Application of choerospondias axillaris peel extract in preparation of medicine for resisting atherosclerosis

Abstract

The invention provides application of a choerospondias axillaris peel extract in preparation of an anti-atherosclerotic drug, and a preparation method of the choerospondias axillaris peel extract comprises the following steps: cutting peels from choerospondias axillaris fruits which are washed and aired, and drying the peels to obtain dried choerospondias axillaris peels; crushing the choerospondias axillaris peel into choerospondias axillaris peel powder, mixing the choerospondias axillaris peel powder with an extraction solvent to obtain a mixed solution, and performing ultrasonic extraction on the mixed solution to obtain an extracting solution; performing centrifugal treatment on the extracting solution to obtain supernate, performing rotary evaporation on the supernate to obtain jujube peel dry powder, and redissolving the jujube peel dry powder in distilled water to obtain the choerospondias axillaris peel extract. The choerospondias axillaris peel extract can relieve the inflammation level in a mouse with atherosclerosis, delay the formation of atherosclerotic plaques, reduce the number of mononuclear / macrophages in the plaques and reduce the adhesion effect of endothelial cells on mononuclear cells, so that the occurrence of atherosclerosis can be effectively delayed.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to application of a jujube peel extract in the preparation of anti-atherosclerotic medicines. Background Art

[0002] The treatment of atherosclerosis-related diseases currently faces multiple challenges. While existing drugs (such as statins and PCSK9 inhibitors) can effectively lower lipids, they are associated with side effects, drug resistance, and economic burdens. Surgical intervention cannot reverse plaque progression and carries the risk of complications. Lifestyle interventions have limited effectiveness in patients with advanced or advanced disease. As a complex disease involving multiple mechanisms intertwined, including lipid metabolism disorders, chronic inflammation, and oxidative stress, single-target drugs are unlikely to effectively intervene in the multiple processes of its development.

[0003] Natural extracts have multi-target synergistic effects, can simultaneously regulate inflammatory pathways, scavenge free radicals, and improve endothelial function, which is more consistent with the systemic pathological characteristics of atherosclerosis. In addition, natural extracts are highly safe and have few side effects, making them suitable for long-term prevention and control, especially for patients who are intolerant to statins or require combination therapy. With their multi-dimensional regulatory advantages and sustainability, natural extracts provide an important strategy for personalized treatment of atherosclerosis and "prevention before disease occurs", and are a key direction to fill the current treatment gap. Therefore, the development of natural extracts for the treatment of atherosclerosis is an important social need. Summary of the Invention

[0004] In view of this, the present invention provides the use of a zizyphus jujuba peel extract in the preparation of anti-atherosclerotic medicines.

[0005] Furthermore, the preparation method of the extract of the Chinese jujube peel comprises the following steps: The peel is removed from the washed and dried Chinese jujube fruit, and the peel is dried to obtain dried Chinese jujube peel; Grinding the dried jujube peel into jujube peel powder, mixing the jujube peel powder with an extraction solvent to form a mixed solution, and performing ultrasonic extraction on the mixed solution to obtain an extract; The extract is centrifuged to obtain a supernatant, the supernatant is rotary evaporated to obtain jujube peel dry powder, and the jujube peel dry powder is redissolved in distilled water to obtain a sour jujube peel extract.

[0006] Furthermore, the extraction solvent is anhydrous methanol.

[0007] Furthermore, the mixing ratio of the jujube peel powder and the extraction solvent is 1:1~1:4.

[0008] Furthermore, the ultrasonic extraction time is 20min~40min.

[0009] Furthermore, the rotation speed of the centrifugal treatment is 3000 rpm to 5000 rpm, and the time of the centrifugal treatment is 8 min to 15 min.

[0010] Furthermore, the temperature of the rotary evaporation is 50°C to 70°C.

[0011] Furthermore, the mixing ratio of the jujube peel dry powder and the distilled water is 80 mg / ml~120 mg / ml.

