A kind of Gentiana macrophylla polysaccharide and its preparation method and application

Abstract

The invention discloses a genus gentiana polysaccharide. The polysaccharide structure is characterized in that the monosaccharide composition is mainly composed of arabinose and galacturonic acid, the molecular weight is 82kDa, the connection mode is that the main chain is arabinose (4)-α-GalpA-(1→4)-α-GalpA-(6-OMe)-(1→5)-α-Araf ...6

Description

Technical Field

[0001] The invention belongs to the technical field of nano-biomedicine, and specifically relates to a genus-rooted gentiana polysaccharide and a preparation method and application thereof. Background Art

[0002] Qinjiao is the dried root of a perennial herbaceous plant of Gentiana (Tourn.) L. of the Gentianaceae family. The 2020 edition of the Chinese Pharmacopoeia stipulates that its legal source is the plants Gentiana macrophylla Pall., Gentiana straminea Maxim., Gentiana crassicaulis Duthie exBurk. or Gentiana dahurica Fisch. Among them, Gentiana dahurica is also called Gentiana dahurica, which is mainly distributed in China, Russia and Mongolia; in China, it is distributed in northern and northwest Sichuan, northwest, north China, northeast and other regions. It grows on the edge of fields, roadsides, river beaches, lakeside sands, ditches, sunny slopes and dry grasslands at an altitude of 870-4500 meters. Gentiana macrophylla was first listed as a medium-grade herb in Shennong's Herbal Classic, and has a history of more than 2,000 years of medicinal use. Gentiana macrophylla is an important traditional Chinese medicine and is also used by many ethnic groups such as Tibetans, Mongolians and Yi people. It has the effects of removing rheumatism, clearing dampness and heat, relieving pain and relieving fever. At present, the anti-inflammatory and anti-rheumatic active ingredients of Gentiana macrophylla are mainly concentrated in its iridoids, flavonoids and triterpenes. There are relatively few reports on the separation, purification, structural characterization and activity research of Gentiana macrophylla polysaccharides.

[0003] At present, studies have shown that polysaccharides have a variety of biological activities, including anti-tumor, antibacterial, anti-cancer, hypolipidemic, antioxidant and immunomodulatory abilities, and have good biocompatibility and high safety. Due to the different preparation methods, polysaccharides are composed of a variety of monosaccharides. Their structure, content and the connection mode of sugar units will cause differences in the spatial structure of polysaccharides, and their different structures have a significant effect on activity. For example, Otu Phyllis Naa Yarley et al. pointed out that the polysaccharide components from the fruit of Zizyphus Jujuba cv. Jinsixiaozao, among which the components containing high content of uronic acid have stronger free radical scavenging activity than the components without uronic acid, indicating that uronic acid is an effective indicator of the antioxidant activity of polysaccharides, which may be attributed to the presence of electrophilic groups such as aldehydes or ketones in polysaccharides to promote the release of hydrogen atoms in O-H bonds. The glycosidic bond linkage of monosaccharides also affects the antioxidant activity, especially the side chain arabinose 1→4 and mannose 1→2 linkages are significantly related to reducing power, while glucose 1→6 and arabinose 1→4 linkages are closely related to the DPPH free radical scavenging effect. At the same time, some studies have shown that molecular modification of polysaccharides (such as sulfation, phosphorylation, acetylation, alkylation, sulfonylation, carboxymethylation, benzoylation, etc.) affects the antioxidant effect of polysaccharides to a certain extent. Summary of the invention

[0004] The invention aims to provide a genus root polysaccharide and a preparation method and application thereof.

[0005] The technical solution of the present invention is:

[0006] A polysaccharide of gentiana macrophylla, wherein the molecular weight of the polysaccharide is 82 kDa, the monosaccharide composition of the polysaccharide of gentiana macrophylla is rhamnose, arabinose, galactose, glucose, mannose, galacturonic acid and glucuronic acid, the main chain of the polysaccharide of gentiana macrophylla is connected by →5)-Araf-(1→ and →4)-GalpA-(1→), and the side chains are α-Araf-(1→, →3,5)-α-Araf-(1→, →3,4)-GalpA-(1→, α-GalpA-(1→ and →1)-Rhap-(2,4→; the structural formula of the polysaccharide of gentiana macrophylla is:

[0007]

[0008] Furthermore, the molar ratio of the main chain residues is 16.0:20.4, the molar ratio of the side chain residues is 14.6:3.2:2.5:8.6:4.6, and n is selected from 100 to 150.

