Arabinogalactan glycan, and preparation method and application thereof

Abstract

The application discloses an arabinogalacturonan polysaccharide, the polysaccharide structural characteristics of which are that monosaccharide composition is mainly arabinose and galacturonic acid, the molecular weight is 23 kDa, the connection mode is (1→3, 5)-alpha-Araf-(1→3, 5)-alpha-Araf-(1→3, 4)-alpha-GalpA-(6-OMe)-(1→ as the main chain, and the arabinose branch, HG domain and terminal galacturonic acid are contained. The preparation method comprises the following steps: pulverizing small gentiana powder, water extraction and alcohol precipitation, removing protein, removing small molecules and utilizing weak anion exchange resin adsorption, and then eluting with a salt solution with a concentration of 0.1 M to obtain high-purity small gentiana polysaccharide. The arabinogalacturonan polysaccharide has good antioxidant activity and immunomodulatory activity, has application value for treating various diseases, and has clear structure and good activity, thereby laying a foundation for developing the medicinal value of the small gentiana polysaccharide.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of nanobiomedicine, and particularly relates to an arabinogalacturonan and a preparation method and application thereof. BACKGROUND

[0002] Gentiana is a perennial herb of Gentiana (Tourn.) L. Gentiana (Sect. Cruciata Gaudin) dry root, and the legal source thereof is stipulated in Chinese Pharmacopoeia 2020 edition as Gentiana macrophylla Pall., G. straminea Maxim., G. crassicaulis Duthie ex Burk. or G. dahurica Fisch. Among them, G. dahurica is also called Dahurian Gentiana, which is distributed in northern and northwestern Sichuan, northwest, north China, northeast and other regions of China. It grows at an altitude of 870-4500 meters on the edge of fields, roadsides, riverbanks, lake edges, sandlands, ditches, sunny slopes and dry grasslands. Gentiana was first recorded in Shennong Bencao Jing and has been used as a medicine for more than 2000 years. Gentiana is an important traditional Chinese medicine with the effects of expelling wind and dampness, clearing damp-heat, relieving arthralgia and reducing virtual heat. At present, the active components of Gentiana for anti-inflammatory and anti-rheumatic activities are mainly concentrated in its iridoid, flavonoids and triterpenes, and there are few reports on the structure and activity of macromolecular polysaccharides of Gentiana.

[0003] At present, the research on polysaccharides of Gentiana is mainly focused on the chemical composition and activity. For example, Chen Kek et al. analyzed the monosaccharide composition of polysaccharides of Gentiana by HPLC method, and the results showed that the polysaccharides of Gentiana contained seven kinds of monosaccharides, i.e. mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose, and their molar ratio was 0.06:0.25:0.03:1.00:1.23:0.87:3.52. The crude polysaccharides isolated from G. straminea were considered to be mainly composed of glucose and had certain anti-inflammatory effect. Zou isolated two pectin-like polysaccharides from the dried rhizomes of G. crassicaulis, which contained galactose (66.2% and 36.1%), galacturonic acid (15.8% and 24.0%) and arabinose (7.8% and 32.0%), respectively, and their molecular weights were 627 kDa and 471.3 kDa, respectively, and they had natural immune regulation effect. However, the definite structure of polysaccharides of Gentiana has not been elucidated so far. Therefore, in order to further develop the application of polysaccharides of Gentiana, it is necessary to conduct in-depth and systematic research on the definite structure and pharmacological activity thereof, so as to develop its medicinal value. SUMMARY

[0004] The present application aims to provide an arabinogalacturonan and a preparation method and application thereof.

[0005] The technical scheme of the present application is:

[0006] An arabinogalactan polysaccharide, the molecular weight of the arabinogalactan polysaccharide is 23 kDa, the monosaccharide composition in the arabinogalactan polysaccharide is arabinose, galacturonic acid, rhamnose, galactose, glucose, mannose and glucuronic acid, the main chain of the arabinogalactan polysaccharide is connected with →3,5)-Araf-(1→ and →3,4)-GalpA-(1→, and the branched chain has α-Araf-(1→, →5)-α-Araf-(1→, α-GalpA-(1→, →4,6)-β-Galp-(1→ and →4)-β-Glcp-(1→; the structural formula of the arabinogalactan polysaccharide is:

[0007]

[0008] Further, the molar ratio of the arabinose, galacturonic acid, rhamnose, galactose, glucose, mannose and glucuronic acid is 50-60:25-35:2-3:5-6:4-5:0.5-1.5:1-2, and the n is selected from 10-30.

