A kind of Tripterygium wilfordii polysaccharide and its application in diabetic retinopathy medicine

Abstract

The present invention provides a trifoliate green polysaccharide and its application in diabetic retinopathy drugs: the aqueous extract of trifoliate green is deproteinized with CaCl2 and depigmented with activated carbon, and then subjected to column chromatography, eluted with a NaCl solution, and the eluate fractions collected are concentrated under reduced pressure, dialyzed using a dialysis bag with a cutoff value of 0.5 KDa, and vacuum freeze-dried. Subsequently, further chromatography is performed using Chromdex 200PG dextran gel, and the fractions are collected and freeze-dried to obtain a homogeneous polysaccharide with an average molecular weight of 36.848 KDa. At the same time, the pharmacological activity of the trifoliate green polysaccharide is evaluated, and it is found that the polysaccharide can reduce free Fe 2+ In addition, animal experiments also confirmed that Tripterygium wilfordii polysaccharide can reduce the retinal Fe 2+ This study can provide a scientific basis for the development of a new natural medicine, Tripterygium wilfordii polysaccharide, for the treatment of DR.

Description

Technical Field

[0001] The present invention relates to the field of traditional Chinese medicine chemistry, and in particular to a trifoliate green polysaccharide and its application in a drug for treating diabetic retinopathy. Background Art

[0002] Diabetic retinopathy (DR) is one of the most common complications of diabetes, severely impacting central vision in its late stages. The latest guidelines from the American Academy of Ophthalmology report indicate that the global prevalence of DR among diabetic patients is approximately 34.6%. The underlying mechanism of this disease is not yet fully understood. Existing first-line DR treatments are primarily monoclonal antibodies based on anti-angiogenic principles. These drugs have a short half-life, and frequent injections increase the risk of endophthalmitis and thromboembolism. Laser or surgical treatments can only partially preserve visual function or reduce the risk of vision loss, but remain ineffective in reversing DR progression.

[0003] In recent years, new pathological studies have provided new targets for the treatment of DR. Studies have found that the imbalance of intracellular iron homeostasis, especially iron overload-induced ferroptosis, plays a key role in the occurrence and development of DR. Hyperglycemia can lead to abnormal expression of iron ion transporters in retinal cells (especially retinal pigment epithelial cells ARPE-19), resulting in the accumulation of free ferrous ions (Fe 2+ ) levels are significantly increased. Excessive Fe 2+ The Fenton reaction catalyzes the production of a large number of highly reactive hydroxyl radicals and other reactive oxygen species (ROS), triggering severe oxidative stress, leading to lipid peroxidation, mitochondrial dysfunction, and cell death. This ultimately damages the retinal blood-retinal barrier, promotes neovascularization, and drives the progression of DR. Therefore, developing drugs that can effectively chelate iron ions, scavenge ROS, and inhibit ferroptosis-related oxidative damage is expected to become a new strategy to intervene in DR at the source.

[0004] Tetrastigma hemsleyanum (Diels et Gilg), an evergreen, herbaceous vine of the genus Tetrastigma in the Vitaceae family, is a common folk herb used by the She ethnic group. Also known as three-leaf vine and snake aconite, it is primarily distributed in Zhejiang and Fujian provinces and is renowned for its unique pharmacological activities of clearing heat and detoxifying, promoting blood circulation and removing blood stasis. Modern pharmacological studies have revealed that polysaccharides (THP) from the roots of Tetrastigma hemsleyanum possess potent antioxidant activity. Polysaccharides are polymers of monosaccharides, typically consisting of a main chain and several side chains connected by glycosidic bonds. Polysaccharides are important signaling molecules that sustain life and are commonly found in microorganisms, algae, plants, and animals. Plant polysaccharides have been reported to exhibit antioxidant, anti-tumor, immunomodulatory, anti-aging, and hypoglycemic and hypolipidemic activities, and have remained a hot topic in nutritional and medical research. However, existing technologies have not yet conducted in-depth research on the structure of Tripterygium wilfordii polysaccharides. Most of the isolated polysaccharides are crude extracts or mixtures with unclear components, and the structure-activity relationship between their chemical structure and biological activity is unclear. More importantly, to date, no published literature has reported the isolation of Tripterygium wilfordii polysaccharides with a specific structure and uniform components, nor has anyone revealed that they can treat diabetic retinopathy by regulating iron homeostasis and inhibiting oxidative stress.

[0005] Therefore, there is an urgent need in this field to develop a new drug with natural origin, clear structure, high safety and clear mechanism of action to effectively address the challenges in DR treatment. Summary of the Invention

[0006] In order to solve the problem that there are no natural drugs that can effectively treat DR, the present invention obtains a trifoliate green polysaccharide and provides a preparation method and pharmaceutical application thereof. The present invention is achieved through the following technical solutions:

[0007] To achieve the above purpose, the present invention provides a trifoliate green polysaccharide THPE2-F, the polysaccharide structural formula of which is as follows:

[0008]

[0009] in, .

