Application of boxthorn leaf extract in preparation of medicine for treating diabetic retinopathy

Abstract

The invention discloses application of a boxthorn leaf extract in preparation of a medicine for treating diabetic retinopathy. The boxthorn leaf refined extract is prepared by adopting specific process steps, and the boxthorn leaf active component can protect ARPE-19 cells damaged by high glucose induction, also has a certain improvement effect on diabetic retinopathy zebrafish, and remarkably improves eye symptoms in a diabetic retinopathy mouse model. The boxthorn leaf extract provided by the invention has the potential of becoming a medicine for treating diabetic retinopathy.

Description

Technical Field

[0001] The present invention relates to a traditional Chinese medicine, and specifically to the application of wolfberry leaf extract in improving diabetic retinopathy, belonging to the field of medical technology. Background Art

[0002] Diabetes is a common disease. In clinical practice, various complications will occur in the middle and late stages of diabetes. Among them, diabetic retinopathy (DR), as one of the diabetic complications, is one of the main causes of blindness. Effectively preventing and treating the occurrence and development of DR has become an urgent social need. The pathogenesis of DR is complex. At present, it is generally believed that the pathogenesis of DR includes microvascular lesions, neuronal lesions, and low to moderate inflammatory reactions induced by hyperglycemia. The clinical treatment of DR mainly includes controlling blood sugar and blood lipids, laser photocoagulation, and intravitreal injection of antibody drugs. In recent years, traditional Chinese medicine has shown obvious characteristics and significant advantages in delaying the progression of DR, reducing its incidence rate, and improving the quality of life of patients due to its mild and lasting effects, multiple components, and multiple targets. Therefore, under the guidance of traditional Chinese medicine theory, the research and development of new drugs for preventing and treating DR has become a current research hotspot.

[0003] Wolfberry leaf (named "Tianjingcao" in "Compendium of Materia Medica") is the dried young leaf of Lycium barbarum Lycium barbarum L. or Lycium chinense L. chinense Mill., and has the effects of tonifying deficiency and replenishing essence, clearing heat, stopping thirst, dispelling wind and improving eyesight; it has various pharmacological effects, such as immunomodulation, anti-tumor, antioxidant, anti-aging, and lowering blood sugar and blood lipids.

[0004] There has been no report on the improvement effect of different active components of wolfberry leaf on DR and its potential mechanism. Summary of the Invention

[0005] Object of the Invention: The object of the present invention is to screen the wolfberry leaf active component with the best curative effect by studying the improvement effect of different active components of wolfberry leaf on DR, so as to provide a theoretical basis for the rational clinical use of wolfberry leaf to treat DR.

[0006] The present invention will use different active components of wolfberry leaf to treat ARPE-19 cells, A / B-type zebrafish, and C57BL / 6J mice, and investigate the protective effect of different active components of wolfberry leaf on ARPE-19 cells damaged by high glucose through methods such as CCK8, qPCR, oxidation kits (MDA, SOD, GSH), and ROS; investigate the protective effect of different active components of wolfberry leaf on the DR zebrafish model by detecting the glucose level in the DR zebrafish body, changes in the diameter of zebrafish retinal blood vessels, qPCR, and oxidation kits (MDA, SOD, GSH), etc., so as to screen out the best active component of wolfberry leaf for improving DR.

[0007] The various active components of the wolfberry leaves described in the present invention are obtained by the following method: Weigh 1 kg of dried wolfberry leaves, add 10 times the mass of distilled water, heat under reflux for extraction twice, with each extraction lasting 1 h, concentrate under reduced pressure, and obtain the aqueous extract of wolfberry leaves after freeze-drying; Weigh 2 kg of dried wolfberry leaves, add 10 times the mass of 80% ethanol, heat under reflux for extraction twice, with each extraction lasting 1 h, to obtain the ethanol extract of wolfberry leaves and the ethanol-extracted medicinal residues; Divide the ethanol extract into two equal parts, concentrate one part under reduced pressure and obtain the ethanol extract of wolfberry leaves after freeze-drying, and for the other part, concentrate it to an appropriate volume and then fully adsorb it with D101 macroporous resin for 24 h, elute it with distilled water, 10% ethanol, and 30% ethanol for 2 - 3 column volumes respectively, discard the eluate, use 70% ethanol as the eluent, elute for 2 - 3 column volumes, collect the eluate, concentrate under reduced pressure, and obtain the refined extract of wolfberry leaves after freeze-drying; Air-dry the ethanol-extracted medicinal residues, add 10 times the mass of distilled water, heat under reflux for extraction twice, each time for 1 h, concentrate under reduced pressure, add absolute ethanol until the ethanol concentration reaches 80%, let it stand overnight at 4°C, discard the supernatant, dry it, to obtain crude polysaccharides, and use the Sevage (chloroform: n-butanol = 4:1) method to remove proteins to obtain refined polysaccharides.

