Use of guanidine compounds in the preparation of a medicament for the treatment of diabetes

Abstract

This invention provides the application of guaijaverin in the prevention and / or treatment of diabetic retinopathy, belonging to the field of biopharmaceutical manufacturing technology. The application of guaijaverin in the prevention and / or treatment of diabetic retinopathy is described. This invention verifies the efficacy of guaijaverin by constructing an in vitro model of high-glucose damage in human retinal microvascular endothelial cells. Guaijaverin significantly improves high-glucose-induced abnormal cell proliferation and reduces membrane lipid peroxidation levels (using malondialdehyde content as an indicator). Further verification was conducted in a mouse model of high-glucose-induced retinopathy, clarifying that guaijaverin has a role in improving diabetic retinopathy. This invention creatively proposes that guaijaverin has a protective effect against diabetic retinopathy, protecting its preventive and / or therapeutic effects on diabetic retinopathy, and further protecting its development and application in products such as drugs for the prevention and / or treatment of diabetic retinopathy.

Description

Technical Field

[0001] This application belongs to the field of biopharmaceutical manufacturing technology, specifically relating to the use of guaijaverin in drugs for the prevention and / or treatment of diabetic retinopathy. Background Technology

[0002] Diabetic retinopathy (DR) caused by high blood sugar is recognized by the World Health Organization as the leading cause of irreversible blindness in working-age populations due to its high rate of blindness and irreversible vision loss. Current first-line treatments for DR (such as intravitreal injection of anti-VEGF drugs and laser photocoagulation) are not only highly invasive and expensive, but also only target lesions that have already occurred in the late stages of the disease. Summary of the Invention

[0003] The purpose of this invention is to provide the use of guavaside in the prevention and / or treatment of diabetic retinopathy.

[0004] This invention provides the use of guavaside or guavaside derivatives in medicaments for the prevention and / or treatment of diabetic retinopathy.

[0005] Preferably, the diabetic retinopathy includes abnormal proliferation of retinal endothelial cells, increased levels of membrane lipid peroxidation, retinal structural damage, and retinal microvascular damage.

[0006] Preferably, the retinal structural damage includes reduced retinal thickness, degenerative changes in retinal neural structures, and retinal microvascular damage.

[0007] Preferably, the retinal thickness includes at least one of the following: full-thickness retinal thickness, inner retinal thickness, middle retinal thickness, and outer retinal thickness.

[0008] Preferably, the degenerative changes in the retinal neural structure include thinning of the retinal neural layer and / or loss of ganglion cells.

[0009] Preferably, the retinal neural layer structure includes an inner nuclear layer and / or an outer nuclear layer.

[0010] Preferably, the retinal microvascular injury includes loss of retinal pericellular cells and / or damage to fine lock-like cell-free capillaries.

[0011] Preferably, the guava glycoside derivatives include the following glycosyl site modified derivatives: fully acetylated guava glycoside, triacetylated guava glycoside, methylated arabinoside, and 6''-acylated guava glycoside.

[0012] Preferably, the drug comprises at least one of the following dosage forms: injection, drops, powder, tablet, capsule, granule, and oral liquid.

[0013] Preferably, the mass percentage of guavaside in the drug is 10% to 98%.

[0014] This invention provides the application of guavaside or guavaside derivatives in pharmaceuticals for the prevention and / or treatment of diabetic retinopathy. The invention validates the in vitro efficacy of guavaside using an in vitro model of high-glucose damage in human retinal microvascular endothelial cells. Results show that guavaside significantly improves high-glucose-induced abnormal cell proliferation and reduces membrane lipid peroxidation levels (as indicated by malondialdehyde content). Further validation was achieved in a mouse model of high-glucose-induced retinopathy, clarifying that guavaside has a role in improving diabetic retinopathy. This invention creatively proposes that guavaside has a protective effect against diabetic retinopathy, protecting its preventive and / or therapeutic effects on diabetic retinopathy, and further protecting its development and application in pharmaceuticals and other products for the prevention and / or treatment of diabetic retinopathy. Attached Figure Description

