A uniformed polysaccharide of alpinia officinarum and a preparation method and application thereof

Abstract

The application provides a uniform polysaccharide of Alpinia oxyphylla, a preparation method and application thereof, and belongs to the technical field of medicines. 3 The uniform polysaccharide of Alpinia oxyphylla contains galactose and glucose; the molar ratio of the galactose and the glucose is 3:22; the average molecular weight of the uniform polysaccharide of Alpinia oxyphylla is 5.26*10 3 The application also provides a preparation method of the uniform polysaccharide of Alpinia oxyphylla and application thereof in preparing immunoregulation drugs. The uniform polysaccharide of Alpinia oxyphylla obtained through the specific preparation method has good effects in regulating immunity, and provides a new choice for preparing traditional Chinese medicine innovative drugs for immune system diseases.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a homogeneous polysaccharide from Alpinia galanga, its preparation method, and its application. Background Technology

[0002] Galangal, specifically the rhizome of *Alpinia officinarum* Hance, belongs to the genus *Alpinia* in the family Zingiberaceae. It is an ancient and commonly used traditional Chinese medicine. The rhizome of galangal has been used for many years in traditional Chinese medicine to warm the stomach, dispel cold, promote digestion, and relieve pain. It is used for abdominal pain due to cold, vomiting due to stomach cold, belching, and acid reflux. Modern pharmacological studies have shown that the flavonoids and diarylheptane compounds in galangal possess bioactive components such as anticancer and antioxidant properties. Besides the small molecules in the galangal rhizome, there is still significant potential for drug development in the biomolecules, especially polysaccharides.

[0003] Immunological diseases are illnesses caused by an imbalance in immune regulation that affects the body's immune response. In a broader sense, immunological diseases also include structural or functional abnormalities of the immune system due to congenital or acquired causes. Clinically, immunomodulators are commonly used for treatment. Immunomodulators are preparations that non-specifically enhance or inhibit immune function, including immunostimulants, immunosuppressants, and immunomodulatory factors. They can regulate excessively high or low immune function to normal levels and are used in immunodeficiency diseases, chronic infections, and as adjuvant therapy for tumors. Traditional Chinese medicine believes that imbalances in Yin and Yang and poor circulation of Qi and blood lead to immune system diseases. It commonly uses herbal medicine and acupuncture, which are relatively gentle and safe. Currently, Western medicine commonly uses immunomodulators including microbial preparations, hormones, and chemically synthesized drugs. However, while treating diseases, these modulators can easily cause a series of side effects such as infections, tumors, and autoimmune diseases, seriously affecting patients' quality of life and prognosis. Therefore, exploring new treatment options from traditional Chinese medicine has unique advantages.

[0004] Chinese patent CN118027233A discloses a method for preparing galangal polysaccharide and its application in the preparation of anti-liver cancer drugs. It includes the extraction and purification steps of galangal polysaccharide and the preparation process for its application in the treatment and / or prevention of anti-liver cancer drugs.

[0005] Chinese patent CN117362466A discloses a method for preparing galangal polysaccharides, describing a polysaccharide extraction process: alkali concentration 0.033 mol / L, extraction temperature 81.3℃, extraction time 94.302 min, and a material-to-liquid ratio of 1:107.292 (g / mL). A verification experiment was conducted using 1.000 g of galangal powder, and the galangal polysaccharide extraction rates were 4.235%, 4.270%, and 4.270%, with an average of 4.258%.

[0006] Chinese patent CN112694539A discloses a galangal polysaccharide and its preparation method, as well as its application as an emulsifier in the preparation of slow-digesting emulsions. It includes the extraction and purification steps of galangal polysaccharide and the preparation process of slow-digesting emulsions.

[0007] Chinese patent CN108997510A discloses the antioxidant application of Alpinia galanga polysaccharide and its separation and purification method, involving the preparation method of crude Alpinia galanga polysaccharide and refined Alpinia galanga polysaccharide, as well as the antioxidant activity of refined Alpinia galanga polysaccharide.

[0008] Chinese patent CN106753955A discloses a health tonic for women that improves physical strength, comprising 5-30 parts by weight of galangal polysaccharide, 10-40 parts of jujube phenylpropanoid, 2-30 parts of chrysanthemum flavonoid, 5-35 parts of lily flavonoid, with the remainder being wine, totaling 1000 parts.

