A typhae pollen acid polysaccharide, and a preparation method and use thereof

Abstract

The application belongs to the technical field of medicine, and particularly relates to a pollen typhae acidic polysaccharide, 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. A brand new pollen typhae acidic polysaccharide TPP-4 is prepared in the application, the acidic polysaccharide can improve vascular integrity, and then can improve the change of vascular endothelial cell permeability caused by inflammation, that is, has a protective effect on the integrity of endothelial cells in an inflammatory state, and can be used for preventing and / or treating blood stasis and bleeding.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to an acidic polysaccharide from Typha pollen, its preparation method, and its uses. Background Technology

[0002] Both blood stasis and bleeding fall under the category of "blood disorders" in Traditional Chinese Medicine (TCM), and while they are mutually exclusive, they can also transform into each other. Blood stasis and bleeding caused by factors such as qi stagnation and blood cold are clinically common in gynecology, internal medicine, and trauma, such as functional uterine bleeding, cerebral hemorrhage, renal hematuria, and retinal hemorrhage. Western medicine treats blood stasis and bleeding primarily with antithrombotic drugs, hormones, or surgery, but these methods have limited efficacy, are subject to tolerance, and are prone to serious side effects such as bleeding.

[0003] Endothelial cells are semi-permeable, selectively regulatory barriers within blood vessels. Composed of endothelial cells arranged continuously in arteries, veins, and lymphatic vessels of various sizes, they participate in numerous physiological processes, including the regulation of vascular tone, maintenance of endothelial integrity, endothelial cell metabolism, platelet activity and interaction, hemostasis regulation, and endothelial injury and repair. With advancements in modern vascular biology, the concept of endothelial cell dysfunction has gained increasing attention, and the reversible regulation of endothelial dysfunction is currently a hot topic in cardiovascular disease research. Blood stasis and bleeding are characterized by impaired or reduced blood flow and activated coagulation function, leading to persistent endothelial cell hypoxia and oxidative stress, thereby inducing inflammatory responses, exacerbating endothelial dysfunction, and damaging vascular wall integrity, resulting in blood extravasation and microcirculatory damage. Eliminating vascular inflammation and improving endothelial function are important approaches to treating blood stasis and bleeding disorders.

[0004] Typha pollen, the dried pollen of Typha angustifolia L., Typha orientalis Presl, or related species in the Typhaceae family, can promote blood circulation, stop bleeding, and relieve urinary tract infections, and is particularly suitable for bleeding caused by blood stasis. Literature review has found that total extracts of Typha pollen can promote angiogenesis and stop bleeding in zebrafish models, and total polysaccharides from Typha pollen 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 focused on small molecule compounds, but research on the structure and efficacy of macromolecular components, especially their effects on promoting blood circulation and stopping bleeding, is still insufficient. Summary of the Invention

[0005] Purpose of the invention:

[0006] The present invention aims to prepare a novel acidic polysaccharide from Typhae pollen and to reveal its potential application prospects in improving vascular integrity.

[0007] Technical solution

[0008] A type of acidic polysaccharide from Typha pollen, characterized in that the polysaccharide comprises rhamnose, galactose, galacturonic acid, arabinose, glucose, xylose, and mannose.

[0009] The aforementioned acidic polysaccharide from Typha pollen is characterized in that the main chain of the polysaccharide comprises rhamnose, galactose, and galacturonic acid; and the branches of the polysaccharide comprise galactose, glucose, arabinose, rhamnose, galacturonic acid, and xylose.

[0010] The aforementioned acidic polysaccharide from Typhae pollen is characterized in that the main chain of the polysaccharide comprises 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; and the branched packages of the polysaccharide comprise 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 aforementioned acidic polysaccharide from Typha pollen is characterized in that the main chain of the polysaccharide is composed of RG-I type pectin polysaccharides consisting 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→); 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 aforementioned acidic polysaccharide from Typha pollen is characterized in that its molecular weight is 1.5 × 10⁻⁶. 5 -4.0×10 5 Da.

[0013] As an optimization method, the acidic polysaccharide, based on molar concentration percentage, consists 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%).