[0015] Figure 1 The results of guavaside ameliorate the abnormal proliferation of HREMCs under high glucose damage conditions were obtained. The blank group consisted of normally cultured HREMCs. The model groups were established using 60 mmol / L and 30 mmol / L D-glucose to create high glucose cell damage models. The treatment groups were treated with 30 μmol / L guavaside under the corresponding high glucose damage conditions. P <0.001; ## P <0.01; Figure 2 The results of measuring the effect of guavaside on the membrane lipid peroxidation level of HREMCs under high glucose damage were obtained. The control group consisted of normally cultured HREMCs; the model group was established using 60 mmol / L D-glucose to create a high glucose cell injury model; and the treatment group was treated with 30 μmol / L guavaside under the corresponding high glucose injury conditions. P <0.01; ## P <0.01; Figure 3 The results show the effects of guavaside on general physiological indicators in DR mice. A is a schematic diagram of the overall dosing regimen; B shows the effect of guavaside on the body weight of DR mice. All data are expressed as mean ± SEM, n = 6; compared with the control group, the model group... P <0.001; Figure 4Results of guavaside in improving blood glucose levels in DR mice; Figure 5 The results show the effect of guavaside on glucose tolerance in DR mice; A represents the blood glucose levels of mice 30 min, 60 min, 90 min and 120 min after injection of glucose solution; B represents the area under the curve in the 0-120 min time period; all data are expressed as mean ± SEM, n = 6. P <0.001; P <0.01; ### P <0.001; ## P <0.01; # P <0.05; Figure 6 The results show the effect of guavaside on retinal thickness in DR mice; A is a schematic diagram of retinal layering; B is a schematic diagram of OCT of the retina in each group of mice; C is the effect of guavaside on the thickness of the inner retina in DR mice; D is the effect of guavaside on the thickness of the middle retina in DR mice; E is the effect of guavaside on the thickness of the outer retina in DR mice; F is the effect of guavaside on the overall retinal thickness in DR mice; all data are expressed as mean ± SEM, n = 6; P <0.001; P <0.01; ### P <0.001; ## P <0.01; # P <0.05, ns indicates no significant difference; # indicates statistical difference between different drug groups and the model group; the scale bar in the figure is 50 μm; Figure 7 The results show the effects of guavaside on the retinal structure of DR mice. A is a schematic diagram of retinal layering; B is a schematic diagram of HE staining of the retina of mice in each group, with the red circle and arrow marking the area of ​​GCL cells; C is the effect of guavaside on the thickness of the inner nuclear layer of the retina of DR mice; D is the effect of guavaside on the thickness of the outer nuclear layer of the retina of DR mice; E is the effect of guavaside on the number of ganglion cells in the retina of DR mice; F is the effect of guavaside on the overall thickness of the retina of DR mice. All data are expressed as mean ± SEM, n = 6. P <0.001; ### P<0.001; ## P <0.01; # P <0.05, ns indicates no significant difference; # indicates statistical difference between different drug groups and the model group; the scale bar in the figure is 50 μm; Figure 8 The results show the effect of guavaside on retinal vessels in DR mice. A is a schematic diagram of PAS staining of the retina of mice in each group; B is the number of cellless vessels; C is the ratio of pericytes to endothelial cells (P / EC%). Red arrows indicate cellless vessels, and yellow arrows indicate pericytes. All data are expressed as mean ± SEM, n = 6; model group and guavaside-treated group, P <0.001; P <0.01; the scale bar in the figure is 50 μm. Detailed Implementation

[0016] This invention provides the use of guavaside or guavaside derivatives in medicaments for the prevention and / or treatment of diabetic retinopathy.

[0017] In this invention, the English name of the guaijaverin is: Guaijaverin, and its molecular formula is: C 20 H 18 O 11 Molecular weight: 434.35, structural formula as follows: Formula I.