[0009] However, there is currently no existing technology for the use of homogeneous polysaccharides from Alpinia galanga in immunotherapy. Therefore, it is necessary to develop a homogeneous polysaccharide from Alpinia galanga, its preparation method, and its use in the preparation of immunomodulatory drugs. Summary of the Invention

[0010] Addressing the shortcomings of existing technologies, this invention aims to provide a homogeneous polysaccharide from Alpinia galanga, its preparation method, and its applications. Cellular experiments have demonstrated that the homogeneous polysaccharide from Alpinia galanga provided by this invention possesses immunomodulatory effects and can be further used to develop innovative traditional Chinese medicine drugs for immunotherapy.

[0011] To achieve the above objectives, the present invention adopts the following technical solution:

[0012] On one hand, the present invention provides a homogeneous polysaccharide of Alpinia galanga, wherein the homogeneous polysaccharide of Alpinia galanga has an average molecular weight of 5.26 × 10⁻⁶. 3 Da.

[0013] The homogeneous polysaccharide of Alpinia galanga contains galactose and glucose; wherein the molar ratio of galactose to glucose is 3:22.

[0014] Furthermore, the homogeneous polysaccharide of Alpinia galanga is composed of terminal glucose, 1,4-linked glucose, 1,6-linked glucose, 1,3,4-linked galactose and 1,4,6-linked glucose.

[0015] The primary structure of the homogeneous polysaccharide from Alpinia galanga is shown in formulas A and B below:

[0016]

[0017] On the other hand, the present invention also provides a method for preparing the above-mentioned homogeneous polysaccharide of Alpinia galanga, comprising the following steps:

[0018] (1) Alpinia galanga was degreased by reflux using an organic solvent and then dried to obtain the degreased residue.

[0019] (2) Take the defatted medicinal residue, add water, heat and extract to obtain the extract;

[0020] (3) The extract was concentrated and then precipitated with alcohol to obtain crude galangal polysaccharide;

[0021] (4) After dissolving the crude galangal polysaccharide, it was purified by an exchange column and eluted with water. The eluent was then detected by the phenol-sulfuric acid method. The eluted part with the highest absorbance was collected and freeze-dried to obtain the homogeneous galangal polysaccharide.

[0022] The organic solvent mentioned in step (1) above is selected from one or more of ethanol, petroleum ether, ethyl acetate, cyclohexane, acetone, dichloromethane, chloroform, diethyl ether, and methanol;

[0023] Preferably, the organic solvent is ethanol;

[0024] More preferably, the ethanol is 90% ethanol;

[0025] More preferably, the 90% ethanol is an aqueous solution of ethanol with a mass fraction of 90%.

[0026] The reflux degreasing process described in step (1) above is performed twice. In the first reflux degreasing, the ratio of galangal to organic solvent is 1:9-11 g / mL, and the reflux time is 2-2.5 h. In the second reflux degreasing, the ratio of galangal to organic solvent is 1:7-8 g / mL, and the reflux time is 1-1.5 h.

[0027] Preferably, for the first reflux degreasing, the ratio of galangal to organic solvent is 1:10 g / mL, and the reflux time is 2 hours; for the second reflux degreasing, the ratio of galangal to organic solvent is 1:8 g / mL, and the reflux time is 1.5 hours.

[0028] The water mentioned in step (2) above is selected from one or more of ultrapure water, deionized water and distilled water; preferably, the water is ultrapure water.

[0029] The water extraction described in step (2) above is a heating and boiling extraction using water as a solvent. The heating extraction is performed twice. In the first heating, the ratio of the residue to water is 1:9-11 g / mL, and the heating and boiling time is 2-2.5 h. In the second heating, the ratio of the residue to water is 1:7-8 g / mL, and the heating and boiling time is 1-1.5 h.

[0030] Preferably, in the first heating, the ratio of medicinal residue to water is 1:10 g / mL, and the heating and boiling time is 2 hours; in the second heating, the ratio of medicinal residue to water is 1:8 g / mL, and the heating and boiling time is 1.5 hours.

[0031] The concentration mentioned in step (3) above refers to concentrating the raw drug to a concentration of 0.5-1.5 mg / mL;

[0032] Preferably, the concentration mentioned in step (3) above is concentrated to a crude drug concentration of 1 mg / mL.

[0033] The alcohol used for alcohol precipitation in step (3) above is an ethanol solution;

[0034] Preferably, the ethanol solution has a mass fraction of 85%-100%; more preferably, the ethanol solution has a mass fraction of 100%.

[0035] In the alcohol precipitation process described in step (3) above, alcohol is added until the mass fraction is 80%.

[0036] The exchange column mentioned in step (4) above is DE-52.

[0037] Thirdly, the present invention also provides the application of the above-mentioned homogeneous polysaccharide of Alpinia galanga in the preparation of immunomodulatory drugs.

[0038] Fourthly, the present invention also provides a pharmaceutical composition comprising the above-mentioned homogeneous polysaccharide of Alpinia officinarum.