[0010] Preferably, the above-mentioned Tripterygium wilfordii polysaccharide is a homogeneous polysaccharide with an average molecular weight of 36.848 KDa.

[0011] Preferably, the preparation steps of the above-mentioned Tripterygium wilfordii polysaccharide are as follows:

[0012] a. Obtaining a water extract of Tripterygium wilfordii by water extraction;

[0013] b. The aqueous extract in step a was deproteinized using CaCl2;

[0014] c. Activated carbon was used for depigmentation and column chromatography, and NaCl solution was used for elution;

[0015] d. The eluate fractions were combined and concentrated under reduced pressure, dialyzed using a 0.5KDa cutoff dialysis bag, and freeze-dried in vacuo;

[0016] e. The mixture was further chromatographed using Chromdex 200PG dextran gel, and the fractions were collected and freeze-dried to obtain the above-mentioned Tripterygium wilfordii polysaccharide.

[0017] Preferably, the water extraction steps are as follows:

[0018] i Take the root tubers of Tripterygium wilfordii, add ultrapure water and heat under reflux for extraction, collect the filtrate after the extraction, and concentrate;

[0019] ii. After cooling, add amylase and stir, then add 95% ethanol while stirring to make the ethanol volume ratio reach 80%, stir and place after ultrasonication, pour off the supernatant, and let the remaining evaporate naturally to dry, leaving a powder block;

[0020] iii. Add ultrapure water to the powder, extract and centrifuge, retain the supernatant and precipitate, add ultrapure water to the precipitate, repeat the above steps, retain the supernatant, combine the supernatants and concentrate.

[0021] Preferably, the above-mentioned CaCl2 deproteinization step is as follows:

[0022] Add CaCl2 and dissolve completely, adjust the pH to 8-9, heat, remove, cool and centrifuge to obtain the supernatant.

[0023] Preferably, the column chromatography uses 0.1-0.3 M NaCl for elution and separation at a rate of 10-20 mL / min.

[0024] Preferably, the above-mentioned dextran chromatography collects the elution fraction in the 80-100 min region, concentrates under reduced pressure, and freeze-dries in vacuum to obtain the Tripterygium wilfordii polysaccharide THPE2-F.

[0025] The present invention also provides an application of Tripterygium wilfordii polysaccharide in a drug for diabetic retinopathy, wherein the drug contains the Tripterygium wilfordii polysaccharide THPE2-F at a pharmacologically effective concentration.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. Novel product structure, high purity, and controlled quality: This invention isolates, purifies, and characterizes a novel, homogeneous polysaccharide (designated THPE2-F) from Tripterygium wilfordii for the first time. Detailed spectroscopic analysis (including 1D / 2D NMR) confirms its precise monosaccharide composition, linkage pattern, and main-side chain structure. This structural clarity lays a solid scientific foundation for subsequent pharmacological studies, structure-activity relationship analysis, industrial production, and the establishment of reliable quality control standards.

[0028] 2. Significant in vitro efficacy, protecting retinal cells: In vitro cell experiments have demonstrated that the obtained Tripterygium wilfordii polysaccharide, at concentrations as low as 50-100 µg / mL, significantly antagonizes the toxic damage of high glucose to human retinal pigment epithelial cells (ARPE19) and significantly improves cell survival. This demonstrates its direct retinal cell protective effect.

[0029] 3. Clear mechanism of action, targeting the core pathological aspects of DR: The present invention has deeply studied the mechanism of action of this polysaccharide and confirmed for the first time that it can significantly reduce the free Fe in ARPE19 cells induced by high glucose. 2+ levels, effectively clearing excess ROS in the cells and replenishing depleted glutathione (GSH). This indicates that the polysaccharide provided by the present invention directly acts on the "iron overload-oxidative stress" core pathological axis of DR, blocking the damage signal at the source.

[0030] 4. In vivo efficacy is definite and effectively improves the DR pathological model: This invention establishes an STZ-induced diabetic rat DR model, confirming that the polysaccharide also has excellent therapeutic effects in living animals. After administration, the retinal tissue pathological changes (such as edema and thinning) of DR rats were significantly improved, the expression of key pathological factors (such as VEGF) was downregulated, and the expression of protective factors (PEDF) was upregulated. More importantly, at the in vivo tissue level, it was once again verified that it can reduce Fe in the retina. 2+ These in vivo data strongly demonstrate the great potential of the polysaccharide of the present invention as a drug for the treatment of DR.