[0018] The guava glycoside is a yellow powder, purchased from Chengdu Mansite Biotechnology Co., Ltd. (extraction source: *Oroxylum indicum*), product number MUST-25022810. The guava glycoside derivatives include the following glycosyl-modified derivatives: fully acetylated guava glycoside, triacetylated guava glycoside, methylated arabinose guava glycoside, and 6''-acylated guava glycoside. Modification of the arabinose hydroxyl group improves the lipophilicity and / or stability of the guava glycoside.

[0019] In this invention, the diabetic retinopathy preferably includes abnormal proliferation of retinal endothelial cells, increased levels of membrane lipid peroxidation, retinal structural damage, and retinal microvascular damage. The retinal structural damage preferably includes decreased retinal thickness, degenerative changes in retinal neural structures, and retinal microvascular damage. The retinal thickness preferably includes at least one of the following: full-thickness retinal thickness, inner retinal thickness, middle retinal thickness, and outer retinal thickness. The degenerative changes in retinal neural structures preferably include thinning of the retinal neural layer and / or loss of ganglion cells. The retinal neural layer structure preferably includes the inner nuclear layer and / or outer nuclear layer. The retinal microvascular damage preferably includes loss of retinal pericellular cells and / or damage to fine, cell-free capillaries.

[0020] In this invention, the diabetic retinopathy is preferably streptozotocin-induced ocular retinopathy.

[0021] In one embodiment of the present invention, a human retinal microvascular endothelial cell hyperglycemic injury model was constructed to evaluate the efficacy of guavaside. The results showed that guavaside could improve hyperglycemia-induced abnormal proliferation of endothelial cells and reduce membrane lipid peroxidation levels. In another embodiment of the present invention, a mouse hyperglycemia-induced retinopathy model was constructed to evaluate the in vivo efficacy of guavaside. The results showed that guavaside could improve the long-term hyperglycemic state in DR mice and effectively improve the impaired glucose tolerance in diabetic mice, improve diabetes-induced retinal thinning, effectively alleviate diabetes-induced retinal neural layer thinning and ganglion cell loss, and has a protective effect against retinal structural damage. It also reduced hyperglycemia-induced retinal pericellular loss and fine clamp cell-free capillary damage, and has a certain protective effect against diabetic microvascular damage.

[0022] In this invention, the drug preferably comprises at least one of the following dosage forms: injection, drops, powder, tablet, capsule, granule, and oral liquid. The drug also includes pharmaceutically acceptable excipients. This invention does not impose any special restrictions on the type of excipients; the type of excipients can be conventionally selected according to the type of oral preparation. This invention does not impose any special restrictions on the preparation method of the oral preparation; oral preparations well known in the art can be used. The mass percentage of guavaside in the drug is preferably 10%~98%, but can also be 20%~90%, 30%~80%, 40%~70%, and more preferably 50%~60%.

[0023] The following detailed description, in conjunction with embodiments, illustrates the application of guavaside provided by the present invention in the prevention and / or treatment of diabetic retinopathy, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0024] Example 1 Cellular Experimental Methods and Results 1. Experimental Methods: 1.1 Culture and passage of human retinal microvascular endothelial cells Human retinal microvascular endothelial cells (HRMECs) were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. Cells were cultured in complete culture medium (Endothelial Cell Medium + 10% fetal bovine serum) at 37°C in a 5% CO2 incubator. When the cells reached 80% confluence, they were carefully collected for passage to ensure exponential cell growth. In all cell experiments, all cells remained mycoplasma-free when the passage number was <25.

[0025] 1.2 Human retinal microvascular endothelial cell DR modeling and guava glycoside treatment HRMECs in the logarithmic growth phase were used for subsequent experiments. Cells were randomly divided into three groups: a control group treated with medium containing 5.5 mmol / L D-glucose; high-glucose model groups treated with medium containing 30 mmol / L and 60 mmol / L D-glucose, respectively; and guava glycoside treatment groups treated with high-glucose medium supplemented with 30 μmol / L guava glycoside for co-incubation. Cells in each group underwent continuous intervention for 24 hours.