[0039] The pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0040] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0041] (1) The homogeneous polysaccharide of Alpinia galanga provided by the present invention comprises galactose and glucose; wherein the molar ratio of galactose to glucose is 3:22, and the average molecular weight is 5.26 × 10⁻⁶. 3 Da.

[0042] (2) The present invention obtains a homogeneous polysaccharide of Alpinia galanga through a specific preparation method, which has a good effect on regulating immunity, and provides a new option for the preparation of innovative traditional Chinese medicine for immune system diseases. Attached Figure Description

[0043] Figure 1 The gel chromatography elution curve of the homogeneous polysaccharide obtained by this invention is shown below.

[0044] Figure 2 The high performance liquid chromatography-gel chromatography (HPGPC) chromatogram of the homogeneous polysaccharide and amylopectin standards obtained in this invention.

[0045] In this diagram, A is the high performance liquid chromatography-gel chromatography (HPGPC) chromatogram of homogeneous polysaccharide from Alpinia galanga, and B is the high performance liquid chromatography-gel chromatography (HPGPC) chromatogram of amylopectin standard.

[0046] Figure 3 The monosaccharide composition test spectrum of homogeneous polysaccharide obtained by the present invention is shown.

[0047] Figure 4 Infrared spectrum of homogeneous polysaccharide from Alpinia galanga obtained in this invention;

[0048] Figure 5 The glycosidic bond test spectrum of the homogeneous polysaccharide obtained by this invention is shown.

[0049] Figure 6 The hydrogen and carbon spectra of the homogeneous polysaccharide obtained by this invention are shown.

[0050] Figure 7 The HSQC and HMBC spectra of homogeneous polysaccharides from Alpinia galanga obtained in this invention;

[0051] In this diagram, A is the HSQC spectrum of homogeneous polysaccharide from Alpinia galanga, and B is the HMBC spectrum of homogeneous polysaccharide from Alpinia galanga.

[0052] Figure 8 The HHCOSY and HHNOESY spectra of homogeneous polysaccharides from Alpinia galanga obtained in this invention;

[0053] Among them, A is the HHCOSY spectrum of homogeneous polysaccharide of Alpinia galanga, and B is the HHNOESY spectrum of homogeneous polysaccharide of Alpinia galanga.

[0054] Figure 9 This is a statistical graph showing the effect of the homogeneous polysaccharide obtained by this invention on the proliferation of RAW 264.7 cells;

[0055] Figure 10 This is a statistical chart showing the effect of homogeneous polysaccharide from Alpinia galanga obtained in this invention on the generation of nitric oxide (NO). Detailed Implementation

[0056] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the invention based on the disclosed content, which should also fall within the scope of protection claimed in this application.

[0057] In this invention, the terms "comprising" or "including," and similar terms, mean that the element preceding the term encompasses the element listed after it, and do not exclude the possibility of encompassing other elements. The terms "inner," "outer," "upper," "lower," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing the invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the invention. When the absolute position of the described object changes, the relative positional relationship may also change accordingly. In this invention, unless otherwise explicitly specified and limited, the term "attached," etc., should be interpreted broadly. For example, it can refer to a fixed connection, a detachable connection, or an integral part; it can refer to a direct connection or an indirect connection through an intermediate medium; it can refer to the internal communication of two elements or the interaction relationship between two elements. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances. The term “about” as used in this invention has a meaning known to those skilled in the art, and preferably refers to the numerical value modified by the term within the range of ±50%, ±40%, ±30%, ±20%, ±10%, ±5%, or ±1%.

[0058] In this invention, the term "pharmaceuticalally acceptable excipient" refers to all pharmaceutical materials, other than the active pharmaceutical ingredient, added to the formulation to address the formation properties, efficacy, stability, and safety of the drug product during manufacturing and formulation preparation. These substances have undergone reasonable safety assessments and are included in the pharmaceutical preparation. Besides acting as excipients, carriers, and improving stability, pharmaceutically acceptable excipients also possess important functions such as solubilization, co-solubilization, and sustained-release. They are crucial components that may affect the quality, safety, and efficacy of the drug. The pharmaceutically acceptable excipients described in this application can be suitable carriers or excipients, emulsifiers, wetting agents, preservatives, stabilizers, antioxidants, adjuvants (e.g., aluminum hydroxide adjuvants, oil adjuvants, Freund's complete adjuvants, and Freund's incomplete adjuvants), etc.

[0059] This invention provides a method for preparing homogeneous polysaccharides from Alpinia galanga, the method comprising the following steps:

[0060] (1) Alpinia galanga was degreased by reflux using an organic solvent and then dried to obtain the degreased residue.