[0014] The method for obtaining the acidic polysaccharide from Typha pollen is characterized by comprising the following steps:

[0015] (1) Extract the pollen by heating and refluxing with water to obtain an aqueous extract;

[0016] (2) The aqueous extract was concentrated to obtain a concentrated solution;

[0017] (3) The concentrated solution was subjected to ethanol precipitation, and the precipitate was obtained by filtration;

[0018] (4) The precipitate was dissolved in water to obtain a sample solution. The protein was removed by the Sevag method and then freeze-dried to obtain total polysaccharides from Typhae pollen.

[0019] (5) Dissolve the total polysaccharide of Typha pollen in water and centrifuge. Separate the supernatant by anion exchange chromatography in series with dextran gel chromatography to obtain acidic polysaccharide of Typha pollen.

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

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

[0022] In step (2), the concentration conditions are as follows: concentration is carried out at 60°C using a vacuum rotary evaporator;

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

[0024] In step (4), the sample solution is mixed with Sevag reagent, shaken thoroughly, vortexed, and then transferred to a separatory funnel 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 with a volume ratio of 1:4. The ratio of sample solution to Sevag reagent is 5:1.

[0025] In step (5), anion exchange chromatography was performed using a DEAE-52 cellulose column. After loading the crude Typhae pollen polysaccharide, it was eluted sequentially with distilled water, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, and 2 mol / L NaCl solutions. The fractions were collected, and the polysaccharide was tracked using the anthrone-sulfuric acid method. The fraction eluted with 0.3 mol / L NaCl solution was collected, concentrated, dialyzed, and freeze-dried. Dextran gel column chromatography was performed using a Sephadex G-100 column. Elution was performed with 0.3 mol / L NaCl solution, and the eluent was collected. The purified fraction was tracked using the anthrone-sulfuric acid method, collected, concentrated, dialyzed, and freeze-dried to obtain the Typhae pollen acidic polysaccharide.

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

[0027] A pharmaceutical composition comprising the aforementioned Typhae pollen acidic polysaccharide and pharmaceutically acceptable excipients.

[0028] The application of the described Typhae pollen acidic polysaccharide or the described pharmaceutical composition in the preparation of drugs for endothelial cell dysfunction and vascular leakage. Furthermore, its effect on improving vascular integrity is mediated by SOX7 nuclear translocation via the VEGFA / PI3K / mTOR pathway, thereby upregulating VE-cadherin expression.

[0029] The application of the aforementioned Typhae pollen acidic polysaccharide or pharmaceutical composition in the preparation of drugs or products for the prevention and / or treatment of blood stasis and bleeding.

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

[0031] Beneficial effects:

[0032] This invention prepared high-purity Typhae pollen acidic polysaccharide TPP-4, which has not been reported in the literature. Surprisingly, TPP-4 can improve vascular integrity, thereby mitigating inflammation-induced changes in vascular endothelial cell permeability, thus protecting the integrity of endothelial cells under inflammatory conditions. TPP-4 can promote SOX7 nuclear translocation and upregulate VE-cadherin expression through the VEGFA / PI3K / mTOR signaling pathway, thereby enhancing vascular integrity and reducing leakage and bleeding. It can be used for regulating vascular inflammation and / or improving microcirculation. Furthermore, utilizing this acidic polysaccharide to treat blood stasis and bleeding syndromes (cardiovascular and cerebrovascular hemorrhage, gynecological bleeding, renal hematuria, and retinal hemorrhage, etc.) represents a novel therapeutic approach.

[0033] Specifically as follows:

[0034] Acidic polysaccharides are fluffy, white, flaky granules with numerous protrusions on the surface, and are low in degree of esterification. The average molecular weight is 1.5 × 10⁻⁶. 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 in zebrafish caused by inflammation 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 mTOR phosphorylation expression, promote SOX7 nuclear translocation expression, and promote the upregulation of VE-cadherin expression; SOX7 knockdown can weaken the effect of TPP-4 on improving vascular integrity; Attached Figure Description

[0035] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the accompanying drawings involved in the embodiments will be briefly described below.

[0036] Figure 1 Characterization of Typhae pollen acidic polysaccharide TPP-4. (A and B) Representative scanning electron microscope images. (C) EDS spectrum. (D) Ultraviolet 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 an ion fragmentation diagram from GC-MS of TPP-4 methylated acidic polysaccharide from Typhae pollen.