[0008] Research shows that 98% of the alkaloids in wolfberry leaves are betaine. Therefore, betaine is used as the alkaloid in wolfberry leaves for further research.

[0009] The present invention can form the refined extract of wolfberry leaves and a pharmaceutically acceptable carrier into a drug in the form of tablets, capsules, granules, or pills.

[0010] When making tablets in the present invention, add the carrier lactose or corn starch to the refined extract of wolfberry leaves, and add magnesium stearate as a lubricant when needed, mix evenly, and then press into tablets.

[0011] When making capsules, mix the refined extract of wolfberry leaves and the carrier lactose or corn starch evenly, granulate, and then fill into capsules to make capsules.

[0012] When making granules in the present invention, mix the refined extract of wolfberry leaves and the diluent lactose or corn starch evenly, granulate, and dry to make granules.

[0013] Beneficial effects: The wolfberry leaf extract provided by the present invention has the following advantages compared with the prior art: This invention screened the improvement effects of different active components of wolfberry leaves on in vitro cell models and zebrafish models of DR, and verified them in in vivo animal models. The experimental results showed that except for wolfberry leaf polysaccharide, the water extract of wolfberry leaves, the alcohol extract of wolfberry leaves, the refined extract of wolfberry leaves, and the active components of wolfberry alkaloids could all increase the survival rate of ARPE-19 cells damaged by high glucose induction, down-regulate the expression levels of ERK, p38, and JNK, improve oxidative stress, and significantly reduce ROS generation. Compared with other groups, the refined extract of wolfberry leaves had a more prominent effect at the same crude drug dose, indicating that the refined extract of wolfberry leaves was the main active component.

[0014] In the DR zebrafish model, except for wolfberry leaf polysaccharide, the water extract of wolfberry leaves, the alcohol extract of wolfberry leaves, the refined extract of wolfberry leaves, and the active components of wolfberry alkaloids could all reduce the glucose content in their bodies, down-regulate the expression levels of ERK, p38, and JNK, improve oxidative stress, and significantly reduce the retinal vessel diameter of zebrafish. Compared with other groups, the refined extract of wolfberry leaves had a more prominent effect at the same crude drug dose, indicating that the refined extract of wolfberry leaves was the main active component.

[0015] Based on the research of in vitro cell models and zebrafish models, by comparing with the water extract and alcohol extract of wolfberry leaves, the results showed that the refined extract of wolfberry leaves had a more prominent effect at the same crude drug dose, indicating that the components of the refined extract of wolfberry leaves were the best active components for improving DR. Therefore, the improvement effect of the refined extract of wolfberry leaves on DR was further verified in the DR mouse model. By detecting the changes in mouse body weight, blood glucose level, retinal diameter, and retinal vessel density, it was proved that the refined extract of wolfberry leaves could significantly increase the retinal vessel diameter of mice and reduce the retinal vessel density of mice. Therefore, the refined extract of wolfberry leaves screened by this invention can be used as a therapeutic and improving drug for DR. This invention can achieve the efficient comprehensive utilization of wolfberry leaf resources. Brief Description of the Drawings

[0016] Figure 1 Effects of different active components of wolfberry leaves on the survival rate (CCK8) of ARPE-19 cells damaged by high glucose induction and the levels of oxidative stress (GSH, SOD, MDA).

[0017] Figure 2 Effects of different active components of wolfberry leaves on the gene expression levels (p38, ERK, JNK) of ARPE-19 cells damaged by high glucose induction.

[0018] Figure 3 Effects of different active components of wolfberry leaves on the ROS content of ARPE-19 cells damaged by high glucose induction. In the figure, (A) representative pictures of the ROS content after administration of each active component; (B) statistical results of the ROS content level.

[0019] Figure 4 The effects of different active components of wolfberry leaves on the glucose content (GLU) and oxidative stress levels (GSH, SOD, MDA) in DR zebrafish

[0020] Figure 5 The effects of different active components of wolfberry leaves on the gene expression levels (p38, ERK, JNK) in DR zebrafish

[0021] Figure 6 The effects of different active components of wolfberry leaves on the retinal vessel diameter in DR zebrafish. In the figure, (A) Representative pictures of zebrafish retinal vessels after administration of each active component; (B) Statistical results of retinal vessel diameter after administration of each active component.

