Pollen typhae acidic polysaccharide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to pollen typhae acidic polysaccharide as well as a preparation method and application thereof. The pollen typhae acidic polysaccharide is characterized in that the structure of the polysaccharide comprises rhamnose, galactose, galacturonic acid, arabinose, glucose, xylose and mannose. The brand-new pollen typhae acidic polysaccharide TPP-4 prepared by the invention can improve the integrity of blood vessels so as to improve the permeability change of vascular endothelial cells caused by inflammation, namely, the acidic polysaccharide has a protective effect on the integrity of the endothelial cells in an inflammatory state, and can be used for preventing and / or treating blood stasis and bleeding syndromes.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a pollen acid polysaccharide and a preparation method and use thereof. Background Art

[0002] Blood stasis and bleeding both belong to the category of "blood syndrome" in traditional Chinese medicine. The two are mutually opposed and can be transformed into each other. Blood stasis and bleeding syndrome caused by qi stagnation, blood coldness and other reasons are common in gynecology, internal medicine, and trauma, such as functional uterine bleeding, cerebral hemorrhage, renal hematuria, fundus hemorrhage and other diseases. Western medicine mainly treats blood stasis and bleeding diseases with antithrombotic drugs, hormone drugs or surgery, which have limited efficacy, tolerance and are prone to serious side effects such as bleeding.

[0003] Endothelial cells are semipermeable, selective regulatory barriers within blood vessels. They are composed of endothelial cells arranged continuously in arteries, veins, and lymphatic vessels of various sizes. They participate in a variety of physiological processes, including the regulation of vascular tone, the maintenance of endothelial integrity, endothelial cell metabolism, platelet activity and interaction, hemostasis regulation, and endothelial injury and repair processes. With the advancement of modern vascular biology, the concept of endothelial cell dysfunction has received increasing attention, and the reversible regulation of endothelial dysfunction is currently a hot topic in cardiovascular disease research. Blood stasis and bleeding syndromes are manifested by impaired or reduced blood flow and activated coagulation function, leading to persistent endothelial cell hypoxia and oxidative stress, thereby inducing an inflammatory response, aggravating endothelial dysfunction and damaging the integrity of the vascular wall, leading to blood extravasation and microcirculatory damage. Eliminating vascular inflammation and improving endothelial function are important ways to treat blood stasis and bleeding diseases.

[0004] Typha angustifolia L., Typha orientalis Presl. or the dried pollen of plants of the same genus, Typha angustifolia L., Typha orientalis Presl., or plants of the same genus, can dissipate blood stasis, stop bleeding, and relieve stranguria, and is particularly suitable for bleeding syndromes caused by blood stasis. Literature research found that the total extract of Typha angustifolia can promote angiogenesis and stop bleeding in zebrafish models, and the total polysaccharide of Typha angustifolia can inhibit the expression of inflammatory factors (IL-6 and TNFα), and improve blood circulation and vascular permeability in diabetic rats. Previous studies on active ingredients have mostly focused on small molecule compounds, but the structure and pharmacological effects of macromolecular components, especially the effects of dissipating blood stasis and stopping bleeding, are still insufficient. Summary of the invention

[0005] Purpose of the invention:

[0006] The present invention aims to prepare a new Typha typhae acidic polysaccharide and reveal its potential application prospect in improving vascular integrity.

[0007] Technical Solution

[0008] A Typhae pollen acidic polysaccharide, characterized in that the structure of the polysaccharide contains rhamnose, galactose, galacturonic acid, arabinose, glucose, xylose, and mannose.

[0009] The Puhuang acidic polysaccharide is characterized in that the main chain of the polysaccharide includes rhamnose, galactose and galacturonic acid; the branches of the polysaccharide include galactose, glucose, arabinose, rhamnose, galacturonic acid and xylose.

[0010] The Puhuang acidic polysaccharide is characterized in that the main chain of the polysaccharide includes 1,3-β-D-Manp, 1,2-α-L-Rhap, 1,2,4-α-L-Rhap, 1,3,4-β-D-Galp, 1,4-β-D-Galp and 1,4-α-D-GalpA; the branch bag of the polysaccharide includes T-β-D-Galp, T-α-D-Glcp, 1,5-α-L-Araf, 1,2-α-L-Rhap, 1,3-α-L-Araf, T-α-L-Araf, T-α-D-GalpA and T-β-D-Xylp.

[0011] The Puhuang acidic polysaccharide is characterized in that the main chain of the polysaccharide is composed of RG-I type pectin polysaccharides of →3)-β-D-Manp-(1→, →3,4)-β-D-Galp-(1→, →2)-α-L-Rhap-(1→, →4)-β-D-Galp-(1→, →4)-α-D-GalpA-(1→ and →2,4)-α-L-Rhap-(1→); and the side chains are mainly composed of β-D-Galp-(1→, α-D-Glcp-(1→, →5-α-L-Araf-(1→, →2)-α-L-Rhap-(1→, →3-α-L-Araf-(1→, α-L-Araf-(1→, α-D-GalpA-(1→ and β-D-Xylp-(1→).

