antidepressant and intestinal flora regulating effects.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A method for preparing Alpinia oxyphylla pectin polysaccharide includes: extracting Alpinia oxyphylla shell and kernel by water extraction and alcohol precipitation, and then separating the Alpinia oxyphylla pectin polysaccharide.

[0010] Specifically, the following steps are included: S1: Take the whole fruit of Alpinia oxyphylla, crush it, extract it by hot water reflux, then concentrate and centrifuge it, add ethanol to precipitate it, and obtain the alcohol precipitate; S2: Dissolve the alcohol precipitate in water, add a protein removal reagent to remove plant protein, add ethanol again to precipitate, separate the precipitate, and obtain Alpinia oxyphylla crude polysaccharide. S3: The crude polysaccharide of Alpinia oxyphylla was separated by column chromatography using a DEAE-52 cellulose column, and two Alpinia oxyphylla polysaccharide solutions were obtained by gradient elution with salt solution. In some embodiments, in step S1, the solid-liquid ratio of the hot water reflux extraction is 1:8 to 1:15, the temperature is 90 to 100°C, and the number of extractions is 1 to 3.

[0011] In some embodiments, in step S2, the protein removal reagent includes Sevag reagent (chloroform: n-butanol = 4:1).

[0012] In some embodiments, in step S3, the salt solution includes a sodium chloride solution, a potassium chloride solution, or a sodium acetate solution.

[0013] In some embodiments, step S3, the gradient elution includes sequential elution with water and salt solutions with concentrations of 0.2 mol / L, 0.4 mol / L, 0.6 mol / L, 0.8 mol / L, and 1.0 mol / L.

[0014] In some examples, step S3, the gradient elution includes sequential elution with water and salt solutions with concentrations of 0.2 mol / L, 0.6 mol / L, and 0.8 mol / L.

[0015] In some embodiments, in step S3, the elution rate of the column chromatography separation is 1.0 mL / min, and 5 mL is collected in one tube, with a minimum of 120 elution tubes.

[0016] In some embodiments, the preparation method further includes: S5: The two Alpinia oxyphylla polysaccharide solutions are concentrated by rotary evaporation, then purified by water dialysis, and the dialysate is concentrated and freeze-dried to obtain the two Alpinia oxyphylla polysaccharides.

[0017] This invention also provides two types of Alpinia oxyphylla pectin polysaccharides prepared by the above preparation method, which are mainly of the HG type pectin structure and are named AOFP1 and AOFP2 according to the elution order.

[0018] In some embodiments, one of the Alpinia oxyphylla pectin polysaccharides is AOFP1, which is obtained by elution with water during a gradient elution process; The monosaccharide composition of AOFP1 is galacturonic acid, xylose, galactose, glucuronic acid, arabinose, and glucose.

[0019] In some examples, the glycosidic bond type of the AOFP1 includes: 1,4-Gal p A, 1,4-Xyl p t-Gal p A、1,2,4-Xyl p t-Glc p A.

[0020] In some examples, the weight-average molecular weight (Mw) of the AOFP1 is 61.87 kDa.

[0021] In some examples, the monosaccharide composition of the AOFP1, measured in molar ratios, is galacturonic acid: xylose: galactose: glucuronic acid: arabinose: glucose = 68.32-73.54: 16.74-19.26: 4.04-5.18: 3.12-3.75: 2.42-3.09: 0.14-0.4.

[0022] In some embodiments, one of the Alpinia oxyphylla pectin polysaccharides is AOFP2, which is obtained by elution with 0.6 mol / L and 0.8 mol / L salt solutions during gradient elution. The monosaccharide composition of AOFP2 is galacturonic acid, mannose, xylose, arabinose, galactose, and glucose.

[0023] In some examples, the weight-average molecular weight (Mw) of the AOFP2 is 67.51 kDa.

[0024] In some examples, the glycoside type of the AOFP2 includes: 1,4-Gal p A、t-Gal p A、t-Ara f 6. In some examples, the monosaccharide composition of the AOFP2, on a molar ratio, is galacturonic acid:mannose:xylose:arabinose:galactose:glucose = 85.37-88.01:6.80-7.96:1.98-2.35:1.20-1.57:1.06-1.92:0.83-0.95.

[0025] In some embodiments, the Alpinia oxyphylla pectin polysaccharide or its derivatives are used to prepare antidepressant drugs or functional foods.

[0026] In some examples, the preparation method of the drug is as follows: adding the Alpinia oxyphylla pectin polysaccharide AOFP1 or AOFP2 to excipients and compressing it into tablets.

[0027] For example, the excipients include starch and sodium carboxymethyl cellulose.

[0028] In some embodiments, the Alpinia oxyphylla pectin polysaccharide or its derivatives are used to prepare drugs or functional foods for treating intestinal flora imbalance.