[0031] 5. Natural origin and high safety: The trifoliate green polysaccharide of the present invention is derived from natural plants that can be used as food and medicine. Compared with chemically synthesized drugs or biological antibody drugs, it usually has better biocompatibility and lower toxic side effects, providing DR patients with a safer and milder long-term treatment option. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0033] Figure 2This is the elution curve of the THPE2 gel filtration column chromatography of the sample of the present invention; wherein A is 96.805, B is 128.21

[0034] Figure 3 The LogMp-T calibration curve of the present invention; wherein Log Mp = -0.1678T + 10.6291; R 2 =0.9916; the horizontal axis is time T, the unit is minute min;

[0035] Figure 4 is the LogMw-T calibration curve of the present invention; wherein Log Mw = -0.1761T + 11.0299; R 2 =0.9927; the horizontal axis is time T, the unit is minute min;

[0036] Figure 5 The LogMn-RT calibration curve of the present invention; wherein LogMn=-0.1674T+10.5361; R 2 =0.9937; the horizontal axis is time T, the unit is minute min;

[0037] Figure 6 This is the HPGPC chromatogram of THPE2-F of the present invention; wherein the peak value is 30.369; the abscissa is the retention time (min), and the ordinate is the detector response value;

[0038] Figure 7 The figure is the UV-visible absorption scanning spectrum of THPE2-F of the present invention; the abscissa is the wavelength WL (nm), and the ordinate is the absorbance A;

[0039] Figure 8 This is the infrared spectrum of THPE2-F of the present invention; A in the figure is 3408 cm -1 , B is 2934 cm -1 , C is 1609cm -1 , D is 1417 cm -1 , E is 1146 cm -1 、F is 1073 cm -1 , G is 1030 cm -1 ; The horizontal axis is the wave number (cm -1 ), the ordinate is the transmittance (%);

[0040] Figure 9 The chromatogram of the standard product of the present invention; the abscissa is the residence time, in min; the ordinate is the milliabsorptivity, in mAU;

[0041] Figure 10The chromatogram of THPE2-F of the present invention; the abscissa is the residence time, in min; the ordinate is the milliabsorptivity, in mAU;

[0042] Figure 11 THPE2-F of the present invention 1 H NMR spectrum;

[0043] Figure 12 THPE2-F of the present invention 1 H NMR spectrum (water peak suppression);

[0044] Figure 13 THPE2-F of the present invention 13 C NMR spectrum; where A is -COO - ;

[0045] Figure 14 DEPT-135 spectrum of THPE2-F of the present invention;

[0046] Figure 15 is the HSQC spectrum of THPE2-F of the present invention;

[0047] Figure 16 is the COSY spectrum of THPE2-F of the present invention;

[0048] Figure 17 is the HMBC spectrum of THPE2-F of the present invention;

[0049] Figure 18 is the NOESY spectrum of THPE2-F of the present invention;

[0050] Figure 19 The effect of different concentrations of Tripterygium wilfordii polysaccharide of the present invention on the survival rate of ARPE19 cells damaged by high glucose for 24 h and 48 h; compared with the Control group, **P<0.01; compared with the HG group, ##P<0.01; the horizontal axis is the treatment time, which is 0 h, 24 h and 48 h respectively; the vertical axis is the absorbance at 450 nm, which is the absorbance value detected by a microplate reader at a wavelength of 450 nm; HG represents ARPE19 cells damaged by high glucose (HG); HG+25 μg / ml (administration group) represents cells damaged by high glucose and then given different concentrations (25, 50, 100, 200 μg / ml) of Tripterygium wilfordii polysaccharide;

[0051] Figure 20The effect of different concentrations of Tripterygium wilfordii polysaccharide of the present invention on the Fe2+ level in ARPE19 cells damaged by high glucose after 48 h of administration; compared with the control group, **P<0.01; compared with the HG group, ##P<0.01; the horizontal axis represents different groups, namely the control group, HG+25 μg / ml, etc. (drug group); the vertical axis represents the ferrous ion Fe2+ level, unit is (pg / μg protein);

[0052] Figure 21 The effect of different concentrations of Tripterygium wilfordii polysaccharide of the present invention on GSH in ARPE19 cells damaged by high glucose after 48 h of administration; compared with the control group, **P<0.01; compared with the HG group, ##P<0.01; the horizontal axis represents different groups, namely the control group, HG+25 μg / ml, etc. (administered groups); the vertical axis represents GSH levels, in (mmol / mL); GSH is glutathione;

[0053] Figure 22 The effect of different concentrations of Tripterygium wilfordii polysaccharide of the present invention on lipid ROS in ARPE19 cells damaged by high glucose after 48 hours of administration; compared with the control group, **P<0.01; compared with the HG group, ##P<0.01; the horizontal axis represents different groups, namely the control group, HG+25 μg / ml, etc. (drug group); the vertical axis represents the lipid ROS level, and the unit is the multiple of the control group;

[0054] Figure 23 This is a graph showing the efficacy of Tripterygium wilfordii polysaccharide on the STZ-induced DR model in SD rats, wherein: A is the body weight after modeling; the horizontal axis is the time after modeling (days), and the vertical axis is the body weight (g); B is the blood glucose level after modeling; the horizontal axis is the time after modeling (days), and the vertical axis is the blood glucose level (mmol / L);

[0055] Figure 24 This is a graph showing the efficacy of Tripterygium wilfordii polysaccharide on the STZ-induced DR model in SD rats, and HE was used to detect retinal histopathological changes; control is the control group, and DR is the DR model group;

