Application of traditional Chinese medicine composition in preparation of medicine for preventing and treating age-related macular degeneration

Abstract

The invention provides application of a traditional Chinese medicine composition in preparation of a medicine for preventing and treating age-related macular degeneration. Compared with the prior art, in-vitro experiments find that laggera pterodonta has a good protection effect on a VEGF-induced RF / 6A cell proliferation model, in animal experiments, the 95% ethanol extraction part of laggera pterodonta has an obvious protection effect on a mouse retinopathy model animal model established through intraperitoneal injection of sodium iodate, the retinopathy process of a mouse can be slowed down, and the application of the laggera pterodonta has a good application prospect. And a significant difference exists between the component values of the model. Therefore, the 95% ethanol extraction part of laggera pterodonta can be used for preparing medicines for treating AMD, and has good development and application prospects.

Description

[0001] This application claims the priority of the Chinese patent application filed with the China Patent Office on June 30, 2023, with application number 202310795078.6 and invention name "Application of Xiu Lingdan in the Preparation of Drugs for Preventing and Treating Age-Related Macular Degeneration", the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The invention belongs to the technical field of traditional Chinese medicines, and in particular relates to an application of a traditional Chinese medicine composition in preparing a medicine for preventing and treating age-related macular degeneration. Background Art

[0003] Laggera Pterodonta (DC.) Benth is a medicinal plant of the genus Laggera of the Asteraceae family. It is also known as lion grass, stinky leaves, and six-angled chrysanthemum. It has good antibacterial and anti-inflammatory effects, and is widely used to treat colds, pharyngitis, bronchitis, malaria and other diseases. The compounds identified from this genus are mainly eucalyptane-type sesquiterpenes and flavonol compounds. The volatile oil components of Laggera Pterodonta also have important biological activities, such as antibacterial, anti-tumor, and antiviral activities. It can also play a therapeutic role in bronchial inflammation, expectorant, liver protection and analgesic effects.

[0004] Age-related macular degeneration (AMD) is the main cause of severe, irreversible visual impairment. The disease can progress from early AMD to intermediate AMD (iAMD), and finally to late AMD. In the past decade, AMD has affected about 30% of the elderly worldwide, mostly occurring in people over 50 years old, and occasionally in people aged 40 to 50 years old. Epidemiological surveys show that AMD causes blindness, accounting for about 8.7% of the world's blindness. There are about 30 million AMD patients worldwide, and about 500,000 people become blind due to AMD each year. The prevalence of early AMD in people over 50 years old in my country is between 1.7% and 9.5%, and the prevalence of late AMD is between 0.2% and 1%. The prevalence of AMD increases with age. The aging population will lead to a significant increase in the prevalence of AMD. Late AMD is a serious disease, and there is an urgent need for drugs to improve the visual function damage associated with late AMD and delay the progression of AMD. AMD has become a hot area for new drug research and development.

[0005] Western medical treatments include photodynamic therapy (PDT), laser photocoagulation, intravitreal injection of anti-vascular endothelial growth factor (VEGF) drugs, surgery (such as removal of neovascular membrane under the retinal membrane, macular transposition surgery, etc.), etc., but they have certain limitations.

[0006] Traditional Chinese medicine believes that the occurrence of AMD is related to liver, spleen, and kidney dysfunction. According to the symptoms of AMD, it is named "blurred vision", "colored vision", "seeing straight as curved", "distorted vision", "seeing big as small", etc. Therefore, providing a product based on Chinese herbal medicine has important practical significance in the prevention and treatment of AMD. Summary of the invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide an application of a Chinese medicine composition in the preparation of a drug for preventing and treating age-related macular degeneration.

[0008] The invention provides an application of Xiulingdan in preparing a medicine for preventing and treating age-related macular degeneration.

[0009] Preferably, the xiulingdan prevents and treats age-related macular degeneration by reducing vascular endothelial growth factor (VEGF)-induced apoptosis of RF / 6A cells.

[0010] In the present invention, preferably, the age-related macular degeneration is exudative age-related macular degeneration.

[0011] The present invention also provides an application of the extract of Chou Ling Dan in preparing a medicine for preventing and treating age-related macular degeneration.

[0012] Preferably, the extract of Xiulingdan is preferably an ethanol extract of Xiulingdan; specifically, in the present invention, the Xiulingdan is a 90% to 95% ethanol extract of Xiulingdan, and more specifically, a 95% ethanol extract of Xiulingdan. The present invention has found through experiments that Xiulingdan can slow down the progression of retinopathy in mice injected with NaIO3 solution intraperitoneally, therefore, the 95% ethanol extract of Xiulingdan of the present invention has a good therapeutic effect on AMD.

