Alsophila spinulosa polysaccharide compound, extraction method and application thereof

Abstract

The invention discloses a polysaccharide compound of Alsophila spinulosa and an extraction method and application thereof, wherein the polysaccharide compound is composed of rhamnose, arabinose, galactose and glucuronic acid. The polysaccharide compound of the invention is derived from mature leaves of Alsophila spinulosa, and is extracted by water extraction and alcohol precipitation, crude polysaccharide impurity removal, DEAE seplife FF ion column separation, and Sephacryl S‑400HR gel column purification. The method is simple and fast to operate, and water extraction and anhydrous ethanol precipitation are mainly used in the extraction and separation process. The process method is environmentally friendly, and the purity of the compound separated by the method is relatively high, all greater than 92%. The extracted polysaccharide compound has been found to have a significant hypoglycemic effect through research, and can be used as a raw material for new drug development and pharmacological activity research, and can also be used for food additives or health care product development.

Description

Technical Field

[0001] The invention relates to a new polysaccharide compound, in particular to an Alsophila spinulosa polysaccharide compound and an extraction method and application thereof. Background Art

[0002] Alsophila spinulosa (Wall. ex Hook.) RMTryon is a famous relict plant belonging to the Alsophila genus of the Cyatheaceae family. It is also known as tree fern, snakewood, etc. It is the only existing woody fern plant, which can reach several meters in height and is known as the "king of ferns". The "Pharmacopoeia of the People's Republic of China" includes dried stem slices of Alsophila spinulosa as medicine, which is called "dragon bone wind" and has great medicinal value. It has the effects of clearing away heat and detoxification, moistening the lungs and relieving cough, removing dampness and dispelling wind, and expelling worm infections, relieving influenza coughs and hepatitis, etc.

[0003] Modern pharmacological studies on Alsophila spinulosa have shown that it has anti-inflammatory, anti-tumor, antibacterial, and antioxidant effects. People use Alsophila spinulosa leaves to boil and drink soup to eliminate phlegm and relieve cough, clear away heat and relieve summer heat. According to research reports, the main secondary metabolites of Alsophila spinulosa include flavonoids, terpenes, steroids, alkaloids, etc. At present, the research on Alsophila spinulosa mainly focuses on the stems of Alsophila spinulosa, while there are few studies on the leaves of Alsophila spinulosa, and there are few reports on the isolation of new polysaccharide compounds in the leaves of Alsophila spinulosa and their in vitro pharmacological activity studies.

[0004] At present, there are relatively few secondary metabolites isolated from Alsophila spinulosa, and most of them are known compounds with low structural novelty. Therefore, the development and separation of new compounds from Alsophila spinulosa is urgently needed. Summary of the invention

[0005] The purpose of the present invention is to provide a Alsophila spinulosa polysaccharide compound and an extraction method and application thereof, which is separated from mature leaves of Alsophila spinulosa, has good hypoglycemic activity, can be used as a natural product to develop new Chinese medicines, and has broad medical application prospects.

[0006] In order to achieve the above object, the present invention provides a polysaccharide compound of Alsophila spinulosa, which is composed of rhamnose, arabinose, galactose and glucuronic acid, and has a structure as shown in Formula I:

[0007]

[0008] Preferably, the molecular weight of the Alsophila spinulosa polysaccharide compound is 17.746 KDa.

[0009] Preferably, the Alsophila spinulosa polysaccharide compound is extracted and separated from mature leaves of Alsophila spinulosa.

[0010] Preferably, the Alsophila spinulosa polysaccharide compound is obtained by sequentially extracting mature Alsophila spinulosa leaves by water extraction and alcohol precipitation, removing impurities from crude polysaccharides, separating by ion column, and purifying by gel column.

[0011] Preferably, the ion column adopts a DEAE seplife FF anion exchange column; or / and the gel column adopts a Sephacryl S-400HR gel column.