[0029] It is worth noting that while there are studies on the extraction and separation of polysaccharides from Alpinia oxyphylla in existing technologies, there are no reports on Alpinia oxyphylla pectin polysaccharides. According to the Chinese Pharmacopoeia, the standard specification for processed Alpinia oxyphylla is the seed with the outer shell removed (including raw and salt-processed products). Therefore, current research on Alpinia oxyphylla-related compound preparations and active ingredients generally uses clean Alpinia oxyphylla (seeds) or salt-processed Alpinia oxyphylla as the main raw material or research object. Therefore, polysaccharides isolated and prepared from Alpinia oxyphylla generally refer to polysaccharides extracted from Alpinia oxyphylla seeds, mainly neutral polysaccharides composed of monosaccharides such as glucose, xylose, and arabinose.

[0030] As is well known, plant seed coats and pericarps (shells) are rich in pectin polysaccharides. These polysaccharides play a key role in maintaining tissue integrity, providing mechanical protection, and regulating water retention. However, the industry currently neglects research on the polysaccharide components in the pericarp of Alpinia oxyphylla, or directly assumes that the polysaccharide components in the pericarp of Alpinia oxyphylla are the same as those in the seeds.

[0031] According to the experimental results of this invention, crude polysaccharides were extracted from Alpinia oxyphylla seeds using a water extraction-alcohol precipitation method. Monosaccharide composition analysis showed that the main components were glucose, xylose, arabinose, and galactose, which is similar to the monosaccharide composition of Alpinia oxyphylla polysaccharides reported in the literature. Then, using Alpinia oxyphylla containing the pericarp as raw material, a novel acidic pectin polysaccharide was mainly obtained, with galacturonic acid as the main monosaccharide component, which is significantly different from the polysaccharide composition reported in the literature. This confirms that the polysaccharide components extracted from Alpinia oxyphylla pericarp and seeds are different. Furthermore, the activity evaluation results showed that the neutral crude polysaccharide extracted from Alpinia oxyphylla seeds and the alcohol extract of Alpinia oxyphylla were significantly less effective than Alpinia oxyphylla pectin polysaccharide AOFP1 in preventing and treating depression in CUMS model mice, suggesting that Alpinia oxyphylla pectin polysaccharide is the main active antidepressant component.

[0032] The beneficial effects of this invention are: The method of this invention uses the whole fruit of Alpinia oxyphylla as material to separate and extract two novel polysaccharide components. Compared with crude extracts obtained by alcohol extraction or polysaccharide components obtained by extracting only the kernel, it has better effects in antidepressant and treatment of intestinal flora disorder, overcoming the prejudice in clinical practice that Alpinia oxyphylla shell has low medicinal value. Attached Figure Description

[0033] Figure 1 This is a DEAE-52 column chromatography image of Alpinia oxyphylla pectin polysaccharide in Example 1 of the present invention; Figure 2 This is the high-performance gel permeation chromatogram of Example 2 of the present invention; Figure 3 The Fourier transform infrared spectra of two Alpinia oxyphylla polysaccharides, AOFP1 and AOFP2, were detected by infrared spectroscopy in Example 2 of this invention. Figure 4 This is a graph showing the results of determining the monosaccharide composition of AOFP1 by high performance liquid chromatography in Example 2 of the present invention; Figure 5 This is a graph showing the results of determining the monosaccharide composition of AOFP2 by high performance liquid chromatography in Example 2 of the present invention; Figure 6 The following is the nuclear magnetic resonance spectrum of AOFP1 in Example 2 of the present invention, wherein Figure A is the carbon spectrum and Figure B is the proton spectrum; Figure 7 This refers to the structural analysis results obtained from the AOFP1 nuclear magnetic resonance detection results in Embodiment 2 of the present invention; Figure 8The following is the nuclear magnetic resonance spectrum of AOFP2 in Example 2 of the present invention, wherein Figure A is the carbon spectrum and Figure B is the proton spectrum; Figure 9 The NMR spectrum and structural analysis results of the AOFP2 chemical structure obtained by NMR detection in Example 2 of this invention are shown. Figure 10 The results of the sucrose preference experiment in mice in Example 3 of this invention; Figure 11 The results of the mouse tail suspension test in Example 3 of this invention; Figure 12 The results of the elevated cruciate maze experiment in mice in Example 3 of this invention; Figure 13 The results of the open field experiment in mice after administration of AOFP1 and AOFP2 in Example 3 of this invention; Figure 14 This is a comparison chart of the levels of inflammatory factors in the brain regions of mice after administration of AOFP1 and AOFP2 in Example 3 of the present invention. Detailed Implementation

[0034] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings, but the scope of protection of the present invention is not limited to the following description.

