A novel structural polysaccharide derived from the roots of *Trifolium repens* and its combination with vitamin E and its applications.

Abstract

This invention discloses a novel structural polysaccharide SYQE3-F derived from the roots of *Tripterygium wilfordii*, its preparation method, pharmaceutical composition, and applications, belonging to the field of biomedical technology. This invention is the first to isolate and purify a homogeneous polysaccharide SYQE3-F from the roots of *Tripterygium wilfordii*, with an average molecular weight of 13.174 kDa. Its main chain consists of →4)-β-D-GlcpA-(1→) and →2)-α-D-Manp-(1→), and its side chains include t-Galp and t-Araf. This invention also discloses a pharmaceutical composition containing this polysaccharide and vitamin E. In vitro and in vivo experiments show that this polysaccharide can effectively break down choroidal occlusion barriers and significantly promote the transport of vitamin E to the target retinal tissue, providing a novel natural drug combination for the clinical treatment of DR.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and natural drug extraction technology, specifically to a novel polysaccharide *Trifolium repens* SYQE3-F, its preparation method, a pharmaceutical composition comprising the polysaccharide and vitamin E, and its application in the prevention and / or treatment of diabetic retinopathy. Background Technology

[0002] Diabetic retinopathy (DR) is one of the most common complications of diabetes and a serious blinding eye disease, falling under the category of "Xiao Ke Mu Bing" (wasting and thirsting eye disease) in Traditional Chinese Medicine (TCM). Obstruction of the eye meridians and stagnation of Qi, blood, and body fluids are important mechanisms in the onset of this eye disease; therefore, unblocking the eye meridians is one of the key principles of TCM treatment for DR.

[0003] *Tetrastigma hemsleyanum* Diels et Gilg, Sanyeqing, SYQ is an evergreen herbaceous vine belonging to the genus *Tetrastigma* in the Vitaceae family. It is one of the "New Eight Zhejiang Herbs" due to its unique pharmacological activities of promoting blood circulation, clearing heat, and detoxifying. The *Compendium of Materia Medica* states, "All vines can unblock meridians and collaterals, for vines twine like a net, their shape resembling veins." Vine-like medicinal plants typically possess the characteristic of readily traveling and unblocking meridians and collaterals.

[0004] Current research on *Trifolium repens* mainly focuses on its tuberous roots, with limited exploration of the chemical composition and pharmacological activity of the rootlets. In the pathological progression of diabetic retinopathy (DR), severe oxidative stress damage and microvascular occlusion are often present, and key antioxidants (such as vitamin E) face significant transport barriers when targeting retinal tissue, leading to poor efficacy of conventional antioxidant therapies. Therefore, there is an urgent need to develop novel drugs or drug compositions that can overcome this transport barrier and effectively alleviate DR lesions. Summary of the Invention

[0005] This invention aims to address the shortcomings of existing treatments for diabetic retinopathy (DR), particularly the problem of impaired vitamin E transport to retinal tissue in the pathological state of DR. It provides a novel polysaccharide SYQE3-F extracted from the roots of Tripterygium wilfordii with a clearly defined structure, and provides its preparation method, pharmaceutical composition, and medicinal uses.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] This invention discloses a novel polysaccharide SYQE3-F derived from the roots of *Trifolium repens*, the structure of which is as follows:

[0008] .

[0009] Preferably, the average molecular weight of the polysaccharide SYQE3-F is 13.174 kDa.

[0010] Preferably, the monosaccharide composition of the above polysaccharide SYQE3-F, by molar percentage, includes: mannose 26.771%, glucuronic acid 26.904%, galacturonic acid 10.163%, galactose 31.550%, and arabinose 4.613%.

[0011] Preferably, the above-mentioned *Trifolium repens* polysaccharide SYQE3-F is glucuronic acid galactomannan, whose main chain is composed of →4)-β-D-GlcpA-(1→ and →2)-α-D-Manp-(1→) and is linked with methylated galacturonic acid. Its side chains include t-Galp and t-Araf, and most of the side chains are linked to the O-3 positions of →2)-α-D-Manp-(1→) in the main chain.

[0012] The sugar residues of the above-mentioned *Trifolium repens* polysaccharide SYQE3-F include 1,2,3-Manp, 1,2-Manp, t-Galp, 1,4-GlcpA, 1,4-GalpA, and t-Araf, and the molar ratio of the sugar residues 1,2,3-Manp, 1,2-Manp, t-Galp, 1,4-GlcpA, 1,4-GalpA, and t-Araf is approximately 8:1:10:6:2:1;

[0013] The 1H and 1C NMR spectra of *Trifolium repens* polysaccharide SYQE3-F are as follows: The anomeric protons and anomeric carbon chemical shifts δ of each sugar residue are as follows:

[0014] The anisotope proton chemical shift of →2,3)-α-D-Manp-(1→ is 5.26 ppm, and the anisotope carbon chemical shift is 98.71 ppm;

[0015] →2)-α-D-Manp-(1→) has an antecedent proton chemical shift of 5.26 ppm and an antecedent carbon chemical shift of 98.71 ppm;

[0016] The anomeric proton chemical shift of α-D-Galp-(1→ is 5.14 ppm, and the anomeric carbon chemical shift is 100.97 ppm;

[0017] The anomeric proton chemical shift of β-L-Araf-(1→ is 5.22 ppm, and the anomeric carbon chemical shift is 102.09 ppm;

[0018] →4)-β-D-GlcpA-(1→ The anterior proton chemical shift is 4.36 ppm, and the anterior carbon chemical shift is 102.66 ppm;

[0019] The anterior proton chemical shift of →4)-α-D-GalpA-6-OMe-(1→ is 5.01 ppm, and the anterior carbon chemical shift is 99.29 ppm.

[0020] Preferably, the above-mentioned polysaccharide SYQE3-F is derived from the root parts of Tetrastigma hemsleyanumbi.