[0012] Another object of the present invention is to provide a method for extracting the Alsophila spinulosa polysaccharide compound, which comprises the following steps:

[0013] (1) Water extraction and alcohol precipitation

[0014] The mature Alsophila spinulosa leaves were extracted with water and ethanol, and the precipitated solids were collected to obtain a crude polysaccharide extract;

[0015] (2) Removal of impurities from crude polysaccharides

[0016] The crude polysaccharide extract is fully dissolved in water, a protease is added thereto for enzymolysis, an organic solvent is added for extraction, an aqueous phase is collected, and then a macroporous resin AB-8 is added, a liquid is collected, and a 3000Da dialysis bag is used for dialysis to remove small molecular components, and the polysaccharide liquid is freeze-dried to obtain a crude Alsophila spinulosa polysaccharide;

[0017] (3) Ion column separation and gel column purification

[0018] The crude Alsophila spinulosa polysaccharide is dissolved in water, filtered, and then separated by an ion column and purified by a gel column. The eluate is collected, concentrated, and freeze-dried to obtain the Alsophila spinulosa polysaccharide compound.

[0019] Preferably, in step (1), the water extraction and alcohol precipitation are precipitated by anhydrous ethanol; or / and, the water extraction temperature is 60° C.; or / and, the water extraction and alcohol precipitation are performed for 4 hours; or / and, the mature Alsophila spinulosa leaves are defatted by anhydrous ethanol.

[0020] Preferably, in step (2), the protease is selected from papain; or / and, the organic solvent extraction is: adding chloroform and n-butanol to the enzymatic hydrolyzate, mixing thoroughly, and collecting the upper aqueous phase; adding petroleum ether to the collected aqueous phase, mixing thoroughly, and collecting the lower aqueous phase.

[0021] More preferably, in step (2), the organic solvent extraction is: adding 1 / 4 volume of chloroform and n-butanol to the enzymatic hydrolyzate, mixing thoroughly, and collecting the upper aqueous phase; adding petroleum ether to the collected aqueous phase, mixing thoroughly, and collecting the lower aqueous phase.

[0022] Preferably, in step (2), dialysis is performed using a 3000 Da dialysis bag for 48 hours to remove small molecule components.

[0023] Preferably, in step (3), the ion column separation uses a DEAE seplife FF anion exchange column, which is eluted with water and 0.1M, 0.2M, and 0.3M NaCl solutions in sequence; or / and, the gel column purification uses a Sephacryl S-400HR gel column.

[0024] More preferably, in step (3), the ion column separation has an elution rate of 4 mL / min.

[0025] Preferably, in step (3), the ion column is used for separation, the eluate is collected, concentrated, dialyzed to a molecular weight cutoff of 3000 Da, freeze-dried, and then purified by gel column.

[0026] Preferably, in step (3), the gel column purification is performed by dissolving the substance obtained by ion column separation in water, centrifuging and taking the supernatant to pass through the gel column at a flow rate of 1 mL / min, eluting with pure water for 1.5 times the column volume, collecting all the eluates, concentrating, and freeze-drying to obtain the Alsophila spinulosa polysaccharide compound ASP.

[0027] Another object of the present invention is to provide the use of the Alsophila spinulosa polysaccharide compound in the preparation of hypoglycemic drugs, foods or health products.

[0028] The Alsophila spinulosa polysaccharide compound and the extraction method and application thereof of the present invention have the following advantages:

[0029] (1) The present invention extracts a new polysaccharide compound from the mature leaves of Alsophila spinulosa. Studies have shown that the polysaccharide compound has a significant hypoglycemic effect and can be used as a raw material for new drug development and pharmacological activity research, and can also be used for food additives or health care product development;

[0030] (2) The polysaccharide compound of the present invention is derived from mature leaves of Alsophila spinulosa, and is extracted by water extraction and alcohol precipitation, crude polysaccharide impurities are removed, DEAE seplife FF ion column is separated, and Sephacryl S-400HR gel column is purified. The method has only four steps, and the operation method is simple and rapid. Water extraction and anhydrous ethanol precipitation are mainly used in the extraction and separation process. The process method is environmentally friendly, and the purity of the compound separated by this method is relatively high, greater than 92%. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the ion column separation diagram of the polysaccharide compound ASP of the present invention.

[0032] Figure 2 It is the ion chromatogram of the polysaccharide compound ASP of the present invention.