[0035] Example 1 This embodiment provides a method for extracting pectin polysaccharide from Alpinia oxyphylla and the polysaccharide itself. The specific method is as follows: 1) Weigh 100 g of *Alpinia oxyphylla* seeds (origin: Hainan, Beijing Lvye Pharmaceutical Co., Ltd.) with the husk intact, pulverize, add 10 times the volume of hot water, reflux for 2 hours, filter using a Buchner funnel, collect the filtrate, and repeat the extraction 3 times. Combine the filtrates, concentrate under reduced pressure using a rotary evaporator, add 8 times the volume of 95% ethanol for precipitation, and separate the alcohol precipitate. After drying, dissolve in water, add 4 times the volume of Sevag reagent (chloroform: n-butanol = 4:1) to the above polysaccharide solution, shake vigorously for 30 minutes to fully emulsify the mixture and form an emulsion, centrifuge at 5000 rpm for 15 minutes at room temperature to remove protein, collect the upper aqueous phase, and repeat this step 3 times. Combine the upper aqueous phases, add 8 times the volume of 95% ethanol again for precipitation, separate the alcohol precipitate, dry, and obtain crude polysaccharide of *Alpinia oxyphylla*.

[0036] 2) DEAE-52 cellulose was soaked in distilled water overnight and then packed into a column. DEAE-52 cellulose was used to separate the crude polysaccharide from Alpinia oxyphylla. Elution was performed sequentially with distilled water, 0.1 mol / L, 0.2 mol / L, 0.4 mol / L, 0.6 mol / L, and 0.8 mol / L NaCl aqueous solutions at a flow rate of 1.0 mL / min. Samples were collected in test tubes, one tube every 5 mL. The absorbance of each tube was measured using the phenol-sulfuric acid method. An elution curve was plotted with the tube number on the x-axis and absorbance on the y-axis. The results are as follows: Figure 1 As shown, two homogeneous components were mainly present, named AOFP1 and AOFP2 in the order of elution. AOFP1 was obtained by elution with distilled water, and AOFP2 was obtained by elution with a 0.6 mol / L~0.8 mol / L NaCl aqueous solution. After concentration, dialysis, and freeze-drying, two homogeneous Alpinia oxyphylla polysaccharide samples were obtained.

[0037] Example 2 This embodiment tests the two types of Alpinia oxyphylla polysaccharides extracted in Example 1. The specific methods are as follows: 1) Molecular weight was determined using high-performance gel permeation chromatography: Establishment of molecular weight calibration curves: Accurately weigh pullulan polysaccharide standards of different molecular weights (analytical standards of molecular weights 180, 666, 6000, 9800, 21900, 46500, 110000, 193000, 353000, 692000, and 1060000), and add them to 0.05 mol / L NaCl aqueous solution to prepare 5 mg / mL dextran standard solutions. Filter the solutions using a 0.22 μm microporous membrane and use them for detection using a high-performance gel permeation chromatography tandem column. Perform linear regression with the logarithm of the relative molecular mass of the standards as the ordinate and the retention time of the corresponding chromatographic peak as the abscissa to obtain the calibration curves.

[0038] Preparation of test sample solution: Accurately weigh 5 mg of each of AOFP1 and AOFP2 samples, add 1 mL of 0.05 mol / L NaCl aqueous solution to the sample to prepare a 5 mg / mL test sample solution, filter it with a 0.22 μm microporous membrane, and then transfer the sample to a 2 mL injection bottle for later use.

[0039] Chromatographic conditions: High-performance gel permeation chromatography (HPLC) was used, with a tandem HPLC column for detection. Instrument: Waters HPLC system; Detector: Waters differential detector; Column: Two polymer-based water-soluble SEC (GFC) columns (8 × 300 mm) in series; Mobile phase: 0.05 mol / L NaCl aqueous solution; Flow rate: 0.50 mL / min; Column temperature: 40 ºC.

[0040] Molecular weight calculation: A linear regression was performed with the logarithm of the relative molecular mass of the standard as the ordinate and the elution volume (V) of the corresponding chromatographic peak as the abscissa to obtain the molecular weight correction curve, as shown below: log(M) = 18.20 2.13V + 0.130V 2 0.00305V 3 Based on the calibration curves of the standards, the molecular weights of AOFP1 and AOFP2 samples were calculated. The results are as follows: Figure 2 As shown in Table 1, the retention time (RT), peak molecular weight (Mp), weight-average molecular weight (Mw), data molecular weight (Mn), peak area, and polydispersity index (Mw / Mn) are as follows. It can be seen that the weight-average molecular weight Mw of AOFP1 is 61.87 kDa, and the Mw of AOFP2 is 67.51 kDa.

[0041] Table 1. Molecular weight determination results of AOFP1 and AOFP2

[0042] 2) Characteristic functional groups were detected using infrared spectroscopy: Determination method: Take 1-2 mg of each dried polysaccharide sample in a mortar, add 200 mg of KBr powder and grind evenly, compress into tablets, and scan the samples using a Fourier transform infrared microscopy spectrometer with a wavelength range of 400-4000 cm⁻¹. -1 Record infrared spectra, such as Figure 3 As shown.