[0012] The Puhuang acidic polysaccharide is characterized in that the molecular weight of the Puhuang acidic polysaccharide is 1.5×10 5 -4.0×10 5 Da.

[0013] As an optimization method, the acidic polysaccharide is composed of rhamnose (28.21%), galactose (20.88%), galacturonic acid (19.65%), arabinose (17.28%), glucose (6.38%), xylose (6.11%) and a small amount of mannose (1.49%) in terms of molar concentration percentage.

[0014] The method for producing Typha typhae acidic polysaccharide is characterized in that the method comprises the following steps:

[0015] (1) extracting the Typhae Pollen medicinal material by heating and refluxing with water to obtain a water extract;

[0016] (2) concentrating the aqueous extract to obtain a concentrated solution;

[0017] (3) subjecting the concentrated solution to ethanol precipitation, and filtering to obtain a precipitate;

[0018] (4) dissolving the precipitate in water to obtain a sample solution, removing the protein therein using the Sevag method, and freeze-drying the solution to obtain Typha Pollen total polysaccharide;

[0019] (5) The Typhae Pollen total polysaccharide is dissolved in water and then centrifuged, and the supernatant is subjected to anion exchange chromatography coupled with dextran gel chromatography to obtain Typhae Pollen acidic polysaccharide.

[0020] The method is further characterized in that the method comprises the following steps:

[0021] In step (1), the Puhuang medicinal material is passed through a No. 7 sieve to remove impurities; the water is pure water, and the amount used is 10 times the amount of L / kg; the extraction temperature is 100° C.; the heating extraction operation is repeated once or multiple times;

[0022] In step (2), the concentration condition is to use a vacuum rotary evaporator to concentrate at 60°C;

[0023] In step (3), the final concentration of ethanol precipitation is 80%; the time of ethanol precipitation is 12 hours;

[0024] In step (4), the sample solution is mixed with the Sevag reagent, shaken thoroughly, vortexed, and then transferred to a separatory funnel and allowed to stand. After standing, the upper layer of liquid is taken, centrifuged, and the supernatant is taken. The above process is repeated until no protein is produced; the Sevag reagent is a mixed solution of n-butanol and dichloromethane, and the volume ratio of the two is 1:4; the ratio of the sample solution to the Sevag reagent is 5:1;

[0025] In step (5), the anion exchange chromatography uses a DEAE-52 cellulose chromatography column, and after the crude Typhae Pollen polysaccharide is loaded, it is eluted with distilled water, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 and 2 mol / L NaCl solutions in sequence, and the fractions are collected, and the polysaccharide is traced using the anthrone-sulfuric acid method. The fraction eluted with the 0.3 mol / L NaCl solution is collected, concentrated, dialyzed and freeze-dried; the dextran gel column chromatography uses a Sephadex G-100 column, eluted with a 0.3 mol / L NaCl solution, and the eluate is collected, and the anthrone-sulfuric acid method is used to trace, and the purified fractions are collected, concentrated, dialyzed and freeze-dried to obtain Typhae Pollen acidic polysaccharide.

[0026] Specifically, the pollen used is the dried pollen of Typha angustifolia L.

[0027] A pharmaceutical composition comprises the Typhaceae acid polysaccharide and pharmaceutically acceptable excipients.

[0028] The use of the Puhuang acid polysaccharide or the pharmaceutical composition in the preparation of drugs for endothelial cell dysfunction and vascular leakage. Further, the improvement of vascular integrity is through SOX7 nuclear translocation mediated by the VEGFA / PI3K / mTOR pathway, thereby upregulating VE-cadherin expression.

[0029] The application of the Typhae Pollen acidic polysaccharide or the pharmaceutical composition in the preparation of a medicine or product for preventing and / or treating blood stasis and bleeding.

[0030] The blood stasis and bleeding symptoms include cardiovascular and cerebrovascular bleeding, gynecological bleeding, renal hematuria and fundus hemorrhage.

[0031] Beneficial effects:

[0032] The present invention prepares a high-purity pollen acidic polysaccharide TPP-4, which has not been reported in the literature. It is surprisingly found that TPP-4 can improve vascular integrity, and further improve the permeability changes of vascular endothelial cells caused by inflammation, that is, it has a protective effect on the integrity of endothelial cells under inflammatory conditions. Among them, TPP-4 can promote the nuclear translocation of SOX7 and upregulate the expression of VE-cadherin through the VEGFA / PI3K / mTOR signaling pathway, thereby enhancing vascular integrity, reducing leakage and bleeding, and is used for regulating vascular inflammation and / or improving microcirculation. Further using the acidic polysaccharide to treat blood stasis and bleeding (cardiovascular and cerebrovascular bleeding, gynecological bleeding, renal hematuria and fundus hemorrhage, etc.) is a new treatment idea.

