Application of effective component formula of Guiaogangrene in treatment of diabetic retinopathy

Abstract

The invention provides an application of an effective component formula of Guiaogangrene in treatment of diabetic retina neuropathy, the extract of the effective component formula forms a hyssopus officinalis extract, and the hyssopus officinalis extract comprises the following main components: carnosol, 8-hydroxythiaflavin, methyl rosmarinate and eugenol glycoside. The invention also provides a preparation method of the eye drops of the effective component formula extract, and the effective component formula extract can obviously reduce the permeability of retinal vessels of diabetic rats and improve the expression level of three inflammatory factors, namely TNF-alpha, IL-6 and IL-1beta, in the blood of rats with retinal neuropathy.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and more particularly to an application of a formula of effective ingredients of Gastrocnemius sacchariflorus in treating diabetic retinopathy. Background Art

[0002] Diabetic retinopathy (DR) refers to retinal microvascular disease in diabetic patients, with clinical features such as intraretinal hemorrhage, hard exudates, cotton-wool spots, macular edema, and microaneurysms. In severe cases, tractional retinal detachment and visual impairment may occur. DR is one of the most common microvascular complications of diabetes, with a high blindness rate, and the incidence rate also ranks first among the working population. Diabetes belongs to the category of "Xiao Ke disease" in traditional Chinese medicine, and diabetic retinopathy belongs to "sparrow eyes", "blurred vision", "blood in the pupil", "cloudy eyes" or sudden blindness. Modern Chinese medicine calls DR "Xiao Ke eye disease". Since ancient times, many predecessors have studied the pathogenesis and treatment of Xiao Ke eye disease, which has also laid the foundation for modern Chinese medicine to understand and treat DR.

[0003] The pathogenesis of DR is generally believed to be closely related to abnormal metabolic pathways, inflammatory damage, and retinal neurodegeneration caused by hyperglycemia. The pathogenesis of DR is complex and is related to abnormalities in multiple glucose metabolic pathways, such as the protein kinase C pathway, the polyol pathway, and the advanced glycation product pathway. All of these pathways can lead to an imbalance in the production and elimination of reactive oxygen species (ROS), which increases the body's oxidative stress level, thereby causing cell apoptosis and abnormalities in the structure and function of retinal cells.

[0004] The pathogenesis of DR has not yet been fully elucidated. Studies have shown that oxidative stress is one of the important cellular mechanisms in diabetic complications, which can induce cell apoptosis and microvascular damage, and ultimately lead to damage to the blood-retinal barrier, thus leading to the occurrence of DR. How to effectively prevent and treat DR is still a difficult problem that needs to be solved. Treatment methods such as laser photocoagulation and the use of anti-vascular endothelial growth factor drugs are effective, but their limitations and the occurrence of adverse reactions cannot be ignored. In recent years, there has been an increasing number of studies on the prevention and treatment of DR with traditional Chinese medicine. Traditional Chinese medicine treatment has played its unique advantages in the treatment of DR and can complement Western medicine treatment. Summary of the invention

[0005] In order to make up for the deficiencies of the prior art, the present invention provides an application of a formula of effective ingredients of Gastrocnemius sutchuenensis in treating diabetic retinopathy.

[0006] The present invention is achieved through the following technical solutions:

[0007] In the first aspect, the present invention provides an application of an effective ingredient formula of Gynura strychnifolia in the treatment of diabetic retinopathy, wherein the effective ingredient formula of Gynura strychnifolia comprises: Hyssopus officinalis extract; the ingredients of Hyssopus officinalis extract comprise: trilobatin, 8-hydroxystyril, methyl rosmarinate, and eugenol glycosides.

[0008] Preferably, the proportions of the Hyssop extract components in the Hyssop extract are: trilobatin (8%-14%), 8-hydroxystyril (2.7%-3.4%), methyl rosmarinate (0.75%-1.49%), and eugenol glycosides (1.25%-1.78%).

[0009] Preferably, the composition of the effective ingredient formula of the Herba Lysimachiae is: including 3%-12% of Hyssopus officinalis extract.