[0021] This invention also provides a method for preparing the above-mentioned *Trifolium repens* polysaccharide SYQE3-F, comprising the following steps: (1) Extraction: Take *Trifolium repens* root powder, add water and reflux to extract, filter, combine the filtrates and concentrate under reduced pressure to obtain a concentrated solution, add ethanol to the above concentrated solution to a final concentration of 80%, let it stand to precipitate, centrifuge to collect the precipitate and dry it to obtain crude *Trifolium repens* polysaccharide; (2) Deproteinization and dialysis: Redissolve the above-mentioned crude *Trifolium repens* polysaccharide, remove the protein using the Sevage method, then perform dialysis, collect the dialysis fluid and dry it to obtain purified *Trifolium repens* polysaccharide; (3) Ion exchange chromatography: Redissolve the above-mentioned purified *Trifolium repens* polysaccharide and load it onto a DEAE Sepharose FF ion exchange chromatography column, perform gradient elution with different concentrations of NaCl solution, collect the 0.5M NaCl eluent, dialyze and dry it; (4) Gel filtration chromatography: The fraction obtained in step (3) was reconstituted and loaded onto a Chromdex gel filtration chromatography column, eluted with water, and the eluted fraction in the 165-185 min region was collected, concentrated and dried to obtain the above-mentioned Trifolium repens polysaccharide SYQE3-F.

[0022] Preferably, the conditions for water reflux extraction in step (1) above are: add 20 times the volume of deionized water, reflux extract at 95°C for 2 hours, and extract 3 times; the conditions for static precipitation are: stand at 4°C for 24 hours.

[0023] Preferably, in step (2) above, the Sevage method for protein removal specifically involves mixing the polysaccharide solution with the Sevage reagent at a volume ratio of 4:1. The Sevage reagent is a mixture of chloroform and n-butanol at a volume ratio of 4:1. The dialysis is performed using a dialysis bag with a molecular weight cutoff of 3.5 kDa for 48 hours.

[0024] The present invention also discloses a pharmaceutical composition comprising the above-mentioned polysaccharide SYQE3-F of Clematis chinensis, vitamin E, and pharmaceutically acceptable excipients or carriers.

[0025] The present invention also discloses the use of the above-mentioned pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of diabetic retinopathy.

[0026] Preferably, the above-described pharmaceutical composition is used in the preparation of a medicament that promotes the transport of vitamin E to retinal tissue.

[0027] Preferably, the above-described pharmaceutical composition is used in the preparation of a medicament for scavenging peroxides; preferably, the peroxides include reactive oxygen species (ROS), malondialdehyde (MDA), and lipid reactive oxygen species (lipid ROS).

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] 1. This invention is the first to isolate and purify a homogeneous polysaccharide SYQE3-F from the roots of *Trifolium repens*, with an average molecular weight of 13.174 kDa. This invention clarifies the precise monosaccharide composition, main-side chain linkage (the main chain consists of →4)-β-D-GlcpA-(1→ and →2)-α-D-Manp-(1→, with most side chains linked at the O-3 positions), and NMR characteristics of this polysaccharide, providing a clear material basis for the study of the "unblocking meridians" efficacy mechanism of vine-type traditional Chinese medicines and the development of natural drugs.

[0030] 2. This invention is the first to discover that the combined use of *Trifolium repens* polysaccharide SYQE3-F and Vitamin E has a significant synergistic effect. In the pathological state of DR, Vitamin E mainly accumulates in the serum, with a low content transported to the retinal tissue (due to transport barriers); while SYQE3-F can effectively break through this choroidal occlusion barrier, significantly promote the transport of Vitamin E to the target site of retinal tissue, and restore the level of Vitamin E in the retina.

[0031] 3. In vitro and in vivo pharmacological experiments have confirmed that SYQE3-F combined with Vitamin E can significantly improve the clearance rate of reactive oxygen species (ROS), malondialdehyde (MDA), and lipid ROS, effectively improving high glucose-induced damage to retinal pigment epithelial cells (APRE-19). In a diabetic mouse model, this combination regimen can significantly reduce linear atrophy and dendritic branching of retinal vessels, effectively alleviating the progression of diabetic retinopathy, and has extremely high clinical translational value. Attached Figure Description

[0032] Figure 1 This is the elution curve of the ion exchange column chromatography of the present invention.

[0033] Figure 2 The elution curve of the SYQ-E3 gel filtration column chromatography for the sample of this invention is shown.

[0034] Figure 3 The liquid chromatogram of the SYQE3-F sample of this invention is shown, with peak A at 17.220, peak B at 24.822, peak C at 28.090, peak D at 39.448, and peak E at 43.608.

[0035] Figure 4 This is a PAS-stained retinal vascular patch of the present invention, in which the red arrow indicates the linear atrophic vascular lumen, 400×, scale bar = 50 μm, where NM is the normal mouse group, DR is the diabetic retinopathy group, DR+Vit E is the diabetic retinopathy group treated with vitamin E, and DR+Vit E+SYQE3-F is the diabetic retinopathy group after administration of the combination composition of the present invention.

[0036] Figure 5 This study compared serum and retinal tissue vitamin E levels in diabetic retinopathy (DR) model mice after the combined use of Tripterygium wilfordii polysaccharide SYQE3-F and vitamin E. A represents the comparison of serum vitamin E levels, and B represents the comparison of retinal vitamin E levels. *P<0.05. NM = normal group, DR = diabetic retinopathy group, vitamin E = vitamin E, SYQ = Tripterygium wilfordii, and units are mol / L.