[0033] The details are as follows:

[0034] Acidic polysaccharides are fluffy, white flakes with many protrusions on the surface and low esterification degree. The average molecular weight is 1.5×10 5 -4.0×10 5 Da; Among them: TPP-4 can improve the changes in vascular endothelial cell permeability caused by inflammation in vitro; TPP-4 can improve the vascular exudation caused by inflammation in zebrafish in vivo; TPP-4 can inhibit the expression of inflammatory factors IL-1β, IL-6 and TNFα, and reduce vascular inflammation; TPP-4 can upregulate the protein expression of VEGFA and PI3K, promote the phosphorylation expression of mTOR, promote the nuclear translocation expression of SOX7, and promote the upregulation of VE-cadherin expression; SOX7 knockdown can weaken the improvement effect of TPP-4 on vascular integrity; BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention more clear, the drawings involved in the embodiments are briefly described below.

[0036] Figure 1 Characterization diagram of Typhaceae acidic polysaccharide TPP-4. (A and B) Representative SEM images. (C) EDS spectrum. (D) UV spectrum. (E) Fourier transform infrared spectrum. (F) Elution curve of Sephadex G-100 column. (G) HPGPC analysis. (H) HPAEC-PAD analysis.

[0037] Figure 2 This is the ion fragmentation pattern of methylation GC-MS of Puhuang acidic polysaccharide TPP-4.

[0038] Figure 3 This is the NMR spectrum analysis of Typha typhae acidic polysaccharide TPP-4. (A) 1 H-NMR spectrum. (B) 13 C-NMR spectrum. (C) HSQC spectrum. (D) 1 H-TOCSY spectrum. (E) HSQC-TOCSY spectrum. (F) NOESY spectrum. (G) HMBC spectrum. (H) Schematic diagram of the structure of TPP-4.

[0039] Figure 4 The protective effect of Typha puerariae acidic polysaccharide TPP-4 on inflammation-induced in vitro vascular permeability. (A) Effect of TPP-4 on HUVECs cell viability (n=6); (B) Effect of TPP-4 on LPS-induced HUVECs cell viability (n=6); (C) Schematic diagram of cell permeability detection; (D) Cell permeability detection; (E) Changes in resistance value during 2h of LPS induction (n=3); (F) Resistance value after 24h of LPS induction (n=3).

[0040] Figure 5 The protective effect of Typha typhinae acidic polysaccharide TPP-4 on inflammation-induced vascular permeability in vivo. (A) Main phenotypes of zebrafish larvae treated with 100 μg / mL LPS (bright field); (B) Vascular leakage of transgenic zebrafish (fluorescence); (C) Statistical graph of bleeding points; (D) Statistical graph of bleeding percentage (n=10).

[0041] Figure 6RNA-Seq reveals potential targets of Pollen Typha acidic polysaccharide TPP-4. (A) Volcano plot of differentially expressed genes. (B) Differentially expressed gene enrichment analysis (left: model group vs control group; right: model group vs TPP-4 group). (C) GSEA analysis. (D) Venn diagram. (E) Heat map of 20 intersection genes.

[0042] Figure 7 TPP-4, an acidic polysaccharide from Puhuang, maintains vascular endothelial integrity by regulating the VEGF signaling pathway. (A) mRNA expression of IL-6 (n=3). (B) mRNA expression of IL-1β (n=3). (C) mRNA expression of TNFα (n=3). (D) Representative images of western blot (n=3). (E) Relative protein expression of VEGFA. (F) Relative protein expression of PI3K. (G) Relative protein expression of mTOR. (H) Relative protein expression of p-mTOR. (I) Relative total protein expression of SOX7. (J) Relative protein expression of VE-cadherin. (K) Representative images of IF (n=5). (L) Statistical graph of F-actin. (M) Statistical graph of VE-cadherin.

[0043] Figure 8 The effect of SOX7 knockdown on the improvement of vascular barrier damage by Puhuang acidic polysaccharide TPP-4. (A) IF of SOX7 nuclear translocation (n=5). (B) Representative images of SOX7 nuclear expression (n=3). (C) Relative protein expression of SOX7 in the nucleus. (D) Relative mRNA expression of shSOX7 after cell transfection (n=3). (E) Representative images of cells transfected with shSOX7 (n=3). (F) Relative protein expression of SOX7 after cells transfected with shSOX7. (G) Representative images of VE-cadherin after SOX7 knockdown (n=3). (H) Relative protein expression of VE-cadherin after SOX7 knockdown. (I) Relative mRNA expression of CDH5 after SOX7 knockdown (n=3). (J) Representative images of VE-cadherin after SOX7 knockdown (n=5). DETAILED DESCRIPTION

[0044] To further illustrate the technical solution of the present invention, the following embodiments will provide specific examples. It should be noted that these embodiments are only for illustrative purposes, and the protection scope of the present invention is not limited thereto, and all equivalent modifications without creative work are included.