[0043] Analysis of characteristic functional groups of AOFP1: Infrared spectroscopy analysis showed that the sample had a characteristic absorption peak of polysaccharides at 3431.74 cm⁻¹. -1 A strong and broad absorption peak exists at 2930.93 cm⁻¹, which is the strong absorption peak of the OH stretching vibration of hydrogen bonds between or within polysaccharide molecules; -1 The nearby moderate-intensity spikes are absorption peaks of the CH stretching vibrations of methyl (-CH3) and methine (-CH2) groups; 1748.83 cm⁻¹ -1 The absorption peak at 1621.24 cm⁻¹ can be attributed to the symmetric stretching vibration of the C=O group of the esterified carboxyl group in uronic acid, while the peak at 1621.24 cm⁻¹ is also significant. -1 The peak at 1450-1200 cm⁻¹ is a characteristic absorption peak of the asymmetric stretching vibration of the free carboxyl group COO- in uronic acid, indicating that the polysaccharide sample is an acidic polysaccharide and contains esterified groups. -1 The absorption peak is at 1417.69 cm⁻¹. -1 1334.11cm -11237.31 cm -1 These are absorption peaks from the angular vibrations of CH, which, along with the stretching vibrations of CH, constitute the characteristic absorption of the sugar ring; 1150.88 cm⁻¹ -1 This represents the stretching vibration of pyranose-COC; 1104.04 cm⁻¹ -1 1047.82 cm -1 and 1015.52 cm -1 The presence of pyranoside was further confirmed at these three sites, and the absorption at these three sites was due to the bending vibration of the CO bond in the COH or COC structure; 954.53 cm⁻¹ -1 The peak indicates a vibrational peak in carbohydrate molecules; 892.19 cm⁻¹ -1 This is a characteristic region of a β-pyranoside bond, suggesting the presence of β-pyranose in the polysaccharide; 833.04 cm -1 The peak represents the CH-angle vibration of the α-terminal epimer of pyranose; 761.47 cm⁻¹ -1 The peaks of symmetrical ring stretching vibrations of the pyran ring are shown.

[0044] Analysis of AOFP2 characteristic functional groups: Infrared spectroscopy analysis showed that the sample had a characteristic absorption peak for polysaccharides: 3433.77 cm⁻¹. -1 A strong and broad absorption peak exists at 2931.17 cm⁻¹, which is the strong absorption peak of the OH stretching vibration of hydrogen bonds between or within polysaccharide molecules; -1 The nearby moderate-intensity peaks are absorption peaks of the CH stretching vibrations of methyl (-CH3) and methine (-CH2) groups; 1747.40 cm⁻¹ -1 The absorption peak at 1622.49 cm⁻¹ can be attributed to the symmetric stretching vibration of the C=O group of the esterified carboxyl group in uronic acid, while the peak at 1622.49 cm⁻¹ is... -1 The peak at 1450-1200 cm⁻¹ is a characteristic absorption peak of the asymmetric stretching vibration of the free carboxyl group COO- in uronic acid, indicating that the polysaccharide sample is an acidic polysaccharide and contains esterified groups. -1 The absorption peak is at 1417.02 cm⁻¹. -1 1332.36 cm -1 1237.21 cm -1 The absorption peaks are due to the angular vibrations of CH, which, along with the stretching vibrations of CH, constitute the characteristic absorption of the sugar ring; 1148.58 cm⁻¹ -1 The stretching vibration of pyranose COC; 1102.05 cm⁻¹ -1 1015.69cm -1 The presence of pyranoside was further confirmed at these two sites, and the absorption at both sites was due to the bending vibration of the CO bond in the COH or COC structure; 952.55 cm -1The peak indicates a vibrational peak in carbohydrate molecules; 893.17 cm⁻¹ -1 This is a characteristic region of a β-pyranoside bond, suggesting that the polysaccharide contains β-pyranose; 834.40 cm -1 The peak represents the CH-angle vibration of the diastereomer of the α-terminal group of pyranose; 762.86 cm⁻¹ -1 The peaks of symmetrical ring stretching vibrations of the pyran ring are shown.

[0045] 3) The monosaccharide composition was determined using high-performance liquid chromatography (HPLC): Preparation of Standards: Weigh out 5 mg each of rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid, glucuronic acid, glucosamine hydrochloride, and glucosamine galactose hydrochloride monosaccharides, and 10 mg of fucose. Dissolve them and bring the volume to 10 mL in a volumetric flask to prepare a standard stock solution. Then, dilute to the following serial dilutions, filter through a 0.22 μm microporous membrane, and transfer to sample vials.

[0046] Preparation of the test sample solution: Take a clean chromatographic vial, accurately weigh 5 mg (±0.05 mg) of polysaccharide sample, add 1 mL of 2M TFA acid solution, and heat at 121 ℃ for 2 hours. Purge with nitrogen and dry. Add 3 mL of methanol to wash, then dry again, repeating the methanol washing 2-3 times. Dissolve in 1 mL of sterile water, transfer to a chromatographic vial for analysis.

[0047] Chromatographic method: Thermo U3000 high-performance liquid chromatography system, Agilent ZORBAX Eclipse XDB-C18 column (4.6). The column was 250 nm long and 5 μm wide. The mobile phase was acetonitrile: phosphate buffer (12 g / L potassium dihydrogen phosphate, pH adjusted to 6.8 with 2 M NaOH) with isocratic elution. The volume ratio of acetonitrile to phosphate buffer was 17:83. The flow rate was 0.8 mL / min. The column temperature was 30 ºC. The detection wavelength was 250 nm. The injection volume was 10 μL.