[0037] Figure 6 This study investigated the effect of the combined use of *Tripterygium wilfordii* polysaccharide SYQE3-F and Vitamin E on the clearance of peroxides (ROS, MDA, and lipid ROS) from Vitamin E in in vitro and in vivo high glucose models. In this study, A represents the effect of the combined use of *Tripterygium wilfordii* polysaccharide SYQE3-F and Vitamin E on ROS clearance; B represents the effect of the combined use of *Tripterygium wilfordii* polysaccharide SYQE3-F and Vitamin E on MDA clearance; and C represents the effect of the combined use of *Tripterygium wilfordii* polysaccharide SYQE3-F and Vitamin E on lipid ROS clearance. *P<0.05, **P<0.01; where NM = normal group, DR = diabetic retinopathy group, Vitamin E, SYQ = *Tripterygium wilfordii*, ROS = reactive oxygen species, MDA = malondialdehyde, and lipid ROS = lipid reactive oxygen species. Detailed Implementation

[0038] The present invention will be described in more detail below with reference to the embodiments. It should be understood that the implementation of the present invention is not limited to the embodiments below, and any modifications or alterations made to the present invention fall within the protection scope of the present invention; and the methods in the following embodiments, unless otherwise specified, are conventional methods in the art.

[0039] Example 1: Extraction, separation and purification of Trifoliate orange polysaccharide SYQE3-F

[0040] First, select fresh, mold-free *Trifolium repens* roots and rinse them repeatedly with pure water to remove surface mud and impurities. Then, dry them in a 60℃ constant temperature drying oven until constant weight to avoid damaging the polysaccharide activity due to high temperature. After drying, pulverize the raw material in a high-speed grinder and pass it through an 80-100 mesh sieve to obtain uniform *Trifolium repens* powder, facilitating the full dissolution of polysaccharides during subsequent extraction. Place a certain amount of powder in an extraction container, add 20 times (g / ml) of deionized water, and reflux at 95℃ for 2 hours, stirring continuously to improve extraction efficiency. After extraction, filter using a Buchner funnel, collect the filtrate, and continue extraction of the residue under the same conditions twice. Combine all filtrates to minimize polysaccharide loss. Place the combined filtrate in a rotary evaporator and concentrate it under reduced pressure at 45-55℃ and vacuum to 1 / 5 of the original volume to obtain a concentrated solution. Then, pre-cooled 95% ethanol was slowly added to the cooled concentrate while stirring until the final ethanol concentration reached 80%. The mixture was then allowed to stand in a 4°C refrigerator for 24 hours to allow the polysaccharides to fully precipitate. Subsequently, the mixture was centrifuged at 4000 r / min for 20 min, the precipitate was collected, and freeze-dried to obtain crude polysaccharide from *Trifolium repens*.

[0041] The crude polysaccharide was then reconstituted with an appropriate amount of deionized water to prepare a polysaccharide solution of a certain concentration. The Sevage method was used to remove proteins: the polysaccharide solution was mixed with Sevage reagent (chloroform: n-butanol = 4:1) at a volume ratio of 4:1, stirred vigorously for 30 minutes, allowed to stand, centrifuged to separate the layers, and the chloroform layer and intermediate protein layer were removed. This process was repeated 3-4 times until no obvious protein layer appeared. The deproteinized polysaccharide solution was placed in a dialysis bag and dialyzed with deionized water (dialysis bag molecular weight cutoff 3.5 kDa) for 48 hours, changing the deionized water every 8 hours to remove small molecule impurities (such as monosaccharides, inorganic salts, etc.). The dialysate was collected, concentrated, and freeze-dried to obtain the purified *Trifolium repens* polysaccharide.

[0042] Ion exchange column chromatography

[0043] (1) Pretreatment of ion exchange chromatography media: Pour the DEAE sepharose FF gel suspension into a Buchner funnel, remove the liquid, and wash with about 3 times the volume of ultrapure water. Repeat the above operation several times until the packing material has no alcohol smell. Transfer it to a beaker, add half to one times the volume of distilled water of the precipitated gel, and stir well in preparation for column packing.

[0044] (2) Column packing and equilibration: After stirring the pretreated DEAE sepharose FF gel suspension, slowly add it to the XK chromatography column (φ3.0×50cm). After complete sedimentation, connect the upper column head to the peristaltic pump and start the pump at a flow rate of 250cm / h to flush the column with ultrapure water until the gel surface is stable, thus completing the column packing.

[0045] (3) Sample loading and elution: The polysaccharide sample was dissolved in an appropriate amount of pure water, centrifuged at 8000 rpm for 10 min to remove the precipitate, filtered through a 0.45 μm microporous membrane, and the filtrate was loaded onto an equilibrated ion exchange chromatography column. The sample loading volume was 30% of the column volume. After all the sample solution entered the chromatography column, it was eluted sequentially according to different salt concentrations (0, 0.1, 0.2, 0.5, 1.0 M NaCl). Each concentration was eluted twice the column volume at a elution rate of 15 ml / min. The eluent was collected by an automatic fraction collector. 100 tubes were collected for each eluent gradient, and 10 ml was collected from each tube. The anthrone-sulfuric acid method was used, and the polysaccharide content in the eluent was tracked and detected by an enzyme-linked immunosorbent assay reader at 630 nm. The polysaccharide elution curve was obtained by plotting the number of tubes on the x-axis and the absorbance on the y-axis. The eluent from each tube with different elution peaks was collected, and the ion exchange chromatography was repeated multiple times. The collected eluent components were combined, concentrated under reduced pressure, dialyzed using a dialysis bag with a filtration cutoff of 3.5 kDa, and then freeze-dried under vacuum to obtain the individual components.