[0033] Figure 3 It is the infrared spectrum of the polysaccharide compound ASP of the present invention.

[0034] Figure 4 The polysaccharide compound ASP of the present invention 1 H-NMR spectrum.

[0035] Figure 5 The polysaccharide compound ASP of the present invention 13 C-NMR spectrum.

[0036] Figure 6 It is the nuclear magnetic resonance HSQC spectrum diagram of the polysaccharide compound ASP of the present invention.

[0037] Figure 7 It is the nuclear magnetic resonance HMBC spectrum diagram of the polysaccharide compound ASP of the present invention.

[0038] Figure 8 It is the nuclear magnetic resonance NOESY spectrum diagram of the polysaccharide compound ASP of the present invention.

[0039] Fig. 9 The figure is the nuclear magnetic resonance COSY spectrum of the polysaccharide compound ASP of the present invention.

[0040] Fig.10 This is a diagram showing the in vitro hypoglycemic activity of the polysaccharide compound ASP of the present invention.

[0041] Fig.11 This is a graph showing the effect of the polysaccharide compound ASP of the present invention on the body weight of mice.

[0042] Fig.12 This is a graph showing the effect of the polysaccharide compound ASP of the present invention on blood sugar in mice. DETAILED DESCRIPTION

[0043] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] It should be noted that if the specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased from the market.

[0045] Example 1

[0046] A polysaccharide compound ASP is extracted and separated from mature leaves of Alsophila spinulosa, and the specific steps are as follows:

[0047] 1) Weigh 1 kg of dry powder of mature Alsophila spinulosa leaves, add anhydrous ethanol and stir to extract fat-soluble pigments and some impurities, then collect the precipitate by centrifugation at 6000g for 10 minutes. Add pure water to the precipitate, extract in a 60°C water bath for 4 hours, centrifuge at 6000g for 10 minutes, and collect the supernatant extract. Extract the precipitate residue again according to the same steps. Combine the two extracts, concentrate by vacuum rotary evaporation to 1 / 10 of the original volume, and add four times the volume of anhydrous ethanol to precipitate overnight. Then centrifuge the mixture at 8000g for 10 minutes to obtain a precipitated solid in the form of crude polysaccharide;

[0048] 2) The precipitate was fully dissolved with pure water, papain was added, and enzymolysis was carried out overnight; then 250 mL of chloroform and n-butanol (4:1, v / v) were added to the aqueous phase, mixed thoroughly, and the upper aqueous phase was collected. Petroleum ether was added to the collected aqueous phase, mixed thoroughly, and the lower aqueous phase was collected. Macroporous resin AB-8 was added to the liquid and allowed to stand overnight, and then the liquid was collected and dialyzed with a 3000Da dialysis bag for 48 hours to remove small molecular components, and the polysaccharide solution was freeze-dried to obtain crude Alsophila spinulosa polysaccharide;

[0049] 3) The crude Alsophila spinulosa polysaccharide obtained in step 2) was dissolved in pure water, filtered, and centrifuged at 10000g for 10 minutes. The supernatant was loaded into a DEAE seplife (26mmX400mm) FF anion exchange column, and then eluted with distilled water and sodium chloride solutions of different concentrations (0.1, 0.2, 0.3mol / L) at a flow rate of 4mL / min. All eluates were collected (15mL / tube) and three eluate components were identified (see Figure 1 ), the eluates from each collection tube corresponding to the same elution peak were combined, and then concentrated, dialyzed (molecular weight cutoff 3000Da, time 48-72h) and freeze-dried;

[0050] 4) Sephacryl S-400HR (26mmX1000mm) was used to further purify the intermediate polysaccharide obtained in step 3). It was dissolved in pure water, centrifuged at 10000g for 10 minutes, and the supernatant was separated and purified by gel chromatography column at a flow rate of 1ml / min; 1.5 column volumes of pure water were used for elution, and one tube was collected for every 12mL, and all the eluate was collected. The polysaccharide compound ASP was obtained by rotary steam concentration to 1 / 5 of the original volume and freeze-drying, and the purity reached more than 92%, and its molecular weight was determined to be 17.746KDa.