[0013] Preferably, the Xiulingdan extract is prepared according to the following steps: extracting Xiulingdan with warm ethanol solution to obtain the Xiulingdan extract.

[0014] In the present invention, the choulingdan is preferably a dry raw material, more preferably a dry above-ground part.

[0015] In the present invention, the volume concentration of ethanol in the ethanol solution is preferably 90% to 95%, more preferably 95%; the mass ratio of the stinky elixir to the ethanol solution is 1:(5 to 20), more preferably 1:(8 to 15), more preferably 1:(8 to 12), and most preferably 1:10; the temperature of the warm immersion extraction is preferably 80°C to 95°C, more preferably 85°C to 90°C, and more preferably 90°C; the time of the warm immersion extraction is preferably 1 to 3 hours, more preferably 1.5 to 3 hours; the number of warm immersion extractions is preferably 1 to 3 times, more preferably 2 to 3 times; the time of each warm immersion extraction is preferably 1 to 3 hours, more preferably 1.5 to 2 hours.

[0016] After warm immersion extraction, centrifugation is preferably performed; when the extraction is performed multiple times, the extracts are preferably combined and centrifuged; the speed of the centrifugation is preferably 1000-5000 r / min, more preferably 2000-4000 r / min, more preferably 2500-3500 r / min, and most preferably 3000 r / min; after centrifugation, the supernatant is concentrated and freeze-dried to obtain the stinky lingdan extract; the concentration temperature is preferably 50°C-70°C, more preferably 55°C-65°C, and more preferably 60°C; the concentration is preferably to a density of 1.05-1.08 g / cm 3 ; Pre-freezing is preferably performed before freeze-drying; the pre-freezing temperature is preferably -40℃~-70℃, more preferably -50℃~-60℃; the pre-freezing time is preferably 8~12h; the freeze-drying time is preferably 15~30h, more preferably 20~26h, and more preferably 24h.

[0017] Preferably, the extract of Chlorophytum corydalis prevents and treats age-related macular degeneration by reducing vascular endothelial growth factor (VEGF)-induced apoptosis of RF / 6A cells.

[0018] In the present invention, preferably, the age-related macular degeneration is exudative age-related macular degeneration.

[0019] In the present invention, the administration method of the choulingdan extract includes but is not limited to one or more of oral administration, injection and external application.

[0020] The present invention also provides an application of a traditional Chinese medicine composition in a medicine for preventing and treating age-related macular degeneration, wherein the traditional Chinese medicine composition comprises Choulingdan.

[0021] According to the present invention, the presence of Choulingdan in the traditional Chinese medicine composition includes but is not limited to raw materials, powder made from raw materials, slices made from raw materials, raw material extracts, raw material fermentations or other forms with the same or similar biological activity.

[0022] According to the present invention, the Chinese medicine composition preferably also includes other pharmaceutically acceptable excipients.

[0023] According to the present invention, the dosage form of the Chinese medicine composition is preferably one or more of decoction, granules, capsules, tablets, oral liquid, pills, tincture, syrup, suppository, gel, spray or injection.

[0024] The present invention provides an application of a Chinese medicine composition in the preparation of a drug for preventing and treating age-related macular degeneration. Compared with the prior art, the present invention found in an in vitro experiment that the 95% ethanol extract of the 100mg ... BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a graph showing the absorbance detection result of the VEGF-induced RF / 6A cell proliferation model in Example 1 of the present invention;

[0026] Figure 2 This is a diagram of mouse eye tissue sections in Example 2 of the present invention. DETAILED DESCRIPTION

[0027] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0028] Patients with wet ARMD are mostly due to deficiency of the liver, spleen and kidney. Spleen deficiency leads to disordered transportation and transformation, endogenous water and dampness, dysfunctional transportation and distribution, combination of dampness and cold, and invasion of the eyes. Kidney Qi mainly regulates the qi transformation function of the internal organs. Kidney Qi deficiency, abnormal qi transformation, poor transportation and distribution, and accumulation in the eyes aggravate the condition. Liver Yin deficiency, liver Qi stagnation, blood stasis in the veins, and untransformed water and dampness will form phlegm in the eyes over time. Therefore, Chinese medicinal materials with the functions of protecting the liver and clearing away heat and detoxification were selected for activity screening, and Chinese herbal medicines with active therapeutic effects on AMD were obtained.