[0026] 1.3 Cell proliferation detection Cell proliferation was assessed using the MTT assay. HREMCs in logarithmic growth phase were seeded in 96-well plates and treated according to pre-defined conditions. After drug administration, 20 μL of MTT solution (5 mg / mL) was added to each well, and the plates were incubated at 37°C in the dark for 4 hours. After incubation, the supernatant was carefully aspirated, and 150 μL of dimethyl sulfoxide (DMSO) was added to each well. The plates were then shaken slowly in the dark for 10 minutes to fully dissolve the generated blue-purple formazan crystals. The absorbance (OD) of each well was then measured using a microplate reader at a wavelength of 490 nm (or 570 nm), and cell viability was calculated using the control group as a baseline.

[0027] 1.4 Detection of malondialdehyde (MDA) content in cells After group intervention, the intracellular malondialdehyde (MDA) content was detected using a malondialdehyde (MDA) kit. The specific steps were as follows: cells were collected into centrifuge tubes, and the supernatant was discarded after centrifugation; extraction buffer was added, and the cells were sonicated (power 200 W, sonication for 3 seconds, interval 10 seconds, repeated 30 times); the cells were centrifuged at 8000 g at 4℃ for 10 min, and the supernatant was collected and measured according to the kit method.

[0028] 2. Experimental Results: 2.1 Guavain improves the abnormal proliferation of human retinal microvascular endothelial cells under high glucose damage environment. A high-glucose injury model of HREMCs was constructed using the above method to explore the effect of guavaside in improving abnormal cell proliferation under high-glucose injury conditions. Figure 1 It was found that, compared with the control group, the proliferation activity of HREMCs in the 60 mmol / L and 30 mmol / L high glucose model groups was significantly increased, successfully constructing a high glucose-induced abnormal proliferation model of retinal microvascular endothelial cells. After intervention with 30 μmol / L guavaside, the cell proliferation activity under high glucose damage at 60 mmol / L and 30 mmol / L was significantly reduced compared with the corresponding model groups, suggesting that guavaside can effectively inhibit the abnormal proliferation of HREMCs induced by high glucose and has a clear intervention effect on high glucose damage at different concentrations.

[0029] 2.2 Guavain improves the level of membrane lipid peroxidation (malondialdehyde content) in human retinal microvascular endothelial cells under high glucose damage environment. Using the aforementioned high-glucose damage model, we further investigated the effect of guavaside in improving high-glucose-induced membrane lipid peroxidation in HREMCs. Figure 2 It was found that, compared with the control group, the malondialdehyde (MDA) content in HREMCs in the 60 mmol / L high glucose model group was significantly increased, suggesting that high glucose can induce a severe oxidative stress response in HREMCs, leading to cell membrane lipid peroxidation damage. After intervention with 30 μmol / L guavaside, the intracellular MDA content was significantly reduced in the high glucose model group and approached the level of the control group, indicating that guavaside can effectively inhibit high glucose-induced membrane lipid peroxidation in HREMCs. In summary, in vitro cell experiments demonstrate that guavaside can improve cell proliferation and damage of human retinal microvascular endothelial cells under high glucose environment.

[0030] Example 2 Mouse experiment 1. Experimental Methods 1.1 Laboratory Animals SPF-grade male C57BL / 6J mice aged 6-8 weeks were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. Prior to the experiment, all mice were subjected to a 12:12 h light / dark adaptation diet for one week in an animal room with controlled temperature and humidity, and were provided with normal food and drinking water daily. All experimental procedures followed the Animal Care and Use Guidelines of the Institute of Zoology, Zhejiang University.