[0038] Figure 3 NMR spectral analysis of the acidic polysaccharide TPP-4 from Typhae pollen. (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 TPP-4 structure.

[0039] Figure 4 The protective effect of TPP-4, an acidic polysaccharide from Typhae pollen, 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 resistivity during 2 hours of LPS induction (n=3); (F) Resistivity after 24 hours of LPS induction (n=3).

[0040] Figure 5 To demonstrate the protective effect of Typhae pollen acidic polysaccharide TPP-4 on inflammation-induced vascular permeability in vivo. (A) Major phenotype of zebrafish larvae treated with 100 μg / mL LPS (bright field); (B) Vascular leakage in transgenic zebrafish (fluorescence); (C) Statistical graph of hemorrhage points; (D) Statistical graph of hemorrhage percentage (n=10).

[0041] Figure 6RNA-Seq was used to reveal potential targets of the acidic polysaccharide TPP-4 from Typhae pollen. (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) Heatmap of 20 intersecting genes.

[0042] Figure 7 The acidic polysaccharide TPP-4 from Typhae pollen maintains vascular endothelial integrity by regulating the VEGF signaling pathway. (A) IL-6 mRNA expression (n=3). (B) IL-1β mRNA expression (n=3). (C) TNFα mRNA expression (n=3). (D) Representative images from 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 image 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 the acidic polysaccharide TPP-4 from Typhae pollen. (A) IF of SOX7 nuclear translocation (n=5). (B) Representative image of SOX7 nuclear expression (n=3). (C) Relative protein expression of SOX7 in the cell nucleus. (D) Relative mRNA expression of shSOX7 after cell transfection (n=3). (E) Representative image of cells transfected with shSOX7 (n=3). (F) Relative protein expression of SOX7 after cell transfection with shSOX7. (G) Representative image 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 image of VE-cadherin after SOX7 knockdown (n=5). Detailed Implementation

[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 for illustrative purposes only, and the scope of protection of the present invention is not limited thereto; all equivalent modifications made without inventive effort are included.

[0045] Unless otherwise stated, all raw materials and reagents used in the following examples are commercially available products or can be prepared according to standard methods. The experimental methods and conditions are all conventional methods recognized in the art, and specific details can be found in relevant experimental manuals or literature. Furthermore, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0046] Example 1

[0047] 1. Preparation of Typha pollen acidic polysaccharide TPP-4

[0048] Pollen from *Typha orientalis* was placed in a No. 7 sieve and sieved horizontally to remove impurities. The treated pollen was then refluxed with 10 times its volume of water for 2 hours, repeated three times. The filtrates were combined after filtration and concentrated under reduced pressure to obtain an aqueous extract of *Typha orientalis*. The aqueous extract was then added to 4 times its volume of anhydrous ethanol and allowed to stand at room temperature overnight to precipitate. The precipitate was collected by filtration under reduced pressure and dried under vacuum to obtain a crude polysaccharide mixture from *Typha orientalis*. Sevag reagent was prepared by mixing chloroform and n-butanol in a ratio of 4:1 (V / V). The crude polysaccharide mixture solution was mixed with Sevag reagent at a ratio of 5:1 and placed in a separatory funnel. The mixture was shaken thoroughly, and the crude polysaccharide extract was recovered. This process was repeated several times until no protein precipitation was observed at the interface between the polysaccharide extract and the Sevag reagent. The supernatant was collected by centrifugation, concentrated under vacuum, and then freeze-dried to obtain crude polysaccharide from *Typha orientalis*.

[0049] 1 g of the crude polysaccharide was dissolved in 20 mL of distilled water and centrifuged at 4500 rpm / min. The supernatant was separated using a DEAE-52 cellulose column, eluted with distilled water, and 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, and 2 mol / L NaCl solutions. The fractions were collected and analyzed using the anthrone-sulfuric acid method. The fraction eluted with 0.3 mol / L NaCl solution was collected, concentrated, dialyzed, and lyophilized. The sample was further purified using a Sephadex G-100 column, eluted with 0.3 mol / L NaCl solution, and detected again using the anthrone-sulfuric acid method. An elution curve was plotted with the tube number on the x-axis and the absorbance on the y-axis. The peaks were merged according to the elution curve. After concentration under reduced pressure and lyophilization, the acidic polysaccharide TPP-4 of Typhae pollen was obtained.