[0061] (2) Take the defatted medicinal residue, add water, heat and extract to obtain the extract;

[0062] (3) The extract was concentrated and then precipitated with alcohol to obtain crude galangal polysaccharide;

[0063] (4) After dissolving the crude galangal polysaccharide, it was purified by a weak anion exchange column and eluted with water. The eluent was then detected by the phenol-sulfuric acid method. The eluted part with the highest absorbance was collected and freeze-dried to obtain the homogeneous galangal polysaccharide.

[0064] In this invention, the concentration used in the preparation method can be achieved by any method known in the art. There are no particular limitations on this concentration, provided that the active ingredient is not destroyed. A common concentration method is evaporation, such as atmospheric pressure evaporation, reduced pressure evaporation, thin-film evaporation, etc., but it is not limited thereto.

[0065] In this invention, the drying process involved can be any one of atmospheric pressure drying, vacuum drying, spray drying, freeze drying, etc., and is not limited thereto.

[0066] The pharmaceutical compositions involved in this invention can be used in various dosage forms, often depending on the route of administration. The pharmaceutical compositions involved in this invention can be administered via multiple routes, such as oral, parenteral, etc. The dosing regimen and dosage depend on various factors, such as the route of administration, the patient's health condition, etc., and can be determined by a physician. Dosage ranges can be determined by those skilled in the art through routine experiments.

[0067] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all chemical reagents used in the embodiments of the present invention were obtained through conventional commercial means. Unless otherwise specified, all contents mentioned below are mass contents. Unless otherwise specified, it is understood that the process was carried out at room temperature.

[0068] The DE-52 column used in the following examples is a cellulose DE-52 column, purchased from Borui Sugar Biotechnology Co., Ltd., model BRS008.

[0069] Example 1

[0070] (1) Take Alpinia officinarum and add 90% ethanol to heat and reflux twice to defatt the herbs. The first defatting ratio is 1g:10mL and the reflux time is 2h. The second defatting ratio is 1g:8mL and the reflux time is 1.5h. After defatting, the herbs are dried to obtain the defatted residue.

[0071] (2) Take the defatted drug residue and add ultrapure water for heating and extraction twice. The first heating extraction has a material-to-liquid ratio of 1g:10mL and a reflux time of 2h. The second heating extraction has a material-to-liquid ratio of 1g:8mL and a heating time of 1.5h. Combine the extracts.

[0072] (3) Concentrate the extract obtained in step (2) to a concentration of 1 mg / mL of crude drug, slowly add 100% ethanol until the final concentration of ethanol is 80%, let stand overnight to obtain precipitate and supernatant, and dry the precipitate at 60℃ to obtain crude Alpinia polysaccharide.

[0073] (4) The crude polysaccharide was redissolved in deionized water and passed through a DE-52 column. The sample was eluted with three times its volume of water at a rate of 15 mL / min. The eluent was scanned at 490 nm using an ELISA reader, and the phenol-sulfuric acid method was used to detect the eluent. A scatter plot of tube number versus absorption was plotted, as shown below. Figure 1 As shown, polysaccharides were collected based on peak positions, and after freeze-drying, the first fraction was collected and renamed AOP-W.

[0074] Test Example 1: Chemical Structural Characteristics of Homogeneous Polysaccharides from Alpinia galanga

[0075] 1.1 Molecular weight detection

[0076] 1.1.1. Experimental supplies:

[0077] The product AOP-W obtained in Example 1, and sodium chloride.

[0078] 1.1.2. Experimental Methods:

[0079] The molecular weight of AOP-W obtained in Example 1 was determined using high-performance gel permeation chromatography (HPGPC) (differential detector: Shimadzu RI-20A, tandem gel column: BoRui Saccharide, BRT105-103-101 (8×300mm), centrifuge: Eppendorf 5424). A 0.05M sodium chloride solution was prepared, filtered through a 0.45μm filter membrane, and sonicated for 10 min. AOP-W sample and amylopectin standard were accurately weighed and dissolved separately in 0.05M sodium chloride to form 5 mg / mL solutions. After centrifugation at 12000 rpm for 10 min, the supernatant was collected and filtered through a 0.22μm microporous membrane for molecular weight determination. The prepared AOP-W and standard solutions were processed using an HPGPC system. The mobile phase was 0.05M sodium chloride, and the flow rate was 0.7 mL / min. Chromatograms were recorded and data analyzed.