[0045] Unless otherwise specified, all raw materials and reagents used in the following examples are conventional commercial products or can be prepared according to standard methods, and the experimental methods and conditions are conventional methods recognized in the art, and specific references can be made to relevant experimental manuals or literature. At the same time, the meanings of all technical and scientific terms herein are consistent with the common understanding of ordinary technicians in the field.

[0046] Example 1

[0047] 1. Preparation of Typha Pollen Acidic Polysaccharide TPP-4

[0048] Take the pollen of Typha acuta and place it in a No. 7 sieve, keep it horizontal and sieve it to remove impurities. Take the treated pollen of Typha acuta, add 10 times the amount of water, reflux extract for 2h, repeat the extraction 3 times, filter and combine the filtrate, and concentrate under reduced pressure to obtain the water extract of Typha acuta. Add 4 times the amount of anhydrous ethanol to the water extract of Typha acuta, let it stand at room temperature overnight, filter under reduced pressure to take the precipitate, and vacuum dry to obtain a crude polysaccharide mixture of Typha acuta. Prepare Sevag reagent according to chloroform: n-butanol = 4:1 (V / V), mix the crude polysaccharide mixture solution with Sevag reagent in a ratio of 5:1, place it in a separating funnel, shake it fully, recover the crude polysaccharide extract, repeat it many times until there is no protein precipitation at the interface between the polysaccharide extract and the Sevag reagent, centrifuge to take the supernatant, vacuum concentrate, and freeze-dry to obtain the crude polysaccharide of Typha acuta.

[0049] Take 1g of the above crude polysaccharide and dissolve it in 20mL of distilled water, centrifuge at 4500rpm / min. The supernatant is separated using a DEAE-52 cellulose chromatography column, eluted with distilled water, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 and 2mol / L NaCl solution, collect fractions, and analyze using the anthrone-sulfuric acid method. The fractions eluted by 0.3mol / L NaCl solution are collected, concentrated, dialyzed and freeze-dried. The above sample is further purified by a Sephadex G-100 chromatographic column, eluted with a 0.3mol / L NaCl solution, and detected again using the anthrone-sulfuric acid method, with the tube number as the horizontal coordinate and the absorbance value as the vertical coordinate, draw an elution curve, and merge the peak tip according to the elution curve, and obtain Typha latifolia Typha latifolia acidic polysaccharide TPP-4 after reduced pressure concentration and freeze-drying.

[0050] 2. Determination of the homogeneity and molecular weight of Typha puerariae acidic polysaccharide TPP-4

[0051] The determination was performed by high performance gel permeation chromatography (HPGPC). This experiment was performed on a Waters 2695 system equipped with an ELSD 6000 detector. A TSK gel G4000 PWXL column (7.8 mm × 30 cm, 10 μm) was used for chromatographic separation. The mobile phase was ammonium acetate solution (20 mM) with a flow rate of 1.0 mL / min. A series of molecular weight dextran standards were used to establish a calibration curve and calculate the molecular weight. As shown in the Sephadex G-100 chromatogram ( Figure 1 F) and HPGPC chromatogram ( Figure 1 G) shows that TPP-4 is a single symmetrical peak, which is a polysaccharide with uniform molecular weight. The average molecular weight is calculated to be 2.5×10 5 Da.

[0052] 3. Characterization of Pollen Typhae Acidic Polysaccharide TPP-4

[0053] Microscopic morphology and spectral analysis

[0054] Take 5 mg of TPP-4 and fix it evenly on the sample holder, evenly coat it with platinum powder, observe each sample at an accelerating voltage of 5.0 kV, and analyze the chemical element composition of the TPP-4 surface with a tandem energy spectrum analyzer. Figure 1 As shown in AC, TPP-4 has a fluffy, white flake appearance with many protrusions on the surface, and the surface element composition does not contain nitrogen, indicating that there is no protein in the polysaccharide.