[0012] Compared with the prior art, the present invention has the following beneficial effects: Experiments have shown that the extract from the peel of the Chinese jujube tree in this application can reduce inflammation levels, delay the formation of atherosclerotic plaques, reduce the number of monocytes and macrophages within plaques, and reduce the adhesion of endothelial cells to monocytes in mice with atherosclerosis. In other words, the extract from the peel of the Chinese jujube tree in this application can effectively delay the onset of atherosclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a graph showing the test results of the Achyranthes australis peel extract in Example 1 of the present invention in alleviating inflammation associated with atherosclerosis; Figure 2 This is a graph showing the test results of the A. jujube peel extract in Example 1 of the present invention in reducing the size of atherosclerotic plaques, wherein A represents the proportion of aortic tree plaque area, and B represents the proportion of aortic root plaque area; Figure 3 This is a diagram showing the improvement effect of the Choerospondias australis peel extract in Example 1 of the present invention on reducing the size of atherosclerotic plaques; Figure 4 This is a graph showing the detection results of the Achyranthes australis peel extract in Example 1 of the present invention in reducing monocytes and macrophages in atherosclerotic plaques; Figure 5 This is a diagram showing the improvement effect of the Choerospondias australis peel extract in Example 1 of the present invention on reducing monocytes and macrophages in atherosclerotic plaques; Figure 6 This is a graph showing the detection results of the extract from the peel of Ziziphus jujuba in Example 1 of the present invention in reducing monocyte adhesion and adhesion molecule expression, wherein A represents the number of monocytes adhered to endothelial cells, B represents the expression of VCAM1 in HUVEC cells, and C represents the expression of ICAM1 in HUVEC cells; Figure 7 Graph showing the improvement effect of the Choerospondias australis peel extract in Example 1 of the present invention on reducing monocyte adhesion and adhesion molecule expression, wherein A is a bright field image and B is a FITC fluorescence image; The following specific embodiments will further illustrate the present invention in conjunction with the above-mentioned drawings. DETAILED DESCRIPTION

[0014] To facilitate understanding of the present invention, the present invention will be described more fully below with reference to the accompanying drawings. The drawings illustrate several embodiments of the present invention. However, the present invention may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the present invention.

[0015] It should be noted that when an element is referred to as being "fixed to" another element, it may be directly on the other element or there may be an intermediate element. When an element is referred to as being "connected to" another element, it may be directly connected to the other element or there may be an intermediate element. The terms "vertical," "horizontal," "left," "right," and similar expressions used herein are for illustrative purposes only.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention pertains. The terms used in this specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0017] In a first aspect, Example 1 of the present invention provides a use of a zizyphus jujuba peel extract in the preparation of an anti-atherosclerotic drug. Specifically, the preparation method of the zizyphus jujuba peel extract comprises the following steps: The peel is removed from the washed and dried Chinese jujube fruit, and the peel is dried to obtain dried Chinese jujube peel; Preferably, the temperature of the drying treatment is 50° C. to 80° C., and the time of the drying treatment is 20 hours to 25 hours. In this embodiment, the temperature of the drying treatment is 60° C., and the time of the drying treatment is 24 hours.

[0018] Grinding the dried jujube peel into jujube peel powder, mixing the jujube peel powder with an extraction solvent to form a mixed solution, and performing ultrasonic extraction on the mixed solution to obtain an extract; Specifically, the jujube peel is crushed to obtain an initial powder, and the initial powder is sieved to obtain the jujube peel powder, and the sieve is a 40-mesh sieve. In this embodiment, the extraction solvent is anhydrous methanol, the mixing ratio of the jujube peel powder to the extraction solvent is 1:2, and the ultrasonic extraction time is 30 minutes.

[0019] The extract is centrifuged to obtain a supernatant, the supernatant is rotary evaporated to obtain a jujube peel dry powder, and the jujube peel dry powder is redissolved in distilled water to obtain a jujube peel extract; Preferably, the speed of the centrifugal treatment is 3000 rpm to 5000 rpm, the time of the centrifugal treatment is 8 min to 15 min, and the temperature of the rotary evaporation is 50° C. to 70° C. In this embodiment, the speed of the centrifugal treatment is 4000 rpm, the time of the centrifugal treatment is 10 min, and the temperature of the rotary evaporation is 60° C. After obtaining the jujube peel dry powder, the mixing ratio of the jujube peel dry powder and the distilled water is 80 mg / ml to 120 mg / ml. In this embodiment, the mixing ratio of the jujube peel dry powder and the distilled water is 100 mg / ml.

[0020] After obtaining the jujube peel extract, the jujube peel extract is subjected to an anti-atherosclerosis test: Reduces inflammation associated with arteriosclerosis: See also Figure 1 , 12 ApoE - / - Mice were divided into a control group and a treatment group and fed a high-fat diet for 8 weeks. During this period, the treatment group received daily gavage of the Choerospondias australis peel extract described in this example (50 μL / 10 g), based on body weight, while the control group received daily gavage of distilled water (50 μL / 10 g), based on body weight. After 8 weeks, serum levels of the inflammation-related factor IL-6 were measured. The results showed that the Choerospondias australis peel extract described in this example could reduce levels of the inflammation-related factor IL-6.