[0031] 1.2 Construction of mouse DR model Before modeling, mice were fasted but allowed free access to water overnight. After accurate weight measurement, a diabetic model was induced using the STZ intraperitoneal injection method. STZ powder was dissolved in pre-cooled 0.1 mol / L sodium citrate buffer (pH 4.5) to prepare a solution with a concentration of 5 mg / mL. To ensure STZ activity, the solution was tightly wrapped in aluminum foil to protect it from light, stored in an ice bath, and injected within 30 minutes of preparation.

[0032] Mice in the model group were intraperitoneally injected with STZ solution at a dose of 50 mg / kg body weight for 5 consecutive days; mice in the control group were intraperitoneally injected with the same volume of blank sodium citrate buffer for 5 consecutive days. Two weeks after the last injection, mice were fasted for 12 hours, and fasting blood glucose (FBG) was measured using a portable blood glucose meter via tail vein sampling. Mice with FBG ≥ 13.9 mmol / L and accompanied by polydipsia, polyphagia, and polyuria were considered to have successfully developed diabetes and were included in subsequent experiments.

[0033] 1.3 Procedures for grouping, administering medication, and intervening in experimental animals Mice were randomly divided into four groups using a random number table: control group (n=6), model group (n=6), low-dose guavaside group (50 mg / kg, n=6), and high-dose guavaside group (100 mg / kg, n=6). Guavaside was suspended in 5% sodium carboxymethyl cellulose (CMC-Na) solution, and mice in each group were administered the drug by gavage once daily at a fixed time. Mice in the control and model groups were administered an equal volume of 5% CMC-Na by gavage. After 12 weeks of continuous administration, all mice were fasted for 12 hours but allowed free access to water. They were then anesthetized by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg), and blood and ocular tissue were collected by enucleation for subsequent experimental testing.

[0034] 1.4 Experimental Apparatus Table 1. Description of Experimental Instruments

[0035] 1.5 Experimental Consumables and Reagents Table 2. Description of Experimental Consumables and Reagents

[0036] 1.6 Intraperitoneal glucose tolerance test (IPGTT) IPGTT was performed on one day during the twelfth week of drug administration in mice. At 17:00 the day before the experiment, mice were transferred to clean cages and fasted for 16 hours (water was allowed). The following morning, each mouse was weighed and labeled. First, fasting basal blood glucose (0 min) was measured, and tail blood was collected for glucose level analysis using a glucometer. Then, glucose solution was injected intraperitoneally at a dose of 2 g / kg body weight. Timing began from the first mouse injected, and each group of mice completed the injection within 15 minutes. Tail blood was collected at 30, 60, 90, and 120 min after injection to measure blood glucose levels. Blood glucose-time curves were plotted based on the blood glucose values ​​at each time point, and the area under the curve (AUC) for the 0-30 min, 0-60 min, 0-90 min, and 0-120 min time periods was calculated using the trapezoidal integral method to evaluate the mice's glucose tolerance and glucose metabolism. After the experiment, mice were returned to normal feeding conditions.

[0037] 1.7 In vivo retinal structure detection in mice 1.7.1 Animal handling and OCT image acquisition In vivo retinal structure examination was performed 12 weeks after guavaside intervention. Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital at a dose of 50 mg / kg. After adequate anesthesia, compound tropicamide eye drops were instilled into both eyes to dilate the pupils, and sterile artificial tears were continuously used to keep the cornea moist to avoid corneal dryness affecting image quality.

[0038] The mouse was secured to a specialized small animal ophthalmology examination platform, and its head and body position were adjusted to ensure that the axial length of the eye was coaxial with the imaging optical path. The probe was gently touched to the corneal surface, and the focus and depth of focus were adjusted until a clear and stable image of the retinal layer structure was obtained.

[0039] Scans were performed using the Phoenix Micron IV small animal ophthalmic imaging system (Phoenix Research Labs, USA). A circular scanning mode was selected to acquire tomographic images of the central retinal region, while the corresponding fundus color images and raw OCT data were saved. After imaging, the mice were placed in a constant-temperature resuscitation incubator until they recovered spontaneously.

