Preparation of Eucommia ulmoides leaf extract and its application in improving age-related macular degeneration

Abstract

This invention, entitled "Preparation of Eucommia ulmoides Leaf Extract and Its Application in Improving Age-Related Macular Degeneration," belongs to the field of traditional Chinese medicine extraction technology. The technical problem to be solved is how to prepare an Eucommia ulmoides leaf extract with higher contents of chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid, aucubin, and geniposide, and with better anti-age-related macular degeneration effects. The key technical points are that the Eucommia ulmoides leaf extract contains 50-100 mg / g of chlorogenic acid, 40-60 mg / g of cryptochlorogenic acid, 35-55 mg / g of neochlorogenic acid, 56-79 mg / g of aucubin, and 65-105 mg / g of geniposide. The Eucommia ulmoides leaf extract obtained by this method can better retain the effective components in the Eucommia ulmoides leaf extract, exhibiting a superior anti-age-related macular degeneration effect.

Description

Technical Field

[0001] This invention relates to the preparation of Eucommia ulmoides leaf extract and its application in improving age-related macular degeneration, belonging to the field of traditional Chinese medicine extraction technology. Background Technology

[0002] For understanding the technical content of this invention: The macula is the area in the center of the human retina where visual cells are most concentrated, making it the most sensitive area for vision. The macula contains a large number of cone cells responsible for vision and color perception. Any lesion affecting the macula will cause a significant decrease in central vision, blurred vision, and distorted vision.

[0003] Age-related macular degeneration (AMD) is a aging-related structural change in the macula. The main manifestation of AMD is a decreased ability of retinal pigment epithelial cells to phagocytose and digest the outer segment disc membranes of photoreceptor cells. This results in the accumulation of undigested disc membrane remnants in the basal cytoplasm, which are then expelled extracellularly and deposited on Bruch's membrane, forming drusen, or causing rupture of Bruch's membrane itself. Choroidal capillaries then pass through the ruptured Bruch's membrane into the subepithelial space of the optic nerve, forming choroidal neovascularization. AMD mostly occurs in people over 45 years of age, and its prevalence increases with age, making it a major cause of blindness in the elderly.

[0004] Relevant patent documents retrieved: This document, published in China (CN115475187A) on December 16, 2022, discloses a freeze-dried formulation containing Eucommia ulmoides extract. The freeze-dried formulation comprises Eucommia ulmoides extract and a binder. The Eucommia ulmoides extract contains 0.004 wt%-95 wt% of the extract by mass. The extract contains lignins, iridoids, phenylpropanoids, and flavonoids. More preferably, the content of each substance in the Eucommia ulmoides extract is as follows: pinoresinol diglucoside 0.1 wt%-0.35 wt%, preferably 0.2 wt%-0.25 wt%; and / or genipin 0.5 wt%-5 wt%, preferably 1 wt%-3.5 wt%; and / or aucubin 0.1 wt%-1.5 wt%, preferably 0.2 wt%-1 wt%; and / or chlorogenic acid 1.7 wt%-5.5 wt%, preferably 2 wt%-5 wt%. %; and / or quercetin 0.006wt%-0.05wt%, preferably 0.01wt%-0.03wt%; and / or rutin 0.01wt%-0.4wt%, preferably 0.2wt%-0.3wt%; and / or isoquercitrin 0.01wt%-0.08wt%, preferably 0.02wt%-0.06wt%; and / or catechin 0.01wt%-0.05wt%, preferably 0.02wt%-0.04wt%.

[0005] This document, published in China (CN114344358A) on April 15, 2022, discloses a Eucommia ulmoides composition, preparation, and application containing aucubin and geniposide. The Eucommia ulmoides composition contains 10... 50% aucubin, 3 30% genipin and 0.05 5% protocatechuic acid. This technology provides a Eucommia ulmoides composition with high content of aucubin and genipin, as well as a drug formulation with Scutellaria baicalensis, overcoming the drawbacks of conventional Chinese medicine preparations where the numerous ingredients make it difficult to determine side effects, and greatly improving the treatment effect of diabetic nephropathy.

[0006] Relevant non-patent literature retrieved: The journal or book title is "Chinese Journal of Experimental Traditional Medical Formulae," and the article title is "A Review of Research on Chemical Constituents, Pharmacological Effects, and Product Development of Eucommia ulmoides, a Plant with Both Medicinal and Edible Properties." The volume number is 30, the issue number is 2, and the publication date is October 31, 2023. This article discloses that Eucommia ulmoides, due to its rich components, has various pharmacological effects such as lowering blood pressure, lowering blood lipids, lowering blood sugar, preventing osteoporosis, anti-tumor, antibacterial and antiviral, anti-inflammatory, antioxidant, and liver-protective effects. Therefore, it has the potential to develop diversified medicinal and edible products.

[0007] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: The existing technology has not provided an extract of Eucommia ulmoides leaves for treating age-related macular degeneration.