[0046] (4) Results of ion exchange column chromatography

[0047] The crude polysaccharide sample was subjected to ion exchange column chromatography, with gradient elution using different salt concentrations of 0, 0.1, 0.2, and 0.5 M NaCl solutions. The polysaccharide content in each tube was determined using the anthrone-sulfuric acid method, and the resulting elution curves are shown below. Figure 1 As shown, the samples collected from left to right are: SYQ-E1 (eluted with 0 M NaCl), SYQ-E2 (eluted with 0.2 M NaCl), SYQ-E3 (eluted with 0.5 M NaCl), and SYQ-E4 (eluted with 1.0 M NaCl). After one cycle of gradient elution, the 2.0 mol / L NaCl eluent was analyzed using the anthrone-sulfuric acid method. No obvious color reaction was observed, indicating that the crude polysaccharide was completely eluted from the ion exchange chromatography column. After polysaccharide content testing and lyophilization, the polysaccharide content of the fraction eluted with 0.2 M NaCl was significantly higher than that of the fractions eluted with 0 M NaCl, 0.1 M NaCl, and 0.5 M NaCl solutions.

[0048] Gel filtration column chromatography

[0049] (1) Pretreatment of gel filtration chromatography medium: Pour the Chromdex 75PG gel suspension into a Buchner funnel, remove the liquid, and wash with about 3 times the volume of ultrapure water. Repeat the above operation several times until the packing material has no alcohol smell. Transfer it to a beaker, add half to one times the volume of distilled water of the precipitated gel, and stir well in preparation for column packing.

[0050] (2) Column packing and equilibration: After stirring the pretreated gel filtration chromatography medium suspension, slowly add it to the XK chromatography column (φ2.6×100cm). After complete sedimentation, connect the upper column head to the chromatography system and flush the column with ultrapure water at a flow rate of 30cm / h until the gel surface is stable to complete the column packing.

[0051] (3) Sample loading and elution: The polysaccharide sample prepared by ion exchange column chromatography was dissolved in an appropriate amount of dH2O, centrifuged at 8000 rpm for 10 min to remove the precipitate, filtered through a 0.45 μm microporous membrane, and the filtrate was loaded onto a pre-equilibrated gel filtration chromatography column at a volume of 1% of the column volume. After all the sample solution entered the chromatography column, it was eluted with ultrapure water at a flow rate of 1.5 ml / min. The eluent was collected by an automatic fraction collector, with 3 ml collected per tube, eluting one column volume. The collection was monitored online using a differential detector, and the eluent with higher peak height and better symmetry in the same elution peak region was collected. The gel filtration column chromatography was repeated multiple times to enrich and purify the polysaccharide. The combined collected eluent fractions were concentrated under reduced pressure and freeze-dried under vacuum to obtain the polysaccharide purified by gel filtration column chromatography.

[0052] (4) Results of gel filtration column chromatography

[0053] Based on the elution curves obtained from ion column chromatography and the molecular weight test results, sample SYQ-E3 was selected as the sample for gel filtration column chromatography. The elution curves obtained after gel filtration column chromatography are as follows: Figure 2 As shown, the eluted fraction from the 165-185 min region was collected, concentrated under reduced pressure, freeze-dried under vacuum, and subjected to repeated gel filtration chromatography to obtain the purified polysaccharide, named SYQE3-F.

[0054] Example 2: Identification of polysaccharide purity and determination of molecular weight distribution

[0055] (1) Establishment of molecular weight calibration curve: Weigh dextran standards of different molecular weights (analytical standards of molecular weights of 180, 666, 6000, 9800, 21900, 46500, 110000, 193000, 353000, 692000, and 1060000), add 0.05M NaCl solution to prepare standard solutions, filter with a 0.22μm microporous membrane for later use, and use the HPGPC method with a high-performance gel permeation chromatography tandem column for detection. Perform linear regression with the logarithm of the relative molecular mass of the standard as the ordinate and the retention time of the corresponding chromatographic peak as the abscissa to obtain the calibration curve.

[0056] (2) Preparation of test sample solution: Weigh the purified polysaccharide sample, add 0.05M NaCl solution to the sample to prepare a 5mg / ml test sample solution, take the supernatant and filter it with a 0.22μm microporous membrane, and then transfer the sample to a 2ml injection bottle for later use.

[0057] (3) Chromatographic method: HPGPC method was adopted, using a high performance liquid chromatograph with a differential detector, and Ohpak SB-804 HQ and Ohpak SB-806 HQ (8×300 mm) tandem columns of polymer matrix water-soluble SEC (GFC) chromatographic column. The mobile phase was 0.05M NaCl solution, the flow rate was 0.5 ml / min, the column temperature was 40 ºC, and the injection volume was 30 μl.

[0058] (4) Results of molecular weight correction curve

[0059] A linear regression was performed with the logarithm of the relative molecular mass of the standard as the ordinate and the elution volume (V) of the corresponding chromatographic peak as the abscissa to obtain the molecular weight correction curve, as shown below:

[0060] Log molecular weight = 1.82e+01 - 2.13e+00V^1 + 1.30e-01V^2 - 3.05e-03V^3, R 2 =0.9998.

[0061] (5) Sample test results

[0062] The molecular weight of each sample was calculated based on the calibration curve of the standard.

[0063] Sample name: SYQE3-F (3.5 kDa dialysis)

[0064] The peak around 43.3 min is the mobile phase salt peak. Excessively high salt peaks and subsequent peaks may contain small molecules. Molecular weight determination by HPGPC showed that the polysaccharide SYQE3-F component was a single symmetrical peak, indicating that SYQE3-F is a homogeneous polysaccharide with an average molecular weight of 13.174 kDa. Further structural analysis of SYQE3-F will be conducted later.

[0065] Example 3: Preparation of Standards for Monosaccharide Composition Determination

[0066] 5 mg each of rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid, glucuronic acid, glucosamine hydrochloride, and glucosamine galactose hydrochloride monosaccharides, and 10 mg of fucose, were dissolved and diluted to 10 ml in a volumetric flask to prepare a standard stock solution (see table below). The solution was then further diluted to the following serial dilution levels, filtered through a 0.22 μm microporous membrane, and then transferred to sample vials.