[0040] 1.7.2 Retinal Layer Determination and Thickness Measurement The retinal structure was divided into zones based on the reflectance characteristics of each tissue layer in OCT images. The stratification criteria are as follows: Full-thickness retinal thickness: the vertical distance from the outer boundary of the Retinal pigment epithelium (RPE) to the inner boundary of the Nerve fiber layer (NFL).

[0041] Inner retinal thickness: from the inner edge of the NFL to the outer boundary of the Inner nuclear layer (INL).

[0042] Intermediate layer thickness: The distance from the outer edge of the INL to the junction between the inner and outer segments of the photoreceptor (IS / OS boundary).

[0043] Outer retinal thickness: from the distal edge of IS / OS to the RPE layer.

[0044] Under uniform magnification and focusing conditions, a standardized scanning section of the central retinal region was selected for measurement. Three to five equidistant measurement points were taken from each animal, and the average value was calculated to reduce errors caused by local anatomical differences.

[0045] Thickness data were quantitatively analyzed using ImageJ software, and statistical processing and graphing were performed using GraphPad Prism 10.3.1.

[0046] 1.8 HE staining Eye tissue fixed in paraformaldehyde was removed, trimmed, and then dehydrated sequentially with graded alcohols, cleared with xylene, and then routinely embedded in paraffin. The embedded paraffin block was sliced ​​into thin sections using a microtome, flattened, attached to glass slides, and baked for later use. The sections were dewaxed sequentially with xylene and graded alcohols until water was removed, then immersed in hematoxylin staining solution for 3 min, washed with water, and then blued with tap water. After slight dehydration with alcohol, eosin staining solution was added for 5 min. After staining, the sections were again thoroughly dehydrated with anhydrous ethanol and cleared with xylene, and finally mounted with neutral resin. After mounting, the sections were observed under an optical microscope and images were acquired. The thickness of the entire retina and each sublayer was quantitatively measured according to anatomical layers using Image-Pro Plus software. Finally, GraphPad 10.3.1 software was used for data statistics and visualization.

[0047] 1.9 PAS staining of mouse retinal digestive smears To observe the retinal microvascular structure and pericyte loss, a combination of retinal digestion and PAS staining was used. Mice were euthanized after the experiment, and the eyeballs were quickly enucleated and fixed in 4% paraformaldehyde for 24 hours. After fixation, the retina was thoroughly rinsed with PBS to remove residual fixative. The cornea, lens, and vitreous tissue were removed. The retinal neuroepithelial layer was carefully dissected using the optic nerve as a fulcrum and rinsed in PBS. The intact retina was placed in 3% trypsin digestion solution and digested at a constant temperature of 37 °C for approximately 2-3 hours (adjusted according to tissue condition). The degree of digestion was observed under a microscope during this time. Digestion was stopped when the nerve tissue gradually dissolved and the vascular network structure became clearly visible. The digested retina was transferred to warm water and gently blown to remove residual nerve tissue, obtaining a translucent vascular network structure. The vascular network was carefully aspirated and placed on an anti-detachment slide, gently spread using liquid tension to ensure the network was fully flattened, and then allowed to air dry and fix. Staining was performed using a PAS staining kit, with the following steps: The dried blood vessel slides were directly immersed in 0.5% periodic acid solution for 15 min for oxidation, followed by washing three times with pure water. Then, Schiff's reagent, restored to room temperature, was added, and the slides were incubated in the dark for 20–30 min. After rinsing with running water for 5 min, the cell nuclei were counterstained with hematoxylin for 30 s. Following rinsing with tap water, brief differentiation with hydrochloric acid solution, and blueing with ammonia, the slides were dehydrated sequentially with a gradient of anhydrous ethanol, cleared with xylene, and finally mounted with neutral resin.

[0048] 1.10 Statistical Analysis All data are expressed as mean ± standard error (SEM). GraphPad Prism 10.3.1 software was used for analysis. Differences between groups were analyzed using one-way ANOVA or unpaired-samples t-test. P A value <0.05 indicates a significant difference.