[0050] 2. Homogeneity and molecular weight determination of Typhae pollen acidic polysaccharide TPP-4

[0051] The determination was performed by high-performance gel permeation chromatography (HPGPC). This experiment was conducted on a Waters 2695 system equipped with an ELSD 6000 detector. Chromatographic separation was performed using a TSK Gel G4000 PWXL column (7.8 mm × 30 cm, 10 μm). The mobile phase was ammonium acetate solution (20 mM), and the flow rate was 1.0 mL / min. A calibration curve was established using a series of dextran standards of varying molecular weights, and the molecular weight was calculated. The chromatogram is shown in the Sephadex G-100 chromatogram. Figure 1 F) and HPGPC chromatograms ( Figure 1 As shown in G), TPP-4 exhibits a single symmetrical peak, indicating it is a polysaccharide with a uniform molecular weight. Its calculated average molecular weight is 2.5 × 10⁻⁶. 5 Da.

[0052] 3. Characterization of the acidic polysaccharide TPP-4 from Typha pollen

[0053] Micromorphology and spectral analysis

[0054] 5 mg of TPP-4 was uniformly fixed onto the sample holder, and platinum powder was uniformly coated onto it. Each sample was observed under an accelerating voltage of 5.0 kV, and the surface chemical elemental composition of the TPP-4 was analyzed using a tandem energy dispersive spectroscopy (EDS) analyzer. Figure 1 As shown in AC, TPP-4 has a fluffy, white, flaky appearance with many protrusions on the surface. The surface elemental composition does not contain nitrogen, indicating that there is no protein in the polysaccharide.

[0055] Mix KBr powder and compress into tablets. Dissolve 20 mg of TPP-4 in water, take a small amount of the liquid and drop it onto the center of the compressed tablet. Heat under an infrared lamp to evaporate the solvent, at 4000–4000 °C. -1 Infrared spectral scanning was performed within the specified range. For example... Figure 1 As shown in D, in the range of 3200–3600 cm -1 A strong and broad peak appears in the region, which can be attributed to the stretching vibration of OH. 2934 cm⁻¹ -1 The peak at 1760–1730 cm⁻¹ is generated by the CH stretching vibration of the methylene group (-CH₂). -1 Characteristic absorption of the carbonyl band was observed at 1630–1600 cm⁻¹, and its intensity was positively correlated with the degree of methylation of the carboxyl group. -1 Produced by the stretching vibration of the methylated carboxyl group, 1410–1400 cm⁻¹ -1 Produced by asymmetric and symmetric stretching of C=O. At 1618cm -1 and 1411cm -1 A strong absorption peak is observed at 1760–1730 cm⁻¹, indicating the presence of free carboxyl groups. However, a strong absorption peak is observed at 1760–1730 cm⁻¹. -1 No absorption peak was observed, indicating that TPP-4 is an acidic polysaccharide with a low degree of esterification.

[0056] Dissolve 20 mg of TPP-4 in water and perform a full-wavelength scan of the polysaccharide solution using a UV spectrophotometer, with a scanning wavelength range of 200–450 nm. Figure 1 As shown in Figure E, TPP-4 showed no absorption at 260 and 280 nm, indicating that the polysaccharide does not contain protein.

[0057] 4. Monosaccharide composition analysis

[0058] A pulsed ampere detector (PAD) (Dionex ICS 5000 system) was used as the detector. Chromatographic separation was performed using a CarboPac PA-20 anion exchange column (3×150 mm). Mobile phase A was water, mobile phase B was 0.1 M NaOH solution, and mobile phase C was 0.1 M NaOH-0.2 M NaAc. The elution gradients were set as follows: 0 min A / B / C (95:5:0, V / V), 26 min A / B / C (85:5:10), 42 min A / B / C (85:5:10, V / V), 42.1 min A / B / C (60:0:40, V / V), 52 min A / B / C (60:40:0, V / V), 52.1 min A / B / C (95:5:0, V / V), and 60 min A / B / C (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). Higher levels of rhamnose, galactose, galacturonic acid, and arabinose suggest that TPP-4 may be a pectin polysaccharide.