[0022] Figure 7 The effects of different active components of wolfberry leaves on the body weight and blood glucose levels in DR mice. In the figure, (A) Changes in mouse body weight levels after administration of each active component; (B) Changes in mouse blood glucose levels after administration of each active component.

[0023] Figure 8 The effects of different active components of wolfberry leaves on the retinal vessel density in DR mice. In the figure, (A) Representative pictures of mouse retinal vessel density after administration of each active component; (B) Statistical results of mouse retinal vessel density after administration of each active component.

[0024] Figure 9 The effects of different active components of wolfberry leaves on the retinal vessel diameter in DR mice. In the figure, (A) Representative pictures of mouse retinal vessel diameter after administration of each active component; (B) Statistical results of mouse retinal vessel diameter after administration of each active component. Detailed implementation manners

[0025] According to the following embodiments, the present invention can be better understood. However, those skilled in the art can easily understand that the specific material ratios, process conditions and their results described in the embodiments are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0026] The reagents used in the embodiments of the present invention include: Reagent Name Manufacturer Fetal Bovine Serum (FBS) Gibco, USA Penicillin BI, Israel Streptomycin BI, Israel DMEM / F12 Medium Gibco, USA CCK8 Detection Kit Beyotime Biotechnology Co., Ltd., Shanghai <![CDATA[H2FDCA]]> Glpbio, USA The primers used in the embodiments of the present invention include:

[0027] The animals and cells used in the embodiments of the present invention include: ARPE-19 cells were derived from the ATCC cell bank and cultured in DMEM / F12 complete medium containing 10% FBS (with 1% double antibody) in a cell incubator at a constant temperature (37°C, 5% CO2), and subcultured every 2 - 3 days.

[0028] Wild type A / B zebrafish and fli1:EGFP zebrafish, 6 months old, were purchased from Nanjing Yaoshunyu Biotechnology Co., Ltd. They were cultured in a 28.5°C circulating filtered water system with a light cycle of 14 h (light):10 h (dark). Zebrafish embryos with normal development 2 h after fertilization were used for subsequent experiments.

[0029] C57BL / 6J mice, 8 weeks old, were purchased from Shanghai Slack Experimental Animal Co., Ltd. Each group was housed separately. The blank group was fed with normal feed, and the other groups were fed with high - sugar and high - fat feed. They had free access to food and water, normal light, and the temperature was controlled at 20 - 25°C. All operations and research processes were carried out in accordance with the "Regulations on the Administration of Laboratory Animals". Research procedures.

[0030] Example 1: Preparation of active components from wolfberry leaves Weigh 1 kg of dry wolfberry leaves, add 10 times the mass of distilled water, and extract by heating under reflux 2 times, each extraction for 1 h. Then concentrate under reduced pressure and freeze - dry to obtain the water extract of wolfberry leaves. Weigh 2 kg of dry wolfberry leaves, add 10 times the mass of 80% ethanol, and extract by heating under reflux 2 times, each extraction for 1 h, to obtain the ethanol extract of wolfberry leaves and the ethanol - extracted medicinal residues. Divide the ethanol extract into two equal parts. One part is concentrated under reduced pressure and freeze - dried to obtain the ethanol extract of wolfberry leaves.

[0031] The other part is concentrated under reduced pressure to an appropriate volume and then fully adsorbed with D101 macroporous resin for 24 h. It is eluted with distilled water, 10% ethanol, and 30% ethanol for 2 - 3 column volumes respectively, and the eluates are discarded. Then, 70% ethanol is used as the eluent, eluted for 2 - 3 column volumes, and the eluate is collected, concentrated under reduced pressure, and freeze - dried to obtain the refined extract of wolfberry leaves. After drying the medicinal residues of wolfberry leaves after ethanol extraction, add 10 times the mass of distilled water, extract by heating under reflux 2 times, each time for 1 h. Concentrate under reduced pressure and then add absolute ethanol until the ethanol concentration reaches 80%. Let it stand overnight at 4°C, discard the supernatant, dry it, to obtain crude polysaccharide. The protein is removed by Sevage (chloroform:n - butanol = 4:1) method to obtain wolfberry polysaccharide. Research shows that 98% of the alkaloids in wolfberry leaves are betaine, so betaine is used as the alkaloid in wolfberry leaves for the next step of research.