[0080] 1.1.3. Experimental Results:

[0081] In the HPGPC chromatogram, the peak of AOP-W was relatively symmetrical and sharp at 40.75 min, indicating the homogeneity of AOP-W. The standard curve plotting and molecular weight calculation were automatically completed by GPC software, and the data are shown in Table 1. The HPGPC chromatograms of AOP-W and the amylopectin standards, along with their linear regression plots, are shown below. Figure 2As shown in the figure, the polydispersity index (PDI) value (Mw / Mn) after calculation is 1.0, indicating that AOP-W in this study is a polysaccharide with relatively low degree of polymerization and high homogeneity.

[0082] Table 1

[0083] Example peak Mw(Da) Mn(Da) Mw / Mn Example 1 Peak1 <![CDATA[5.26×10 3 ]]> <![CDATA[5.25×10 3 ]]> 1.00

[0084] 1.2 Monosaccharide Composition Test

[0085] 1.2.1. Experimental supplies:

[0086] The solid sample AOP-W obtained in Example 1 contained trifluoroacetic acid (ACROS), 15 mM sodium hydroxide solution (Alfa Aesar), and 100 mM sodium acetate (ThermoFishe).

[0087] Ion chromatograph (ThermoFisher, ICS5000), electric thermostatic drying oven (Lichen Technology, 101-1BS), nitrogen evaporator (Lichen Technology, UGC-24M), electronic balance (Sartorius BS, 210S), centrifuge (ThermoFisher, D-37520), pipette (DRAGONLAB, 19050983).

[0088] Fucose (X29D7Y27768), galactosamine (GalN), rhamnose (H10S9Z69863), arabinose (S15A10G85850), glucosamine (GlcN), galactose (E1927035), glucose (Q18F10N80946), xylose (A22S6X3606), mannose (C17D9H775) 86), D-fructose (J01J10R89818), D-ribose (H26F10Z81556), galacturonic acid (K02A9B66077), L-guluronic acid (S200115AG1), glucuronic acid (K14M10S82777), D-mannuronic acid (S200108AM1). All monosaccharide standards were obtained from Borui Sugar Biotechnology.

[0089] 1.2.2. Experimental Methods:

[0090] AOP-W samples and appropriate amounts of 15 monosaccharide standards (fucose, galactosamine, rhamnose, arabinose, glucosamine, galactose, glucose, xylose, mannose, D-fructose, D-ribose, galacturonic acid, L-guluronic acid, glucuronic acid, D-mannuronic acid) were dissolved in 3M TFA solution, hydrolyzed in a metal bath at 120℃ for 3 h, dried under nitrogen, redissolved in double-distilled water and centrifuged. The supernatant was subjected to high-performance anion exchange chromatography (HPAEC, ICS5000, ThermoFisher system).

[0091] Chromatographic column: Dionex Carbopac™ PA20 (3mm × 150mm) (Thermo Scientific, ICS5000); Mobile phase: A: MilliQ water; B: 15mM NaOH; C: 15mM NaOH & 100mM NaOAc; Flow rate: 0.3mL / min; Elution gradient: 0 min A / B / C (98.8:1.2:0, V / V), 20 min A / B / C (98.8:1.2:0, V / V), 20.1 min A / B / C (50:50:0, V / V), 30 min A / B / C (50:50:0, V / V), 30.1 min A / B / C (0:0:100, V / V), 46 min Phase A / Phase B / Phase C (0:0:100, V / V), 46.1 min; Phase A / Phase B / Phase C (0:100:0, V / V), 50 min; Phase A / Phase B / Phase C (0:100:0, V / V), 50.1 min; Phase A / Phase B / Phase C (98.8:1.2:0, V / V), 80 min; Phase A / Phase B / Phase C (98.8:1.2:0, V / V). Detector: Electrochemical detector (EC). Monosaccharide standards were used for quantification. Data were processed using Chromeleon 7.2.9 software.

[0092] 1.2.3. Experimental Results:

[0093] The relevant graphs of the experimental results are shown below. Figure 3 The chromatogram of AOP-W mainly showed two strong peaks at 14.48 min and 16.48 min. When compared with the chromatogram of the mixed standard, these two peaks could be identified as galactose (Gal) and glucose (Glc), respectively, with a molar ratio of Gal to Glc of approximately 0.12:0.88.

[0094] 1.3 Infrared Spectroscopy Analysis

[0095] Accurately weigh 1 mg of dried AOP-W, add 198 mg of potassium bromide, mix, compress into tablets, and perform infrared spectroscopy analysis using an FT-IR 650 (Tianjin Port East, China). The infrared spectrum is measured at 400-4000 cm⁻¹. -1 Scan and record data within the range.