[0009] Another technical scheme of the present application is:

[0010] A preparation method of an arabinogalactan polysaccharide, the method comprises:

[0011] (1) Crude preparation: the dried rootstock of small gentian is crushed to obtain small gentian powder, the small gentian powder is extracted by refluxing with ethanol, after removing small molecules, the small gentian water extract is obtained by refluxing with hot water, and then ethanol is added for precipitation to obtain crude polysaccharide of small gentian;

[0012] (2) Refining: the crude polysaccharide of small gentian is loaded onto a weak anion exchange resin, after elution with 2 times the column volume of water to remove impurities, a polysaccharide solution is obtained by elution with a 0.1M salt solution, and the arabinogalactan polysaccharide is obtained by desalting, concentration under reduced pressure and freeze-drying.

[0013] Further, in step (1), the weight-to-volume ratio of the small gentian powder to the ethanol is 1g:7.26mL.

[0014] Further, in step (2), the matrix of the weak anion exchange resin is any one of agarose, dextran, polymethylacrylamide matrix, and the surface bonded phase is diethylaminoethyl.

[0015] The third technical scheme of the present application is:

[0016] Use of an arabinogalactan in the preparation of a medicament for the treatment of a disease caused by oxidative damage.

[0017] Use of an arabinogalactan in the preparation of an immunomodulatory medicament.

[0018] The application provides an arabinogalactan and a preparation method and application thereof, and has the beneficial effect that high-purity polysaccharides are obtained by extraction and purification in Gentiana barbata roots, and the structural unit and basic composition are obtained through structure identification. It is found through free radical scavenging experiments, RAW264.7 oxidative damage and immunomodulatory models that the arabinogalactan obtained in the application has good antioxidant and immunomodulatory activity. The method of the application is simple in operation, short in time consumption and low in cost. The small Gentiana barbata polysaccharides prepared by the method have high purity, clear components and definite structure, and have potential application values, and lay a foundation for developing the medicinal value of the small Gentiana barbata polysaccharides. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 An infrared spectrum of the arabinogalactan according to the application;

[0020] Figure 2 A one-dimensional nuclear magnetic resonance spectrum of the arabinogalactan according to the application 1 H-NMR, 13 C-NMR);

[0021] Figure 3 A two-dimensional nuclear magnetic resonance spectrum of the arabinogalactan according to the application 1 H- 1 HCOSY diagram, 1 H- 13 C HSQC diagram, 1 H- 13 C HMBC diagram, 1 H- 1 H NOESY diagram);

[0022] Figure 4 A result diagram of a free radical scavenging experiment of the arabinogalactan according to the application;

[0023] Figure 5 A diagram of the influence of the arabinogalactan according to the application on the oxidative damage protection activity of RAW 264.7;

[0024] Figure 6 A diagram of the influence of the arabinogalactan according to the application on the NO release level. DETAILED DESCRIPTION

[0025] In order to make the above objectives, features and advantages of the present application more apparent, the technical solutions of the present application will be further described below in conjunction with embodiments. However, the present application is not limited to the listed embodiments, and should also include any known changes within the scope of the claimed rights of the present application.

[0026] Reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described can be included in at least one implementation of the present application. Appearances of "in one embodiment" at various places in this specification do not necessarily all refer to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.

[0027] Example 1

[0028] The present embodiment demonstrates a method for preparing an arabinogalactan glycan according to the following steps:

[0029] Step 1: Extraction

[0030] Commercially available Gentiana macrophylla root rhizomes were ground and fine powder was collected. 688 g of the fine powder was added to 5 L of 95% ethanol and extracted by refluxing at 90°C for 2 h. The filtrate was collected and the extraction was repeated twice. The filtrate was added to 5 L of deionized water at a solid-to-liquid ratio of 1:10 and extracted by boiling at 100°C for 2 h. The extraction was repeated twice and the filtrate was collected by centrifugation. The filtrate was then filtered.