[0048] Sample calculation results: Quantification was performed using the external standard method, and a standard curve was established by preparing standard samples of different concentrations.

[0049] The content of each component in a solid sample (ug / mg) = C V F / M Where C is the concentration read by the instrument, in μg / mL; V is the volume of the sample extract, in mL; F is the dilution factor; and M is the total amount of sample weighed, in mg.

[0050] The monosaccharide detection results of Alpinia oxyphylla pectin polysaccharide are shown in Table 2 and Figure 4-5As shown, the weight-average molecular weight of AOFP1 is 61.87 kDa, and the molar ratio of galacturonic acid: xylose: galactose: glucuronic acid: arabinose: glucose in AOFP1 is 73.54: 16.74: 4.04: 3.12: 2.42: 0.14. The weight-average molecular weight of AOFP2 is 67.51 kDa, and the molar ratio of galacturonic acid: mannose: xylose: arabinose: galactose: glucose in AOFP2 is 88.01: 6.80: 1.98: 1.20: 1.06: 0.95.

[0051] Table 2. Monosaccharide composition results of AOFP1 and AOFP2

[0052] 4) Methylation detection was used to analyze the types and proportions of monosaccharide residues, glycosidic bonds, and their ratios: Methylation detection method: Weigh 5 mg of polysaccharide sample, dissolve in 1 mL of primary water, add 200 µL of 0.2 mol / L 2-morpholinoethanesulfonic acid (MES) solution, then add 500 µL of 200 mg / mL carbodiimide, and react at room temperature for 2 h. Add 1 mL of 2 mol / L imidazole and 1 mL of 70 mg / mL NaBD4, and react for 3 h. Add 300 μL of glacial acetic acid to terminate the reaction. Dialyze the sample for 48 h, and after dialysis, freeze-dry the sample for methylation treatment. Dissolve 1 mg of the freeze-dried sample in 1 mL of DMSO, add 30 mg of NaOH, and incubate for 30 min. Add 250 μL of iodomethane solution, purge with nitrogen, and react in the dark for 1 h. Add another 250 μL of iodomethane solution and react for 1 h. Add 1 mL of water and 2 mL of dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. Wash three times with water, collect the lower dichloromethane phase and dry under nitrogen. Add 1 mL of 2M TFA, react at 121℃ for 120 min, and dry under nitrogen at 30℃. Add 1 mL of freshly prepared 1 M NaBD4 (ammonia solution). Incubate magnetically at room temperature for 2.5 h, add 300 μL of acetic acid to terminate the reaction, and dry under nitrogen. Dry twice with 2 mL of 5% (vol / vol) acetic acid in methanol at 40℃ under nitrogen, then twice with 2 mL of methanol under nitrogen at 40℃. Add 1.5 mL of acetic anhydride, vortex to mix, react at 100℃ for 2.5 h, add 2 mL of water and let stand for 10 min. Add 1 mL of dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. Wash three times with water, collect the lower dichloromethane phase, and analyze it.

[0053] Chromatographic parameters: The chromatographic system used was an Agilent gas chromatography system (Agilent 7890A; Agilent Technologies, USA), HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm, Agilent J&W Scientific, Folsom, CA, USA), with high-purity helium (purity not less than 99.999%) as the carrier gas. The flow rate was 1.0 mL / min, and the injection port temperature was 260℃. The injection volume was 1 μL, split injection, split ratio 10:1, and solvent delay 2.2 min. Temperature program: 50℃ held for 1.0 min, increased to 130℃ at 50℃ / min, increased to 230℃ at 3℃ / min, and held for 2 min.

[0054] Mass spectrometry parameters: The mass spectrometry system used is an Agilent 5977B quadrupole mass spectrometer (Agilent Technologies, USA), equipped with an electron impact ionization (EI) source and a MassHunter workstation. The EI source has an inlet temperature of 230°C, a quadrupole temperature of 150°C, and an electron energy of 70 eV. The scanning mode is full scan (SCAN), with a mass scan range (m / z) of 30-600.

[0055] Sample determination results: Based on the relative retention time and mass spectrum of each chromatographic peak, and by comparing with literature data and the database of the Complex Carbohydrates Research Center at the University of Georgia, the sugar residue type corresponding to each chromatographic peak was analyzed. The content of each component was calculated using the peak area corresponding to the sugar residue type. The sample results are summarized in Tables 3 and 4. Table 3. Major sugar residue types and proportions of AOFP1

[0056] Table 4. Major sugar residue types and proportions of AOFP2

[0057] 5) The chemical structure of the polysaccharide was determined using nuclear magnetic resonance (NMR) detection: Nuclear magnetic resonance (NMR) detection method: The lyophilized sample was dissolved in 0.5 mL of D₂O, and one-dimensional NMR was measured using a 600 MHz Bruker NMR spectrometer. 1 H-NMR, 13 C-NMR, DEPT-135, and 2D NMR (COSY, HSQC, HMBC, NOESY). Calibration: HDO hydrogen δH = 4.70 ppm, TMS carbon δC = 0.00 ppm. Detection results are as follows: Figure 6(AOFP1) and Figure 9 (AOFP2) (Two-dimensional NMR spectra of HSQC, COSY, HMBC, NOESY, HSQC-TOCSY, etc. are not listed due to space limitations).