[0051] The structure of the polysaccharide compound ASP was studied by methylation to determine the connection positions between the monosaccharide residues in the polysaccharide. The structure is shown in Table 1. The NMR data of the polysaccharide compound ASP is shown in Table 2.

[0052] Table 1 Methylation data of polysaccharide compound ASP

[0053]

[0054] Note: PMAAs are partially methylated sugar alcohol acetyl ester derivatives.

[0055] Table 2 NMR data of polysaccharide compound ASP

[0056]

[0057] Note: Galp stands for galactopyranoside; Araf stands for arabinofuranoside; GlcpA stands for glucuronide; Rhap stands for rhamnoside.

[0058] like Figure 2 , which is the ion chromatogram of the polysaccharide compound ASP of the present invention, it can be seen that the polysaccharide compound ASP is composed of rhamnose (Rha), arabinose (Ara), galactose (Gal), and glucuronic acid (Glc-UA).

[0059] like Figure 3 As shown, it is the infrared spectrum (IR) diagram of the polysaccharide compound ASP of the present invention, at 3428.93 cm -1 A major broad stretching peak appears at 2934.37 cm-1, which is caused by some hydroxyl functional groups. -1 The weak band at 1634.01 cm is caused by some CH stretching vibrations, and these two peaks characterize polysaccharides. -1 The absorption peak at 1401.83cm is the characteristic absorption of aldehyde acid, indicating that the polysaccharide is an acidic polysaccharide. -1 The peak at 1075.37 cm is related to the CH bending vibration. -1 The absorption peak at 800-950cm is related to the stretching vibration of CO. -1 There are several weak absorption signals at ASP, indicating the presence of α- and β-type glycosidic bonds.

[0060] like Figure 4 As shown in the figure, the hydrogen spectrum signal of the polysaccharide is mainly concentrated between δ3.0 and 5.5 ppm, and multiple coupling signal peaks are identified in the anomeric hydrogen signal region of δ4.3 to 5.4 ppm, indicating that the polysaccharide contains a variety of sugar residues, and the chemical shifts of the corresponding anomeric hydrogen are δ5.37, 5.20, 5.12, 5.02, 4.71, 4.63, 4.57, 4.46 and 4.39 ppm, etc. The non-anomeric hydrogen signals are mainly concentrated in the region of δ3.1 to 4.2 ppm.

[0061] like Figure 5 As shown, multiple signal peaks were identified in the anomeric carbon region of 95-110 ppm. 13 CNMR spectroscopy and Figure 6 The cross peaks in the anomeric region of the HSQC spectrum determined that the anomeric signals present in the sample were: δ4.39 / 103.39, δ4.46 / 102.66, δ5.37 / 108.36, δ4.57 / 103.75, δ4.63 / 103.7, δ5.12 / 98.26, δ5.2 / 109.25, δ5.02 / 107.98, δ4.57 / 96.15 and δ4.71 / 100.75, which were recorded as sugar residues A, B, C, D, E, F, G, I, K and L, respectively. Combined with the polysaccharide bonding structure (methylation) information and the anomeric signal, it was inferred that sugar residue A was β-D-Galp-(1→, residue B was →3,6)-β-D-Galp-(1→, residue C was α-L-Araf-(1→, residue D was →6)-β-D-Galp-(1→, residue E was →4)-β-D-Galp-(1→, residue F was α-D-GlcpA-(1→, residue G was →4)-α-D-GlcpA-(1→, residue I was →3)-α-D-Galp-(1→, residue K was →3,4,6)-β-D-Galp-(1→, and residue L was β-Rhap-(1→).

[0062] like Figure 7 As shown, the cross peak at δ103.39 / 3.46 (B H3 / A C1) shows the connectivity between β-D-Galp-(1→ and →3,6)-β-D-Galp-(1→. The correlation at δ4.46 / 69.26 (B H1 / B C6) indicates that there is a loop self-linkage within the →3,6)-β-D-Galp-(1→ units. The peak at δ4.46 / 75.77 (B H1 / E C4) shows the connection between →3,6)-β-D-Galp-(1→ and →4)-β-D-Galp-(1→. Finally, at δ4.63 / 81.32 (E The cross peak at H1 / IC3) indicated the connection between →4)-β-D-Galp-(1→ and →3)-α-D-Galp-(1→. Since the cross peak signal in the HMBC spectrum was relatively weak, we combined it with the NOESY spectrum to further confirm the residue connection sequence in the polysaccharide.