[0067]

[0068] Preparation of test sample solution

[0069] (1) Solid sample extraction

[0070] Take a clean chromatographic vial, weigh 2.5 mg of the polysaccharide sample, add 1 mL of 2M TFA acid solution, and heat at 110℃ for 4 hours. Purge with nitrogen and dry. Add 1 mL of methanol to wash, then dry again, repeating the methanol washing 2-3 times. Dissolve in 1 mL of sterile water, transfer to a chromatographic vial for analysis.

[0071] (2) Liquid sample extraction

[0072] Take an appropriate amount of the supernatant and concentrate by rotation or dry under nitrogen. Add 1 ml of 2M TFA solution and heat at 110 °C for 4 hours. Purge with nitrogen and dry. Wash with methanol, then dry again, repeating the methanol washing 2-3 times. Dissolve in sterile water and transfer to a chromatographic vial for analysis.

[0073] PMP Derivatization

[0074] Take 0.2 mL of monosaccharide standard solution or polysaccharide hydrolysate into a stoppered conical centrifuge tube, add 0.2 mL of 0.5 mol / L sodium hydroxide solution and 0.5 mL of 0.5 mol / L PMP methanol solution, vortex to mix, and react in a 70 ℃ water bath for 2 h. After the reaction is complete, add 0.2 mL of 0.5 mol / L hydrochloric acid to neutralize the added sodium hydroxide, add 1 mL of chloroform, vortex extract 3 times to remove excess PMP, discard the chloroform layer, and take 0.3 mL and dilute with water to 1 mL.

[0075] Chromatographic methods

[0076] The Thermo U3000 liquid chromatography system was used, with a ZORBAX EclipseXDB-C18 column (4.6*250mm, 5μm). The mobile phase was acetonitrile: phosphate buffer (12g / L potassium dihydrogen phosphate, pH adjusted to 6.8 with 2M NaOH) isocratic elution at a volume ratio of 17:83. The flow rate was 0.8ml / min, the column temperature was 30ºC, the detection wavelength was 250nm, and the injection volume was 10μl.

[0077] Correction curve results

[0078] The project employs the external standard method for quantification, establishing a standard curve by preparing standard samples of different concentrations. The linear relationship information is as follows:

[0079] .

[0080] Polysaccharide composition calculation

[0081] SYQE3-F mass: 2.63 mg; total molar amount of all components: 0.544 mol.

[0082]

[0083] Example 4: Nuclear Magnetic Resonance Detection and Structural Identification

[0084] The lyophilized sample was dissolved in 0.5 ml of D2O and measured using a 600 MHz Bruker NMR spectrometer. One-dimensional NMR (1H-NMR, 13C-NMR) and two-dimensional NMR (HSQC, COSY, TOCSY, HMBC, NOESY) were performed. Calibration: HDO hydrogen δH = δ 4.70 ppm, TMS carbon δC = δ 0.00 ppm.

[0085] To further obtain structural feature information of polysaccharide sample SYQE3-F, one-dimensional NMR spectroscopy (HSQC, COSY, TOCSY, HMBC, NOESY) was performed to obtain H and C chemical shift information of each major sugar residue and to infer the connection sequence between each sugar residue.

[0086] One-dimensional proton nuclear magnetic resonance (¹H-NMR) spectroscopy was used to further identify the glycosidic bond configuration of the polysaccharide samples. Most of the proton signals in the polysaccharide spectrum were in the δ 3.0–5.5 ppm range, with the δ 4.3–5.5 ppm range typically representing the anodic proton (H-1) resonance region. In the ¹H-NMR spectra of the polysaccharide samples, a large number of proton resonance signals were concentrated in the δ 3.0–5.5 ppm region, exhibiting significant signal overlap. Multiple anodic hydrogen signals were found in the anodic region (δ 4.3–5.5 ppm), indicating the presence of various sugar residues with some overlap, making them difficult to distinguish. Other hydrogen signals were concentrated in the δ 4.3–3.0 ppm region, with significant signal overlap, making them difficult to assign. Compared to the ¹H-NMR spectrum, the ¹³C-NMR spectrum of the polysaccharide samples had fewer spectral lines, with multiple anodic carbons present in the δ 90–110 ppm anodic carbon region, indicating the presence of various sugar residues in the polysaccharide samples.

[0087] In the monosaccharide composition test and methylation analysis results, the monosaccharide composition was mainly composed of Man, Gal, GlcA, and GalA. The methylation analysis results mainly contained 1,2,3-Manp, 1,2-Manp, t-Galp, 1,4-GlcpA, 1,4-GalpA, and t-Araf sugar residues. Based on the monosaccharide composition and methylation analysis results of the polysaccharide samples, analysis of 1H-NMR, 13C-NMR, HSQC, COSY, and TOCSY NMR spectra revealed multiple anodic signals. The polysaccharide samples showed significant cross-peaks in multiple anodic regions in the HSQC and COSY spectra. Combined with literature reports, these findings can be used for structural analysis.

[0088] (1) A strong cross-peak signal exists in the HSQC anodic region at δ 5.26 / 98.71 ppm (H-1 / C-1). Cross-peaks of anodic hydrogens at δ 5.26 / 4.16 ppm (H-1 / H-2) and δ 5.26 / 4.04 ppm (H-1 / H-2) can be found in the COSY spectrum. Combining the methylation analysis results and literature reports, it can be inferred that the anodic signal belongs to the sugar residue of α-Manp. First, the H-1 chemical shift of the sugar residue was determined to be δ 5.26 ppm using HSQC and COSY. Then, the H-2, H-3, H-4, and H-5 signals can be deduced from the cross-peaks in the COSY and TOCSY spectra. The H-2, H-3, H-4, and H-5 chemical shifts of this sugar residue are assigned to δ 4.16 ppm, δ 3.88 ppm, δ 3.67 ppm, and δ 3.80 ppm, respectively. The H-6a and H-6b shifts of the sugar ring were assigned using HSQC spectra, with values ​​of δ 3.68 ppm and δ 3.78 ppm, respectively. After assigning the chemical shifts of the hydrogen atoms on the sugar ring, the chemical shifts of C-1 to C-6 on the sugar ring were assigned using HSQC correlation spectra, with values ​​of δ 98.71 ppm, δ 78.75 ppm, δ 75.96 ppm, δ 68.79 ppm, δ 69.33 ppm, and δ 59.62 ppm, respectively. The chemical shifts of C-1, C-2, and C-3 towards the lower field indicate that the residues have been substituted at positions C-1, C-2, and C-3 of the sugar ring. Based on literature reports, the sugar residues are inferred to be →2,3)-α-D-Manp-(1→, labeled M1,2,3. Following a similar method, the chemical shifts of the sugar residues corresponding to the COSY signal δ 5.26 / 4.04 ppm (H-1 / H-2) were derived using HSQC, COSY, and TOCSY. The chemical shifts of C-1 and C-2 towards the lower field indicate that the residues have been substituted at positions C-1 and C-2 of the sugar ring. Based on literature reports, the sugar residues are inferred to be →2)-α-D-Manp-(1→, labeled M1,2.