[0008] In solving the above problems or overcoming the above defects, the present invention encountered the following difficulties and obstacles: Existing processing techniques have significant shortcomings: traditional hot air drying causes heat-sensitive components to decompose and oxidize in large quantities due to high temperatures, with chlorogenic acid loss exceeding 40%, weakening the pharmacodynamic material basis. Furthermore, the lack of concentration before drying results in a low final content of active ingredients. While existing freeze drying can theoretically reduce component damage, it suffers from large ice crystals, a drying cycle exceeding 48 hours, lack of optimization for the tissue structure of Eucommia ulmoides leaves, and a lack of concentration before freeze drying, resulting in a low retention rate of active ingredients. High energy consumption also limits its industrialization. Summary of the Invention

[0009] The purpose of this invention is to provide: An application of Eucommia ulmoides leaf extract in improving age-related macular degeneration, and related technologies, to solve the following technical problems or combinations thereof: developing an Eucommia ulmoides leaf processing technology that integrates efficient extraction, scientific concentration and optimized freeze-drying; efficiently retaining active ingredient groups, improving freeze-drying efficiency and increasing the content of active ingredients; and applying Eucommia ulmoides leaf extract for the first time in the treatment of age-related macular degeneration.

[0010] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0011] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0012] Definitions of pharmaceutical terms can be found in the reference "Pharmacopoeia of the People's Republic of China (2020 Edition)" (China Medical Science and Technology Press, May 2020, First Edition).

[0013] Unless otherwise specified, conventional methods within the scope of the art, such as filtration, HPLC, mouse acclimatization, intraperitoneal injection of mice, anesthesia of mice, blood collection from mouse orbital fossa, removal of eyeball tissue, homogenization, etc., shall be used.

[0014] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0015] In a first aspect, the present invention provides the application of Eucommia ulmoides leaf extract in the preparation of products that improve age-related macular degeneration.

[0016] Furthermore, the Eucommia ulmoides leaf extract contains 50-100 mg / g of chlorogenic acid, 40-60 mg / g of cryptochlorogenic acid, 35-55 mg / g of neochlorogenic acid, 56-79 mg / g of aucubin, and 65-105 mg / g of genipin.

[0017] The chlorogenic acid content is selected from any value within the range of 50-100 mg / g, such as the following values ​​or any range between two: 50 mg / g, 50.5 mg / g, 51 mg / g, 51.5 mg / g, 52 mg / g, 52.5 mg / g, 53 mg / g, 53.5 mg / g, 54 mg / g, 54.5 mg / g, 55 mg / g, 55.5 mg / g, 56 mg / g, 56.5 mg / g, 57 mg / g, 57.5 mg / g, 58 mg / g, 58.5 mg / g, 59 mg / g, 59.5 mg / g, 60 mg / g. , 60.5mg / g, 61mg / g, 61.5mg / g, 62mg / g, 62.5mg / g, 63mg / g, 63.5mg / g, 64mg / g, 64.5mg / g, 65mg / g, 65.5mg / g, 66mg / g, 66.5mg / g, 6 7mg / g, 67.5mg / g, 68mg / g, 68.5mg / g, 69mg / g, 69.5mg / g, 70mg / g, 70.5mg / g, 71mg / g, 71.5mg / g, 72mg / g, 72.5mg / g, 73mg / g, 73.5m g / g, 74mg / g, 74.5mg / g, 75mg / g, 75.5mg / g, 76mg / g, 76.5mg / g, 77mg / g, 77.5mg / g, 78mg / g, 78.5mg / g, 79mg / g, 79.5mg / g, 80mg / g, 80.5mg / g, 81mg / g, 81.5mg / g, 82mg / g, 82.5mg / g, 83mg / g, 83.5mg / g, 84mg / g, 84.5mg / g, 85mg / g, 85.5mg / g, 86mg / g, 86.5mg / g, 87 mg / g, 87.5mg / g, 88mg / g, 88.5mg / g, 89mg / g, 89.5mg / g, 90mg / g, 90.5mg / g, 91mg / g, 91.5mg / g, 92mg / g, 92.5mg / g, 93mg / g, 93.5mg / g, 94mg / g, 94.5mg / g, 95mg / g, 95.5mg / g, 96mg / g, 96.5mg / g, 97mg / g, 97.5mg / g, 98mg / g, 98.5mg / g, 99mg / g, 99.5mg / g, 100mg / g.

[0018] The cryptochlorogenic acid content is selected from any value within the range of 40-60 mg / g, such as the following values ​​or any range between two: 40 mg / g, 40.5 mg / g, 41 mg / g, 41.5 mg / g, 42 mg / g, 42.5 mg / g, 43 mg / g, 43.5 mg / g, 44 mg / g, 44.5 mg / g, 45 mg / g, 45.5 mg / g, 46 mg / g, 46.5 mg / g, 47 mg / g, 47.5 mg / g, 48 mg / g, 48.5 mg / g. mg / g, 49mg / g, 49.5mg / g, 50mg / g, 50.5mg / g, 51mg / g, 51.5mg / g, 52mg / g, 52.5mg / g, 53mg / g, 53.5mg / g, 54mg / g, 54 .5mg / g, 55mg / g, 55.5mg / g, 56mg / g, 56.5mg / g, 57mg / g, 57.5mg / g, 58mg / g, 58.5mg / g, 59mg / g, 59.5mg / g, 60mg / g.