[0059] 5. Glycosyl fragment analysis

[0060] Carboxyl reduction reaction: 100 mg TPP-4 was dissolved in pre-cooled imidazole hydrochloride solution (pH = 7.00, 1 M). NaBD4 was then added and the reaction was carried out overnight on ice. The next day, acetic acid was added dropwise to neutralize excess NaBD4. The reaction product was collected after dialyzing, concentrated, and lyophilized. The reaction product was then redissolved in distilled water, and a mixed solution containing 200 μL of 2-(N-morpholino)ethanesulfonic acid (MES, pH = 4.75, 0.2 M) and 400 μL of carbodiimide reagent (500 mg / mL) was added, and the reaction was carried out overnight. The product was divided into two equal portions and reacted separately with NaBD4 / NaBH4 (70 mg / mL). The above steps were repeated several times to ensure complete reduction of the carboxyl group.

[0061] Methylation reaction: The carboxyl-reduced sample was completely dried and dissolved in anhydrous DMSO, then NaOH powder was added, and the mixture was stirred under nitrogen for 3 h. Subsequently, CH3I solution was added for overnight reaction, and the reaction was terminated with water. Fully methylated TPP-4 was then subjected to hydrolysis (TFA, 2 M, 115 °C), reduction (1 M NaBD4-2 M NH4OH, 2 h), and acetylation (pyridine-acetic anhydride, 100 °C oil bath, 1 h). Finally, the corresponding partially methylated aldose acetates (PMAAs) were determined by GC-MS (Agilent 7890-7000C system). Chromatographic separation was performed using an Agilent HP5-MS capillary column (30 m × 0.25 mm × 0.25 μm). The injection temperature and detector temperature were 200 °C and 230 °C, respectively. The column temperature program was as follows: initial temperature 100℃, held for 3 min, increased to 200℃ at a rate of 20℃ / min, held for 2 min, increased to 230℃ over the next 10 min, held for 5 min, and then increased to 280℃ over the next 8 min. Nitrogen (>99.999%) was used as the carrier gas at a flow rate of 1 mL / min. A total of 11 PMAAs were identified through careful comparison with standard spectra. Table 1 summarizes the relative abundance (Mol%) and main ion fragments (Mol%) of each glycosyl bond. Figure 2 Xylose bonds may degrade during acid-catalyzed acetylation, therefore xylose-associated PMAAs were 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 spectroscopy ( 1 HNMR and 13 CNMR)( Figure 3 A and B) and two-dimensional spectra (including TOCSY, NOESY, HSQC, and HMBC) Figure 3 C–G) were measured using an NMR spectrometer (600 MHz, Bruker, Rheinstetten, Germany) at room temperature. 1 H-NMR, 13 C-NMR spectroscopy analysis, using HSQC, 1 H-TOCSY, HSQC-TOCSY, NOESY, and HMBC spectra assigned the H and C atoms to TPP-4. The combined analysis results of the polysaccharide's monosaccharide composition and infrared spectroscopy, etc., show that... Figure 3As shown in H, the structure of TPP-4 is →(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 of RG-I type pectin polysaccharide, 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 formed by connecting bit 1 of 1,3-β-D-Manp to bit 4 of 1,3,4-β-D-Galp, bit 1 of 1,3,4-β-D-Galp to bit 3 of another 1,3,4-β-D-Galp, bit 1 of 1,3,4-β-D-Galp to bit 2 of 1,2-α-L-Rhap, and bit 1 of 1,2-α-L-Rhap to bit 1,4-β-D-Galp. 4 bits, 1 bit of 1,4-β-D-Galp connects to 2 bits of 1,2-α-L-Rhap, 1 bit of 1,2-α-L-Rhap connects to 4 bits of 1,4-α-D-GalpA, 1 bit of 1,4-α-D-GalpA connects to 4 bits of 1,2,4-α-L-Rhap, 1 bit of 1,2,4-α-L-Rhap connects to 4 bits of 1,4-α-D-GalpA, 1,4- The first bit of α-D-GalpA is connected to the fourth bit of 1,2,4-α-L-Rhap; T-β-D-Galp is connected to the third bit of 1,3,4-β-D-Galp; T-α-L-Araf is connected to the second bit of 1,2,4-α-L-Rhap; T-α-D-GalpA is connected to the second bit of 1,2,4-α-L-Rhap; and T-β-D-Xylp is connected to the third bit of 1,2,4-α-L-Rhap. - The 2nd position of Rhap forms 4 branches; T-α-D-Glcp connects to the 5th position of 1,5-α-L-Araf, the 1st position of 1,5-α-L-Araf connects to the 2nd position of 1,2-α-L-Rhap, the 1st position of 1,2-α-L-Rhap connects to the 3rd position of 1,3-α-L-Araf, and the 1st position of 1,3-α-L-Araf connects to the 4th position of 1,3,4-β-D-Galp on the main chain.