[0056] Figure 25 The figure shows the efficacy of the trifoliate green polysaccharide of the present invention on the DR model of SD rats induced by STZ, and the retinal tissue pathological changes were detected by VEGF immunohistochemistry; wherein control is the control group and DR is the DR model group;

[0057] Figure 26This is a diagram showing the efficacy of Tripterygium wilfordii polysaccharide on the STZ-induced SD rat DR model. Immunofluorescence was used to detect the changes in the levels of retinal pigment epithelium-derived factor (PEDF) in the retinal tissue after the administration of Tripterygium wilfordii polysaccharide (THP) to the DR model of SD rats. The control group was the control group, the DR model group was the DR model group, and the DR+THP (drug administration group) was the retinal tissue of the DR model rats after the intervention of Tripterygium wilfordii polysaccharide (THP). The GCL (Ganglion Cell Layer) is composed of the cell bodies of ganglion cells. The axons of these cells form the optic nerve and transmit the visual information received by the retina to the brain. The INL (Inner Nuclear Layer) is the inner nuclear layer, which contains the cell bodies of bipolar cells, horizontal cells and amacrine cells, etc. These cells play an important role in the conduction and regulation of visual signals in the retina and participate in the initial processing and transmission of light signals. The ONL (Outer Nuclear Layer) is the outer nuclear layer, which is mainly composed of the cell bodies of rods and cones. Rods and cones are the photoreceptor cells of the retina, which are responsible for the visual functions such as sensing weak light and strong light and distinguishing colors respectively. Pigment Epithelial: The retinal pigment epithelium is a layer of epithelial cells located between the outer segments of retinal photoreceptor cells and the choroid. It has multiple functions, such as nourishing photoreceptor cells, phagocytizing membrane discs detached from the outer segments of photoreceptors, participating in the transport and metabolism of substances in the retina, maintaining the outer retinal barrier, etc. It is essential for the normal function and structural stability of the retina.

[0058] Figure 27 This is a graph showing the efficacy of Tripterygium wilfordii polysaccharide on the STZ-induced DR model in SD rats. It shows the effect of THP administration on the Fe2+ level in the DR retinal tissue; **P<0.01 compared with the control group. The horizontal axis represents different groups, namely the control group, the DR model group, and the DR+THP (administration group); the vertical axis represents the ferrous ion Fe2+ level, in pg / μg protein.

[0059] Figure 28 This figure shows the efficacy of Tripterygium wilfordii polysaccharide on the STZ-induced DR model in SD rats. The figure also shows the effect of THP administration on ROS in retinal tissues of DR rats. **P < 0.01 compared with the control group. The horizontal axis represents the different groups: control group, DR model group, and DR + THP (treatment group); the vertical axis represents the percentage of tissue reactive oxygen species (RS) (relative to the control group). DETAILED DESCRIPTION

[0060] The present invention will be described in more detail below with reference to the following examples. It should be understood that the present invention is not limited to the following examples, and any modifications or variations made to the present invention fall within the scope of protection of the present invention. The methods in the following examples are all conventional methods in the art unless otherwise specified.

[0061] Example 1:

[0062] Tripterygium wilfordii polysaccharide extraction

[0063] Sanyeqing was collected from Qujiang District, Quzhou City, Zhejiang Province, and was identified by Pu Jinbao, a researcher at Zhejiang Institute of Traditional Chinese Medicine, as the dried tuberous root of the grape family plant Sanyeqing (Tetrastigma hemsleyanum Diels et Gilg).

[0064] (1) Take 4000 g of the above-mentioned root tubers of Tripterygium wilfordii, add 5 times of ultrapure water and perform heating reflux extraction twice, each time for 3 h. After the extraction, collect the filtrate and concentrate it to 3 L using a double-layer steam boiler.

[0065] (2) After cooling, add 300 g of amylase and stir at 37 °C for 3 h. Then add 95% ethanol while stirring until the ethanol volume ratio reaches 80%. After ultrasonication for 1 h, stir and then place at 4 °C for 24 h. Then pour out most of the supernatant and let the rest evaporate and dry naturally, leaving a powder block.

[0066] (3) Then, add 6 times the amount of ultrapure water of the original medicinal material, that is, 24 L of water, to the powder block, extract at 80℃ for 3 hours, centrifuge at 4000 rpm / min for 10 minutes, keep the supernatant and precipitate, then add 6 times the amount of ultrapure water of the original medicinal material to the precipitate, extract at 80℃ for 3 hours, centrifuge at 4000 rpm / min for 10 minutes, keep the supernatant, combine the two supernatants and concentrate to a volume of 8L.

[0067] (4) Add 5% volume of CaCl2 to dissolve completely, adjust the pH to 8-9 with NaOH, heat in a water bath to 85℃, take out and cool to room temperature, then centrifuge, centrifuge at 4000 rpm / min for 20 min to obtain the supernatant, repeat this step 3 times to remove the protein.

[0068] (5) Then, add activated carbon to the solution, shake well, heat in a 60°C water bath for 30 min, and filter. Combine the filtrates for subsequent treatment.