[0019] The chlorogenic acid content is selected from any value within the range of 35-55 mg / g, such as the following values ​​or any range between two: 35 mg / g, 35.5 mg / g, 36 mg / g, 36.5 mg / g, 37 mg / g, 37.5 mg / g, 38 mg / g, 38.5 mg / g, 39 mg / g, 39.5 mg / g, 40 mg / g, 40.5 mg / g, 41 mg / g, 41.5 mg / g, 42 mg / g, 42.5 mg / g, 43 mg / g, 43.5 mg / g. mg / g, 44mg / g, 44.5mg / g, 45mg / g, 45.5mg / g, 46mg / g, 46.5mg / g, 47mg / g, 47.5mg / g, 48mg / g, 48.5mg / g, 49mg / g, 49 .5mg / g, 50mg / g, 50.5mg / g, 51mg / g, 51.5mg / g, 52mg / g, 52.5mg / g, 53mg / g, 53.5mg / g, 54mg / g, 54.5mg / g, 55mg / g.

[0020] The aucubin content is selected from any value within the range of 56-79 mg / g, such as the following values ​​or any range between two: 56 mg / g, 56.5 mg / g, 57 mg / g, 57.5 mg / g, 58 mg / g, 58.5 mg / g, 59 mg / g, 59.5 mg / g, 60 mg / g, 60.5 mg / g, 61 mg / g, 61.5 mg / g, 62 mg / g, 62.5 mg / g, 63 mg / g, 63.5 mg / g, 64 mg / g, 64.5 mg / g, 65 mg / g, 65.5 mg / g, 6 ... g / g, 66.5mg / g, 67mg / g, 67.5mg / g, 68mg / g, 68.5mg / g, 69mg / g, 69.5mg / g, 70mg / g, 70.5mg / g, 71mg / g, 71.5mg / g, 72mg / g, 72.5mg / g, 73mg / g, 73.5mg / g, 74mg / g, 74.5mg / g, 75mg / g, 75.5mg / g, 76mg / g, 76.5mg / g, 77mg / g, 77.5mg / g, 78mg / g, 78.5mg / g, 79mg / g.

[0021] The genipin acid content is selected from any value within the range of 65-105 mg / g, such as the following values ​​or any range between the two: 65 mg / g, 65.5 mg / g, 66 mg / g, 66.5 mg / g, 67 mg / g, 67.5 mg / g, 68 mg / g, 68.5 mg / g, 69 mg / g, 69.5 mg / g, 70 mg / g, 70.5 mg / g, 71 mg / g, 71.5 mg / g, 72 mg / g, 72.5 mg / g. , 73mg / g, 73.5mg / g, 74mg / g, 74.5mg / g, 75mg / g, 75.5mg / g, 76mg / g, 76.5mg / g, 77mg / g, 77.5mg / g, 78mg / g, 78.5mg / g, 79mg / g, 79.5mg / g, 80mg / g, 80.5mg / g, 81mg / g, 81.5mg / g, 82mg / g, 82.5mg / g, 83mg / g, 83.5mg / g, 84mg / g, 84.5mg / g, 85mg / g, 85.5mg / g, 86mg / g, 86.5mg / g, 87mg / g, 87.5mg / g, 88mg / g, 88.5mg / g, 89mg / g, 8 9.5mg / g, 90mg / g, 90.5mg / g, 91mg / g, 91.5mg / g, 92mg / g, 92.5mg / g, 93mg / g, 93.5mg / g, 94mg / g, 94.5mg / g, 9 5mg / g, 95.5mg / g, 96mg / g, 96.5mg / g, 97mg / g, 97.5mg / g, 98mg / g, 98.5mg / g, 99mg / g, 99.5mg / g, 100mg / g, 1 00.5mg / g, 101mg / g, 101.5mg / g, 102mg / g, 102.5mg / g, 103mg / g, 103.5mg / g, 104mg / g, 104.5mg / g, 105mg / g.

[0022] Furthermore, the chlorogenic acid content in the Eucommia ulmoides leaf extract is 80.5-94.5 mg / g, the cryptochlorogenic acid content is 43-50 mg / g, the neochlorogenic acid content is 38-43 mg / g, the aucubin content is 58-74 mg / g, and the geniposide content is 65-105 mg / g.

[0023] The chlorogenic acid content is selected from any value within the range of 80.5-94.5 mg / g, such as the following values ​​or any range between the two: 80.5 mg / g, 81 mg / g, 81.5 mg / g, 82 mg / g, 82.5 mg / g, 83 mg / g, 83.5 mg / g, 84 mg / g, 84.5 mg / g, 85 mg / g, 85.5 mg / g, 86 mg / g, 86.5 mg / g, 87 mg / g, 87.5 mg / g, 88 mg / g, 88.5 mg / g, 89 mg / g, 89.5 mg / g, 90 mg / g, 90.5 mg / g, 91 mg / g, 91.5 mg / g, 92 mg / g, 92.5 mg / g, 93 mg / g, 93.5 mg / g, 94 mg / g, 94.5 mg / g.