[0029] The following experiments, if no specific conditions are specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The APIs or excipients, reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially. Unless otherwise specified, all percentages, ratios, proportions or parts are by weight.

[0030] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the present invention.

[0031] The raw materials and reagents used in the application of the traditional Chinese medicine composition provided by the present invention in the preparation of drugs for preventing and treating AMD can all be purchased from the market.

[0032] Preparation Example: Preparation of 95% ethanol extraction of xiulingdan

[0033] Take 500g of the dry aerial part of the stinking dan, crush it through a No. 2 sieve, add 10 times the amount of 95% ethanol solution, and extract it twice at 90℃, each time for 1.5h. After the extracts are combined, centrifuge at 3000r / min for 15min, and concentrate the supernatant at 60℃ to a density of 1.05-1.08g / cm 3 , pre-freeze at -60℃ for 10h, and then freeze-dry for 24h to obtain 95% ethanol extract of Xiulingdan.

[0034] Example 1: Anti-VEGF induces proliferation activity of monkey choroid-retinal cells (RF / 6A)

[0035] The test drug of this example was obtained according to the preparation method of the preparation example of the present invention. Other materials and reagents are shown in Table 1; the instruments and equipment used are shown in Table 2.

[0036] Table 1 Materials and reagents

[0037]

[0038] Table 2 Instruments and equipment

[0039]

[0040]

[0041] Cell culture:

[0042] RF / 6A cells were subcultured and inoculated into culture flasks and cultured in an incubator at 37°C, 5% CO2 and saturated humidity. When the cells in the culture flasks were basically full (the cells were kept in the logarithmic growth phase), the culture medium was discarded, and an appropriate amount of DPBS solution (pH 7.0-7.3, calcium-free and magnesium-free thermofisher.cn) was added to gently wash and discarded. Then, 1 mL of 0.25% trypsin solution (0.25% Trypsin-EDTA) was added to digest for 1 min, 4 mL of culture medium containing 10% serum (MEM complete culture medium) was added and pipetted to terminate the action of trypsin, and the cells were transferred to a centrifuge tube, centrifuged at 1000r for 5 min, the supernatant was discarded, and culture medium containing 10% serum (MEM complete culture medium) was added and carefully pipetted into single cells.

[0043] The cells were counted using an automatic cell counter and then diluted with complete culture medium to an appropriate cell density (5 × 10 4 100 μL of DPBS was added to the edge wells, and the cells were cultured in a 37°C, 5% CO2 incubator.

[0044] Cell modeling and drug treatment

[0045] Preparation of VEGF: According to the instructions, DPBS containing 0.1% bovine serum albumin was used to prepare a 100 μg / mL stock solution, and diluted to a 4 μg / mL secondary stock solution with serum-free MEM medium before use.

[0046] Drug preparation: In order to keep the total system (200 μL) of all groups consistent, the preparation is as follows: DMSO is used to prepare the mother solution of Xiulingdan (concentration is 25 mg / mL), and the secondary mother solution of Xiulingdan (concentration is 100 μg / mL) is prepared with MEM medium without serum. The experiment includes three groups, namely:

[0047] Blank control group: Normally cultured cells, added with 200 μL of MEM medium without serum;

[0048] Model group: 100 μL MEM medium without serum + 100 μL VEGF (4 μg / mL);

[0049] Drug treatment group: 50 μL MEM medium without serum + 50 μL of the second stock solution of Xiulingdan (100 μg / mL) + 100 μL VEGF (4 μg / mL).

[0050] The cells were cultured in a CO2 incubator for 72 hours. After the modeling and drug administration, MTS was applied for 2 hours, and the absorbance was detected at 490 nm. The results are shown in the figure below. Figure 1 shown.

[0051] Depend on Figure 1 It can be seen that in RF / 6A cells, the cell viability can be improved to a certain extent by treating them with different concentrations (1, 2 μg / mL) of VEGF for 72 hours. Therefore, the subsequent experiments used 2 μg / mL of VEGF for 72 hours as the condition for model preparation.

[0052] In the VEGF-induced RF / 6A cell proliferation model, the protection rate of 25 μg / mL 95% ethanol extract of Xiulingdan was 48.97%, and it was preliminarily identified as an active sample.

[0053] Example 2: Effects of intraperitoneal injection of sodium iodate on mice

[0054] SPF grade C57BL / 6 healthy mice, 8 weeks old, male, were placed in natural light and fed with free drinking water and diet for 1 week. The animal conditions were observed and the mice with poor conditions, sparse hair, and body weight outside the range of 18-22g were eliminated.