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

[0068]

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

[0070] In vitro studies on its protective effect on vascular permeability

[0071] HUVECs were seeded in 96-well plates for 24 hours, and then incubated for another 24 hours 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). 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] Cell permeability was measured using Transwell plates with a pore size of 0.4 μm. HUVECs were measured at 1 × 10⁻⁶ wells. 5 The cells were cultured overnight in the upper chamber at a density suitable for incubation. Afterward, they were incubated for 24 hours in medium containing LPS (1 μg / mL) and a series of concentrations of TPP-4 (100, 200, 400, 800 μg / mL). 20 μL of FITC-dextran solution (30 μg / mL) was added to each upper chamber. EX / EM The fluorescence intensity of the lower cavity culture medium was detected at 490 / 520 nm.

[0073] HUVECs were cultured using the above procedure, and transmembrane resistance was measured. The results showed that TPP-4 reduced FITC-glucan leakage and increased resistance, thereby improving HUVEC permeability. Figure 4 CF).

[0074] 8. In vivo studies on its protective effect against vascular permeability

[0075] Embryos of Tg(flk1:GFP; gata-1:Dsred) 3 days after fertilization were randomly divided into groups. The model group and the treatment group were subjected to 100 μg / mL LPS to induce vascular integrity disruption. The treatment groups were then incubated with a series of concentrations of TPP-4 (200, 400, and 800 μg / mL) for 24 hours. Images of each larva were taken using THUNDER Imaging Systems (Leica Biosystems, Heidelberg, Germany). Results showed that larval exposure to 100 μg / mL LPS resulted in tail injury, with hemorrhages observed around the posterior main vein (PCV). Figure 5 A). Tg(flk1:GFP;gata-1:Dsred) is a double transgenic zebrafish strain with green fluorescence in its blood vessels and red fluorescence in its erythrocytes. TPP-4 can reduce the leakage of erythrocytes from the perivascular space of the periocular vessel (PCV) and intersegmental vessel (ISV) into the extravascular space. Figure 5 BD).

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

[0077] Potential targets revealed by RNA-seq

[0078] The whole-genome expression profiles of HUVECs with and without LPS and TPP-4 were analyzed using bioinformatics on the corresponding cloud platforms. The edgeR package was used to identify differentially expressed genes (DEGs), and the selection criteria were set as |log2 FC|>1.5, P<0.05.

[0079] Compared to the control group, the model group showed 55 upregulated DEGs and 60 downregulated DEGs. Compared to the model group, the TPP-4 group showed 84 upregulated DEGs and 102 downregulated DEGs. Figure 6 A). Enrichment analysis revealed that LPS-affected biological processes can be categorized into calcium-related pathways, inflammatory pathways, lipid metabolism, vascular integrity-related pathways, and other processes, primarily concentrated in inflammatory and lipid metabolism pathways. Simultaneously, the pharmacological effects of TPP-4 also depend on these processes, significantly impacting pathways related to vascular integrity. Figure 6 B).