[0063] like Figure 8As shown, sugar residue A-H1 has a cross peak at a δ value of 4.39 / 3.46ppm. Residue B-H1 has a cross peak at a δ value of 4.46 / 4.17 (4.09) ppm, and has a cross peak with residue E-H4 at a δ value of 4.46 / 3.67ppm. Residue C-H1 has a cross peak with residue D-H6 at a δ value of 5.37 / 3.9ppm, and has a cross peak with residue K-H3 at a δ value of 5.37 / 3.72ppm. D-H1 has a cross peak with B-H6 at a δ value of 4.57 / 4.17ppm. Residue E-H1 has a cross peak with residue I-H3 at a δ value of 4.63 / 4.15ppm. Residue F-H1 has a cross peak with residue K-H4 at a δ value of 5.12 / 3.82ppm. Residue I-H1 has a cross peak with residue K-H6 at δ value of 5.02 / 3.84 ppm. Finally, residue K-H1 has a cross peak with residue G-H4 at δ value of 4.57 / 3.52 ppm.

[0064] Based on the one-dimensional and two-dimensional nuclear magnetic resonance information and the methylation results, it is inferred that the structure of the polysaccharide compound ASP of the present invention is as shown in Formula I, which is mainly composed of →3,6)-β-D-Galp-(1→ and a small amount of →6)-β-D-Galp-(1→, →4)-β-D-Galp-(1→, etc. connected to each other to form a main chain, and the side chain is mainly composed of β-D-Galp-(1→ connected to the C3 position of the sugar residue →3,6)-β-D-Galp-(1→ and α-L-Araf-(1→, α-D-GlcpA-(1→ respectively connected to the C3 and C4 positions of the sugar residue →3,4,6)-β-D-Galp-(1→.

[0065]

[0066] Example 2 Analysis of in vitro hypoglycemic activity of polysaccharide compound ASP

[0067] 1. Experimental reagents

[0068] α-glucosidase (α-GC) activity detection kit (Solarbio, catalog number: BC2550), acarbose (Solarbio, catalog number: SA8820), α-glucosidase (Solarbio, catalog number: G8823).

[0069] 2. Experimental instruments

[0070] Analytical balance (Shenyu Longteng), multifunctional microplate reader (Thermo Fisher), water purifier (Merck).

[0071] 3. Experimental methods

[0072] Acarbose was used as a positive control; a 1U / mL α-glucosidase solution was used to prepare a solution with a concentration of 0.1 mg / mL, and a 1U / mL α-glucosidase solution was used as a negative control; the inhibitory ability of acarbose on α-glucosidase was comprehensively analyzed. The polysaccharide compound ASP sample obtained by separation and purification in Example 1 was prepared into 0.5, 1, 2, 3, and 4 mg / mL using 1U / mL α-glucosidase solution. Before the experiment, the samples and controls were reacted in a 37°C water bath for 10 minutes, and then the inhibition rate of the samples was detected using an α-glucosidase kit, with 3 replicates per group.

[0073] 4. Experimental results

[0074] like Fig.10 As shown, the inhibition rate of the polysaccharide compound ASP on α-glucosidase decreases with the increase of polysaccharide concentration in the range of 0.5-4 mg / mL. At a concentration of 0.5 mg / mL, the inhibition rate of the polysaccharide compound ASP on α-glucosidase is 89.85%, reaching 90.1% of the positive control acarbose. The results show that the polysaccharide has a significant blood sugar lowering ability in vitro.

[0075] Example 3 Detection of hypoglycemic activity of polysaccharide compound ASP in vivo

[0076] 1. Experimental reagents

[0077] Streptozotocin STZ (Phygene), citric acid (Kermiou reagent), sodium citrate (damas-beta), blood glucose test strips (Jiangsu Yuyue Kailite).