[0069] Polysaccharide separation and purification: ion exchange column chromatography

[0070] (1) Pretreatment of ion exchange chromatography medium: 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 filler has no alcohol smell. Transfer it to a beaker, add distilled water with a volume of one-half to one times the volume of the sedimented gel, and stir well for column loading.

[0071] (2) Column packing and equilibration: Stir the pretreated DEAE sepharose FF gel suspension evenly and slowly add it to the XK chromatography column (φ3.0×50cm). After complete sedimentation, connect the upper column head to a 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. This completes the column packing.

[0072] (3) Loading and elution: The polysaccharide sample was dissolved with an appropriate amount of dH2O, centrifuged at 8000 rpm for 10 min, the precipitate was removed, and the sample was filtered with a 0.45 μm microporous filter membrane. The filtrate was loaded onto the equilibrated ion exchange chromatography column. The loading volume was 30% of the column volume. After all the sample liquid entered the chromatography column, it was eluted and separated in different salt concentrations (dH2O, 0.2 M NaCl, 0.5 M NaCl, 1.0 M NaCl). Each concentration was eluted with 2 times the column volume. The elution rate was 15 mL / min. The fractions were collected by an automatic fraction collector. Each eluate was collected in 100 tubes, and 10 mL was collected in each tube. The anthrone-sulfuric acid method was used. The polysaccharide content in the eluate was tracked and detected by an enzyme reader at 630 nm. The number of tubes was used as the horizontal axis and the absorbance was used as the vertical axis to obtain the polysaccharide elution curve. The eluates of each tube with different elution peaks were collected separately. The ion exchange column chromatography was repeated several times. The eluate fractions collected were combined and concentrated under reduced pressure, dialyzed using a 0.5 KDa dialysis bag, and freeze-dried in vacuum to obtain components of each group.

[0073] (4) Ion exchange column chromatography results

[0074] The crude polysaccharide sample was chromatographed on an ion exchange column and gradient eluted with different salt concentrations of dH2O, 0.2M NaCl, 0.5M NaCl, and 1.0M NaCl solutions. The polysaccharide content in each tube was determined by the anthrone-sulfuric acid method. The elution curve was plotted as shown in the figure. Figure 1As shown, samples were collected from left to right in the following order: THP-E1 (eluted with dH2O), THP-E2 (eluted with 0.2M NaCl), THP-E3 (eluted with 0.5M NaCl), and THP-E4 (eluted with 1.0M NaCl). After one cycle of gradient elution, the 2.0 mol / L NaCl eluate was assayed using the anthrone-sulfuric acid method, revealing no obvious color reaction, indicating complete elution of the crude polysaccharide from the ion exchange column. Polysaccharide content was measured, dialyzed, freeze-dried, and weighed, and the 0.2M NaCl elution fraction showed significantly higher polysaccharide content than the fractions eluted with dH2O, 0.5M NaCl, and 1.0M NaCl solutions.

[0075] Polysaccharide separation and purification: gel filtration column chromatography

[0076] (1) Pretreatment of gel filtration chromatography medium: Pour the gel filtration chromatography medium Chromdex 200PG 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 filler has no alcohol smell. Transfer it to a beaker, add distilled water with a volume of one-half to one times the volume of the sedimented gel, and stir well for column loading.

[0077] (2) Column loading and equilibration: Stir the pretreated gel filtration chromatography medium suspension evenly and slowly add it to the XK chromatography column (φ1.6×100cm). 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 loading.

[0078] (3) Sampling and elution: The polysaccharide sample prepared by ion exchange column chromatography was dissolved with an appropriate amount of dH2O, centrifuged at 8000 rpm for 10 min, the precipitate was removed, and filtered with a 0.45 μm microporous filter membrane. The filtrate was loaded onto the equilibrated gel filtration chromatography, with the loading amount being 1% of the column volume. After all the sample liquid entered the chromatography column, it was eluted and separated with ultrapure water at a flow rate of 1.8 mL / min. The eluate was collected by an automatic fraction collector, 4.5 mL per tube, and eluted with 1 column volume. The eluate was collected online with a differential detector, and the eluate with a high peak height and good symmetry in the same elution peak area was collected. The gel filtration column chromatography was repeated several times to enrich the purified polysaccharide. The collected eluate fractions were combined, concentrated under reduced pressure, and freeze-dried in vacuum to obtain the polysaccharide separated and purified by gel filtration column chromatography.

[0079] (4) Gel filtration column chromatography results

[0080] According to the elution curve and molecular weight test results obtained by ion column chromatography, the component sample THP-E2 was selected as the sample for gel filtration column chromatography. After gel filtration column chromatography, the elution curves obtained were: Figure 2As shown, the elution fractions in the 80-100 min region were collected, concentrated under reduced pressure, and freeze-dried in vacuo. After repeated application of the sample to gel filtration column chromatography, the isolated and purified polysaccharide was obtained and named THPE2-F.