[0024] The cryptochlorogenic acid content is selected from any value within the range of 43-50 mg / g, such as the following values ​​or any range between the two: 43 mg / g, 43.5 mg / g, 44 mg / g, 44.5 mg / g, 45 mg / g, 45.5 mg / g, 46 mg / g, 46.5 mg / g, 47 mg / g, 47.5 mg / g, 48 mg / g, 48.5 mg / g, 49 mg / g, 49.5 mg / g, and 50 mg / g.

[0025] The content of neochlorogenic acid is selected from any value in the range of 38-43 mg / g, such as the following values ​​or any range between the two: 38 mg / g, 38.5 mg / g, 39 mg / g, 39.5 mg / g, 40 mg / g, 40.5 mg / g, 41 mg / g, 41.5 mg / g, 42 mg / g, 42.5 mg / g, 43 mg / g.

[0026] The aucubin content is selected from any value within the range of 58-74 mg / g, such as the following values ​​or any range between two: 58 mg / g, 58.5 mg / g, 59 mg / g, 59.5 mg / g, 60 mg / g, 60.5 mg / g, 61 mg / g, 61.5 mg / g, 62 mg / g, 62.5 mg / g, 63 mg / g, 63.5 mg / g, 64 mg / g, 64.5 mg / g. mg / g, 65mg / g, 65.5mg / g, 66mg / g, 66.5mg / g, 67mg / g, 67.5mg / g, 68mg / g, 68.5mg / g, 69mg / g, 69 .5mg / g, 70mg / g, 70.5mg / g, 71mg / g, 71.5mg / g, 72mg / g, 72.5mg / g, 73mg / g, 73.5mg / g, 74mg / g.

[0027] The genipin acid content is selected from any value within the range of 65-105 mg / g, such as the following values ​​or any range between the two: 65 mg / g, 65.5 mg / g, 66 mg / g, 66.5 mg / g, 67 mg / g, 67.5 mg / g, 68 mg / g, 68.5 mg / g, 69 mg / g, 69.5 mg / g, 70 mg / g, 70.5 mg / g, 71 mg / g, 71.5 mg / g, 72 mg / g, 72.5 mg / g. , 73mg / g, 73.5mg / g, 74mg / g, 74.5mg / g, 75mg / g, 75.5mg / g, 76mg / g, 76.5mg / g, 77mg / g, 77.5mg / g, 78mg / g, 78.5mg / g, 79mg / g, 79.5mg / g, 80mg / g, 80.5mg / g, 81mg / g, 81.5mg / g, 82mg / g, 82.5mg / g, 83mg / g, 83.5mg / g, 84mg / g, 84.5mg / g, 85mg / g, 85.5mg / g, 86mg / g, 86.5mg / g, 87mg / g, 87.5mg / g, 88mg / g, 88.5mg / g, 89mg / g, 8 9.5mg / g, 90mg / g, 90.5mg / g, 91mg / g, 91.5mg / g, 92mg / g, 92.5mg / g, 93mg / g, 93.5mg / g, 94mg / g, 94.5mg / g, 9 5mg / g, 95.5mg / g, 96mg / g, 96.5mg / g, 97mg / g, 97.5mg / g, 98mg / g, 98.5mg / g, 99mg / g, 99.5mg / g, 100mg / g, 1 00.5mg / g, 101mg / g, 101.5mg / g, 102mg / g, 102.5mg / g, 103mg / g, 103.5mg / g, 104mg / g, 104.5mg / g, 105mg / g.

[0028] As a specific embodiment of the present invention, the chlorogenic acid content in the Eucommia ulmoides leaf extract is 80.5 mg / g, the cryptochlorogenic acid content is 45 mg / g, the neochlorogenic acid content is 38 mg / g, the aucubin content is 58 mg / g, and the geniposide content is 65 mg / g.

[0029] As a specific embodiment of the present invention, the chlorogenic acid content in the Eucommia ulmoides leaf extract is 80.5 mg / g, the cryptochlorogenic acid content is 43 mg / g, the neochlorogenic acid content is 39 mg / g, the aucubin content is 68 mg / g, and the genipin content is 86 mg / g.

[0030] Preferably, and as a specific embodiment of the present invention, the chlorogenic acid content in the Eucommia ulmoides leaf extract is 94.5 mg / g, the cryptochlorogenic acid content is 50 mg / g, the neochlorogenic acid content is 43 mg / g, the aucubin content is 74 mg / g, and the genipin content is 105 mg / g.

[0031] Furthermore, the Eucommia ulmoides leaf extract is a freeze-dried powder of Eucommia ulmoides leaf extract.

[0032] Furthermore, the preparation method of the Eucommia ulmoides leaf extract includes the following steps: Eucommia ulmoides leaves are mixed with water, warm-soaked for extraction, filtered, and the filtrate is collected and freeze-dried to obtain Eucommia ulmoides leaf extract.

[0033] Furthermore, the weight of the water is 6-10 times the weight of the Eucommia ulmoides leaves; the temperature of the warm soaking extraction is 60-90℃, the time of the warm soaking extraction is 40-80 minutes, and the number of times the warm soaking extraction is repeated is 1-3 times.

[0034] The ratio of the weight of the water to the weight of the Eucommia ulmoides leaves can be selected from any ratio between 6 and 10, such as 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, or any range between the two mentioned above.