[0089] (2) A strong cross-peak signal of 4.36 / 102.66 ppm (H-1 / C-1) was found near the region of 4.30~4.42 ppm in the proton spectrum and 101~103 ppm in the carbon spectrum in the HSQC anomaly region. A cross-peak signal of 4.36 / 3.27 ppm was also found in the COSY spectrum for anomaly hydrogens in this region. Based on the methylation analysis results and literature reports, it can be inferred that the anomaly signal mainly belongs to the β-GlcpA sugar residue. The H-1 chemical shift of the sugar residue was determined to be 4.36 ppm using HSQC and COSY. Similarly, using COSY and HSQC, and considering the cross-peak of 3.64 / 174.62 ppm in the HMBC spectrum, the H-5 signal was obtained as 3.64 ppm and the C-6 signal as 174.62 ppm. The chemical shifts of C-1 and C-4 to lower fields indicate that the sugar residues have been substituted at the C-1 and C-4 positions of the sugar ring. Based on literature reports, the sugar residues are inferred to be →4)-β-D-GlcpA-(1→, labeled as GlcA1,4.

[0090] (3) A strong cross-peak signal δ 5.14 / 100.97 ppm was found in the HSQC anodic region, and a strong cross-peak signal δ 5.14 / 3.69 ppm (H-1 / H-2) was found in the COSY spectrum. Based on the methylation analysis results and literature reports, it can be inferred that the anodic signal mainly belongs to the sugar residue α-Galp. Using COSY, TOCSY and HSQC for deduction, combined with the methylation analysis results and literature reports, it is inferred that this sugar residue is α-D-Galp-(1→, labeled as Gt, and its chemical shift is assigned in the table below.

[0091]

[0092] (4) Through monosaccharide composition determination and polysaccharide methylation analysis, it was found that the polysaccharide sample also contained the sugar residue 1,4-GalpA. A distinct characteristic signal was observed on the HSQC spectrum at δ 3.72 / 52.85 ppm and δ 5.07 / 70.59 ppm. Combined with the methylation analysis results and literature reports, this is consistent with the characteristics of methylated galacturonic acid sugar residues. Furthermore, a cross-peak of δ 5.01 / 3.67 ppm was found on the COSY spectrum, an anterior cross-peak of δ 5.01 / 99.29 ppm (H-1 / C-1) was found on the HSQC spectrum, and a cross-peak of δ 5.07 / 171.01 ppm was found on the HMBC spectrum, yielding a chemical shift of δ 5.07. ppm, and then using COSY and HSQC for derivation, combined with literature reports, the chemical shifts of C-1 and C-4 shifted to the lower field, indicating that the residue was substituted at the C-1 and C-4 positions of the sugar ring, and H-5 underwent a large lower field shift. This is because the introduction of the methoxy group caused the chemical shifts of each proton in the sugar ring to shift. Combining the polysaccharide methylation results and literature reports, it is inferred that the sugar residue is →4)-α-GalpA-6-OMe-(1→, labeled GE1,4).

[0093] (5) Cross peaks of δ 5.22 / 102.09 ppm were found in the HSQC anomaly region. Based on the deduction of HSQC and COSY, combined with polysaccharide methylation analysis and literature reports, this is consistent with the Araf signal characteristics. It is inferred that the sugar residue is β-L-Araf-(1→, labeled as Aβt, and its chemical shift is assigned in the table above.

[0094] According to the polysaccharide methylation analysis results, the polysaccharide sample also contained 1,6-Galp, 1,3,6-Galp, t-GlcpA and other linkages. Since the content of these sugar residues was less than 3%, the signals in NMR were very weak, and their 1H and 13C could not be assigned.