[0081] Polysaccharide purity identification and molecular weight distribution determination

[0082] (1) Establishment of molecular weight calibration curve: Weigh dextran standards of different molecular weights (molecular weight 1000, 5000, 12000, 25000, 50000, 80000, 150000, 270000, 410000, 670000 series analytical standards), add 0.05M NaCl solution to prepare 5 mg / mL dextran standard solution, filter with 0.22 μm microporous filter membrane for later use, adopt high performance gel permeation chromatography (HPGPC method), use high performance gel permeation chromatography tandem column for detection, use the logarithm of the relative molecular mass of the standard as the vertical axis and the retention time of the corresponding chromatographic peak as the horizontal axis for linear regression to obtain the calibration curve.

[0083] (2) Preparation of test sample solution: Weigh the purified polysaccharide sample, add 0.05 M NaCl solution to the sample to prepare a 5 mg / mL test sample solution, take the supernatant and filter it with a 0.22 μm microporous filter membrane, and then transfer the sample to a 2 mL injection vial for later use.

[0084] (3) Chromatographic method: The HPGPC method was used, using a high performance liquid chromatograph equipped with a differential detector, and a polymer-based water-soluble SEC (GFC) column OHpak SB-803 HQ, Ohpak SB-804 HQ, and Ohpak SB-805 HQ (8×300 mm) in series for detection. The mobile phase was 0.05 M NaCl solution, the flow rate was 0.65 ml / min, the column temperature was 40 ºC, and the injection volume was 30 μL.

[0085] (4) Identification of polysaccharide purity and determination of molecular weight distribution.

[0086] Molecular weight calibration curve results

[0087] refer to Figure 3-Figure 5 , linear regression was performed with the logarithmic value of the relative molecular mass of the standard as the ordinate and the retention time (T) of the corresponding chromatographic peak as the abscissa to obtain the calibration curves of LogMp-T (peak molecular weight), LogMw-T (weight average molecular weight), and LogMn-T (number average molecular weight), as shown below:

[0088] LogMp-T calibration curve equation: Log Mp = -0.1678T + 10.6291, R² = 0.9916

[0089] LogMw-T calibration curve equation: Log Mw = -0.1761T + 11.0299, R² = 0.9927

[0090] LogMn-T calibration curve equation: Log Mn = -0.1674T + 10.5361, R² = 0.9937.

[0091] refer to Figure 6 HPGPC molecular weight analysis revealed a single, symmetrical peak for the polysaccharide THPE2-F component, indicating that THPE2-F is a homogeneous polysaccharide. Based on the standard calibration curve, the average molecular weight of the THPE2-F sample was calculated to be 36.848 kDa (see table below). THPE2-F was selected for further structural analysis. The peak near 48.0 min indicates a salt peak in the mobile phase. Peaks following the salt peak may represent small oligosaccharides.

[0092] Table 1 Molecular weight results of sample THPE2-F

[0093]

[0094] UV absorption spectrum of polysaccharide samples

[0095] (1) Sample processing

[0096] Accurately weigh 5 mg of polysaccharide sample, transfer it to a 5 ml volumetric flask, add water to make up to volume, and prepare a 1 mg / mL polysaccharide solution as the sample determination solution.

[0097] (2) Sample measurement

[0098] This project uses a UV-visible spectrophotometer to qualitatively analyze polysaccharide solutions. Using pure water as a blank control, the polysaccharide solution was scanned on the UV-visible spectrophotometer within a wavelength range of 190–400 nm, with scan intervals of 1 nm. In a UV-visible spectrophotometer, when light of different wavelengths is continuously irradiated onto a sample solution of a certain concentration, the corresponding absorption intensities at different wavelengths are obtained. Using wavelength (λ) as the horizontal axis and absorption intensity (A) as the vertical axis, an absorption spectrum curve can be plotted for the substance.

[0099] (3) Polysaccharide UV absorption spectrum scanning results

[0100] The UV absorption spectrum of polysaccharide samples is as follows Figure 7 As shown, the results showed that there were no obvious absorption peaks at 260 nm and 280 nm, indicating that the polysaccharide sample did not contain nucleic acid and protein components.

[0101] Infrared spectroscopy test of polysaccharide samples

[0102] (1) Weigh 1-2 mg of each dried polysaccharide sample in a mortar, add 200 mg of KBr powder and grind evenly, press into tablets, and use a Fourier transform infrared spectrometer to scan the sample in the wavelength range of 4000-400 cm -1 , record the infrared spectrum.

[0103] (2) Results and analysis

[0104] The structural characteristics of polysaccharide THPE2-F were analyzed by FT-IR. -1 Infrared spectrum scanning is performed within the range, and the results are as follows Figure 8 As shown. The infrared spectrum shows the typical characteristics of polysaccharides. Figure 8 It can be seen that at 3408cm -1 The broad and strong absorption peak at 2934 cm is the stretching vibration of OH in sugars. -1 The smaller absorption peak at 1400-1200 cm is the stretching vibration of methyl or methylene CH. -1 The peaks between them should be CH angle vibrations of sugars. From the above, it can be determined that the sample is a polysaccharide. -1 The free carboxyl group COO in uronic acid - The characteristic absorption peak of asymmetric stretching vibration is 1417 cm -1 Protonated carboxyl group COO - Symmetric stretching vibration characteristic absorption peak. 1146 cm -1 、1073cm -1 、1030 cm -1 The three absorption peaks indicate that the polysaccharide sample contains pyranose rings. The above infrared test results show that the polysaccharide sample is an acidic polysaccharide.