[0035] The temperature for the warm immersion extraction can be selected from any temperature between 60-90℃, such as 60℃, 61℃, 62℃, 63℃, 64℃, 65℃, 66℃, 67℃, 68℃, 69℃, 70℃, 71℃, 72℃, 73℃, 74℃, 75℃, 76℃, 77℃, 78℃, 79℃, 80℃, 81℃, 82℃, 83℃, 84℃, 85℃, 86℃, 87℃, 88℃, 89℃, 90℃, or any range between the above two.

[0036] The extraction time can be selected from any time between 40 and 80 minutes, such as 40 minutes, 41 minutes, 42 minutes, 43 minutes, 44 minutes, 45 minutes, 46 minutes, 47 minutes, 48 ​​minutes, 49 minutes, 50 minutes, 51 minutes, 52 minutes, 53 minutes, 54 minutes, 55 minutes, 56 minutes, 57 minutes, 58 minutes, 59 minutes, 60 minutes, 61 minutes, 62 minutes, 63 minutes, 64 minutes, 65 minutes, 66 minutes, 67 minutes, 68 minutes, 69 minutes, 70 minutes, 71 minutes, 72 minutes, 73 minutes, 74 minutes, 75 minutes, 76 minutes, 77 minutes, 78 minutes, 79 minutes, 80 minutes, or any range between the above two.

[0037] The number of times the warm immersion extraction is repeated can be selected from 1 time, 2 times or 3 times.

[0038] Furthermore, the weight of the water is 8 times the weight of the Eucommia ulmoides leaves; the temperature of the warm soaking extraction is 90°C, the time of the warm soaking extraction is 40 minutes, and the warm soaking extraction is repeated twice.

[0039] Secondly, the present invention provides a product for improving age-related macular degeneration, comprising the Eucommia ulmoides leaf extract described in the above-mentioned applications.

[0040] Compared with the prior art, the beneficial effects of the present invention include: 1. This invention provides an extract of Eucommia ulmoides leaves containing specific amounts of chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid, genipin, and aucubin. This extract can effectively reduce the levels of TNF-α, IL-6, and MDA in the eyes of mice with age-related macular degeneration, and increase GSH levels and SOD activity.

[0041] 2. This invention provides a specific method for preparing Eucommia ulmoides leaf extract, which effectively increases the content of chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid, genipin, and aucubin in Eucommia ulmoides leaf extract by using specific warm maceration extraction and drying methods. Attached Figure Description

[0042] Figure 1 The bar chart shows the level of TNF-α in the mouse eyeball; ** indicates a significant difference compared to the model group and p < 0.01; ## indicates a significant difference compared to the S4 group and p < 0.01; && indicates a significant difference compared to the JY group and p < 0.01.

[0043] Figure 2The bar chart shows the level of IL-6 in the mouse eyeball; ** indicates a significant difference compared to the model group and p < 0.01; ## indicates a significant difference compared to the S4 group and p < 0.01; && indicates a significant difference compared to the JY group and p < 0.01.

[0044] Figure 3 The graph shows the horizontal bar graph of MDA in the mouse eyeball; ** indicates a significant difference compared with the model group and p < 0.01; ## indicates a significant difference compared with the S4 group and p < 0.01; && indicates a significant difference compared with the JY group and p < 0.01.

[0045] Figure 4 The graph shows the horizontal bar graph of GSH in the mouse eyeball; ** indicates a significant difference compared with the model group and p < 0.01; ## indicates a significant difference compared with the S4 group and p < 0.01; && indicates a significant difference compared with the JY group and p < 0.01.

[0046] Figure 5 The bar chart shows the SOD activity level in the mouse eyeball; ** indicates a significant difference compared to the model group and p < 0.01; ## indicates a significant difference compared to the S4 group and p < 0.01; && indicates a significant difference compared to the JY group and p < 0.01.

[0047] Figure 6 Photographs of mouse eyeball sections stained with hematoxylin and eosin (HE); where A represents the normal group, B represents the model group, C represents the positive drug group, D represents the S1 group, E represents the S9 group, F represents the S4 group, G represents the JY group, GCL represents the ganglion cell layer, INL represents the inner nuclear layer, ONL represents the outer nuclear layer, and RPE represents the retinal pigment epithelium; in A to G, the scale bar represents a length of 50 μm. Detailed Implementation

[0048] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0049] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0050] Example 1 A method for preparing Eucommia ulmoides leaf extract, comprising the following steps: Dried Eucommia ulmoides leaves were mixed with water and extracted by warm soaking at a certain temperature for a period of time, with the extraction repeated 1-3 times. The extracts were then filtered, the filtrates were combined, and freeze-dried at -40℃ to obtain Eucommia ulmoides leaf extract.

[0051] The effects of four factors on the quality of the obtained Eucommia ulmoides leaf extract were investigated. Nine parallel experiments were conducted, numbered S1, S2, S3, S4, S5, S6, S7, S8, and S9.