[0080] GSEA analysis showed that LPS treatment inhibited cell adhesion (CAM) processes, while TPP-4 treatment upregulated ( Figure 6 C). Using Venn diagrams to obtain intersection genes ( Figure 6 (D) Among them, IL-11 has been shown to be an anti-inflammatory and protective factor, reducing oxidant-induced endothelial damage and capillary leakage. SOX7 is a transcription factor specifically expressed in endothelial cells and plays a crucial role in vertebrate cardiovascular development. SOX7 directly binds to the promoter region of VE-cadherin, thereby enhancing endothelial barrier function. Literature review found that during angiogenesis, the VEGF signaling pathway upregulates SOX7 expression through mTOR. Simultaneously, SOX7 also acts as a positive feedback regulator of the VEGF pathway. The VEGFA / PI3K / mTOR axis is closely related to vascular inflammation and protection against EC damage. Notably, 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 disruption. Enrichment analysis showed that lipid metabolism regulation plays a crucial role in the therapeutic effect of TPP-4. Figure 6As shown in Figure E, LPS treatment decreased the expression of both IL-11 and SOX7, while TPP-4 treatment increased their expression. Therefore, it can be hypothesized that SOX7 and lipid metabolism regulation both function as downstream cascade reactions of the VEGFA / PI3K / mTOR axis, jointly affecting VE-cadherin expression.

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

[0082] LPS treatment increases the mRNA expression levels of inflammatory cytokines, while TPP-4 administration leads to a decrease in these expression levels. Figure 7 This suggests that TPP-4 may alleviate LPS-induced inflammation, thereby creating a favorable microenvironment for subsequent vascular integrity repair. TPP-4 enhances the protein expression levels of VEGFA and PI3K, thereby promoting mTOR phosphorylation, increasing the total protein expression of SOX7, and further upregulating the protein expression of VE-cadherin, thus improving vascular integrity. Figure 7 DJ). VE-cadherin can bind to F-actin to mediate cell adhesion, thereby indirectly affecting cell permeability. Immunofluorescence suggests that F-actin and VE-cadherin can be aggravated by TPP-4 (…). Figure 7 KM). For example Figure 8 As shown in Figure 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, with most SOX7 signaling concentrated in the nucleus. Simultaneously, increased SOX7 expression in the nucleus was observed. TPP-4 promoted the nuclear translocation of SOX7 and enhanced its nuclear expression, consistent with the overall trend observed in total protein expression. Figure 8 AC).

[0083] Knockdown of SOX7 can affect the ameliorative effect 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 investigating 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, under SOX7 knockdown, administration of TPP-4 failed to improve vascular barrier damage (DF). Figure 8 GJ).

[0085] In summary, this invention purified and characterized a novel acidic Typha angustifolia L. pollen, TPP-4. TPP-4 is a pectin polysaccharide containing an RG-I backbone that protects the integrity of endothelial cells under inflammatory conditions. TPP-4 can promote SOX7 nuclear translocation and upregulate VE-cadherin expression through the VEGFA / PI3K / mTOR signaling pathway, thereby enhancing vascular integrity and reducing leakage and bleeding.

[0086] The embodiments of this invention are merely illustrative of the invention to facilitate understanding of its principles and applications, and do not constitute a limitation on its scope of protection. Any modifications, variations, or equivalent substitutions made based on the technical concept of this invention, without departing from its core ideas, should be included within the scope of protection of this invention.

Claims

1. The application of Typha pollen acidic polysaccharide or pharmaceutical composition in the preparation of drugs for treating endothelial cell dysfunction and vascular leakage. Its features are, The main chain of the Typhae pollen acidic polysaccharide is mainly composed of RG-I type pectin polysaccharides 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→); 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→); The molecular weight of the acidic polysaccharide from Typhae pollen is 1.5 × 10⁻⁶. 5 -4.0×10 5 Da; The pharmaceutical composition comprises the acidic polysaccharide of Typhae pollen and pharmaceutically acceptable excipients.

2. The use of Typha pollen acidic polysaccharide or pharmaceutical composition in the preparation of drugs for the prevention and / or treatment of blood stasis and bleeding, characterized in that, The main chain of the Typhae pollen acidic polysaccharide is mainly composed of RG-I type pectin polysaccharides 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→); 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→); The molecular weight of the acidic polysaccharide from Typhae pollen is 1.5 × 10⁻⁶. 5 -4.0×10 5 Da; The pharmaceutical composition comprises the acidic polysaccharide of Typhae pollen and pharmaceutically acceptable excipients.

3. The application according to claim 2, characterized in that, The blood stasis and bleeding symptoms include cardiovascular and cerebrovascular bleeding, gynecological bleeding, renal hematuria, and retinal hemorrhage.

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