[0055] Take KBr powder and mix it to make a tablet. Take 20 mg TPP-4 and dissolve it in water. Take a small amount of liquid and drop it in the middle of the pressed tablet. Heat it under an infrared lamp to evaporate the solvent. -1 Infrared spectrum scanning is performed within the range. Figure 1 D, at 3200~3600cm -1 A strong and broad peak appears in the region, which can be attributed to the stretching vibration of OH. -1 The peak at 1760-1730cm is generated by the CH stretching vibration of the methylene group (-CH2). -1 The characteristic absorption of carbonyl band is observed at 1630~1600cm -1 Produced by the stretching vibration of the methylated carboxyl group, 1410-1400cm -1 Produced by asymmetric and symmetric stretching of C=O. -1 and 1411cm -1 There is a strong absorption peak at 1760-1730cm -1 No absorption peak was observed at the position, indicating that TPP-4 is an acidic polysaccharide with a low degree of esterification.

[0056] Take 20 mg of TPP-4 and dissolve it in water. Use a UV spectrophotometer to scan the polysaccharide solution at the full wavelength range of 200-450 nm. Figure 1 As shown in E, TPP-4 has no absorption at 260 and 280 nm, indicating that the polysaccharide does not contain protein.

[0057] 4. Monosaccharide composition analysis

[0058] A pulsed amperometric detector (PAD) (Dionex ICS 5000 system) was used as a detector. A CarboPac PA-20 anion exchange column (3×150 mm) was used for chromatographic separation. The mobile phase A was water, the mobile phase B was 0.1 M NaOH solution, and the mobile phase C was 0.1 M NaOH-0.2 M NaAc. The elution gradient was set as follows: 0 min A phase / B phase / C phase (95:5:0, V / V), 26 min A phase / B phase / C phase (85:5:10), 42 min A phase / B phase / C phase (85:5:10, V / V), 42.1 min A phase / B phase / C phase (60:0:40, V / V), 52 min A phase / B phase / C phase (60:40:0, V / V), 52.1 min A phase / B phase / C phase (95:5:0, V / V), 60 min A phase / B phase / C phase (95:5:0, V / V). The flow rate was set to 0.5 mL / min. The results showed that TPP-4 was composed of rhamnose (28.21%), galactose (20.88%), galacturonic acid (19.65%), arabinose (17.28%), glucose (6.38%), xylose (6.11%) and a small amount of mannose (1.49%). Figure 1 H). The higher levels of rhamnose, galactose, galacturonic acid, and arabinose indicated that TPP-4 might be a pectic polysaccharide.

[0059] 5. Glycosyl fragment analysis

[0060] Carboxyl reduction reaction: 100 mg TPP-4 was dissolved in a precooled hydrochloric acid imidazole solution (pH = 7.00, 1 M). Subsequently, NaBD4 was added and reacted on ice overnight. The next day, acetic acid was added dropwise to neutralize the excess NaBD4, and the reaction product was collected after dialysis, concentrated, and freeze-dried. Then, the reaction product was redissolved in distilled water, and a mixed solution containing 200 μL 2-(N-morpholino)ethanesulfonic acid (MES, pH = 4.75, 0.2 M) and 400 μL carbodiimide reagent (500 mg / mL) was added and reacted overnight. It was divided into two equal parts and reacted with NaBD4 / NaBH4 (70 mg / mL) respectively. Repeat the above steps several times to ensure that the carboxyl group is completely reduced.

[0061] Methylation reaction: The carboxyl-reduced sample was completely dried and dissolved in anhydrous DMSO, and then NaOH powder was added and stirred for 3 h under nitrogen. In addition, CH3I solution was added for overnight reaction and terminated with water. The fully methylated TPP-4 was then subjected to hydrolysis reaction (TFA, 2 M, 115 ° C), reduction reaction (1 M NaBD4-2 M NH4OH, 2 h) and acetylation reaction (pyridine-acetic anhydride, 100 ° C oil bath, 1 h). Finally, the corresponding partially methylated alditol acetates (PMAAs) were determined by GC-MS (Agilent 7890-7000C system). Chromatographic separation was performed using an Agilent HP5-MS capillary column (30m×0.25mm×0.25μm). The injection temperature and detector temperature were 200 ° C and 230 ° C, respectively. The column temperature program was as follows: initial temperature was 100 °C for 3 min, then increased to 200 °C at a rate of 20 °C / min, held for 2 min, then increased to 230 °C in the next 10 min, held for 5 min, and then increased to 280 °C in the next 8 min, using nitrogen (>99.999%) as carrier gas with a flow rate of 1 mL / min. A total of 11 PMAAs were identified by careful comparison with the standard spectra. Table 1 summarizes the relative abundance (Mol%) and main ion fragments ( Figure 2 ). The xylose bond might be degraded during the acid-catalyzed acetylation process, so xylose-associated PMAA was not detected.