[0055] 1. Main reagents

[0056] Su Mian Xin II Cumshot-Anesthetic

[0057] 0.9% Sodium Chloride Injection

[0058] Sodium iodate powder-modeling agent

[0059] CMC Sodium

[0060] 2. Modeling and Experiments

[0061] 2.1 Investigation of modeling agent dosage and modeling time:

[0062] C57 mice were randomly divided into 3 groups according to the random number table method: blank group, modeling agent (low) and modeling agent (high), with 12 mice in each group. NaIO3 solutions with concentrations of 6.25 mg / mL (high) and 3.75 mg / ml (low) were prepared with normal saline and intraperitoneally injected at a dose of 8 mL / kg per mouse.

[0063] (1) Normal group: normal mice, raised normally, with free access to food and water;

[0064] (2) Modeling agent (low): Prepare a NaIO3 solution with a concentration of 3.75 mg / ml (low) using normal saline and inject it intraperitoneally at a dose of 8 mL / kg per mouse.

[0065] (3) Modeling agent (high): A NaIO3 solution with a concentration of 6.25 mg / mL (high) was prepared with normal saline and injected intraperitoneally at a dose of 8 mL / kg per mouse.

[0066] 2.2 Fundus examination, microscopic examination and sectioning:

[0067] Each group was examined on the 7th, 14th and 28th day after model establishment.

[0068] a. First, prepare 10% Su Mian Xin II solution with normal saline, and inject it intraperitoneally at a dose of 0.01 mL / g per mouse. The mouse will show anesthesia symptoms 3 to 5 minutes after intraperitoneal injection of the anesthetic. After anesthesia, place the mouse horizontally prone on the experimental table, quickly remove the mouse eyeball with elbow forceps and put it into Davidson eyeball fixative, and avoid damaging the posterior sclera of the mouse eyeball during the removal of the mouse eyeball, and send the sample for inspection.

[0069] 2.3 Test results:

[0070] The main retinal lesions in the model group of this experiment were damage and degeneration of the pigment epithelial cells, abnormal inner and outer nuclear layer cells, cell reduction, disordered arrangement, and wavy appearance. In addition, drusen were deposited between the retinal drusen and the pigment epithelial cells. The results indicated that the model was successfully established.

[0071] As the modeling time prolonged, the severity of the lesions increased. At 7 days after modeling, the lesion severity of the low-dose model group was slightly lower than that of the high-dose model group. At 14 days and 28 days after modeling, there was no significant difference between the low-dose model group and the high-dose model group. The modeling was successful, and both the low-dose and high-dose models produced lesions at 7 days. Therefore, low-dose was used for modeling in the subsequent modeling, and detection was performed at 7 days, 14 days, and 28 days, respectively.

[0072] 2.4 In vivo activity screening of test drugs:

[0073] 2.4.1 Drug preparation

[0074] Based on an adult's body weight of 60 kg, and a mouse's body weight of 20 g, the metabolic ratio between mice and humans is 10 times. Based on the daily oral dosage of Chiao Ling Dan of 9 to 15 g, the low dose - 3 times the clinical dose is calculated as follows:

[0075] Low-dose dosage (raw drug): 15g÷60kg×10*3=2.5*3g / kg=7.5g / kg; 0.1mL (volume) was administered orally to each mouse every day for every 0.01kg (body weight), so the low-dose preparation concentration was: 7.5g / kg×0.01kg÷0.1mL=0.75g / mL;

[0076] Converted into dry cream: 0.75g / mL*14.58%=0.11g / ml.

[0077] 2.4.2 Modeling, grouping and drug administration:

[0078] A NaIO3 solution with a concentration of 3.75 mg / ml was prepared with normal saline and injected intraperitoneally at a dose of 8 mL / kg per mouse.

[0079] The modeled C57 mice were randomly divided into a treatment group, a positive drug group, and a model group according to the random number table method, with 12 mice in each group, and a normal group of 8 mice was set up as a control.

[0080] Treatment group: Oxidative stress mice, 95% ethanol sample of Xiulingdan converted to a low dose and then gavaged;

[0081] Model group: oxidative stress mice, intragastric administration of distilled water;

[0082] Positive drug group: oxidative stress mice, intragastric administration of lutein tablet aqueous solution;

[0083] Normal group: normal mice, raised normally, with free access to food and water;

[0084] Each group was examined on the 7th, 14th and 28th day after model establishment.