[0010] Preferably, a formula of effective ingredients of Guizhi syringa is used in the treatment of diabetic retinopathy, wherein the formula of effective ingredients of Guizhi syringa, 8-hydroxystyril, methyl rosmarinic acid, and eugenol can significantly reduce the permeability of retinal blood vessels in diabetic rats; and significantly improve the expression levels of three inflammatory factors, TNF-α, IL-6, and IL-1β, in the blood of rats with retinal neuropathy.

[0011] In a second aspect, the present invention provides a method for preparing an eye drop with a formula of effective ingredients of saccharomyces cerevisiae. The method comprises the following steps: taking 200 ml of water for injection, heating the mixture to 80°C to 90°C, weighing 0.7 g of K-30 and dispersing the mixture therein, stirring the mixture, cooling the mixture, filtering the mixture, and using the mixture as agent A for later use; taking 50.0 g of hyssop extract, 7.3 g of sodium chloride, and 2.7 g of benzalkonium bromide and sequentially adding the mixture to 200 ml of water for injection, dissolving the mixture, adding 5 g of activated carbon for decolorization and impurity removal, heating the mixture to a slight boil for 10 minutes, cooling the mixture, filtering the mixture, and using the mixture as agent B for later use; slowly adding agent B to agent A, placing the mixture in a 1000 ml volumetric flask, diluting the mixture to the mark with water for injection, shaking the mixture, adjusting the pH value to 7.0, sterilizing the mixture at 115°C for 30 minutes, and packaging the mixture.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] The present invention provides a formula of effective ingredients of Gastrodia ulmoides and a medicine prepared by using the formula, wherein the medicine has an excellent therapeutic effect on diabetic retinopathy. In addition, the permeability of retinal blood vessels of diabetic rats can be significantly reduced; and the expression levels of three inflammatory factors, TNF-α, IL-6 and IL-1β, in the blood of rats with retinal neuropathy can be significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The present invention will be further described below in conjunction with the accompanying drawings.

[0015] Figure 1 Comparison of body weight changes of rats in each group;

[0016] Figure 2 Comparison of blood glucose changes in rats in each group;

[0017] Figure 3 Comparison of cell-free capillary quantity analysis;

[0018] Figure 4 Comparison of retinal Evans blue dye leakage;

[0019] Figure 5 Comparison of TNF-α, IL-6 and IL-1β expression levels. DETAILED DESCRIPTION

[0020] The present invention is described in further detail below.

[0021] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0022] Example 1: Preparation of a formulation of an active ingredient for treating diabetic retinopathy

[0023] The formula of Guizhi Sugarcanthus contains 3%-12% Hyssopus officinalis extract; the Hyssopus officinalis extract contains: trilobate carnosine, 8-hydroxystyril, methyl rosmarinic acid, and eugenol. The proportions of the Hyssopus officinalis extract in the Hyssopus officinalis extract are: trilobate carnosine (8%-14%), 8-hydroxystyril (2.7%-3.4%), methyl rosmarinic acid (0.75%-1.49%), and eugenol (1.25%-1.78%).

[0024] Take 200 ml of water for injection, heat it to 80°C-90°C, weigh 0.7 g of K-30 and disperse it in it, stir, cool, filter, and use it as Agent A for later use; take 50.0 g of Hyssopus officinalis extract, 7.3 g of sodium chloride, and 2.7 g of benzalkonium bromide and add them to 200 ml of water for injection in sequence, dissolve, add 5 g of activated carbon for decolorization and impurity removal, heat to a slight boil for 10 minutes, cool, filter, and use it as Agent B for later use; slowly add Agent B to Agent A, place it in a 1000 mL volumetric flask, dilute to the scale with water for injection, shake well, adjust the pH value to 7.0, sterilize at 115°C for 30 minutes, and package it.

[0025] Example 2: Application of the formula of the effective component of Guizhi sugar carbuncle in the treatment of diabetic retinopathy

[0026] 1. Materials

[0027] 1.1 Experimental animals

[0028] Male SD rats (weight 200±20g) were purchased from Shanghai Slake Laboratory Animal Technology Co., Ltd. They were kept in a special pathogen-free environment with a temperature of (25±2)℃, a humidity of (55±15)%, and a light-dark cycle of 12h. They were acclimatized for 7 days before the start of the experiment and were fed with free access to water and rat food for 1 week.