[0062] Table 1. Glycosyl bond composition of carboxyl-reduced TPP-4

[0063]

[0064] 6. NMR analysis

[0065] 100 mg of TPP-4 was dissolved in D2O for NMR analysis. One-dimensional spectrum ( 1 HNMR and 13 CNMR)( Figure 3 A and B) and two-dimensional spectroscopy (including TOCSY, NOESY, HSQC and HMBC) ( Figure 3 C-G) were measured by NMR spectrometer (600 MHz, Bruker, Rheinstetten, Germany) at room temperature. 1 H-NMR, 13 C-NMR spectroscopy, using HSQC, 1 The H-TOCSY, HSQC-TOCSY, NOESY and HMBC spectra assigned the H and C of TPP-4, and the analysis results of the monosaccharide composition and infrared spectrum of the polysaccharide were combined, such as Figure 3H, the structure of TPP-4 is a RG-I type pectin polysaccharide with →(3)-β-D-Manp-(1→, →3,4)-β-D-Galp-(1→, →2)-α-L-Rhap-(1→, →4)-β-D-Galp-(1→, →4)-α-D-GalpA-(1→ and →2,4)-α-L-Rhap-(1→ as the main chain, and the side chains are mainly composed of β-D-Galp-(1→, α-D-Glcp-(1→, →5-α-L-Araf-(1→, →2)-α-L-Rhap-(1→, →3-α-L-Araf-(1→, α-L-Araf-(1→, α-D-GalpA-(1→ and β-D-Xylp-(1→;

[0066] That is, the main chain is connected by the 1st position of 1,3-β-D-Manp to the 4th position of 1,3,4-β-D-Galp, the 1st position of 1,3,4-β-D-Galp to the 3rd position of another 1,3,4-β-D-Galp, the 1st position of 1,3,4-β-D-Galp to the 2nd position of 1,2-α-L-Rhap, the 1st position of 1,2-α-L-Rhap to the 4th position of 1,4-β-D-Galp. 4, the 1st position of 1,4-β-D-Galp is linked to the 2nd position of 1,2-α-L-Rhap, the 1st position of 1,2-α-L-Rhap is linked to the 4th position of 1,4-α-D-GalpA, the 1st position of 1,4-α-D-GalpA is linked to the 4th position of 1,2,4-α-L-Rhap, the 1st position of 1,2,4-α-L-Rhap is linked to the 4th position of 1,4-α-D-GalpA, and the 1st position of 1,4- The 1st position of α-D-GalpA is connected to the 4th position of 1,2,4-α-L-Rhap; T-β-D-Galp is connected to the 3rd position of 1,3,4-β-D-Galp, T-α-L-Araf is connected to the 2nd position of 1,2,4-α-L-Rhap, T-α-D-GalpA is connected to the 2nd position of 1,2,4-α-L-Rhap, and T-β-D-Xylp is connected to 1,2,4-α-L -Rhap forms four side chains at the 2 position; T-α-D-Glcp is connected to the 5 position of 1,5-α-L-Araf, the 1 position of 1,5-α-L-Araf is connected to the 2 position of 1,2-α-L-Rhap, the 1 position of 1,2-α-L-Rhap is connected to the 3 position of 1,3-α-L-Araf, and the 1 position of 1,3-α-L-Araf is connected to the 4 position of 1,3,4-β-D-Galp on the main chain.

[0067] Table 2. NMR spectrum assignment of TPP-4

[0068]

[0069] 7. Protective effect of TPP-4 on inflammation-induced vascular permeability

[0070] In vitro study on the protective effect on vascular permeability

[0071] HUVECs cells were seeded in 96-well plates for 24 hours and then incubated with 100 μL of different concentrations of TPP-4 (0, 12.5, 25, 50, 100, 200, 400 and 800 μg / mL) and LPS (1 μg / mL) for 24 hours. The results showed that TPP-4 did not affect cell proliferation regardless of whether HUVECs were exposed to an inflammatory environment ( Figure 4 A and B).

[0072] Transwell plates with a pore size of 0.4 μm were used for cell permeability assay. HUVECs were plated at 1×10 5 The cells were cultured overnight at a density of 1 μg / mL in the upper chamber, and medium containing LPS (1 μg / mL) and a series of TPP-4 concentrations (100, 200, 400, 800 μg / mL) was added and incubated for 24 hours. 20 μL of FITC-dextran solution (30 μg / mL) was added to each upper chamber. EX / EM The fluorescence intensity of the culture medium in the lower chamber was detected at 490 / 520 nm.

[0073] The above procedure was adopted to culture HUVECs and measure the cell transmembrane resistance. The results showed that TPP-4 could reduce FITC-dextran leakage and increase the resistance value, thereby improving the permeability of HUVECs ( Figure 4 CF).