[0105] Monosaccharide composition determination

[0106] (1) Standard product configuration

[0107] Weigh 5 mg each of rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid, glucuronic acid, glucosamine hydrochloride, and galactosamine hydrochloride, and 10 mg of fucose, dissolve, and dilute to 10 mL in a volumetric flask to prepare a standard stock solution. Dilute each solution to a desired volume using the gradient dilution method shown in Table 2 below. Pass through a 0.22 μm filter membrane and transfer to a sample vial.

[0108] Table 2 Monosaccharide mixed standard gradient concentration information

[0109]

[0110] (2) Preparation of test sample solution

[0111] (i) Solid sample extraction

[0112] In a clean chromatographic vial, accurately weigh 5 mg (±0.05 mg) of polysaccharide sample and add 1 mL of 2M TFA solution. Heat at 121°C for 2 hours. Flow nitrogen through the vial and dry. Rinse with 3 mL of methanol, dry again, and repeat the methanol wash 2-3 times. Dissolve the sample in 5 mL of sterile water and transfer to a chromatographic vial for analysis.

[0113] (ii) Liquid sample extraction

[0114] Take an appropriate amount of the supernatant and swirl concentrate or dry it with nitrogen. Add 1 mL of 2M TFA solution and heat at 121°C for 2 hours. Purge with nitrogen and dry it. Rinse with methanol and dry it again. Repeat the methanol wash 2-3 times. Dissolve it in sterile water and transfer it to a chromatographic vial for analysis.

[0115] (3) PMP derivatization

[0116] Take 0.2 mL of monosaccharide standard solution or polysaccharide hydrolyzate in a stoppered conical-bottom 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 mix, and react in a 70°C water bath for 1 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 and vortex extract three times to remove excess PMP. After discarding the chloroform layer, take 0.3 mL and add water to make up to 1 mL.

[0117] (4) Chromatographic method

[0118] ThermoU3000 liquid chromatography system, the chromatographic column is ZORBAX EclipseXDB-C18, the mobile phase is acetonitrile: phosphate buffer (potassium dihydrogen phosphate 12 g / L, 2M NaOH adjusted to pH 6.8) isocratic elution, the volume ratio of acetonitrile to phosphate buffer is 17:83, the flow rate is 0.8 ml / min, the column temperature is 30°C, the detection wavelength is 250 nm, and the injection volume is 10 μL.

[0119] (5) Experimental results

[0120] Calibration curve results

[0121] like Figure 9-10 As shown, the project uses the external standard method for quantification, and a standard curve is established by preparing standard samples of different concentrations. The linear relationship information is shown in Tables 3 and 4 below:

[0122] Table 3 Summary of track information

[0123] .

[0124] Table 4 Summary of track information

[0125] .

[0126] NMR analysis of polysaccharide samples

[0127] In order to further obtain the structural characteristics of the polysaccharide sample THPE2-F, one-dimensional nuclear magnetic resonance 1H-NMR, 13C-NMR, DEPT-135 and two-dimensional nuclear magnetic resonance COSY, HSQC, HMBC, NOESY were measured. The spectra are shown in Figure 2. Figures 11-18 As shown, all H and C chemical shift information of each major sugar residue was obtained to infer the connection order between each sugar residue.

[0128] Based on the monosaccharide composition of the polysaccharide samples, the results of polysaccharide methylation analysis, and one-dimensional and two-dimensional nuclear magnetic resonance information analysis, it was inferred that the preliminary structure of the polysaccharide samples was a glucuronic acid galactomannan (Glycoglucuronomannan) with →4)-β-D-GlcpA-(1→ and →2)-α-D-Manp-(1→) as the main chains, with side chains connected at the O-3 position of most →2)-α-D-Manp-(1→. The side chains mainly contained t-Galp, and there were also small amounts of 1,4-Galp, 1,4-Galp, and t-Araf sugar structure fragments.

[0129] Activity detection of Tripterygium wilfordii polysaccharide samples on diabetic retinopathy