[0052] Extract S1: Eucommia ulmoides leaves were mixed with 6 times their weight of water and extracted at 60°C for 40 minutes, once. The filtrate was filtered and dried under reduced pressure at room temperature to obtain Eucommia ulmoides leaf extract S1.

[0053] Extract S2: Eucommia ulmoides leaves were mixed with 6 times their weight of water and extracted at 75°C for 60 minutes, twice. The extracts were filtered, the filtrates were combined, and dried under reduced pressure at room temperature to obtain Eucommia ulmoides leaf extract S2.

[0054] Extract S3: Eucommia ulmoides leaves were mixed with 6 times their weight of water and extracted at 90°C for 80 minutes, repeated 3 times. The extracts were filtered, combined, and dried under reduced pressure at room temperature to obtain Eucommia ulmoides leaf extract S3.

[0055] Extract S4: Mix Eucommia ulmoides leaves with 8 times their weight of water and extract by warm soaking at 90°C for 40 min, repeating the extraction twice. Filter, combine the filtrates, and dry under reduced pressure at room temperature to obtain Eucommia ulmoides leaf extract S4.

[0056] Extract S5: Mix Eucommia ulmoides leaves with 8 times their weight of water and extract by warm soaking at 60°C for 60 min, repeating the extraction 3 times. Filter, combine the filtrates, and dry under reduced pressure at room temperature to obtain Eucommia ulmoides leaf extract S5.

[0057] Extract S6: Mix Eucommia ulmoides leaves with 8 times their weight of water and extract by warm soaking at 75°C for 80 minutes, repeating the extraction once. Filter the filtrate and dry under reduced pressure at room temperature to obtain Eucommia ulmoides leaf extract S6.

[0058] Extract S7: Mix Eucommia ulmoides leaves with 10 times their weight of water and extract by warm soaking at 75°C for 40 minutes, repeating the extraction three times. Filter, combine the filtrates, and dry under reduced pressure at room temperature to obtain Eucommia ulmoides leaf extract S7.

[0059] Extract S8: Mix Eucommia ulmoides leaves with 10 times their weight of water and extract by warm soaking at 90°C for 60 min, repeating the extraction once. Filter the filtrate and dry under reduced pressure at room temperature to obtain Eucommia ulmoides leaf extract S8.

[0060] Extract S9: Mix Eucommia ulmoides leaves with 10 times their weight of water and extract by warm soaking at 60°C for 80 min, repeating the extraction twice. Filter, combine the filtrates, and dry under reduced pressure at room temperature to obtain Eucommia ulmoides leaf extract S9.

[0061] The contents of chlorogenic acid, aucubin, and genipin in the obtained Eucommia ulmoides leaf extracts S1-S9 were determined by HPLC. The experimental results are shown in Table 1 below: Table 1

[0062] As shown in Table 1, the preparation method S4 yielded chlorogenic acid content of 94.5 mg / g, aucubin content of 74 mg / g, and geniposide content of 105 mg / g. The sum of these three contents reached 273.5 mg / g, which is the highest value among the nine groups of experiments. Therefore, it was determined that using the combination of water volume, soaking time, soaking temperature, and extraction times of method S4 can achieve better extraction effects for chlorogenic acid, aucubin, and geniposide.

[0063] Furthermore, using combinations of water volume, soaking time, soaking temperature, and extraction times as employed in experiments S4, S1, and S9, different drying methods were adjusted to investigate the effects of different drying methods on Eucommia ulmoides leaf extract. Specifically, the following parallel experiments were conducted: Extract S460: Eucommia ulmoides leaves were mixed with 8 times their weight of water and extracted at 90°C for 40 minutes, twice. The extracts were filtered, the filtrates were combined, and dried under reduced pressure at 60°C to obtain Eucommia ulmoides leaf extract S460.

[0064] Extract S470: Eucommia ulmoides leaves were mixed with 8 times their weight of water and extracted at 90°C for 40 minutes, twice. The extracts were filtered, the filtrates were combined, and dried under reduced pressure at 70°C to obtain Eucommia ulmoides leaf extract S470.

[0065] Extract S480: Eucommia ulmoides leaves were mixed with 8 times their weight of water and extracted at 90°C for 40 minutes, twice. The extracts were filtered, the filtrates were combined, and dried under reduced pressure at 80°C to obtain Eucommia ulmoides leaf extract S480.

[0066] Extract S490: Eucommia ulmoides leaves were mixed with 8 times their weight of water and extracted at 90°C for 40 minutes, twice. The extracts were filtered, the filtrates were combined, and dried under reduced pressure at 90°C to obtain Eucommia ulmoides leaf extract S490.

[0067] The contents of chlorogenic acid, cryptochlorogenic acid, and neochlorogenic acid in the obtained Eucommia ulmoides leaf extracts S460, S470, S480, and S490 were determined by HPLC. The experimental results are shown in Table 2 below. Table 2

[0068] As shown in Table 2, the freeze-drying process can significantly and effectively increase the content of chlorogenic acid, cryptochlorogenic acid and neochlorogenic acid in Eucommia ulmoides leaf extract.

[0069] Example 2 Evaluation of the therapeutic effects of Eucommia ulmoides leaf extracts prepared by different processes on age-related macular degeneration.