[0032] Example 2: Activity screening based on cell models 1. Experimental method 1.1 High - glucose - induced injury of ARPE - 19 cells Preparation method of active component solution of wolfberry leaves: Weigh 1 mg of freeze-dried powder of different active components of wolfberry leaves (aqueous extract of wolfberry leaves, ethanol extract of wolfberry leaves, refined extract of wolfberry leaves, wolfberry polysaccharide, and betaine) prepared in Example 1, dissolve it in 1 mL of DMEM / F12 medium, and configure it into a 1 mg / mL solution. Dilute it to the required concentration with DMEM / F12 medium during administration, and filter and sterilize it with a 22 μm filter membrane.

[0033] Culture ARPE-19 cells in DMEM / F12 medium containing 10% FBS and 1% penicillin / streptomycin at 37 °C and 5% CO2. The model group was treated with 300 mM glucose and cultured with the corresponding drug added to the medium for 48 h to complete the modeling.

[0034] 1.2 Detection of the protective effect of active components of wolfberry leaves on ARPE-19 cells damaged by high glucose induction using a kit When culturing, inoculate the cells into a 96-well plate, and divide them into a control group (Con group), a model group (Mod group), a high-dose group of aqueous extract of wolfberry leaves (ST-H group), a low-dose group of aqueous extract of wolfberry leaves (ST-L group), a high-dose group of ethanol extract of wolfberry leaves (CT-H group), a low-dose group of ethanol extract of wolfberry leaves (CT-L group), a high-dose group of refined extract of wolfberry leaves (FT-H group), a low-dose group of refined extract of wolfberry leaves (FT-L group), a high-dose group of wolfberry polysaccharide (DT-H group), a low-dose group of wolfberry polysaccharide (DT-L group), a high-dose group of betaine (SWJ-H group), and a low-dose group of betaine (SWJ-L group). The high dose is 10 μg / mL, and the low dose is 1 μg / mL. After the culture is completed, discard the medium, add 10% CCK8 solution and incubate at 37 °C for 1 h, measure the absorbance at 450 nm, and calculate the cell survival rate.

[0035] When culturing the cells, inoculate the cells into a 6-well plate, and divide them into a Con group, a Mod group, an ST-H group, an ST-L group, a CT-H group, a CT-L group, an FT-H group, an FT-L group, a DT-H group, a DT-L group, a SWJ-H group, and a SWJ-L group. The high dose is 10 μg / mL, and the low dose is 1 μg / mL. After the culture is completed, collect the adherent cells, centrifuge at 800 g / min for 5 min at 4 °C, wash the cells twice with PBS, centrifuge again, add 300 ml of PBS, turn on the ultrasound for 2 s, turn it off for 8 s, and break the cells in an ice-water bath for 2 min. Centrifuge at 4000 rpm / min for 10 min at 4 °C, and detect the intracellular GSH level and the activities of SOD and MDA according to the kit instructions.

[0036] 1.3 Detection of the effect of active components of wolfberry leaves on the gene expression level of ARPE-19 cells damaged by high glucose induction by qPCR (1) Total RNA extraction When culturing cells, inoculate the cells into a 6-well plate and divide them into the Con group, Mod group, ST group, CT group, FT group, DT group, and SWJ group. The dosing dose is 10 μg / mL. Collect the cells into an Ep tube, centrifuge at 1000 rpm for 5 min, and discard the supernatant. Add 1 mL of Trizol reagent to each tube and vigorously pipette for 15 s to mix well. Let it stand at room temperature for 2 - 3 min, then centrifuge at 4°C for 1 - 2 h. Add 200 μL of chloroform, vortex for about 15 s until the liquid shows no obvious stratification, and centrifuge at 12000 g for 15 min. Pipette 500 μL from the supernatant into a nuclease-free Ep tube, add 500 μL of isopropanol, vortex to mix well, place on ice, incubate for 5 - 10 min, and centrifuge at 12000 g for 10 min. Discard the supernatant, add 1 mL of 75% ethanol prepared with DEPC water (pre-cooled at 4°C in advance), gently flick to mix, and centrifuge at 7500 g for 5 min. Discard the supernatant, dry the remaining ethanol in a fume hood, add 25 μL of DEPC water, gently flick to mix, and place on ice. After measuring the concentration with a ultra-micro ultraviolet spectrophotometer, store it in a -80°C refrigerator for standby.