[0078] 2. Experimental Animals

[0079] SPF grade Balb / C mice, weighing 30±5 g, were purchased from Hangzhou Qizhen Experimental Animal Technology Co., Ltd.

[0080] 3. Experimental methods

[0081] 3.1) Modeling drug preparation

[0082] The preparation of citrate buffer is as follows:

[0083] ① Weigh 2.1g citric acid (Mw: 210.14) and add 100mL double distilled water to fully dissolve to prepare solution A;

[0084] ② Weigh 2.94 g of sodium citrate (Mw: 294.10) and add 100 mL of double distilled water to fully dissolve to prepare solution B;

[0085] ③Mix A and B solutions in proportion (1:1.32 or 1:1), measure and adjust pH to 4.2-4.5 with a pH meter, filter with a 0.22μm or 0.45μm filter membrane to remove impurities, and prepare STZ citric acid buffer. Use sodium citrate (or double distilled water) to adjust the acidity, and citric acid to adjust the alkalinity. Reduce or enlarge the proportion according to actual needs, and try to prepare and use it immediately to prevent pH drift caused by oxidation.

[0086] The preparation of STZ solution is as follows:

[0087] 30 mg of STZ was accurately weighed and wrapped in tin foil, and completely dissolved in 2 mL of the above-prepared citric acid buffer on ice in a dark place, and the concentration of the obtained STZ solution was 15 mg / mL. In the modeling experiment, the newly prepared STZ solution was quickly injected intraperitoneally within 5 minutes, with an injection dose of 150 mg / kg, that is, the intraperitoneal injection volume was 0.1 mL / 10 g.

[0088] 3.2) Modeling and grouping

[0089] This experiment requires 24 SPF Balb / C mice weighing 30±5g. They were randomly divided into 3 groups using a completely randomized design: healthy control group, model group, and polysaccharide treatment group, with 8 mice in each group. The mice in the model group and polysaccharide treatment group were fasted for 12h before modeling. The diabetic model was established by the following method: 150mg / kg STZ solution was injected intraperitoneally on the first day, and 50mg / kg STZ solution was injected on the second and third days. The success of the modeling was determined by testing the fasting blood glucose 72h after modeling.

[0090] 3.3) Pharmacological intervention

[0091] After the model was successfully established, the drug was administered, and the polysaccharide compound ASP 200 mg / kg was administered orally every day, and the healthy control group and the model group were given normal saline. The drug was administered for 28 consecutive days, and the body weight and fasting blood sugar were measured once a week. After the last administration, the animals were fasted but not watered, and were killed by anesthesia.

[0092] 4. Experimental results

[0093] The experimental data were tested using one-way analysis of variance (ANOVA) to determine whether the difference was significant. P < 0.05 indicated a significant difference, and P < 0.01 indicated an extremely significant difference.

[0094] The most obvious symptom of diabetes is "three mores and one less". The weight of diabetic mice will gradually decrease over time. Fig.11As shown, this is a graph showing the effect of the polysaccharide compound ASP of the present invention on the body weight of mice. It can be seen from the figure that the body weight of mice in the healthy control group increased slightly during the intervention period, the body weight of mice in the model group gradually decreased during the intervention period, and the body weight of mice in the polysaccharide treatment group first decreased and then gradually increased during the intervention period. It can be seen that the polysaccharide treatment group mice played a role in resisting the weight loss caused by diabetes, indicating that the polysaccharide compound ASP of the present invention can improve the weight loss of diabetic mice to a certain extent.

[0095] like Fig.12 As shown, during the intervention period, the blood glucose level in the polysaccharide treatment group showed a gradual downward trend. At the 28th week, the fasting blood glucose value in the polysaccharide treatment group was significantly different from that in the model group (P<0.01), while the model group always maintained a high blood glucose level when given distilled water. It can be seen that the polysaccharide compound ASP of the present invention can reduce the blood glucose of diabetic mice to a certain extent.