[0070] To investigate the therapeutic effects of Eucommia ulmoides leaf extracts prepared by different methods on age-related macular degeneration, Eucommia ulmoides leaf extracts were prepared using the following four methods.

[0071] Extract S1: Eucommia ulmoides leaves were mixed with 6 times their weight of water and extracted at 60°C for 40 minutes, once. The filtrate was filtered and freeze-dried at -40°C to obtain Eucommia ulmoides leaf extract S1.

[0072] Extract S4: Eucommia ulmoides leaves were mixed with 8 times their weight of water and extracted at 90°C for 40 minutes, twice. The extracts were filtered, the filtrates were combined, and freeze-dried at -40°C to obtain Eucommia ulmoides leaf extract S4.

[0073] Extract S9: Mix Eucommia ulmoides leaves with 10 times their weight of water and extract by warm soaking at 60℃ for 80 min, repeating the extraction twice. Filter, combine the filtrates, and freeze-dry at -40℃ to obtain Eucommia ulmoides leaf extract S9.

[0074] Extract JY: Eucommia ulmoides leaves were mixed with 8 times their weight of water and extracted at 90°C for 40 minutes, twice. The extracts were filtered, the filtrates were combined, and dried under reduced pressure at 60°C to obtain Eucommia ulmoides leaf extract JY (i.e., Eucommia ulmoides leaf extract S460).

[0075] The contents of chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid, genipin and coral glycoside in the above-mentioned Eucommia ulmoides leaf extract were determined by HPLC, and the results are shown in Table 3 below.

[0076] Table 3

[0077] Laboratory animals: Healthy SPF-grade C57 / BL6J mice (male, 6-8 weeks old), purchased from Spiford Biotechnology Co., Ltd.

[0078] After one week of acclimatization, all mice were randomly divided into 7 groups of 12 mice each. The groups were: normal group, model group, positive control group, S1 group, S4 group, S9 group, and JY group.

[0079] Except for the normal group, mice in other groups were intraperitoneally injected with a saline solution of sodium iodate (35 mg / kg body weight of mice, 0.01 mL solution / g body weight of mice) to establish an age-related macular degeneration model. The normal group was intraperitoneally injected with the same volume of saline solution to complete the modeling. The mice were administered the drug by gavage for 7 consecutive days after modeling.

[0080] Administration: The normal control group and the model group were fed a maintenance diet for mice without dietary restrictions. The positive control group received AREDS2 (from Bausch & Lomb Ophthalmic Surgical Products, USA; a combination of lutein and vitamins; two tablets per day for a 60kg adult, equivalent to 0.003 tablets / 10g mouse body weight, or 0.2mL / 10g mouse body weight) via gavage, in addition to the treatment given to the model group. Groups S1, S4, S9, and JY were administered Eucommia ulmoides leaf extract S1, S4, S9, and JY via gavage, respectively, at a dose of 400mg of Eucommia ulmoides leaf extract per kilogram of mouse body weight daily for 7 consecutive days.

[0081] After the medication process is completed, fasting is allowed for one day, but water is permitted. Be prepared to conduct various tests.

[0082] Indicator detection and effect evaluation: 1. Determination of inflammatory factors and oxidative stress markers in mouse eyes Detection Method: Mice were anesthetized by isoflurane inhalation. Blood was drawn from the orbital cavity. After blood collection, eyeball tissue was harvested, and 20-30 mg of the tissue was accurately weighed. Ten times the weight of PBS buffer was added, and the mixture was homogenized using an automated tissue homogenizer at 60 Hz for 90 s. The homogenate was then centrifuged at 800 × g for 15 min at 4 °C. The supernatant was collected and stored at -80 °C. Following the kit instructions, the levels of TNF-α, IL-6, MDA, GSH, and SOD activity in the mouse eyes were measured. The mean and standard deviation for 12 mice (n=12) were calculated. The TNF-α level in the mouse eyes is shown below. Figure 1 As shown. The level of IL-6 in the mouse eyeball is as follows. Figure 2 As shown. The level of MDA in the mouse eyeball is as follows. Figure 3 As shown. The level of GSH in the mouse eyeball is as follows. Figure 4 As shown in the figure. The SOD activity level in the mouse eyeball is as follows: Figure 5 As shown.

[0083] It is evident that the Eucommia ulmoides leaf extracts prepared using extracts S1, S4, and S9 significantly improved the levels of TNF-α, IL-6, MDA, GSH, and SOD activity in the eyes of model mice. However, the effect of Eucommia ulmoides leaf extract JY was relatively poor.

[0084] 2. Preparation of mouse eyeballs hematoxylin-eosin (HE staining) sections Method for preparing paraffin sections: After obtaining intact mouse eyeballs, fix them in fixative for 24 hours, and trim away excess tissue structures. The trimmed eyeballs are then dehydrated sequentially with a gradient of ethanol: 75% ethanol for 4 hours, 85% ethanol for 2 hours, 90% ethanol for 1 hour, and 95% ethanol for 1 hour. Finally, they are immersed twice in anhydrous ethanol, 130 minutes each time. The dehydrated eyeballs are then cleared three times in xylene, 30 minutes each time. Next, they are immersed three times in molten paraffin at 65°C, 1 hour each time. The molten paraffin is placed in an embedding frame, and before it solidifies, the tissue is cooled on a freezing stage. After the paraffin solidifies, the paraffin block is removed and trimmed. The trimmed paraffin block is then sectioned on a microtome to a thickness of 4 μm. The sections are floated on a 40°C warm water spreader to flatten the tissue. The tissue is then lifted onto a glass slide, flattened, and placed in a 60°C oven to bake. After the water evaporates and the paraffin melts, the sections are removed and stored at room temperature for later use.