[0096] like Figure 4 As shown, in AOP-W, from 3600-3200cm -1 The broad and strong absorption band belongs to the stretching vibration absorption peak of OH. Furthermore, at 1643 cm⁻¹... -1 The high absorption band at this point is likely due to absorbed water. (1024cm) -1 The absorption peak at 852 cm⁻¹ is likely caused by the angular vibration of OH, and these data indicate the carbohydrate nature of AOP-W. Furthermore, the FT-IR spectrum shows an absorption peak at 852 cm⁻¹. -1 There is a distinct peak at this point, which is likely caused by the deformation vibration of CH in the α-conformation. This indicates the presence of α-linked sugar residues in AOP-W.

[0097] 1.4 Glycosidic bond analysis:

[0098] AOP-W was methylated, hydrolyzed, and acetylated to obtain partially O-methylated sugar alcohol acetate (PMAA) derivatives. The products were analyzed by gas chromatography-mass spectrometry (GC-MS, 1300-1700 Thermo). The chromatographic column was an HP-INNOWAX (30m × 0.32mm × 0.25μm). The temperature program was as follows: initial temperature 140℃, increased to 230℃ at a rate of 1℃ / min, injection port temperature 250℃, detector temperature 250℃. Gas flow rate: 1mL / min. Identification was performed using a spectral library search based on retention time and fragment ions.

[0099] like Figure 5As shown in Table 2, the presence of 2,3,6-Me3-Glcp (68.4%), 2,3-Me2-Glcp (4.5%), and 2,3,4-Me3-Glcp (10.4%) derivatives indicates that the glucose units of AOP-W are mainly bound at O-4, O-4,6, and O-6. The presence of 2,3,4,6-Me4-Glcp (10.3%) indicates a high proportion of non-reducing terminal Glcp. In summary, AOP-W consists of a backbone of (1→4) linked Glcp units, with glucose terminals substituted at the O-6 position. The presence of 2,6-Me2-Galp (6.4%) indicates that the galactose units of AOP-W are mainly bound at O-3,4. Due to the lack of PMAA derivative standards, methylation analysis could not be accurately quantified. Rough calculations of the molar ratio of each residue show that the molar ratio of terminal glucose (10.3%) is approximately equal to the total proportion (10.9%) of branched residues (1→4,6)-linked Glcp (4.5%) and (1→3,4)-linked Galp (6.4%).

[0100] Table 2

[0101]

[0102] 1.5 Nuclear Magnetic Spectroscopy Analysis:

[0103] Weigh an appropriate amount of AOP-W sample, dissolve it in heavy water, and freeze-dry it. Repeat this process twice to ensure sufficient exchange of active hydrogens. Then, dissolve the sample again in heavy water to prepare an 80 mg / mL test solution. 1D and 2D NMR spectra were measured at 25°C using a 600 MHz NMR spectrometer (Avance, Bruker, USA). Data were processed using Bruker TopSpin 4.1.3 software. Chemical shifts were referenced to the internal standard deuterated acetone (δ¹⁰). C 30.89).

[0104] like Figure 6 As shown, the anodic proton resonance region was first analyzed. 1 1H-NMR showed a strong peak at δ 5.31 pointing to the anterior hydrogen of Glcp-(1→). Several cyclic proton signals were present in the δ 3.2–4.4 region. Figure 6 A). 13 The C-NMR spectrum showed multiple signal peaks at δ 100–103, which were attributed to the carbons of galactose and glucose residues. Figure 6 B), and DEPT 135 13 The three inverted peaks at δ61-62 in the C spectrum point to the -CH2 group on the C-6 of the sugar residue. Figure 6C), with the displacements from high to low representing T-α-Glcp (reduction end -α-Glcp), 4-α-Glcp, and 4,6-α-Glcp, respectively.

[0105] HSQC chart ( Figure 7 In the α-D-Glcp unit, the two signals at δ5.26 / 101.33 and δ5.31 / 100.89 point to the H-1 / C-1 correlation of the α-D-Glcp unit, while the two correlation peaks at δ5.13 / 93.33 and δ4.58 / 97.01 originate from the reduction end of the Glcp unit. Furthermore, the signal at δ4.98 / 103.24 in the anotron resonance region indicates the H-1 / C-1 correlation of β-D-Galp.