[0029] 1.2 Main drugs and reagents

[0030] Guizhi anthrax eye drops (prepared according to the steps of Example 1, homemade); citric acid, formamide, sodium citrate, Evans blue dye, purchased from Macklin Reagent; enzyme-linked immunosorbent assay (ELISA) kit, purchased from Shanghai Via Biotechnology Co., Ltd.; streptozotocin, Merck; paraformaldehyde, trypsin, purchased from Shanghai Yisheng Biotechnology Co., Ltd.

[0031] 2 Experimental methods

[0032] 2.1 Animal model preparation and grouping

[0033] Mix 0.1mol·L-1 citric acid solution and sodium citrate solution in a ratio of 14:11, adjust the pH to 4.4, and use it to prepare streptozotocin solution. Weigh an appropriate amount of streptozotocin powder and dissolve it in a certain concentration in the dark. 40 rats that have fasted overnight were intraperitoneally injected with STZ at a dose of 65mg·kg-1 to induce a diabetes model. Another 20 rats were intraperitoneally injected with sodium citrate buffer as a normal control group. Seven days after modeling, blood was collected from the tail vein to measure the fasting blood glucose concentration (FBG). FBG ≥ 16.7mmol·L-1 (250mg·dL-1) was considered a successful diabetes model.

[0034] 2.2 Method of administration

[0035] The diabetic rats with successful modeling were randomly divided into a model group and a Guizhi sugar carbuncle group according to their body weight and FBG, with 20 rats in each group. Two months after STZ intraperitoneal injection, the Guizhi sugar carbuncle group was given 40 mg kg -1 The dose of 50 mg mL was given to rats by dropping into both eyes. -1 The rats in the normal group and the model group were given isotonic solution of Guizhi sugar cane; the normal group and the model group were given physiological saline instilled into both eyes; twice a day, 6 hours apart, for 1 month. During the experiment, the blood glucose and body weight of the rats were recorded. After the end of the administration, 6 rats in each group underwent the retinal acellular capillary experiment. 6 rats in each group underwent the Evans blue dye leakage experiment. The remaining 8 rats were anesthetized, and the serum and retina were collected and stored at -80℃.

[0036] 2.3 Observation indicators and methods

[0037] 2.3.1 Analysis of the number of cell-free capillaries

[0038] The right eye of the rat was removed and fixed with 4% paraformaldehyde for 24 hours, and then washed with PBS four times. The retina was peeled off and then digested with 3% trypsin at 37°C for 1 hour to separate the retinal blood vessels from the retinal tissue. In order to quantify the acellular capillaries in the retina, the separated retinal blood vessels were stained with HE, and the retinal acellular capillaries in the field of view were observed with an inverted microscope. The total number of acellular capillaries in 3 fields of view was calculated, and the average value represented the number of acellular capillaries in the retinal sample.

[0039] 2.3.2 Retinal Evans blue dye leakage assay

[0040] Evans blue dye was dissolved in PBS to prepare a solution with a concentration of 20 g·L-1. The solution was injected intraperitoneally into rats at a concentration of 10 μL·g-1. After 2 hours of circulation, the left ventricle was irrigated with PBS to remove the Evans blue dye in the rat's systemic circulation. The eyeball was removed, the retinal tissue was peeled off, and it was placed in a vacuum drying oven at 55°C for 5 hours. The mass of the dried retinal tissue was weighed. 120 μL of formamide was added and the Evans blue dye was extracted in a 70°C water bath for 18 hours. The extract was centrifuged at 10 000 r·min-1 for 1 hour, and 100 μL of the supernatant of the extract was taken. The absorbance value was measured at a wavelength of 620 nm using an enzyme marker, and the standard curve was substituted to calculate the amount of Evans blue dye. The ratio of the amount of Evans blue dye to the mass of the dried retinal tissue was calculated to obtain the amount of Evans blue dye leakage in the retinal tissue (ng·g-1).

[0041] 2.3.3 ELISA

[0042] Rat blood was centrifuged at 4 000 r·min-1 for 10 min, and the upper serum was removed. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) in the serum were detected according to the ELISA kit method.