[0058] NMR Result Analysis: Based on the monosaccharide composition, methylation results, and one-dimensional and two-dimensional NMR information analysis of the polysaccharide sample, the AOFP1 polysaccharide sample is identified as a complex polysaccharide. It can be inferred that it is mainly composed of an HG domain with a main chain of →4)-α-D-GalpA-6-OMe-(1→, a small number of side chain fragments from the RG-I domain, and glucuronic acid xylan with →4)-β-D-Xylp-(1→, as the main chain. Possible structural elements of AOFP1 include... Figure 7 As shown, the AOFP2 polysaccharide sample can be largely inferred to be composed primarily of numerous HG domains. Possible structural elements of AOFP2 are as follows: Figure 8 As shown.

[0059] Comparative Example 1 This comparative example provides a method for extracting polysaccharides solely from Alpinia oxyphylla seeds, and the polysaccharides themselves are as follows: 100g of shelled Alpinia oxyphylla kernels (origin: Hainan, Beijing Lvye Pharmaceutical Co., Ltd.) were weighed, pulverized, and then extracted with 10 times their volume of hot water under reflux for 2 hours. The mixture was filtered through a Buchner funnel, and the filtrate was collected. This extraction was repeated 3 times. The filtrates were combined and concentrated under reduced pressure using a rotary evaporator. Eight times their volume of 95% ethanol was added for precipitation, and the ethanol precipitate was separated. After drying, the precipitate was dissolved in water. Four times their volume of Sevag reagent (chloroform: n-butanol = 4:1) was added to the polysaccharide solution, and the mixture was vigorously shaken for 30 minutes to fully emulsify the mixture and form an emulsion. The emulsion was centrifuged at 5000 rpm for 15 minutes at room temperature to remove proteins. The upper aqueous phase was collected, and this step was repeated 3 times. The upper aqueous phases were combined, and eight times their volume of 95% ethanol was added again for precipitation. The ethanol precipitate was separated, dried, and the crude polysaccharide from Alpinia oxyphylla seeds was obtained. The monosaccharide composition was determined by high performance liquid chromatography, as shown in Table 5. Table 5

[0060] Comparative Example 2 This comparative example provides an alcohol extract obtained from the whole fruit of Alpinia oxyphylla using a conventional alcohol extraction method, as detailed below: Weigh 100 g of unshelled Alpinia oxyphylla (origin: Hainan, Beijing Lvye Pharmaceutical Co., Ltd.), dry to constant weight, pulverize, and then add carbon tetrachloride (solid-to-liquid ratio 1:10) for degreasing and decolorization. After treatment, dry in a 40℃ oven for 3 hours to obtain degreased Alpinia oxyphylla powder. Add the degreased Alpinia oxyphylla powder to a 70% ethanol solution (solid-to-liquid ratio 1:10) for extraction, remove the supernatant, and repeat the extraction twice. Collect the filtrate and remove the ethanol by rotary evaporation to obtain the Alpinia oxyphylla ethanol extract.

[0061] Experimental Example 1 This experiment established a mouse model of depression to examine the effects of the two types of Alpinia oxyphylla pectin polysaccharides in Example 1, the crude polysaccharide of Alpinia oxyphylla seeds (AZ) in Comparative Example 1, and the alcoholic extract of Alpinia oxyphylla (AC) in Comparative Example 2 on the improvement of depression induced by chronic unpredictable stimuli. The specific methods are as follows: Establishment and administration of a mouse model of depression induced by chronic unpredictable mild stress (CUMS): Eighty-four healthy male C57 / BL6J mice weighing 22–25 g at 8 weeks of age were randomly divided into 7 groups: normal control group (NC, administered only with distilled water by gavage), CUMS model group (CUMS, administered only with distilled water by gavage), AOFP1 administration group (CUMS + AOFP1, 200 mg / kg / day), AOFP2 administration group (CUMS + AOFP2, 200 mg / kg / day), positive control group (CUMS + Sertraline, reference dose), AZ administration group (CUMS + AZ, 200 mg / kg / day), and AC administration group (CUMS + AC, 200 mg / kg / day), with 12 mice in each group. After 7 days of acclimatization, the experiment began. Mice in the NC control group were housed normally without any stimulation, while the other groups were used to establish the CUMS animal model. Two to three randomized stimuli were selected daily, with no repetition within two days, for three consecutive weeks. Stimuli included restraint for 2 hours, water deprivation for 24 hours, food deprivation for 24 hours, swimming in 4°C ice water for 5 minutes, moist bedding for 24 hours, cage tilting at 45° for 24 hours, cage shaking at 45°C for 5 minutes, day-night reversal for 24 hours, foot shock (1 mA), and noise (120 dB) for 10 minutes. Mice in the treatment groups showed significant abnormal mood changes, with significantly prolonged sleep latency and significantly shortened sleep duration. The presence of depression accompanied by insomnia was considered a successful model. From day 22 of model establishment, oral administration of drugs was initiated, and stimulation continued. Control and model group mice were administered an equal volume of water by gavage, while other groups were administered the corresponding drugs by gavage, once daily for 7 days. Behavioral tests were performed after the drug administration period ended.