[0074] 8. In vivo study on the protective effect on vascular permeability

[0075] Tg (flk1:GFP; gata-1:Dsred) embryos were randomly divided into groups 3 days after fertilization. The model group and the drug-treated group were treated with 100 μg / mL LPS to induce modeling, resulting in the destruction of vascular integrity. A series of concentrations of TPP-4 (200, 400, 800 μg / mL) were added to the drug-treated groups and incubated for another 24 hours. THUNDER Imaging Systems (Leica Biosystems, Heidelberg, Germany) was used to photograph and observe the pattern of each larva. The results showed that larvae exposed to 100 μg / mL LPS could form tail injuries, and bleeding spots could be observed around the posterior cardinal vein (PCV) ( Figure 5 A). Tg(flk1:GFP; gata-1:Dsred) is a double transgenic zebrafish strain, in which blood vessels show green fluorescence and red blood cells show red fluorescence. TPP-4 can reduce the leakage of red blood cells outside the PCV and intersegmental vessels (ISV) Figure 5 BD).

[0076] 9. Study on the mechanism of action of TPP-4 in improving vascular integrity

[0077] Potential targets revealed by RNA-seq

[0078] The global gene expression profiles of HUVECs treated with and without LPS and TPP-4 were detected, and bioinformatics analysis was performed on the corresponding cloud platform. The edgeR package was used to find differentially expressed genes (DEGs), and the screening criteria were set as |log2 FC|>1.5, P<0.05.

[0079] Compared with the control group, 55 up-regulated DEGs and 60 down-regulated DEGs were found in the model group. Compared with the model group, 84 up-regulated DEGs and 102 down-regulated DEGs were found in the TPP-4 group ( Figure 6 A). Enrichment analysis found that the biological processes affected by LPS can be divided into calcium ion-related pathways, inflammatory pathways, lipid metabolism, vascular integrity-related pathways and other processes, mainly concentrated in inflammatory pathways and lipid metabolism. At the same time, the pharmacological effects of TPP-4 also depend on the above processes and have a significant impact on pathways related to vascular integrity ( Figure 6 B).

[0080] GSEA analysis showed that LPS treatment inhibited the cell adhesion (CAMs) process, and TPP-4 was upregulated after treatment ( Figure 6 C). Use Venn diagram to obtain intersection genes ( Figure 6 D). Among them, IL-11 has been shown to be an anti-inflammatory and protective factor that can reduce oxidant-induced endothelial damage and reduce capillary leakage. SOX7 is a transcription factor specifically expressed in endothelial cells and plays a vital role in vertebrate cardiovascular development. SOX7 directly binds to the promoter region of VE-cadherin, thereby enhancing endothelial barrier function. Literature research found that during angiogenesis, the VEGF signaling pathway can upregulate the expression of SOX7 through mTOR. At the same time, SOX7 also acts as a positive feedback regulator of the VEGF pathway. The VEGFA / PI3K / mTOR axis is closely related to vascular inflammation and ECs damage protection. It is worth noting that mTOR is also a key gene regulating lipid biosynthesis and metabolism. Lipid metabolism disorders can induce VE-cadherin internalization, which has been shown to be associated with barrier damage. Enrichment analysis showed that lipid metabolism regulation plays a vital role in the therapeutic effect of TPP-4. As Figure 6As shown in E, the expression of IL-11 and SOX7 was decreased after LPS treatment, while it was increased after TPP-4 treatment. Therefore, it can be assumed that both SOX7 and lipid metabolism regulation act as downstream cascades of the VEGFA / PI3K / mTOR axis and jointly affect VE-cadherin expression.

[0081] TPP-4 maintains vascular endothelial integrity by regulating VEGF signaling pathway

[0082] LPS treatment increased the mRNA expression levels of inflammatory factors, while TPP-4 administration led to a decrease in these expressions ( Figure 7 AC). This suggests that TPP-4 has the potential to reduce LPS-induced inflammation, thereby creating a favorable microenvironment for the subsequent repair of vascular integrity. TPP-4 enhanced the protein expression levels of VEGFA and PI3K, thereby promoting the phosphorylation of mTOR, increasing the total protein expression of SOX7, and further upregulating the protein expression of VE-cadherin, thereby improving vascular integrity ( Figure 7 DJ). VE-cadherin can bind to F-actin to mediate cell-to-cell adhesion, thereby indirectly affecting cell permeability. Immunofluorescence suggests that F-actin and VE-cadherin can be improved by TPP-4 ( Figure 7 KM). Figure 8 As shown in A, SOX7 was expressed in both the cytoplasm and nucleus of the control and model groups. After TPP-4 administration, cytoplasmic expression was significantly reduced, and most of the SOX7 signal was concentrated in the nucleus. At the same time, increased SOX7 expression in the nucleus was observed. TPP-4 promoted the nuclear translocation of SOX7 and enhanced its nuclear expression, which was consistent with the overall trend of total protein expression observed ( Figure 8 AC).