[0096] In summary, the polysaccharide compound ASP extracted from the mature leaves of Alsophila spinulosa in the present invention is extracted by water extraction and alcohol precipitation, crude polysaccharide impurity removal, DEAE seplife FF ion column separation, and Sephacryl S-400HR gel column purification in sequence, and the new polysaccharide compound ASP is successfully extracted and separated. The extraction and separation method is simple and rapid. Pure water extraction and anhydrous ethanol precipitation are mainly used in the extraction and separation process. The process method is environmentally friendly, and the compound separated by the method has a high purity. The polysaccharide compound ASP extracted from the mature leaves of Alsophila spinulosa in the present invention has good hypoglycemic activity, can be used as a raw material for new drug development and pharmacological activity research, and can also be used for food additives or health care product development.

[0097] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. A polysaccharide compound from Alsophila spinulosa, characterized in that: The Alsophila spinulosa polysaccharide compound is composed of rhamnose, arabinose, galactose and glucuronic acid, and its structure is shown in Formula I: Formula I.

2. The Alsophila spinulosa polysaccharide compound according to claim 1, characterized in that: The molecular weight of the Alsophila spinulosa polysaccharide compound is 17.746 KDa.

3. The Alsophila spinulosa polysaccharide compound according to claim 1, characterized in that: The Alsophila spinulosa polysaccharide compound is extracted and separated from mature leaves of Alsophila spinulosa.

4. The Alsophila spinulosa polysaccharide compound according to claim 3, characterized in that: The Alsophila spinulosa polysaccharide compound is obtained by sequentially extracting mature Alsophila spinulosa leaves by water extraction and alcohol precipitation, removing impurities from crude polysaccharides, separating by ion columns, and purifying by gel columns.

5. The Alsophila spinulosa polysaccharide compound according to claim 4, characterized in that: The ion column adopts DEAEseplife FF anion exchange column; or / and, the gel column adopts Sephacryl S-400HR gel column.

6. A method for extracting polysaccharide compounds from Alsophila spinulosa as claimed in any one of claims 1 to 5, characterized in that: The extraction method comprises the following steps: (1) Water extraction and alcohol precipitation The mature Alsophila spinulosa leaves were extracted with water and ethanol, and the precipitated solids were collected to obtain a crude polysaccharide extract; (2) Removal of impurities from crude polysaccharides The crude polysaccharide extract is fully dissolved in water, a protease is added thereto for enzymolysis, an organic solvent is added for extraction, an aqueous phase is collected, and then a macroporous resin AB-8 is added, a liquid is collected, and a 3000Da dialysis bag is used for dialysis to remove small molecular components, and the polysaccharide liquid is freeze-dried to obtain a crude Alsophila spinulosa polysaccharide; (3) Ion column separation and gel column purification The crude Alsophila spinulosa polysaccharide is dissolved in water, filtered, and then separated by an ion column and purified by a gel column. The eluate is collected, concentrated, and freeze-dried to obtain the Alsophila spinulosa polysaccharide compound.

7. The extraction method according to claim 6, characterized in that In step (1), the water extraction and alcohol precipitation adopts anhydrous ethanol precipitation; Or / and, the water extraction and alcohol precipitation are performed at a temperature of 60°C; Or / and, the water extraction and alcohol precipitation have an extraction time of 4 hours; Or / and, the mature Alsophila spinulosa leaves are defatted using anhydrous ethanol.

8. The extraction method according to claim 6, characterized in that In step (2), the protease is selected from papain; Or / and, the organic solvent extraction is: adding chloroform and n-butanol to the enzymatic hydrolyzate, mixing thoroughly, and collecting the upper aqueous phase; adding petroleum ether to the collected aqueous phase, mixing thoroughly, and collecting the lower aqueous phase.

9. The extraction method according to claim 6, characterized in that In step (3), the ion column separation uses a DEAE seplife FF anion exchange column and is eluted with water and 0.1M, 0.2M, and 0.3M NaCl solutions in sequence; Or / and, the gel column purification adopts Sephacryl S-400HR gel column.

10. Use of the Alsophila spinulosa polysaccharide compound according to any one of claims 1 to 5 in the preparation of hypoglycemic drugs.

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