[0021] Reduces atherosclerotic plaque size: See also Figure 2 and Figure 3 , 12 ApoE - / - Mice were divided into a control group and a treatment group and fed a high-fat diet for 8 weeks. During this period, the treatment group was gavaged daily with the Chinese Ziziphus jujuba peel extract described in this example (50 μL / 10 g) according to body weight, and the control group was gavaged daily with distilled water (50 μL / 10 g) according to body weight. After 8 weeks, the plaque size of the aortic tree and aortic root sections was detected by Oil Red O staining. The results showed that the Chinese Ziziphus jujuba peel extract in this example can reduce plaque formation. It should be noted that in Figure 3 In the figure, the red area is the plaque area.

[0022] .Reduce monocytes / macrophages in atherosclerotic plaques: See also Figure 4 and Figure 5 , 12 ApoE - / -Mice were divided into a control group and a treatment group and fed a high-fat diet for 8 weeks. During this period, the treatment group was gavaged daily with the Choerospondias australis peel extract described in this example (50 μL / 10 g) according to body weight, and the control group was gavaged daily with distilled water (50 μL / 10 g) according to body weight. After 8 weeks, the area of ​​monocytes / macrophages in aortic root sections was detected by immunofluorescence. The results showed that the Choerospondias australis peel extract in this example can reduce the area of ​​monocytes / macrophages in plaques. It should be noted that in Figure 5 In the figure, CD31 indicates an endothelial cell marker, MOMA-2 indicates a monocyte / macrophage marker, DAPI indicates a cell nuclear marker, and Merge indicates a superposition effect.

[0023] Reduce monocyte adhesion and adhesion molecule expression: See also Figure 6 and Figure 7 Human umbilical vein endothelial cells (HUVEC) were seeded in 96-well cell culture plates at a seeding density of 5,000 cells / well. After cell attachment, the extract from the peel of Ziziphus jujuba described in this example was added for 24 hours of pre-protection. The supernatant was discarded, and culture medium containing 20 μg / L TNF-α was added for 6 hours of stimulation. A human monocytic leukemia cell line (THP-1) was collected and cultured in BCECF application solution (10 μM) for 1 hour. THP-1 cells were then added to the 96-well plates seeded with HUVEC and cultured for 1 hour. The cells were observed under a fluorescence microscope, and the number of monocytes adhering to the endothelial cells was counted.

[0024] HUVECs were seeded separately in 6-well plates and cultured normally to an appropriate density. The old culture medium was discarded and the cells were washed with 1× PBS. The treated group was treated with the extract from the peel of Ziziphus jujuba described in this example and cultured for 24 hours. The control and model groups were cultured with an equal volume of purified water for 24 hours. Following the incubation period, the model and treated groups were treated with culture medium containing 20 μg / L TNF-α (TNF-α indicates tumor necrosis factor α-induced inflammation), while the control group was treated with an equal volume of 1× PBS. Six hours later, the expression levels of vascular cell adhesion molecule 1 (VCAM1) and intercellular adhesion molecule 1 (ICAM1) in the cells were measured.

[0025] It should be noted that Figure 1 、 Figure 2 、 Figure 4 Biological replicates n=6, * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, and P<0.05 indicates statistically significant. Figure 6 Biological replicates n=3, * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, P<0.05 indicates statistically significant difference, Figure 6In the table, - indicates no addition, + indicates addition, and the percentage (%) indicates the volume ratio of the peel water extract in the cell culture medium. Figure 7 NC represents the negative control, TNF-α represents tumor necrosis factor-α-induced adhesion, Peel represents the aqueous extract of the peel of Choerospondias australis, and the percentage (%) represents the volume ratio of the peel aqueous extract in the cell culture medium.

[0026] Example 2 of the present invention provides the use of an extract from the peel of Ziziphus jujuba in the preparation of an anti-atherosclerotic drug, which differs from the use described in Example 1 in that: In the process of preparing the jujube peel extract, the mixing ratio of the jujube peel powder and the extraction solvent is 1:1.

[0027] Example 3 of the present invention provides the use of an extract from the peel of Ziziphus jujuba in the preparation of an anti-atherosclerotic drug, which differs from the use described in Example 1 in that: In the process of preparing the jujube peel extract, the mixing ratio of the jujube peel powder and the extraction solvent is 1:4.