[0083] Knockdown of SOX7 may affect the improvement of TPP-4 on vascular barrier damage

[0084] The role of mTOR in endothelial inflammatory injury has been widely validated, and inhibition or blockade of mTOR leads to a dose-dependent decrease in VE-cadherin expression. Therefore, we focused on studying the role of SOX7 in maintaining vascular integrity under inflammatory conditions. Western blot and qPCR analysis confirmed the successful knockdown of SOX7 in HUVECs. Cells transfected with shSOX7-2 were then used for further experiments ( Figure 8 DF). The results showed that the administration of TPP-4 failed to improve vascular barrier damage in the case of SOX7 knockdown ( Figure 8 GJ).

[0085] In summary, the present invention purifies and characterizes a novel acidic pollen polysaccharide TPP-4 from the pollen of Typha angustifolia L. TPP-4 is a pectin polysaccharide containing an RG-I skeleton, which has a protective effect on the integrity of endothelial cells under inflammatory conditions. TPP-4 can promote the nuclear translocation of SOX7 and upregulate the expression of VE-cadherin through the VEGFA / PI3K / mTOR signaling pathway, thereby enhancing vascular integrity and reducing leakage and bleeding.

[0086] The embodiments of the present invention are only used to illustrate the content of the invention, so as to facilitate the understanding of the principles and applications of the invention, and do not constitute a limitation on the scope of protection thereof. Any modification, deformation or equivalent replacement based on the technical concept of the present invention without departing from its core idea shall be included in the scope of protection of the present invention.

Claims

1. A Typhae acidic polysaccharide, characterized in that: The structure of the polysaccharide includes rhamnose, galactose, galacturonic acid, arabinose, glucose, xylose, and mannose.

2. The Typhae Pollen acidic polysaccharide according to claim 1, characterized in that: The backbone of the polysaccharide includes rhamnose, galactose and galacturonic acid; the branches of the polysaccharide contain galactose, glucose, arabinose, rhamnose, galacturonic acid and xylose.

3. A Typhae acidic polysaccharide according to claim 2, characterized in that: The main chain of the polysaccharide includes 1,3-β-D-Manp, 1,2-α-L-Rhap, 1,2,4-α-L-Rhap, 1,3,4-β-D-Galp, 1,4-β-D-Galp and 1,4-α-D-GalpA; the branch bag of the polysaccharide includes T-β-D-Galp, T-α-D-Glcp, 1,5-α-L-Araf, 1,2-α-L-Rhap, 1,3-α-L-Araf, T-α-L-Araf, T-α-D-GalpA and T-β-D-Xylp.

4. A Typhae acidic polysaccharide according to claim 3, characterized in that: The main chain of the polysaccharide is mainly composed of RG-I type pectin polysaccharides, namely, →3)-β-D-Manp-(1→, →3,4)-β-D-Galp-(1→, →2)-α-L-Rhap-(1→, →4)-β-D-Galp-(1→, →4)-α-D-GalpA-(1→ and →2,4)-α-L-Rhap-(1→); and the side chains are mainly composed of β-D-Galp-(1→, α-D-Glcp-(1→, →5-α-L-Araf-(1→, →2)-α-L-Rhap-(1→, →3-α-L-Araf-(1→, α-L-Araf-(1→, α-D-GalpA-(1→ and β-D-Xylp-(1→).

5. A Typhae acidic polysaccharide according to claim 4, characterized in that: The molecular weight of Typha tamarind acid polysaccharide is 1.5×10 5 -4.0×10 5 Da.

6. A method for preparing the Typhaceae acidic polysaccharide according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: (1) extracting the Typhae Pollen medicinal material by heating and refluxing with water to obtain a water extract; (2) concentrating the aqueous extract to obtain a concentrated solution; (3) subjecting the concentrated solution to ethanol precipitation, and filtering to obtain a precipitate; (4) dissolving the precipitate in water to obtain a sample solution, removing the protein therein using the Sevag method, and freeze-drying the solution to obtain Typha Pollen total polysaccharide; (5) The Typhae Pollen total polysaccharide is dissolved in water and then centrifuged, and the supernatant is subjected to anion exchange chromatography coupled with dextran gel chromatography to obtain Typhae Pollen acidic polysaccharide.

7. A pharmaceutical composition, comprising the Typhaceae acidic polysaccharide according to any one of claims 1 to 5 and pharmaceutically acceptable excipients.

8. Use of the Typhaceae acidic polysaccharide according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 7 in the preparation of a drug for treating endothelial cell dysfunction and vascular leakage.

9. Use of the Typhaceae acidic polysaccharide according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 7 in the preparation of a drug or product for preventing and / or treating blood stasis and bleeding.

10. The use according to claim 9, characterized in that: The blood stasis and bleeding syndromes include cardiovascular and cerebrovascular bleeding, gynecological bleeding, renal hematuria and fundus hemorrhage.

Images