[0031] Step 2: Preliminary purification

[0032] The filtrate was concentrated to about 1 L by ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa. The concentrated solution was precipitated by adding 4 L of anhydrous ethanol while stirring. The solution was then precipitated overnight in a refrigerator at 4°C. The precipitate was collected by centrifugation at 3500 rpm for 15 min. The precipitate was then dissolved and loaded onto a macroporous weakly basic anion exchange resin. The eluate was collected and concentrated under reduced pressure. Protein was removed by adding 40 mL of Sevage reagent (chloroform: n-butanol = 5:1) to the solution. The solution was thoroughly mixed in a separatory funnel for 10 min and then centrifuged. The upper aqueous layer was collected and the protein and organic reagent layers were discarded. The above steps were repeated six times. The organic solvent was removed by rotary evaporation. The solution was dialyzed in a 3500 Da dialysis bag for three days.

[0033] Step 3: Refining

[0034] Purification was performed using a chromatography column from Suzhou Huitong Chromatography & Purification Co., Ltd., with agarose DEAE packing material from Suzhou Saifen Technology Co., Ltd. as the matrix. A 50 mg / mL sample solution was prepared from the polysaccharide sample, filtered through a membrane, and injected into the preparative liquid chromatography system at a medium-pressure flow rate of 8 mL / min. The 0.1 M salt eluent was collected, yielding an arabinose-galacturonic acid polysaccharide (Gentiana macrophylla). The sample was dialyzed using a 3.5 kDa dialysis bag, centrifuged, and freeze-dried. After lyophilization, the sample was weighed and stored in a cool, dry place.

[0035] The arabinose-galacturonic acid polysaccharide (Gentiana macrophylla polysaccharide) prepared by the above method has a molecular weight of 23 kDa and a monosaccharide composition percentage of rhamnose:arabinose:galactose:glucose:mannose:galacturonic acid:glucuronic acid = 2.65:53.50:6.35:4.56:1.03:30.76:1.14. The main chain of Gentiana macrophylla polysaccharide is →3,5)-Araf-(1→ and →3,4) -GalpA-(1→) is the linker, the molar ratio of main chain residues is 28.0:20.8, and the side chains are α-Araf-(1→, →5)-α-Araf-(1→, α-GalpA-(1→, →4,6)-β-Galp-(1→ and →4)-β-Glcp-(1→), with a molar ratio of side chain residues of 11.6:12.3:13.4:4.3:4.6. The structural formula of Gentiana macrophylla polysaccharide is:

[0036]

[0037] Analysis revealed that the protein content of this arabinose-galacturonic acid polysaccharide was 1.05%, and the total sugar content was 82.27%. The monosaccharide composition was mainly composed of galacturonic acid and arabinose, and also contained glucose and galactose.

[0038] 1. Infrared analysis was performed on the products obtained in Example 1 to obtain their main functional groups and partial linkage configuration characteristics. Please refer to the results below. Figure 1 , Figure 1 This is the infrared spectrum of an arabinose-galacturonic acid polysaccharide according to the present invention. Figure 1 As shown, arabinose-galacturonic acid polysaccharides are polysaccharides mainly composed of α-glycosidic bonds, with some β-glycosidic bonds interspersed. Methylation analysis can provide the types and proportions of sugar residues in the polysaccharide molecule; the results are shown in Table 1. As shown in Table 1, in the arabinose-galacturonic acid polysaccharides described in this invention, GalA linkages include T-GalA and 1,3,4-GalA, while Ara linkages include T-Ara, 1,5-Ara, and 1,3,5-Ara. Two-dimensional nuclear magnetic resonance analysis can determine different sugar residues and their linkage modes; for specific carbon and hydrogen assignments, please refer to [reference needed]. Figure 2 , Figure 3Table 2 shows the chemical shift assignments of the 1H and 1C NMR spectra of an arabinose-galacturonic acid polysaccharide. Figure 2 This is a one-dimensional nuclear magnetic resonance spectrum of an arabinose-galacturonic acid polysaccharide described in this invention. 1 H-NMR, 13 C-NMR); Figure 3 This is a two-dimensional nuclear magnetic resonance spectrum of an arabinose-galacturonic acid polysaccharide described in this invention. 1 H- 1 H COSY diagram, 1 H- 13 C HSQC diagram, 1 H- 13 CHMBC diagram, 1 H- 1 By combining the H NOESY diagram with the methylation results, the sugar chain structure was finally deduced.