[0028] Example 4 of the present invention provides the use of an extract from the peel of Ziziphus jujuba in the preparation of an anti-atherosclerotic drug, which differs from the use described in Example 1 in that: During the preparation of the Choerospondias australis peel extract, the ultrasonic extraction time is 20 minutes.

[0029] Example 5 of the present invention provides the use of an extract from the peel of Ziziphus jujuba in the preparation of an anti-atherosclerotic drug, which differs from the use described in Example 1 in that: During the preparation of the Choerospondias australis peel extract, the ultrasonic extraction time is 40 minutes.

[0030] Comparative Example 1 of the present invention provides an application of an extract from the peel of Ziziphus jujuba in the preparation of an anti-atherosclerotic drug, which differs from the application described in Example 1 in that: In the process of preparing the jujube peel extract, the mixing ratio of the jujube peel powder and the extraction solvent is 1:0.5.

[0031] Comparative Example 2 of the present invention provides an application of an extract from the peel of Ziziphus jujuba in the preparation of an anti-atherosclerotic drug, which differs from the application described in Example 1 in that: In the process of preparing the jujube peel extract, the mixing ratio of the jujube peel powder and the extraction solvent is 1:8.

[0032] Comparative Example 3 of the present invention provides the use of an extract from the peel of Ziziphus jujuba in the preparation of an anti-atherosclerotic drug, which differs from the use described in Example 1 in that: During the preparation of the Choerospondias australis peel extract, the ultrasonic extraction time is 5 minutes.

[0033] Comparative Example 4 of the present invention provides an application of an extract from the peel of Ziziphus jujuba in the preparation of an anti-atherosclerotic drug, which differs from the application described in Example 1 in that: During the preparation of the Choerospondias australis peel extract, the ultrasonic extraction time is 60 minutes.

[0034] Based on the aforementioned relevant experimental methods, the specific effects of the Chinese jujube peel extract in Examples 1 to 5 and Comparative Examples 1 to 4 of the present application on the relevant indicators of anti-atherosclerosis were tested, and the test results are shown in Table 1 below: Table 1 , As can be seen from the table above, by setting the preparation parameters in the preparation method of the Chinese Zizyphus jujuba peel extract in this application, the improvement effect of the Chinese Zizyphus jujuba peel extract on atherosclerosis can be significantly improved. Experiments have shown that the Chinese Zizyphus jujuba peel extract in this application can reduce inflammation levels, delay atherosclerotic plaque formation, reduce the number of monocytes / macrophages in plaques, and reduce the adhesion of endothelial cells to monocytes in mice with atherosclerosis. In other words, the Chinese Zizyphus jujuba peel extract in this application can effectively delay the occurrence of atherosclerosis.

[0035] Throughout this specification, reference to terms such as "one embodiment," "some embodiments," "examples," "specific examples," or "some examples" means that a specific feature, structure, material, or characteristic described in conjunction with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0036] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. Application of the extract of Ziziphus jujuba peel in the preparation of anti-atherosclerotic drugs.

2. The use according to claim 1, characterized in that The preparation method of the Chinese jujube peel extract comprises the following steps: The peel is removed from the washed and dried Chinese jujube fruit, and the peel is dried to obtain dried Chinese jujube peel; Grinding the dried jujube peel into jujube peel powder, mixing the jujube peel powder with an extraction solvent to form a mixed solution, and performing ultrasonic extraction on the mixed solution to obtain an extract; The extract is centrifuged to obtain a supernatant, the supernatant is rotary evaporated to obtain jujube peel dry powder, and the jujube peel dry powder is redissolved in distilled water to obtain a sour jujube peel extract.

3. The use according to claim 2, characterized in that The extraction solvent is anhydrous methanol.

4. The use according to claim 2, characterized in that The mixing ratio of the jujube peel powder to the extraction solvent is 1:1 to 1:

4.

5. The use according to claim 2, characterized in that The ultrasonic extraction time is 20 min to 40 min.

6. The use according to claim 5, characterized in that The ultrasonic extraction time is 30 minutes.

7. The use according to claim 2, characterized in that The rotation speed of the centrifugal treatment is 3000 rpm to 5000 rpm, and the time of the centrifugal treatment is 8 min to 15 min.

8. The use according to claim 2, characterized in that The temperature of the rotary evaporation is 50°C to 70°C.

9. The use according to claim 8, characterized in that The temperature of the rotary evaporation was 60°C.

10. The use according to claim 2, characterized in that The mixing ratio of the jujube peel dry powder and the distilled water is 80 mg / ml to 120 mg / ml.

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