[0049] 2. Experimental Results: 2.1 Effects of guavaside on general physiological indicators in DR mice Mice body weight was recorded weekly during the 12-week administration period, and mice in all groups maintained good mental condition throughout the experiment. Compared with the control group, the model group mice showed a statistically significant decrease in body weight, indicating that the DR mice exhibited diabetic symptoms, and the diabetic model was successfully established. Compared with the model group, both the low-dose (50 mg / kg) and high-dose (100 mg / kg) groups of guava glycoside showed partial improvement in body weight. Figure 3 (B) There was no statistically significant difference overall. These results indicate that guavaside intervention tends to improve the general metabolic status of diabetic mice.

[0050] 2.2 Guavain improves blood glucose levels in DR mice To reduce the interference of circadian rhythms on blood glucose fluctuations, this study evaluated the effect of guavaside on glucose metabolism in diabetic mice by measuring fasting blood glucose levels in each group after fasting at fixed time intervals.

[0051] Compared with the control group, the fasting blood glucose levels in the model group mice remained elevated after modeling, and the difference was statistically significant, indicating that the diabetes model was successfully established. After 12 weeks of intervention with guavaside, the fasting blood glucose levels in the guavaside 50 mg / kg group and the guavaside 100 mg / kg group were significantly lower than those in the model group. Figure 4 This indicates that guavaside can effectively improve persistent hyperglycemia.

[0052] Further IPGTT experiments were conducted to evaluate the body's ability to clear glucose load. Results showed that the blood glucose levels in the model group were significantly higher than those in the control group at all time points after glucose load, exhibiting elevated peak blood glucose levels and delayed clearance, indicating impaired glucose tolerance. Compared with the model group, the guava glycoside intervention group showed an overall downward shift in the blood glucose curve, with varying degrees of reduction in blood glucose levels at each time point. Figure 5 (A). Area under the curve analysis for the 0-120 min time period showed that the area under the curve in the 50 mg / kg and 100 mg / kg guava glycoside groups was significantly lower than that in the model group, and the high-dose groups showed better recovery of glucose tolerance. Figure 5 (B) This suggests that guavaside can significantly improve glucose clearance and alleviate glucose intolerance.

[0053] The above indicates that guavaside can improve the long-term hyperglycemic state in DR mice and effectively improve the impaired glucose tolerance in diabetic mice, and exhibits a certain dose-dependent effect.

[0054] 2.3 Guavaside improves retinal thickness in DR mice To evaluate the effects of guavaside on retinal structural changes in DR mice, this study used OCT to perform in vivo imaging analysis of the retinas of mice in each group. Based on the retinal anatomical layers (… Figure 6 (A) Thickness measurements were performed on the entire retina, as well as the inner, middle, and outer layers.

[0055] OCT images showed that, compared with the control group, the overall retinal structure of mice in the model group was thinner. Figure 6(Middle B). Quantitative analysis results showed that the thickness of the inner, middle, outer, and overall retina was significantly reduced in the model group. After intervention with guavaside, the thickness of the inner, middle, outer, and overall retina in the guavaside 50 mg / kg and guavaside 100 mg / kg groups was significantly increased compared with the model group. Among them, the thickness of the inner, middle, and overall retina was significantly improved. Stratified comparison showed that retinal thinning in diabetic model mice mainly affected the inner and middle layers, and guavaside could effectively alleviate structural damage in these areas in a dose-dependent manner. Figure 6 (CF).

[0056] In summary, OCT results indicate that guavaside can improve diabetes-induced retinal thinning and has a protective effect against retinal structural damage.