[0037] (2) cDNA synthesis Synthesize cDNA according to the kit instructions. The synthesis system is shown in Table 4. The reverse transcription program is as follows: Incubate the system at 42°C for 15 min and inactivate at 95°C for 3 s. Store the product in a -20°C refrigerator for standby.

[0038] Table 4 cDNA synthesis system Reagent Volume (μL) RNA 5 Random Primer 0.5 Anchored Oligo(dT)18 Primer 0.5 E-Mix 1 R-Mix 1 gDNA Remover(2×) 10 RNase-free Water 2 (3) PCR amplification The PCR amplification system is shown in Table 5. Mix the reaction system well, add it to a 96-well PCR plate, centrifuge, and measure with a PCR machine. The PCR reaction conditions are shown in Table 6. Use β-actin as an internal reference. After measuring the CT value of the sample, calculate the relative expression levels of p38, JNK, and ERK in the sample using the 2-ΔΔCT method.

[0039] Table 5 PCR amplification system Reagent Volume (μL) cDNA 1 SYBR Green(2×) 25 Forward primer 1 Reverse primer 1 RNase-free Water 22 Table 6 PCR reaction conditions

[0040] 1.4 Effect of active components of wolfberry leaves on the ROS content of high glucose-induced ARPE-19 cells When culturing, the cells were inoculated into 96-well plates and divided into Con group, Mod group, ST-H group, ST-L group, CT-H group, CT-L group, FT-H group, FT-L group, DT-H group, DT-L group, SWJ-H group, and SWJ-L group. The high dose was 10 μg / mL, and the low dose was 1 μg / mL. After culturing, the medium was discarded, and the medium containing 1 / 1000 H2FCDA was added and incubated at 37 °C for 30 min. Fluorescence microscopy was used to take pictures at an excitation wavelength of 488 nm, and ImageJ was used to analyze the fluorescence intensity.

[0041] 1.5 Statistical analysis GraphPad Prism 9.4.0 software was used for data analysis and plotting. All results were expressed as mean ± standard deviation (SD). One-way ANOVA was used for inter-group data. A P value < 0.05 indicated a statistically significant difference. (*P < 0.05; **P < 0.01; ***P < 0.001 compared with the model group; #P < 0.05; ##P < 0.01; P < 0.001 compared with the blank group).

[0042] 2 Experimental results 2.1 Protective effect of active components of wolfberry leaves on high glucose-induced damaged ARPE-19 cells This invention investigated the protective effect of different active components of wolfberry leaves on high glucose-induced damaged ARPE-19 cells. The CCK8 detection kit and MDA, SOD, and GSH detection kits were combined with an enzyme-labeled instrument to detect the effects of different active components of wolfberry leaves on the survival rate and oxidative stress level of high glucose-induced damaged ARPE-19 cells.

[0043] The detection results are shown in Figure 1 , where the vertical coordinates represent the cell survival rate, GSH level, MDA, and SOD activities respectively. After high glucose damage, the survival rate, GSH content, and SOD activity of ARPE-19 cells were significantly decreased compared with the blank group, and the MDA activity was significantly increased. After administration, except for wolfberry leaf polysaccharide, each active component could significantly increase the survival rate, GSH content, and SOD activity of ARPE-19 cells and significantly decrease the MDA activity. Among them, the refined extract of wolfberry leaves had the most prominent protective effect on high glucose-induced damaged ARPE-19 cells.

[0044] 2.2 Effect of active components of wolfberry leaves on gene expression levels of high glucose-induced damaged ARPE-19 cells This invention investigated the effect of active components of wolfberry leaves on the gene expression levels of high glucose-induced damaged ARPE-19 cells. The expression levels of p38, ERK, and JNK genes in the MAPK pathway were analyzed by qPCR.

[0045] The qPCR results are shown in Figure 2 , and the vertical axis represents the relative expression level of mRNA. After high-glucose-induced injury, the relative expression levels of p38, ERK, and JNK mRNAs were significantly decreased compared with the blank group. After administration, except for Lycium barbarum leaf polysaccharide, the relative expression levels of p38, ERK, and JNK mRNAs in other components all increased to some extent. In comparison, the effect of the refined extract of Lycium barbarum leaf was significantly better than that of other active components.

[0046] 2.3 Effect of active components of Lycium barbarum leaf on the ROS content in ARPE-19 cells induced by high glucose This invention investigated the effect of active components of Lycium barbarum leaf on the ROS content in ARPE-19 cells induced by high glucose. The ROS content in ARPE-19 cells was detected by adding a fluorescent probe (H2FCDA) and combining with a fluorescence microscope.