[0106] 1 H / 1 In the 1H COSY spectrum, the relevant peaks at δ 5.31 / 3.55, 3.55 / 3.9, 3.9 / 3.48, and 3.48 / 3.76 are attributed to H-1 / H-2, H-2 / H-3, H-3 / H-4, and H-4 / H-5, respectively. These proton chemical shifts are further confirmed by NOESY spectroscopy. Furthermore, the protons corresponding to C-1 through C-5 can be determined based on the coupling peaks in the HSQC spectrum: [The text abruptly ends here, so the translation stops as well.] H / C 5.31 / 100.89 and its related cyclic carbon δ H / C 3.55 / 72.91, 3.90 / 74.56, 3.48 / 78.31, 3.76 / 72.53. Among these, the C-4 (78.31 ppm) signal shifted to a lower field compared to the unsubstituted residues, indicating that the anomer's δ... H / C 5.31 / 100.89(G') originates from α-(1-4)-linked Glcp residues. Similarly, δ H / C 5.26 / 101.33(G) belongs to terminal α-linked Glcp residues. δ H / C 4.88 / 99.89(G”) and δ H / C 4.92 / 99.10(G”') belong to 1,4,6-α-Glcp residues and 1,6-α-Glcp residues, respectively, which means that there is a linear 1,4-α-glucan side chain at the O-6 position.

[0107] →4)-Glcp's reduction terminus H in 1 H / 1 Easily identifiable in H COSY spectra, δ H / C 3.41-3.86 / 61.6-79.5 refers to the hydrocarbons at the reduction end of →4)-α-D-Glcp(Gr), while 3.12-3.85 / 61.67-78.73 refers to the hydrocarbons of →4)-β-D-Glcp(Gr').

[0108] Based on HSQC and COSY spectra, anomeric hydrocarbon δ H / C 4.98 / 103.24 belongs to the 1,3,4-β-D-Galp unit, and H-1 / C-1 to H-5 / C-5 are respectively δ H / C 4.98 / 103.24, 3.54 / 73.55, 3.77 / 77.85, 3.45 / 82.27 and 3.75 / 73.04, C-3 (77.82) and C-4 (82.27) indicate that β-D-Galp is substituted at the O-3 and O-4 positions.

[0109] In HMBC, the strong correlation peaks at δ5.31 / 78.31 and δ5.31 / 77.10 indicate the presence of →4)-α-D-Glcp-(1→4,6)-α-D-Glcp-(1→ and →4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→), and the signal at δ3.48 / 103.24 indicates the presence of →3,4)-β-D-Galp-(1→ and →4)-α-D-Glcp-(1→). Therefore, the skeleton of AOP-W is →3,4)-β-D-Galp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4,6)-α-D-Glcp-(1→).

[0110] The coupling between H-1 at δ5.26 and C-3 at δ77.85 of α-D-Glcp-1→ indicates that the terminal Glcp is connected to O-3 of α-D-Galp-1→.

[0111] In the NOESY spectrum, δ 5.31 / 3.45 (G'-H-1 / Gal H-4) indicates the connection of the fragments →4)-α-D-Glcp-(1→ and O-4 of →3,4)-β-D-Galp-(1→, and the strong correlation peak at δ 4.88 / 3.48 indicates the connection of the fragments →4,6)-α-D-Glcp-(1→ and →4)-α-D-Glcp-(1→, and δ 5.26 / 3.7 indicates that the terminal Glcp residue is attached to O-6 of the →6)-α-D-Glcp-(1→ unit.

[0112] Test Example 2: Evaluation of Immunomodulatory Activity

[0113] 2.1 Study on the effect on RAW 264.7 cell viability

[0114] 2.1.1 Reagents and Instruments

[0115] Reagents: AOP-W obtained in Example 1, fetal bovine serum, penicillin, streptomycin, and DMEM medium.

[0116] Instruments: 0.0001 g balance, benchtop refrigerated centrifuge (ST1R Plus, Thermoscientific), multi-functional microplate reader (VICTOR NIVO, PerkinElmer), 10 μL pipette.

[0117] Experimental cells: RAW264.7 cells.

[0118] 2.1.2 Experimental Methods

[0119] RAW267.4 macrophages were cultured in high-glucose DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. Cells were grown in a humid environment at 37°C and 5% CO2. The effect of AOP-W on the proliferation of RAW264.7 macrophages was determined by the MTT assay; cells in the exponential phase were used for bioactivity studies.

[0120] RAW 264.7 cells were planted at a density of 5 × 10⁶ cells per well. 4 Cells were cultured at concentrations of [number] cells in 96-well plates. After attachment to the plates, cells were treated with different concentrations of AOP-W (0-800 μg / mL). After 24 hours of treatment, 20 μL / well MTT (5 mg / mL) was added, and the cells were incubated for another 4 hours. Then, the supernatant was carefully removed, and 100 μL of DMSO was added. After incubation in the dark for 15 minutes, the mixture was measured at 570 nm using a microplate reader. Cell viability was calculated by comparison with the culture medium control.

[0121] 2.1.3 Experimental Results

[0122] RAW 264.7 cells are a recognized tool for studying the immunomodulatory mechanisms of macrophages. Results showed that AOP-W at concentrations up to 400 μg / mL had no cytotoxic effect on RAW 264.7 cells. However, AOP-W concentrations in the range of 25-100 μg / mL promoted macrophage proliferation. Statistical results are as follows: Figure 9 As shown.