[0057] 2.4 Effects of guavaside on retinal structure in DR mice To further verify the protective effect of guavaside on retinal structure, HE staining was used to observe morphological changes in the retinal tissue of mice in each group. In the control group, the retinal structure was clear, with regular arrangement of layers and uniform cell distribution. Compared with the control group, the overall thickness of the retina in the model group was significantly thinner, with looser cell arrangement and a reduced cell number in both the inner and outer nuclear layers. Simultaneously, the number of ganglion cell layers was significantly decreased; the area marked by the red arrow represents the ganglion cell layer. Figure 7 (B) Quantitative analysis showed that the thickness of both the inner and outer nuclear layers in the model group was significantly lower than that in the control group, the total retinal thickness was significantly reduced, and the number of ganglion cells was also significantly decreased, suggesting that significant degenerative changes occur in the retinal neural structure under diabetic conditions. After intervention with guavaside, the retinal structure in each dose group was significantly improved compared with the model group, with a significant increase in the thickness of both the inner and outer nuclear layers, an increase in the total retinal thickness, and a significant increase in the number of ganglion cells. Among them, the guavaside 100 mg / kg group showed more significant improvement, with some indicators approaching the level of the control group ( Figure 7 (CF).

[0058] The above results indicate that guavaside can effectively alleviate diabetes-induced thinning of the retinal nerve layer and loss of ganglion cells, and has a protective effect against retinal structural damage.

[0059] 2.5 Guavain improves retinal capillary damage in DR mice To systematically evaluate the protective effect of guavaside on diabetic retinal microvascular homeostasis, this study performed microvascular patch staining and quantitative analysis on retinal tissues of mice in each group. In the control group, the microvascular network structure was intact, with relatively uniform distribution of endothelial cells and pericytes. After modeling, the model group showed loss of pericytes (yellow arrows) and increased formation of fine, cell-free capillaries (red arrows). After guavaside intervention, the damage to fine, cell-free capillaries in the 50 mg / kg and 100 mg / kg groups was reduced compared to the model group. Figure 8 (A) The number of cell-free capillaries in the model group mice was significantly higher than that in the control group, and the pericyte-to-endothelial cell ratio (P / EC) was decreased, consistent with the microvascular pathology characteristics of diabetes. After intervention with guavaside, the number of cell-free capillaries in the 50 mg / kg and 100 mg / kg groups was significantly lower than that in the model group; at the same time, the P / EC ratio in both groups rebounded to above 0.5, and the difference compared with the model group was statistically significant. Figure 8 (Middle BC). The above results indicate that guavaside can reduce high glucose-induced retinal pericellular loss and fine clamp-like cell-free capillary damage, and has a certain protective effect against diabetic microvascular damage.

[0060] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of guavaside or guavaside derivatives in drugs for the prevention and / or treatment of diabetic retinopathy.

2. The application according to claim 1, characterized in that, The diabetic retinopathy includes abnormal proliferation of retinal endothelial cells, elevated levels of membrane lipid peroxidation, retinal structural damage, and retinal microvascular damage.

3. The application according to claim 2, characterized in that, The retinal structural damage includes reduced retinal thickness, degenerative changes in retinal neural structures, and retinal microvascular damage.

4. The application according to claim 3, characterized in that, The retinal thickness includes at least one of the following: full-thickness retinal thickness, inner retinal thickness, middle retinal thickness, and outer retinal thickness.

5. The application according to claim 3, characterized in that, The degenerative changes in the retinal neural structure include thinning of the retinal neural layer and / or loss of ganglion cells.

6. The application according to claim 5, characterized in that, The retinal neural layer structure includes an inner nuclear layer and / or an outer nuclear layer.

7. The application according to claim 3, characterized in that, The retinal microvascular injury includes loss of retinal pericellular cells and / or damage to fine, cell-free capillaries.

8. The application according to claim 1, characterized in that, The guava glycoside derivatives include the following glycosyl-modified derivatives: fully acetylated guava glycoside, triacetylated guava glycoside, methylated arabinose guava glycoside, and 6''-acylated guava glycoside.

9. The application according to any one of claims 1 to 8, characterized in that, The drug includes at least one of the following dosage forms: injection, drops, powder, tablet, capsule, granule, and oral liquid.

10. The application according to claim 9, characterized in that, The drug contains guavaside at a mass percentage of 10% to 98%.

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