[0043] 2.4 Test results

[0044] 2.4.1 Body weight and fasting blood glucose concentration

[0045] The body weight of the rats in the normal control group increased steadily throughout the experiment, while the body weight of the diabetic rats did not change significantly (P<0.001); in addition, the fasting blood glucose concentration of the diabetic rats stabilized after the 7th day and was significantly higher than that of the rats in the normal control group (P<0.001). However, the results showed that the administration of 40 mg kg-1 of Guizhi Tanghu Eye Drops did not affect the body weight and fasting blood glucose concentration of diabetic rats. Figure 1 , Figure 2 .

[0046] 2.4.2 Analysis results of the number of cell-free capillaries

[0047] Compared with the normal control group, the number of retinal acellular capillaries in the model group diabetic rats increased significantly (P<0.001); compared with the model group, the number of retinal acellular capillaries in the diabetic rats treated with Guizhi Tangu eye drops at 40 mg kg-1 was significantly reduced (P<0.05). Figure 3 .

[0048] 2.4.3 Retinal Evans blue dye leakage assay

[0049] Compared with the normal control group, the leakage of Evans blue dye in the retina of the diabetic rats in the model group increased significantly, indicating that the permeability of the retinal blood vessels of diabetic rats increased after STZ modeling. Compared with the model group, the leakage of Evans blue dye in the group treated with Guizhi Tangwu eye drops at 40 mg kg-1 was significantly reduced (P<0.05), indicating that the administration of Guizhi Tangwu eye drops can effectively reduce the permeability of blood vessels and improve the vascular function of the DR model. Figure 4 .

[0050] 2.4.4 ELISA

[0051] Compared with the normal control group, the expression of TNF-α, IL-6 and IL-1β in the serum of the model group diabetic rats was significantly increased; compared with the model group, the administration of 40 mg kg-1 Guizhi Tanghu eye drops can effectively reduce the expression of TNF-α, IL-6 and IL-1β in the serum of diabetic rats. Figure 5 .

Claims

1. An application of a formula of effective ingredients of Gastrodia elata in treating diabetic retinopathy, characterized in that: The formula of effective ingredients of the saccharomyces lycopersicum comprises: Hyssopus officinalis extract; the ingredients of the Hyssopus officinalis extract include: trilobate caryophyllein, 8-hydroxystyril, methyl rosmarinic acid, and eugenol glycosides.

2. The use according to claim 1, characterized in that: The proportions of the Hyssop extract components in the Hyssop extract are: trilobate caryophyllene (8%-14%), 8-hydroxystyril (2.7%-3.4%), methyl rosmarinate (0.75%-1.49%), and eugenol glycosides (1.25%-1.78%).

3. The use according to claim 1, characterized in that: The effective ingredient formula of the Chinese medicine for treating ulcers is composed of 3%-12% of Hyssopus officinalis extract.

4. A method for preparing eye drops with a formula of effective ingredients for saccharomyces cerevisiae, characterized in that: Take 200 ml of water for injection, heat it to 80°C-90°C, weigh 0.7 g of K-30 polyvinyl pyrrolidone and disperse it therein, stir, cool, filter, and use it as Agent A for later use; take 50.0 g of Hyssopus officinalis extract, 7.3 g of sodium chloride, and 2.7 g of benzalkonium bromide and add them to 200 ml of water for injection in sequence, dissolve them, add 5 g of activated carbon for decolorization and impurity removal, heat to a slight boil for 10 minutes, cool, filter, and use it as Agent B for later use; slowly add Agent B to Agent A, place it in a 1000 mL volumetric flask, dilute to the scale with water for injection, shake well, adjust the pH value to 7.0, sterilize at 115°C for 30 minutes, and package it.

5. Use of a formula of effective ingredients of Gyuzhi sugar carbuncle in treating diabetic retinopathy, characterized in that: The formula of the effective ingredients of the Radix Angelicae Pubescentis can significantly reduce the permeability of the retinal blood vessels of diabetic rats and significantly improve the expression levels of three inflammatory factors, TNF-α, IL-6 and IL-1β, in the blood of rats with retinal neuropathy.

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