[0130] like Figures 19-22 As shown in the figure, this experimental group also investigated the protective effect of different concentrations of the above-mentioned isolated uniform trifoliate polysaccharide samples (25, 50, 100, 200 μg / mL) on high glucose-induced ARPE19 cell damage after treatment for 24 h and 48 h, and detected the corresponding Fe 2+, LDH, MDA, GSH and lipid ROS levels. First, we used CCK-8 experiments to investigate the effect of time on the efficacy of Tripterygium wilfordii polysaccharide. The results showed that the cell survival rate after 48 h of Tripterygium wilfordii polysaccharide treatment was higher than that after 24 h. Subsequently, in the investigation of the concentration of Tripterygium wilfordii polysaccharide, it was found that when the concentration of Tripterygium wilfordii polysaccharide reached 50 and 100 μg / mL (treated for 48 h), the cell survival rate was statistically different from that of the high glucose (HG) group without drug treatment (##P<0.01). In addition, we also investigated the effects of Tripterygium wilfordii polysaccharide (25, 50, 100 μg / mL) on the levels of Fe2+, GSH (Glutathione), and ROS (Reactive Oxygen Species) in ARPE19 cells damaged by high glucose after 48 h of administration. The results showed that when the concentration of Tripterygium wilfordii polysaccharide reached 100 μg / mL, compared with the HG group, GSH and lipid ROS, Fe 2+ The differences in the levels were statistically significant (##P<0.01), indicating that the uniform trifoliate green polysaccharide can reduce the free Fe 2+ Reversal of high glucose-induced ARPE19 cell damage.

[0131] like Figure 23-Figure 28 As shown, based on the above in vitro experiments, it was found that Tripterygium wilfordii polysaccharide (THP) can reduce free Fe 2+ The level of reversal of high glucose-induced ARPE19 cell damage was also investigated in the in vivo DR model by our research group. 2+ The level was tested. Figure 23 As shown in the figure, blood glucose levels in SD rats were significantly different 7 days after intraperitoneal injection of streptozotocin (STZ), and body weight gradually became more obvious with the passage of time ( Figure 23 HE staining showed that the nerve fiber layer of the DR group was significantly edematous, and the thickness of the inner nuclear layer and outer nuclear layer was thinner. Immunohistochemistry showed that VEGF was highly expressed in the capillaries of the retinal pigment epithelium in the DR group. The above blood glucose, body weight, HE and immunohistochemistry results confirmed that the DR model was successfully established ( Figure 23-Figure 25 ). Figure 26-Figure 28 It showed that Tripterygium wilfordii polysaccharide could increase the level of pigment epithelium-derived factor (PEDF) in the retinal tissue of DR model and reduce Fe 2+ and ROS levels, indicating that the extracted Tripterygium wilfordii polysaccharide samples can reduce Fe 2+ Horizontal mitigation of DR process.

[0132] The above are all preferred embodiments of the present application, and are not intended to limit the scope of protection of the present application. Therefore, any equivalent changes made based on the structure, shape, and principle of the present application should be included in the scope of protection of the present application.

Claims

1. A trifoliate green polysaccharide THPE2-F, characterized in that: The polysaccharide structural formula is as follows: Wherein, R=α-D-Galp-(1→,→4)-α-D-Galp-(1→,→6)-α-D-Galp-(1→or α-L-Araf-(1→, and the trifoliate green polysaccharide is a homogeneous polysaccharide with an average molecular weight of 36.848KDa.

2. The trifoliate green polysaccharide according to claim 1, characterized in that The preparation steps of the trifoliate green polysaccharide are as follows: a. Obtaining a water extract of Tripterygium wilfordii by water extraction; b. The aqueous extract in step a was deproteinized using CaCl2; c. Activated carbon was used for depigmentation and column chromatography, and NaCl solution was used for elution; d. The eluate fractions were combined and concentrated under reduced pressure, dialyzed using a 0.5KDa cutoff dialysis bag, and freeze-dried in vacuo; e. Further chromatography was performed using Chromdex 200PG dextran gel, and the collected fractions were freeze-dried to obtain the trifoliate green polysaccharide according to claim 1.

3. The trifoliate green polysaccharide according to claim 2, characterized in that: The water extraction steps are as follows: i Take the root tubers of Tripterygium wilfordii, add ultrapure water and heat under reflux for extraction, collect the filtrate after the extraction, and concentrate; ii. After cooling, add amylase and stir, then add 95% ethanol while stirring to make the ethanol volume ratio reach 80%, stir and place after ultrasonication, pour off the supernatant, and let the remaining evaporate naturally to dry, leaving the powder block; iii. Add ultrapure water to the powder, extract and centrifuge, retain the supernatant and precipitate, add ultrapure water to the precipitate, repeat the above steps, retain the supernatant, combine the supernatants and concentrate.

4. The trifoliate green polysaccharide according to claim 2, characterized in that The CaCl2 deproteinization steps are as follows: Add CaCl2 and dissolve completely, adjust the pH to 8-9, heat, remove, cool and centrifuge to obtain the supernatant.

5. The trifoliate green polysaccharide according to claim 2, characterized in that: The column chromatography adopts 0.1-0.3M NaCl for elution and separation, and the elution rate is 10-20mL / min.

6. The trifoliate green polysaccharide according to claim 2, characterized in that: The dextran chromatography collects the elution components in the 80-100 min region, concentrates under reduced pressure, and freeze-dries in vacuum to obtain the trifoliate green polysaccharide THPE2-F.

7. Use of Tripterygium wilfordii polysaccharide in the preparation of a drug for diabetic retinopathy, characterized in that: The medicine contains the trifoliate green polysaccharide THPE2-F according to claim 1.

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