[0062] Sugar water preference experiment: Mice were first trained for 48 hours to familiarize themselves with the taste of a 0.1 M sucrose solution, but were then prohibited from drinking the solution for the next 24 hours. Afterwards, the sucrose solution and pure water were placed in two identical water bottles, and the mice's consumption was observed and recorded over 6 hours. Sucrose preference was expressed as the ratio of sucrose consumption to pure water consumption. The experimental results are as follows... Figure 10 As shown (significance) # P<0.05, ## P<0.01, ### (P<0.001) Compared with the normal control group, the CUMS model group mice had significantly reduced sucrose consumption. AOFP1 and AOFP2 intervention significantly increased sucrose preference in the CUMS model group mice, while the AZ and AC groups also increased sucrose preference to some extent, but the increase was about 50% of that of AOFP1 and AOFP2.

[0063] Tail suspension test: Adhesive tape was attached to the tip of the mouse's tail (approximately 1 cm), and the tail was securely fixed to a horizontal wooden board 50 cm above the ground. During the 6-minute test, the mouse's active suspension behavior (manifested as limb movement) and immobile state (defined as limb inactivity) were recorded. The experimental results are as follows: Figure 11 As shown, compared with the normal control group, the CUMS model group mice had a significantly increased resting time in the tail suspension test. AOFP1 and AOFP2 interventions significantly shortened the resting time of the CUMS model group mice, while the AC and AZ interventions also shortened the resting time of the CUMS model group mice, but the degree of shortening was limited, approximately 70% of that of AOFP1 and AOFP2.

[0064] Elevated Cross Maze Experiment: The elevated cross maze consists of two open arms (10 cm × 50 cm), two closed arms (10 cm × 40 cm × 50 cm), and a central open area (10 cm × 10 cm) in a cross shape connecting the four limbs. The central platform is 50 cm above the ground. Mice are placed in the central area with their heads facing one of the open arms. Data is recorded using a computer-connected analysis system, including the number of times mice enter the open arm area, the time spent in the open arm area, and the distance traveled in the open arm area within 5 minutes. The percentage of distance traveled in the open arm area, the percentage of time spent in the open arm area, the number of times mice enter the open arm area, and the resting time are also calculated. The formulas are as follows: Percentage of time spent in the open arm area = (Time spent in the open arm area + Time spent in the closed arm area) × 100%; Percentage of distance traveled in the open arm area = Distance traveled in the open arm area / (Distance traveled in the open arm area + Distance traveled in the closed arm area) × 100%. Experimental results are as follows: Figure 12As shown, compared with the normal control group, the number of times mice entered the open arm region and the dwell time in the CUMS model group were significantly reduced. AOFP1 intervention significantly increased the number of times mice entered the open arm region and the dwell time in the CUMS model group, with a significantly better effect than the AOFP2 group, AZ group, and AC group. This indicates that the anti-anxiety / depression effect is AOFP1 > AOFP2 ≈ AZ > AC.

[0065] Open field experiment: Animals were placed in the test chamber for 30 minutes before the test to acclimatize. The open field measured 50 cm × 50 cm with 50 cm high side walls. The interior of the chamber was white, and the bottom area of ​​the open field was divided into 25 small chambers, including 9 central squares and 16 outer squares. Data were collected and analyzed using a computer-connected analysis system. After the mice acclimatized in the chambers for 1 minute, their activities were recorded for 5 minutes. The number of times the central area was visited, the percentage of distance traveled in the central area, the percentage of time spent in the central area, the resting time, the total distance traveled, and the average speed were selected as evaluation indicators. After each mouse's experiment, the chamber was wiped with 75% ethanol to prevent interference from residual odors from the previous mouse. Data were collected and relevant indicators were calculated. Experimental results are as follows: Figure 13 As shown, compared with the normal control group, the total open field distance and the number of times mice entered the central region were significantly reduced in the CUMS model group. AOFP1 and AOFP2 interventions significantly increased the total open field distance and the number of times mice entered the central region in the CUMS model group.

[0066] Detection of inflammatory factors in mouse brain: The levels of pro-inflammatory factors TNF-α and IL-1β in the brain tissue of mice in each group were measured according to the ELISA kit instructions. Experimental results are as follows: Figure 14 As shown, compared with the normal control group, the levels of pro-inflammatory factors TNF-α and IL-1β in the brain tissue of CUMS model mice were significantly increased. AOFP1 and AOFP2 intervention significantly reduced the levels of pro-inflammatory factors TNF-α and IL-1β in the brain tissue of CUMS model mice.