[0085] 2.4.3 Slicing:

[0086] a. First, prepare 10% Su Mian Xin II solution with normal saline, and inject it intraperitoneally at a dose of 0.01 mL / g per mouse. The mouse will show anesthesia symptoms 3 to 5 minutes after the intraperitoneal injection of the anesthetic. After anesthesia, the mouse is placed horizontally on the experimental table, the eyeball is removed, and the fascia, fat and muscle tissue surrounding the eyeball are quickly removed. The process of removing the mouse eyeball avoids damaging the posterior sclera of the mouse eyeball. The complete eyeball tissue of the mouse is placed in Davidson eyeball fixative and fixed for 24 hours. The sample is sent for inspection, sectioning and reading, and the eyeball tissue section is obtained as shown below Figure 2 shown.

[0087] Lesion evaluation criteria: Lesions are scored from 0.5 to 4 points from mild to severe. Mild or very mild lesions are scored as 0.5 points, mild lesions are scored as 1 point, moderate lesions are scored as 2 points, severe lesions are scored as 3 points, extremely severe lesions are scored as 4 points, and no lesions are scored as 0 points.

[0088] 3. Conclusion:

[0089] The normal retinal tissue consists of the following layers from the inside to the outside: inner limiting membrane, nerve fiber layer, ganglion cell layer, inner plexiform layer, inner nuclear layer, outer plexiform layer, outer nuclear layer, external limiting membrane, photoreceptor layer and pigment epithelium. The retina of mice in the KB group had distinct layers, the inner and outer granular layer cells were neatly arranged, the thickness was uniform, the pigment epithelium was intact, and no obvious lesions were observed.

[0090] The cells of the inner nuclear layer of the retina of the mice in the treatment group were slightly abnormal; the cells of the outer nuclear layer of the retina were occasionally moderately abnormal, and the cells of the retinal pigment epithelium were slightly degenerated. Drus warts began to appear between the retinal drusen and the pigment epithelium cells of the mice. The cells of the inner and outer nuclear layers were reduced, arranged neatly, and had uniform thickness, while the arrangement of the pigment epithelium cells was disordered.

[0091] The cells of the inner nuclear layer of the retina of the model group mice were moderately abnormal; the cells of the outer nuclear layer of the retina were moderately abnormal, of which one case had extremely severe abnormalities in the outer nuclear layer of the retina, and the cells of the retinal pigment epithelial layer were moderately degenerated. Drus warts were deposited between the retinal Bruch's membrane and the pigment epithelial cells of the mouse eyes. The cells of the inner and outer nuclear layers were reduced, arranged in a disordered and wavy shape. The pigment epithelial cells were damaged and degenerated, and drusen were deposited between the retinal Bruch's membrane and the pigment epithelial cells.

[0092] The degree of eye lesions in the treatment group was milder than that in the model group, indicating that the 95% ethanol extract of Xiulingdan has a certain protective effect on macular lesions in mice and can delay the development of macular lesions.

[0093] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of Chou Ling Dan in the preparation of drugs for preventing and treating age-related macular degeneration.

2. Application of the extract of Chou Ling Dan in the preparation of drugs for preventing and treating age-related macular degeneration.

3. The use according to claim 2, characterized in that: The stinky lingdan extract is prepared according to the following steps: The choulingdan is extracted by warm soaking with ethanol solution to obtain the choulingdan extract.

4. The use according to claim 3, characterized in that: The volume concentration of ethanol in the ethanol solution is 90% to 95%; the mass ratio of the stinky elixir to the ethanol solution is 1:(5 to 20).

5. The use according to claim 3, characterized in that: The temperature of the warm immersion extraction is 80° C. to 95° C.; the time of the warm immersion extraction is 1 to 3 hours; and the number of warm immersion extractions is preferably 1 to 3 times.

6. The use according to claim 3, characterized in that: After warm immersion extraction, centrifugation is performed, and the supernatant is concentrated and freeze-dried to obtain the extract of the stinky lingdan.

7. The use according to claim 1, characterized in that: The age-related macular degeneration is exudative age-related macular degeneration.

8. The use according to claim 1, characterized in that: The xiulingdan extract prevents and treats age-related macular degeneration by reducing the apoptosis of RF / 6A cells induced by vascular endothelial growth factor.

9. Use of a Chinese medicine composition in a drug for preventing and treating age-related macular degeneration, characterized in that: The traditional Chinese medicine composition comprises Chou Ling Dan.

10. The use according to claim 9, characterized in that: The dosage form of the Chinese medicine composition is selected from one or more of decoction, granules, capsules, tablets, oral liquids, pills, tinctures, syrups, suppositories, gels, sprays or injections.

Images