[0039]

[0040]

[0041] Table 1

[0042]

[0043] Table 2

[0044] 2. The in vitro antioxidant activity of the products obtained in Example 1 was studied.

[0045] (1) Determination of ABTS, DPPH and hydroxyl radical scavenging capacity

[0046] Please see the results. Figure 4 , Figure 4 This is a graph showing the results of an experiment on the free radical scavenging activity of an arabinose-galacturonic acid polysaccharide described in this invention. Figure 4 As shown, the polysaccharide from Gentiana macrophylla exhibits excellent free radical scavenging ability, which is concentration-dependent.

[0047] (2) Activity protection against oxidative damage of RAW264.7

[0048] Oxidative damage was induced in RAW264.7 cells by adding hydrogen peroxide to a final concentration of 1.2 mM. Cell viability was then calculated using a CCK-8 assay at 450 nm. (See results below.) Figure 5 , Figure 5 This diagram illustrates the effect of an arabinose-galacturonic acid polysaccharide, as described in this invention, on the protective activity against oxidative damage in RAW 264.7. Figure 5As shown, the polysaccharide from Gentiana macrophylla exhibits excellent protective activity against oxidative damage, and this protective effect is dose-dependent. When the concentration of the polysaccharide increases from 10 μg / mL to 500 μg / mL, its protective effect against oxidative damage to RAW 264.7 initially increases and then decreases slightly.

[0049] 3. The immunomodulatory activities of the products obtained in Example 1 were studied.

[0050] Immunomodulatory activity analysis (NO content determination)

[0051] The NO content promoted by NO reagent kit was measured to determine the NO content released from RAW264.7. (See results below.) Figure 6 , Figure 6 This is a graph showing the effect of an arabinose-galacturonic acid polysaccharide described in this invention on NO release levels. Figure 6 As shown, GDP-1 exhibits certain immunomodulatory activity at concentrations of 10, 50, and 500 μg / mL; however, at concentrations of 100 and 200 μg / mL, it demonstrates very strong immunostimulatory activity.

[0052] Compared with the prior art, the beneficial effects of the present invention are as follows: The arabinose-galacturonic acid polysaccharide, its preparation method and application described in the present invention not only have a clear structure and good activity, which can lay the foundation for developing the medicinal value of Gentiana macrophylla polysaccharide, but also have good antioxidant activity and immunomodulatory activity, and have the application value of preparing drugs for the treatment of various diseases.

[0053] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A polysaccharide from Gentiana macrophylla, characterized in that: The molecular weight of the *Gentiana macrophylla* polysaccharide is 23 kDa. The monosaccharide composition of the *Gentiana macrophylla* polysaccharide is arabinose, galacturonic acid, rhamnose, galactose, glucose, mannose, and glucuronic acid. The main chain of the *Gentiana macrophylla* polysaccharide is linked by →3,5)-Araf-(1→ and →3,4)-GalpA-(1→), and the side chains are α-Araf-(1→, →5)-α-Araf-(1→, α-GalpA-(1→, →4,6)-β-Galp-(1→ and →4)-β-Glcp-(1→). The molar ratio of arabinose, galacturonic acid, rhamnose, galactose, glucose, mannose and glucuronic acid is 53.50:30.76:2.65:6.35:4.56:1.03:1.

14.

2. The use of the Gentiana macrophylla polysaccharide according to claim 1 in the preparation of a medicament for treating diseases caused by oxidative damage.

3. The application of the Gentiana macrophylla polysaccharide according to claim 1 in the preparation of immunomodulatory drugs.

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