[0095] By analyzing the coupling signals of anomeric hydrogens with carbons on each sugar residue in the HMBC long-range correlation spectrum, or the coupling signals of anomeric carbons with hydrogens on each sugar residue, and considering that two protons at the connection sites of adjacent sugar residues tend to generate strong NOE signals due to their close spatial positions, the interconnection sequence between sugar residues can be further inferred using the HMBC long-range correlation spectrum and the NOESY spectrum. The HMBC correlation spectrum and NOESY spectrum of the polysaccharide sample are shown. The following coupling signals can be found from the figure: (1) In the HMBC spectrum, the H-1 (δ 5.26 ppm) of sugar residue M1,2,3 and the C-4 (δ 77.07 ppm) of sugar residue GlcA1,4 have a correlation signal peak (M1,2,3 H-1 / GlcA1,4 C-4), and the H-4 (δ 3.71 ppm) of sugar residue GlcA1,4 and the C-1 (δ 98.71 ppm) of sugar residue M1,2,3 have a correlation signal peak (GlcA1,4 H-4 / M1,2,3 C-1). In the NOESY spectrum, the H-1 (δ 5.26 ppm) of sugar residue M1,2,3 and the H-4 (δ 3.71 ppm) of sugar residue GlcA1,4 have a cross peak (M1,2,3 H-1 / M1,2,3 C-1). H-1 / GlcA1,4 H-4), indicating the presence of →2,3)-α-D-Manp-(1→ and →4)-β-D-GlcpA-(1→ connection, the connection site is located at position O-4, and related signal peaks GlcA1,4 H-1 / M1,2,3 C-2 and M1,2,3 H-2 / GlcA1,4 C-1 were found in the HMBC spectrum, indicating the presence of →4)-β-D-GlcpA-(1→ and →2,3)-α-D-Manp-(1→ connection, the connection site is located at position O-2; (2) In the HMBC spectrum, the H-1 (δ 5.14 ppm) of sugar residue Gt and the C-3 (δ 75.96 ppm) of sugar residue M1,2,3 have related signal peaks (Gt H-1 / M1,2,3 C-3), and M1,2,3 H-3 / Gt exists in the HMBC spectrum. In the C-1, NOESY spectrum, the H-1 (δ 5.14 ppm) of sugar residue Gt and the H-3 (δ 3) of sugar residues M1,2,3 are compared.(88 ppm) There is a cross peak (Gt H-1 / M1,2,3 H-3), indicating that there is an α-D-Galp-(1→ and →2,3)-α-D-Manp-(1→ connection, the connection site is located at O-3 position; (3) The relevant signal peak GlcA1,4 H-1 / M1,2 C-2 was found in the HMBC spectrum, and the cross peak GlcA1,4 H-1 / M1,2 H-2 of the sugar residues in the NOESY spectrum indicates that there is a →4)-β-D-GlcpA-(1→2)-α-D-Manp-(1→ connection; (4) The sugar residues M1,2,3 and the C-2 of sugar residues M1,2,3 have a relevant signal peak M1,2,3 H-1 / M1,2,3 C-2 in the HMBC spectrum, and the cross peak M1,2,3 H-1 / M1,2,3 is present in the NOESY spectrum. H-2 indicates the presence of a connection between →2,3)-α-D-Manp-(1→ and →2,3)-α-D-Manp-(1→, with the connection site located at position O-2; (5) In the HMBC spectrum, the C-1 of sugar residues M1,2 and the C-2 of sugar residues M1,2,3 have a related signal peak M1,2 H-1 / M1,2,3 C-2, and the NOESY spectrum has a cross peak M1,2 H-1 / M1,2,3H-2, indicating the presence of a connection between →2)-α-D-Manp-(1→ and →2,3)-α-D-Manp-(1→, with the connection site located at position O-2; (6) In the HMBC spectrum, the C-1 of sugar residues M1,2,3 and the C-4 of sugar residues GE1,4 have a related signal peak M1,2,3 H-1 / GE1,4C-4, and the NOESY spectrum has a cross peak M1,2,3 H-1 / GE1,4 H-4 indicates the presence of →2,3)-α-D-Manp-(1→ and →4)-α-D-GalpA-6-OMe-(1→ connection, with the connection site located at position O-4; (7) In the HMBC spectrum, there is a correlation signal peak GE1,4 H-1 / GE1,4 C-4 between sugar residue GE1,4 and sugar residue GE1,4 C-4, and there is a cross peak GE1,4 H-1 / GE1,4 H-4 in the NOESY spectrum, indicating the presence of →4)-α-D-GalpA-6-OMe-(1→4)-α-D-GalpA-6-OMe-(1→ connection; (8) In the NOESY spectrum, there is a cross peak (Aβt) between sugar residue Aβt H-1 (δ 5.22 ppm) and sugar residue M1,2,3 H-3 (δ 3.88 ppm). H-1 / M1,2,3 H-3) indicates the presence of a β-L-Araf-(1→ and →2,3)-α-D-Manp-(1→ link, with the linker located at position O-3.

[0096] The polysaccharide methylation analysis results showed that the molar ratio of sugar residues 1,2,3-Manp (26.24%), 1,2-Manp (3.53%), t-Galp (34.08%), 1,4-GlcpA (20.66%), 1,4-GalpA (6.53%), and t-Araf (3.21%) in the polysaccharide sample was approximately 8:1:10:6:2:1. It is speculated that the polysaccharide sample is mainly composed of a polysaccharide backbone composed of sugar residues →4)-β-D-GlcpA-(1→ and →2)-α-D-Manp-(1→).

[0097] Based on the monosaccharide composition, methylation analysis, and one-dimensional and two-dimensional NMR information of the polysaccharide sample, the preliminary structure of the polysaccharide sample is inferred to be a glucuronic acid galactomannan with →4)-β-D-GlcpA-(1→ and →2)-α-D-Manp-(1→) as the main chain and a small amount of methylated galacturonic acid attached. Side chains are attached at the O-3 positions of most of the →2)-α-D-Manp-(1→) chains. The side chains mainly contain t-Galp and a small amount of t-Araf. The possible structural model is shown below:

[0098] .

[0099] Example 5: Pharmacodynamic evaluation of the combination of Trifolium repens polysaccharide SYQE3-F and Vitamin E in alleviating diabetic retinopathy: Animal model construction and grouping.

[0100] Healthy mice were selected and a diabetic retinopathy (DR) mouse model was established by intraperitoneal injection of streptozotocin (STZ). After successful modeling, mice were randomly divided into three groups: a DR model group (DR), a DR+Vit E monotherapy group, and a DR+Vit E+SYQE3-F combination group. Normal mice (NM) and NM+Vit E groups were established as controls. Intervention was performed continuously at the prescribed doses. PAS staining of retinal vessels was performed.