[0009] Furthermore, the molar ratio of rhamnose, arabinose, galactose, glucose, mannose, galacturonic acid and glucuronic acid is 8-9:30-35:4-5:2-3:0.5-1:40-60:1-3.

[0010] Another technical solution of the present invention is:

[0011] A method for preparing Gentiana macrophylla polysaccharide, the method comprising:

[0012] (1) Crude preparation: dried rhizomes of Gentiana macrophylla are taken and crushed to obtain Gentiana macrophylla powder. The Gentiana macrophylla powder is subjected to ethanol reflux extraction to remove small molecules, and then subjected to hot water reflux extraction to obtain Gentiana macrophylla water extract, which is then precipitated by adding ethanol to obtain Gentiana macrophylla crude polysaccharide;

[0013] (2) Purification: The crude polysaccharide of Gentiana macrophylla is loaded onto a weak anion exchange resin, eluted with 2 column volumes of water, and then eluted with 0.1 M and 0.2 M saline solutions, respectively. The polysaccharide eluted with the 0.2 M saline solution is collected, concentrated under reduced pressure and freeze-dried to obtain purified polysaccharide of Gentiana macrophylla.

[0014] Furthermore, in step (1), the weight volume ratio of the Gentiana macrophylla powder to the ethanol is 1 g: 5-20 mL.

[0015] Furthermore, in step (2), the matrix of the weak anion exchange resin is any one of agarose, dextran, and polymethyl methacrylate matrix, and the surface bonding phase is diethylaminoethyl.

[0016] The third technical solution of the present invention is:

[0017] The invention discloses an application of Gentiana macrophylla polysaccharide in preparing medicine for treating diseases caused by oxidative damage.

[0018] Application of Gentiana macrophylla polysaccharide in the preparation of immunomodulatory drugs.

[0019] The present invention provides a polysaccharide of Gentiana macrophylla and a preparation method and application thereof, and the beneficial effects are as follows: a high-purity polysaccharide is obtained by extracting and purifying the rhizome of Gentiana macrophylla, and its structural unit and basic composition are obtained through structural identification. Through free radical scavenging experiments, RAW264.7 oxidative damage and immunomodulatory models, it is found that the polysaccharide of Gentiana macrophylla obtained by the present invention has good antioxidant and immunomodulatory activities. The method of the present invention is not only simple to operate, but also time-consuming and low-cost. The polysaccharide of Gentiana macrophylla prepared by the method has high purity, clear components, clear structure, and multiple potential application values, laying a foundation for developing the medicinal value of polysaccharide of Gentiana macrophylla. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is an infrared spectrum of the Gentiana macrophylla polysaccharide described in the present invention;

[0021] Figure 2 This is a one-dimensional nuclear magnetic resonance spectrum of a polysaccharide of Gentiana macrophylla described in the present invention ( 1 H-NMR, 13 C-NMR);

[0022] Figure 3 The two-dimensional nuclear magnetic resonance spectrum of the polysaccharide of Gentiana macrophylla described in the present invention is ( 1 H- 1 H COSY diagram, 1 H- 13 CHSQC diagram, 1 H- 13 C HMBC diagram, 1 H- 1 H NOESY diagram);

[0023] Figure 4 This is a result diagram of a free radical scavenging experiment of Gentiana macrophylla polysaccharide described in the present invention;

[0024] Figure 5 This is a graph showing the effect of a polysaccharide from Gentiana macrophylla on the oxidative damage protection activity of RAW 264.7;

[0025] Figure 6 This is a diagram showing the effect of the Gentiana macrophylla polysaccharide described in the present invention on the NO release level. DETAILED DESCRIPTION

[0026] In order to make the above-mentioned objects, features and advantages of the present invention more clearly understood, the technical solutions of the present invention are further described below in conjunction with embodiments. However, the present invention is not limited to the embodiments listed, and should also include any other known changes within the scope of the rights claimed by the present invention.