[0047] The detection results of the ROS content are shown in Figure 3 , and the vertical axis represents the average fluorescence intensity. After high-glucose-induced injury, the ROS fluorescence intensity increased significantly compared with the blank group ( Figure 3 A). After administration, except for Lycium barbarum leaf polysaccharide, each active component significantly decreased the ROS fluorescence intensity, and the refined extract of Lycium barbarum leaf had the best effect.

[0048] Example 3 Activity screening based on zebrafish model 3 Experimental methods Zebrafish embryos at 3 dpf were cultured in 6-well plates, with 30 embryos in each well. The model and drug administration groups were cultured in circulating water containing 100 mg / mL, and were divided into Con group, Mod group, metformin (MET) group, ST-H group, ST-L group, CT-H group, CT-L group, FT-H group, FT-L group, DT-H group, DT-L group, SWJ-H group, and SWJ-L group. The administration dose of MET was 200 μg / ml, and the crude drug doses of the remaining drug administration groups were converted. The high dose was 40 μg / mL, and the low dose was 20 μg / mL. They were co-cultured for three days, and the water was changed at a fixed time every day.

[0049] 3.1 Detection of the effect of active components of Lycium barbarum leaf on glucose content and oxidative stress level in DR zebrafish by kit After the wild A / B-type zebrafish embryos were cultured according to the above method, the zebrafish embryos were collected into Ep tubes for homogenization, centrifuged at 4000 rpm for 10 min, and the supernatant was taken. The intracellular GLU, GSH levels and SOD, MDA activities were detected according to the kit instructions.

[0050] 3.2 Detection of the effect of active components of Lycium barbarum leaf on gene expression level in DR zebrafish by qPCR The cultured zebrafish embryos were collected into Ep tubes. After adding 1 mL of Trizol to each tube, they were homogenized, centrifuged at 12,000×g for 10 min at 4°C, and the supernatant was taken. The remaining operations were the same as described in 2.3.

[0051] 3.3 Effects of active components of wolfberry leaves on retinal blood vessels of DR zebrafish 3dpf fli1:EGFP zebrafish embryos were cultured according to the method described in 2.4, and 0.003% phenylthiourea (PTU) was added to the circulating water to inhibit the melanogenesis of zebrafish embryos. After culturing, the zebrafish embryos were collected, fixed on glass slides, the retinal blood vessels of zebrafish were photographed using a fluorescence microscope, and the diameter of the retinal blood vessels was measured using ImageJ.

[0052] 3.4 Statistical analysis Data analysis and plotting were performed using GraphPad Prism 9.4.0 software. All results were expressed as mean ± standard deviation (SD). One-way ANOVA was used for inter-group data. A P value < 0.05 indicated a statistically significant difference. (*P < 0.05; **P < 0.01; ***P < 0.001 compared with the model group; #P < 0.05; ##P < 0.01; P < 0.001 compared with the blank group).

[0053] 4 Experimental results 4.1 Effects of active components of wolfberry leaves on glucose content and oxidative stress level in DR zebrafish This invention investigated the effects of active components of wolfberry leaves on blood glucose level and oxidative stress level in the DR zebrafish model. The effects of different active components of wolfberry leaves on glucose content and oxidative stress level in DR zebrafish were detected by a GLU detection kit and MDA, SOD, and GSH detection kits combined with an enzyme-linked immunosorbent assay (ELISA) reader.

[0054] The detection results are shown in Figure 4 , where the vertical coordinates represent glucose content, GSH level, MDA, and SOD activities respectively. Compared with the blank group of zebrafish, the GSH content and SOD activity in DR zebrafish were significantly decreased, while the glucose content and MDA activity were significantly increased. After administration, except for wolfberry leaf polysaccharide, each active component could significantly increase the GSH content and SOD activity in DR zebrafish and significantly decrease the glucose content and MDA activity. Compared according to the converted crude drug dose, the dosage of the refined extract of wolfberry leaves was lower than that of the water extract and alcohol extract, but the effect was the most prominent.