[0067] Experiment Example 2 This experiment validated the regulatory effects of two Alpinia oxyphylla pectin polysaccharides, AOFP1 and AOFP2, on the gut microbiota of depressed mice. The specific method was as follows: Mouse intestinal contents were collected, flash-frozen in liquid nitrogen, and stored at -80℃. Metagenomic sequencing of the mouse intestinal contents was performed by a company. The sequencing results were analyzed using α-diversity, and the Shannon and Simpson indices were calculated. The results showed that compared with the normal control group, the Shannon and Simpson indices at the genus and species levels of the gut microbiota in the CUMS model group mice were decreased. AOFP1 and AOFP2 intervention significantly increased the Shannon and Simpson indices, suggesting that AOFP1 and AOFP2 intervention can improve the species richness and evenness of the gut microbiota. Furthermore, LEfSe analysis showed that bacteria from the Trichophyceae family... s_Lachnospiraceae bacterium , s_Lachnospiraceae bacterium MD308 , s_Lachnospiraceae bacterium MD335 , s_Lachnospiraceae bacterium A4 The bacterial strains were significantly enriched in the AOFP1 and AOFP2 intervention groups and colonized in vivo. s_Lachnospiraceae bacterium The bacterial strains significantly reduced the resting time of CUMS model mice in the tail suspension test and increased the number of times and the dwell time of CUMS model mice in the open arm area in the elevated cruciate maze test, suggesting that AOFP1 and AOFP2 can exert antidepressant effects by promoting the proliferation of the above bacterial strains.

[0068] The above description is merely a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the concept described herein through the above teachings or related technologies or knowledge. Modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present invention should be within the protection scope of the appended claims.

Claims

1. A method for preparing Alpinia oxyphylla pectin polysaccharide, characterized in that, include: The pericarp and kernel of Alpinia oxyphylla were extracted using a water extraction and alcohol precipitation method, and then the Alpinia oxyphylla pectin polysaccharide was obtained by separation.

2. The preparation method according to claim 1, characterized in that, The preparation method specifically includes the following steps: S1: Take the whole fruit of Alpinia oxyphylla, crush it, extract it by hot water reflux, then concentrate and centrifuge it, add ethanol to precipitate it, and obtain the alcohol precipitate; S2: Dissolve the alcohol precipitate in water, add a protein removal reagent to remove plant protein, add ethanol again to precipitate, separate the precipitate, and obtain Alpinia oxyphylla crude polysaccharide. S3: The crude polysaccharide of Alpinia oxyphylla was separated by column chromatography using a DEAE-52 cellulose column, and two Alpinia oxyphylla polysaccharide solutions were obtained by gradient elution with salt solution.

3. The preparation method according to claim 2, characterized in that: In step S3, the gradient elution includes sequential elution with water and sodium chloride solutions with concentrations of 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mol / L.

4. A kind of Alpinia oxyphylla pectin polysaccharide, characterized in that: It is prepared by the preparation method according to any one of claims 1-3.

5. The Alpinia oxyphylla pectin polysaccharide according to claim 4, characterized in that: One of the aforementioned Alpinia oxyphylla pectin polysaccharides is AOFP1, which is obtained by elution with water during a gradient elution process; The monosaccharide composition of AOFP1 is galacturonic acid, xylose, galactose, glucuronic acid, arabinose, and glucose.

6. The Alpinia oxyphylla pectin polysaccharide according to claim 5, characterized in that: Based on a molar ratio, the monosaccharide composition of the AOFP1 is galacturonic acid: xylose: galactose: glucuronic acid: arabinose: glucose = 68.32-73.54: 16.74-19.26: 4.04-5.18: 3.12-3.75: 2.42-3.09: 0.14-0.

4.

7. The Alpinia oxyphylla pectin polysaccharide according to claim 4, characterized in that: One of the aforementioned Alpinia oxyphylla pectin polysaccharides is AOFP2, which is eluted sequentially by water and sodium chloride solutions with concentrations of 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mol / L during a gradient elution process. The monosaccharide composition of AOFP2 is galacturonic acid, mannose, xylose, arabinose, galactose, and glucose.

8. The Alpinia oxyphylla pectin polysaccharide according to claim 7, characterized in that: Based on a molar ratio, the monosaccharide composition of the AOFP2 is galacturonic acid:mannose:xylose:arabinose:galactose:glucose = 85.37-88.01:6.80-7.96:1.98-2.35:1.20-1.57:1.06-1.92:0.83-0.

95.

9. The application of Alpinia oxyphylla pectin polysaccharide as described in any one of claims 4-8, characterized in that: The pectin polysaccharide of Alpinia oxyphylla or its derivatives are used to prepare antidepressant drugs or functional foods.

10. The application of Alpinia oxyphylla pectin polysaccharide as described in any one of claims 4-8, characterized in that: The pectin polysaccharide of Alpinia oxyphylla or its derivatives are used to prepare drugs or functional foods for treating intestinal flora imbalance.

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