[0027] The term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0028] Example 1

[0029] This implementation case shows a method for preparing a polysaccharide from Gentiana macrophylla according to the following steps:

[0030] Step 1: Extract

[0031] The commercially available dried rhizomes of Gentiana macrophylla Pall. were crushed and fine powder was collected. 688 g of the fine powder was taken, 5 L of 95% ethanol was added, and the mixture was refluxed at 90 °C for 2 h. The residue was filtered, and the operation was repeated twice. The residue was added with 5 L of deionized water according to the solid-liquid ratio of 1:10, and extracted with boiling water at 100 °C for 2 h. The extraction was repeated 2 times. The filtrates obtained from the two extractions were centrifuged and combined, and then filtered.

[0032] Step 2: Preliminary purification

[0033] An ultrafiltration membrane with a molecular weight cut-off specification of 10 kDa was used for ultrafiltration and concentration until about 1 L. 4 L of absolute ethanol was added to the concentrated solution for alcohol precipitation, and it was stirred while adding. The mixture was precipitated overnight in a refrigerator at 4 °C, centrifuged (3500 rpm for 15 min), and the precipitate was taken to obtain the crude polysaccharide of Gentiana macrophylla Pall. After re-dissolution, the crude polysaccharide solution of Gentiana macrophylla Pall. was loaded onto a macroporous weakly basic anion exchange resin, and the flowing-through fraction was collected and concentrated under reduced pressure. 40 mL of Sevage reagent (chloroform:n-butanol = 5:1) was added to the above solution to remove proteins. The mixture was shaken well in a separatory funnel for 10 min, centrifuged, and the upper aqueous solution was taken, and the protein layer and the organic reagent layer were discarded. The above steps were repeated 6 times, and then concentrated by rotary evaporation to completely remove the organic solvent. It was continuously dialyzed with a 3500 Da dialysis bag for three days.

[0034] Step 3: Refinement

[0035] A chromatography column from Suzhou Huitong Chromatographic Separation and Purification Co., Ltd. was used for purification with the agarose DEAE filler from Suzhou Sepax Technologies Co., Ltd. as the matrix. The polysaccharide sample was made into a sample solution of 50 mg / mL, filtered through a membrane and then injected. The flow rate of medium-pressure preparative liquid chromatography was 8 mL / min, and the 0.2 M salt eluate was collected, which was the polysaccharide of Gentiana dahurica Fisch. It was dialyzed with a 3.5 kDa dialysis bag, centrifuged, and the sample was freeze-dried. After freeze-drying, the sample was weighed and stored in a cool and dry place.

[0036] The polysaccharide of Gentiana dahurica Fisch. prepared by the above method, which is mainly composed of arabinose and galacturonic acid, has a molecular weight of 82 kDa, and the molar ratio of monosaccharide composition is rhamnose: arabinose: galactose: glucose: mannose: galacturonic acid: glucuronic acid = 8.41:33.90:4.67:2.27:0.61:48.48:1.66. The main chain of the polysaccharide of Gentiana dahurica Fisch. is linked by →5)-Araf-(1→ and →4)-GalpA-(1→, and the molar ratio of the main chain residues is 16.0:20.4. The branches include α-Araf-(1→, →3,5)-α-Araf-(1→, →3,4)-GalpA-(1→, α-GalpA-(1→ and →1)-Rhap-(2,4→, and the molar ratio of the branch residues is 14.6:3.2:2.5:8.6:4.6. The structural formula of the polysaccharide of Gentiana dahurica Fisch. is:

[0037]

[0038] The protein content of a polysaccharide from Gentiana macrophylla was 0.81%, the total sugar content was 79.7%, and the monosaccharide composition was mainly galacturonic acid and arabinose, and also contained glucose and galactose.