[0055] 4.2 Effects of active components of wolfberry leaves on gene expression levels in DR zebrafish This invention examines the effects of the active components of wolfberry leaves on the gene expression levels of DR zebrafish. The expression levels of p38, ERK, and JNK genes in the MAPK pathway were analyzed by qPCR. The qPCR results are shown in Figure 5 , and the vertical axis represents the relative expression of mRNA. Compared with the blank group of zebrafish, the relative mRNA expression levels of p38, ERK, and JNK in DR zebrafish were significantly decreased. After administration, except for wolfberry leaf polysaccharide, the relative mRNA expression levels of p38, ERK, and JNK in each component increased to varying degrees. According to the converted crude drug dose comparison, the dosage of the refined extract of wolfberry leaves was lower than that of the water extract and alcohol extract, but the effect was the most prominent.

[0056] Among them, the comprehensive effect of the refined extract of wolfberry leaves was significantly better than that of other active components.

[0057] 4.3 Effects of the active components of wolfberry leaves on the retinal vessel diameter of DR zebrafish This invention examines the effects of the active components of wolfberry leaves on the retinal vessel diameter of DR zebrafish. Fluorescent microscope was used to take pictures of the retinal vessels of zebrafish, and ImageJ was used to analyze the changes in the retinal vessel diameter of zebrafish.

[0058] The measurement results of the retinal vessel diameter are shown in Figure 6 , and the vertical axis represents the retinal vessel diameter of zebrafish. Compared with the blank group, the retinal vessel diameter of DR zebrafish increased significantly ( Figure 6 A). After administration, except for wolfberry leaf polysaccharide, each active component significantly decreased the retinal vessel diameter of zebrafish.

[0059] After verification by in vitro cell models and zebrafish models, among the active components of wolfberry leaves, the refined extract of wolfberry leaves had the best comprehensive effect. Therefore, we selected the water extract of wolfberry leaves (ST), the alcohol extract of wolfberry leaves (CT), and the refined extract of wolfberry leaves (FT) for further research.

[0060] Example 4 Activity verification based on mouse models 5 Experimental methods Establishment of DR mouse models After 7 days of adaptive feeding, 8-week-old mice were divided into groups: Con group, Mod group, metformin (MET) group, ST-H group, ST-L group, CT-H group, CT-L group, FT-H group, and FT-L group. The blank group was fed with normal diet, and the model group and drug administration groups were fed with high-sugar and high-fat diet. The body weight of the mice was measured weekly. After 8 weeks of continuous feeding, the mice in the model group and drug administration groups were intraperitoneally injected with 1% STZ solution (STZ dissolved in 0.1 mol / L sodium citrate solution) (60 mg / kg) for 3 consecutive days. 72 hours after the injection, the blood glucose level of the mice was measured. Fasting blood glucose ≥ 16.7 mM was considered successful modeling. The mice in the blank group were injected with the same volume of sodium citrate buffer solution. After modeling, the mice were continuously fed with high-sugar and high-fat diet and administered drugs for 8 weeks, and the corresponding drugs were intragastrically administered daily. The blood glucose level of the mice was measured weekly. The MET administration dose was 400 mg / kg / d, and the crude drug doses of the other drug administration groups were converted. The high dose was 4.5 g / kg / d, and the low dose was 2.25 g / kg / d.

[0061] 5.1 Effects of Different Active Components of Lycium barbarum Leaves on Retinal Thickness and Retinal Vascular Density in DR Mice 24 hours after the last drug administration, the mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (dose: 50 mg / kg, 0.5 mL / 100 g). The retinal thickness and retinal vascular density of the mice were detected by Spectralis OCT and OCTA, and the results of retinal thickness and retinal vascular density were analyzed by ImageJ.

[0062] 5.2 Statistical Analysis GraphPad Prism 9.4.0 software was used for data analysis and plotting. All results were expressed as mean ± standard deviation (SD). One-way analysis of variance (One-way ANOVA) was used for inter-group data. When P < 0.05, the difference was considered statistically significant. (Compared with the model group, *P < 0.05; **P < 0.01; ***P < 0.001; compared with the blank group, #P < 0.05; ##P < 0.01; P < 0.001).

[0063] 6 Experimental Results 6.1 Effects of Active Components of Lycium barbarum Leaves on Body Weight and Blood Glucose Level in DR Mice This invention investigated the effects of active components of Lycium barbarum leaves on the body weight and blood glucose level of DR mice. During the feeding process, the body weight change of the mice was monitored weekly, and the blood glucose level change of the mice was monitored by a blood glucose meter after the completion of STZ injection.