[0101] Eyeballs were harvested from each group of mice, and the retinas were separated for PAS staining. Figure 4 As shown, the retinal vascular network in the normal NM group exhibited gradual dendritic branching; in the DR group, the occluded retinal capillaries showed nucleus disappearance and linear atrophy of the lumen. There was no significant difference between the DR+Vit E group and the DR group; however, the Vit E+SYQE3-F combination group showed a significant reduction in retinal vascular dendritic branching and a marked improvement in atrophic lumen, indicating that the combination regimen effectively alleviated the progression of retinal disease. Vit E transport level detection.

[0102] The levels of Vitamin E in the serum and retinal tissue of mice in each group were measured. Figure 5As shown, serum vitamin E levels in the DR+Vit E group were abnormally high compared to the NM+Vit E group, while levels in retinal tissue were significantly lower, indicating a vitamin E transport disorder in the DR model, where vitamin E accumulates in serum and cannot reach the target site. After combined intervention with SYQE3-F, serum vitamin E accumulation decreased, and vitamin E levels in retinal tissue were significantly restored, demonstrating that SYQE3-F can effectively break choroidal occlusion and promote vitamin E transport to retinal tissue. Peroxide clearance rate detection.

[0103] The clearance rates of ROS, MDA, and lipid ROS were measured in in vivo models and in vitro high-glucose-damaged retinal pigment epithelial cells (APRE-19). Figure 6 As shown, the clearance rate of the DR group was significantly lower than that of the normal group. The improvement in the vitamin E-only group was limited; while the combination of SYQE3-F and vitamin E significantly improved the clearance rates of ROS, MDA and lipid ROS compared with the vitamin E-only group (*P<0.05, **P<0.01).

[0104] Example 6: Preparation of pharmaceutical compositions and formulations

[0105] The polysaccharide SYQE3-F obtained in Example 1 was mixed with vitamin E (Vit E) at an effective therapeutic dose ratio. Pharmaceutically acceptable excipients (such as microcrystalline cellulose, magnesium stearate, crospovidone, etc.) were added, and the mixture was prepared into an oral solid dosage form (such as tablets or capsules) using conventional formulation processes (such as wet granulation and tableting or direct capsule filling). This composition can be used clinically for the prevention or treatment of diabetic retinopathy.

[0106] In summary, the novel polysaccharide SYQE3-F extracted in this invention, when used in combination with Vitamin E, effectively promotes the intracellular and targeted transport of Vitamin E through a "network-clearing" mechanism, exerting excellent antioxidant and retinal microvascular protection effects, and providing a novel and highly effective drug combination regimen for the clinical treatment of diabetic retinopathy.

[0107] The above are all preferred embodiments of this application, and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.

Claims

1. A novel structural polysaccharide SYQE3-F derived from the roots of *Trifolium repens*, characterized in that, The structure of the polysaccharide SYQE3-F is as follows: 。 2. The novel structural polysaccharide SYQE3-F derived from the roots of *Trifolium repens* as described in claim 1, characterized in that, The polysaccharide SYQE3-F has an average molecular weight of 13.174 kDa.

3. The novel structural polysaccharide SYQE3-F derived from the roots of *Trifolium repens* as described in claim 1, characterized in that, The monosaccharide composition of the polysaccharide SYQE3-F, by molar percentage, includes: mannose 26.771%, glucuronic acid 26.904%, galacturonic acid 10.163%, galactose 31.550%, and arabinose 4.613%.

4. The novel structural polysaccharide SYQE3-F derived from the roots of *Trifolium repens* as described in claim 1, characterized in that, The polysaccharide SYQE3-F is glucuronic acid galactomannan, whose main chain is composed of →4)-β-D-GlcpA-(1→ and →2)-α-D-Manp-(1→, and the side chain contains t-Galp and a small amount of t-Araf.

5. The novel structural polysaccharide SYQE3-F derived from the roots of *Trifolium repens* as described in claim 1, characterized in that, The polysaccharide SYQE3-F is derived from the root parts of Tetrastigma hemsleyanum.

6. A method for preparing the novel structural polysaccharide SYQE3-F derived from the roots of *Trifolium repens* as described in any one of claims 1-5, characterized in that, Includes the following steps: a. Extraction: Take the root powder of Tripterygium wilfordii, add water and reflux to extract, filter, combine the filtrates and concentrate under reduced pressure to obtain concentrated solution, add ethanol, let stand to precipitate, centrifuge to collect the precipitate and dry to obtain crude polysaccharide of Tripterygium wilfordii. b. Deproteinization and dialysis: The crude polysaccharide of *Trifolium repens* is reconstituted, the protein is removed, and then dialysis is performed to obtain the refined polysaccharide of *Trifolium repens*. c. Ion exchange chromatography: The purified *Trifolium repens* polysaccharide was reconstituted and eluted with NaCl solutions of different concentrations in a gradient, then dialyzed and dried; d. Gel filtration chromatography: The fraction obtained in step c is reconstituted and loaded onto a gel filtration chromatography column, eluted with water, concentrated and dried to obtain the polysaccharide SYQE3-F.

7. The method as described in claim 6, characterized in that, The conditions for water reflux extraction in step a are as follows: add 20 times the volume of deionized water, reflux at 95°C for 2 hours, and extract 3 times; the conditions for static precipitation are to stand at 4°C for 24 hours.

8. The method as described in claim 6, characterized in that, In step b, the deproteinization is performed using the Sevage method, specifically: the polysaccharide solution and the Sevage reagent are mixed at a volume ratio of 4:1, and the Sevage reagent is a mixture of chloroform and n-butanol at a volume ratio of 4:1; the dialysis is performed using a dialysis bag with a molecular weight cutoff of 3.5 kDa for 48 hours.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the novel structural polysaccharide SYQE3-F derived from the roots of *Trifolium repens* as described in any one of claims 1-5, and vitamin E; the pharmaceutical composition further comprises pharmaceutically acceptable excipients or carriers.

10. Use of the pharmaceutical composition of claim 9 in the preparation of a medicament for the prevention and / or treatment of diabetic retinopathy.

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