[0039] 1. The products obtained in Example 1 were subjected to infrared analysis to obtain their main functional groups and some connection configuration characteristics. For the results, please refer to Figure 1 , Figure 1 The infrared spectrum of the polysaccharide of Gentiana macrophylla described in the present invention is shown in FIG. Figure 1 As shown in Table 1, the polysaccharide of Gentiana macrophylla is mainly composed of α-type glycosidic bonds, with some β-type glycosidic bonds in the middle; methylation analysis can provide the types and proportions of sugar residues in the polysaccharide molecules, and the results are shown in Table 1. As shown in Table 1, in the polysaccharide of Gentiana macrophylla described in the present invention, the connection mode of GalA is mainly 1,4-GalA, and contains a certain amount of T-GalA, and the connection mode of Ara is T-Ara and 1,5-Ara. Two-dimensional nuclear magnetic resonance analysis can determine different sugar residues and their connection modes, and their specific carbon-hydrogen attribution can be found in Table 1. Figure 2 , Figure 3 and Table 2, which is a chemical shift assignment table of nuclear magnetic resonance hydrogen and carbon spectra of Gentiana macrophylla polysaccharide, Figure 2 This is a one-dimensional nuclear magnetic resonance spectrum of a polysaccharide of Gentiana macrophylla described in the present invention ( 1 H-NMR, 13 C-NMR); Figure 3 The two-dimensional nuclear magnetic resonance spectrum of the polysaccharide of Gentiana macrophylla described in the present invention is ( 1 H- 1 H COSY diagram, 1 H- 13 C HSQC diagram, 1 H- 13 CHMBC diagram, 1 H- 1 H NOESY map), combined with the methylation results, the sugar chain structure was finally inferred.

[0040]

[0041]

[0042] Table 1

[0043]

[0044] Table 2

[0045] 2. The products obtained in Example 1 were respectively subjected to in vitro antioxidant activity studies.

[0046] (1) Determination of ABTS, DPPH, and hydroxyl radical scavenging abilities

[0047] Please refer to the results Figure 4 , Figure 4 This is a result diagram of a free radical scavenging experiment of a polysaccharide of Gentiana macrophylla described in the present invention. Figure 4 As shown, the polysaccharide from Gentiana macrophylla has a good free radical scavenging ability and is concentration-dependent.

[0048] (2) Analysis of the active protection against oxidative damage of RAW264.7

[0049] Hydrogen peroxide was added to RAW264.7 cells to a final concentration of 1.2 mM for oxidative damage, and then the absorbance was measured at 450 nm using a CCK-8 kit to calculate cell survival rate. Figure 5 , Figure 5 This is a graph showing the effect of a polysaccharide from Gentiana macrophylla on the protective activity of RAW 264.7 against oxidative damage. Figure 5 As shown in the results, the polysaccharide from Gentiana macrophylla exhibited a good protective effect against oxidative damage, and showed a certain dose-dependent relationship. When the concentration of the polysaccharide increased from 10 μg / mL to 500 μg / mL, its protective rate against oxidative damage of RAW 264.7 also increased, and it had a certain proliferation effect.

[0050] 3. The immunomodulatory activity of the products obtained in Example 1 was studied respectively.

[0051] Immunomodulatory activity analysis (NO content determination)

[0052] The NO kit was used to measure the NO content of RAW264.7 released by it. Figure 6 , Figure 6 This is a graph showing the effect of a polysaccharide from Gentiana macrophylla on NO release level described in the present invention. Figure 6 As shown, when its mass concentration is 10, 50, 500 μg / mL, it has certain immunomodulatory activity and is concentration-dependent.

[0053] Compared with the prior art, the beneficial effects of the present invention are: the genus macrophylla polysaccharide and its preparation method and application described in the present invention, the obtained genus macrophylla polysaccharide not only has a clear structure and good activity, which can lay a foundation for developing the medicinal value of genus macrophylla polysaccharide, but also has good antioxidant activity and immunomodulatory activity, and has application value in preparing drugs for the treatment of various diseases.

[0054] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A polysaccharide from Gentiana macrophylla, Features: The molecular weight of the polysaccharide of Gentiana macrophylla is 82 kDa, and the monosaccharide composition of the polysaccharide of Gentiana macrophylla is rhamnose, arabinose, galactose, glucose, mannose, galacturonic acid and glucuronic acid. The main chain of the polysaccharide of Gentiana macrophylla is connected by →5)-Araf-(1→ and →4)-GalpA-(1→, and the side chains are α-Araf-(1→, →3,5)-α-Araf-(1→, →3,4)-GalpA-(1→, α-GalpA-(1→ and →1)-Rhap-(2,4→; the structural formula of the polysaccharide of Gentiana macrophylla is: Among them, the molar ratio of the main chain residues is 16.0:20.4, the molar ratio of the side chain residues is 14.6:3.2:2.5:8.6:4.6, and n is selected from 100 to 150; the molar ratio of rhamnose, arabinose, galactose, glucose, mannose, galacturonic acid and glucuronic acid is 8 to 9:30 to 35:4 to 5:2 to 3:0.5 to 1:40 to 60:1 to 3.

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

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

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