[0085] HE staining method: The paraffin sections were immersed twice in xylene for 20 minutes each time, then twice in anhydrous ethanol for 10 minutes each time. Next, they were immersed in 95% ethanol for 5 minutes, 90% ethanol for 5 minutes, 80% ethanol for 5 minutes, and 70% ethanol for 5 minutes each time, and finally hydrated with distilled water. The sections were then stained with hematoxylin for 5 minutes, rinsed with distilled water, differentiated with 1% hydrochloric acid ethanol for 30 seconds, rinsed with distilled water, and finally blued with 0.6% ammonia solution, and rinsed with running water. The sections were then stained with eosin for 1 minute. Finally, the sections were immersed twice each in 95% ethanol, anhydrous ethanol, and xylene for 5 minutes each time. After dehydration, the sections were air-dried and mounted with neutral resin.

[0086] HE-stained sections of mouse eyeballs from the normal group, model group, positive drug group, S1 group, S4 group, S9 group, and JY group are shown below. Figure 6 As shown. Compared with the normal group ( Figure 6 Compared to A in the model group, the model group ( Figure 6In group B), the number of cells in the RPE layer of mice was reduced, and the tight cell connectivity was disrupted. In some mice, the RPE layer of the retina separated from the upper cells. INL and ONL showed irregular wavy edges in some areas, with significant thickness differences at different locations. The cells at the edges were loosely arranged, and even the two cell layers interleaved. The intercellular spaces were enlarged and disordered, and the ONL was significantly thinner with a significant reduction in cell number. After drug administration, the ONL density improved in all groups (S1, S9, S4, and JY), and the intercellular spaces decreased to varying degrees, with JY showing the least reduction in intercellular spaces.

[0087] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. The application of Eucommia ulmoides leaf extract in the preparation of products for improving age-related macular degeneration, wherein the Eucommia ulmoides leaf extract contains chlorogenic acid at a content of 50-100 mg / g, cryptochlorogenic acid at a content of 40-60 mg / g, neochlorogenic acid at a content of 35-55 mg / g, aucubin at a content of 56-79 mg / g, and genipin at a content of 65-105 mg / g; The preparation method of the Eucommia ulmoides leaf extract includes the following steps: Eucommia ulmoides leaves are mixed with water, warm-soaked for extraction, filtered and the filtrate is collected and freeze-dried to obtain Eucommia ulmoides leaf extract. The weight of the water is 6-10 times the weight of the Eucommia ulmoides leaves; the temperature of the warm soaking extraction is 60-90℃, the time of the warm soaking extraction is 40-80 min, and the number of times the warm soaking extraction is repeated is 1-3. The freeze-drying temperature is -40℃.

2. The application according to claim 1, characterized in that, The Eucommia ulmoides leaf extract contains chlorogenic acid at a content of 80.5-94.5 mg / g, cryptochlorogenic acid at a content of 43-50 mg / g, neochlorogenic acid at a content of 38-43 mg / g, aucubin at a content of 58-74 mg / g, and genipin at a content of 65-105 mg / g.

3. The application according to claim 1, characterized in that, The Eucommia ulmoides leaf extract contains 80.5 mg / g of chlorogenic acid, 45 mg / g of cryptochlorogenic acid, 38 mg / g of neochlorogenic acid, 58 mg / g of aucubin, and 65 mg / g of genipin; or The content of chlorogenic acid is 80.5 mg / g, cryptochlorogenic acid is 43 mg / g, neochlorogenic acid is 39 mg / g, aucubin is 68 mg / g, and genipin is 86 mg / g; or The contents of chlorogenic acid are 94.5 mg / g, cryptochlorogenic acid is 50 mg / g, neochlorogenic acid is 43 mg / g, aucubin is 74 mg / g, and genipin is 105 mg / g.

4. The application according to claim 3, characterized in that, The Eucommia ulmoides leaf extract contained chlorogenic acid (94.5 mg / g), cryptochlorogenic acid (50 mg / g), neochlorogenic acid (43 mg / g), aucubin (74 mg / g), and genipin (105 mg / g).

5. The application according to claim 1, characterized in that, The Eucommia ulmoides leaf extract is a freeze-dried powder of Eucommia ulmoides leaf extract.

6. The application according to claim 1, characterized in that, The weight of the water is 8 times the weight of the Eucommia ulmoides leaves; the temperature of the warm soaking extraction is 90°C, the time of the warm soaking extraction is 40 min, and the warm soaking extraction is repeated twice.

7. A product for improving age-related macular degeneration, characterized in that, This includes the Eucommia ulmoides leaf extract as described in any one of claims 1-6.

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