[0123] 2.2 Studies on the effects of RAW 264.7 cells on nitric oxide (NO) secretion

[0124] 2.2.1 Reagents and Instruments:

[0125] Reagents: AOP-W obtained in Example 1, Griess reagent (Beyotime Biotechnology), and NaNO2 standard.

[0126] Instruments: 0.0001 g balance, benchtop refrigerated centrifuge (ST1R Plus, Thermoscientific), multi-functional microplate reader (VICTOR NIVO, PerkinElmer), 96-well plate, 10 μL pipette.

[0127] Experimental cells: RAW 264.7 cells.

[0128] 2.2.2 Experimental Methods:

[0129] RAW 264.7 cells in the exponential phase were fed with 5 × 10⁻⁶ cells. 3 The sample was seeded at a density of 200 μL / well in a 96-well plate. After treatment with AOP-W for 24 h, the supernatant was collected, and 100 μL of Griess reagent was added to each sample. After incubation for 10 min, the absorbance of the mixture was measured at 492 nm using a microplate reader. A standard curve for NO content determination was plotted using NaNO2 standard.

[0130] 2.2.3 Experimental Results:

[0131] Macrophages kill pathogens directly through phagocytosis and indirectly through the secretion of pro-inflammatory factors. The effect of AOP-W on nitric oxide (NO) production was determined. Figure 10 As shown, NO levels significantly increased after 24 hours of AOP-W treatment compared to the control group (P<0.01).

[0132] The results showed that AOP-w could promote the secretion of the pro-inflammatory factor NO by macrophage RAW264.7, demonstrating good immune-enhancing activity.

[0133] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A homogeneous polysaccharide from Alpinia galanga, characterized in that: The homogeneous polysaccharide of Alpinia galanga contains galactose and glucose.

2. The homogeneous polysaccharide of Alpinia galanga according to claim 1, characterized in that: The molar ratio of galactose to glucose is 3:

22.

3. The homogeneous polysaccharide of Alpinia galanga according to claim 1, characterized in that: The average molecular weight of the homogeneous polysaccharide from Alpinia galanga is 5.26 × 10⁻⁶. 3 Da.

4. The homogeneous polysaccharide of Alpinia galanga according to claim 1, characterized in that: The homogeneous polysaccharide of Alpinia galanga is composed of terminal glucose, 1,4-linked glucose, 1,6-linked glucose, 1,3,4-linked galactose, and 1,4,6-linked glucose; the primary structure of the homogeneous polysaccharide of Alpinia galanga is shown in formulas A and B below:

5. The method for preparing homogeneous polysaccharide from Alpinia galanga according to any one of claims 1-4, characterized in that: Includes the following steps: (1) Alpinia galanga was degreased by reflux using an organic solvent and then dried to obtain the degreased residue. (2) Take the defatted medicinal residue, add water, heat and extract to obtain the extract; (3) The extract was concentrated and then precipitated with alcohol to obtain crude galangal polysaccharide; (4) After dissolving the crude galangal polysaccharide, the product was purified and eluted using an exchange column. The eluent was tested, and the eluted portion with the highest absorbance was collected and freeze-dried to obtain the homogeneous galangal polysaccharide.

6. The preparation method according to claim 5, characterized in that: The reflux degreasing process described in step (1) is performed twice. In the first reflux degreasing, the ratio of galangal to organic solvent is 1:9-11 g / mL, and the reflux time is 2-2.5 h. In the second reflux degreasing, the ratio of galangal to organic solvent is 1:7-8 g / mL, and the reflux time is 1-1.5 h.

7. The preparation method according to claim 5, characterized in that: The water-heating extraction mentioned in step (2) is a water-based extraction by heating and boiling. The extraction is performed twice. In the first extraction, the ratio of the residue to water is 1:9-11 g / mL, and the extraction time is 2-2.5 h. In the second extraction, the ratio of the residue to water is 1:7-8 g / mL, and the extraction time is 1-1.5 h.

8. The preparation method according to claim 5, characterized in that: The exchange column mentioned in step (4) is DE-52.

9. The use of the homogeneous polysaccharide of Alpinia galanga according to any one of claims 1-4 or the homogeneous polysaccharide of Alpinia galanga prepared by the preparation method according to any one of claims 5-8 in the preparation of immunomodulatory drugs.

10. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the homogeneous polysaccharide of Alpinia galanga according to any one of claims 1-4 or the homogeneous polysaccharide of Alpinia galanga prepared by the preparation method according to any one of claims 5-8.

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