[0064] The results of the changes in body weight and blood glucose level of DR mice are shown in Figure 7 , Figure 7 (A) The abscissa represents the feeding date, and the ordinate represents the body weight of the mice;Figure 7 (B)The abscissa represents the feeding date, and the ordinate represents the blood glucose level of the mice. Compared with the blank group, the body weight of the mice in the model group was relatively high. After injecting STZ, the body weight of the mice in the model group and the drug administration group began to decline, while the body weight of the mice in the blank group continued to increase steadily. After injecting STZ, the blood glucose level of the mice in the model group and the drug administration group was much higher than that of the mice in the blank group. After drug administration, the blood glucose level of the DR mice decreased steadily.

[0065] 6.2 Effects of Active Components of Lycium barbarum L. Leaves on Retinal Thickness and Retinal Vascular Density in DR Mice This invention investigated the effects of active components of Lycium barbarum L. leaves on retinal thickness and retinal vascular density in DR mice. Spectralis OCT and OCTA were used to detect the retinal thickness and retinal vascular density of the mice, and ImageJ was combined to analyze the changes in retinal thickness and retinal vascular density of the mice.

[0066] The measurement results of retinal thickness and retinal vascular density are shown in Figure 8 and Figure 9 , with the ordinate representing the retinal thickness and retinal vascular density of the mice respectively. Compared with the blank group, the retinal thickness of the DR mice decreased significantly ( Figure 8 ), and the retinal vascular density increased significantly. After drug administration, each active component increased the retinal thickness of the mice and decreased the retinal vascular density.

[0067] Through the research and comparison of the improvement effects of different active components of Lycium barbarum L. leaves on diabetic retinopathy, it was found that the refined extract of Lycium barbarum L. leaves was the active component with the best effect. Therefore, the refined extract of Lycium barbarum L. leaves can be separated and applied alone in the field of treating DR to achieve the efficient comprehensive utilization of Lycium barbarum L. leaf resources.

[0068] The above embodiments are only for illustrating the technical concept and characteristics of the present invention, and their purpose is to enable those who are familiar with this technology to understand the content of the present invention and implement it. It cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit and essence of the present invention should be covered within the protection scope of the present invention.

Claims

1. Application of wolfberry leaf extract in the preparation of drugs for treating hyperglycemia.

2. Application of wolfberry leaf extract in the preparation of drugs for treating diabetes.

3. Application of wolfberry leaf extract in the preparation of drugs for the treatment of diabetic retinopathy.

4. The use according to any one of claims 1 to 3, characterized in that: The extract comprises wolfberry leaf water extract, wolfberry leaf alcohol extract, wolfberry polysaccharide, wolfberry alkaloid and wolfberry leaf refined extract.

5. The use according to claim 4, characterized in that: The wolfberry leaf refined extract is prepared by the following method: weighing dried wolfberry leaves, adding ethanol, heating and refluxing extraction to obtain wolfberry leaf ethanol extract, concentrating under reduced pressure to a suitable volume, using D101 macroporous resin for full adsorption, eluting with distilled water, 10% ethanol, and 30% ethanol respectively, discarding the eluent, and then eluting with 70% ethanol as the eluent, collecting the eluent, concentrating under reduced pressure, and freeze-drying to obtain the wolfberry leaf refined extract.

6. The use according to claim 4, characterized in that: The wolfberry leaf water extract is prepared by the following method: weighing dried wolfberry leaves, adding distilled water, heating and refluxing for extraction, combining the extracts, concentrating under reduced pressure, and freeze-drying to obtain the wolfberry leaf water extract.

7. The use according to claim 4, characterized in that: The wolfberry leaf alcohol extract is prepared by the following method: Weighing dried wolfberry leaves, adding ethanol, heating and refluxing for extraction, combining wolfberry leaf alcohol extracts, concentrating under reduced pressure, and freeze-drying to obtain wolfberry leaf alcohol extract; After drying the residue after ethanol extraction, distilled water is added, and the mixture is heated under reflux for extraction to obtain an extract. After reduced pressure concentration, a certain amount of anhydrous ethanol is added, the mixture is allowed to stand overnight, the supernatant is discarded, and the mixture is dried to obtain crude polysaccharides. The protein is removed by the Sevage method to obtain wolfberry polysaccharides.

8. The use according to claim 4, characterized in that: The wolfberry alkaloid is betaine.

9. The use according to any one of claims 1 to 3, wherein the wolfberry leaf extract and a pharmaceutically acceptable carrier are made into a medicine in the form of tablets, capsules, granules, pills, mixtures, oral liquids, or ointments.

10. The use according to claim 3, wherein the diabetic retinopathy is caused by type I diabetes or type II diabetes.

Images