Use of ziziphus jujuba mill peel extract in preparation of medicine for resisting atherosclerosis

Abstract

This invention provides the application of *Ziziphus jujuba* peel extract in the preparation of anti-atherosclerotic drugs. The preparation method of *Ziziphus jujuba* peel extract includes: removing the peel from washed and dried *Ziziphus jujuba* fruits; drying the peel to obtain dried *Ziziphus jujuba* peel; pulverizing the dried peel into peel powder; mixing the peel powder with an extraction solvent to form a mixture; ultrasonically extracting the mixture to obtain an extract; centrifuging the extract to obtain a supernatant; rotary evaporating the supernatant to obtain dried peel powder; and redissolving the dried peel powder in distilled water to obtain the *Ziziphus jujuba* peel extract. The *Ziziphus jujuba* peel extract can reduce inflammation levels, delay atherosclerotic plaque formation, reduce the number of monocytes / macrophages within plaques, and decrease endothelial cell adhesion to monocytes in mice with atherosclerosis, thus effectively delaying the occurrence of atherosclerosis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of jujube bark extract in the preparation of anti-atherosclerotic drugs. Background Technology

[0002] The treatment of atherosclerosis-related diseases currently faces multiple challenges. Existing drugs (such as statins and PCSK9 inhibitors) can effectively lower lipids, but they have problems with side effects, drug resistance, or economic burden. Surgical intervention cannot reverse plaque progression and is accompanied by the risk of complications. Lifestyle interventions have limited effectiveness for patients in the middle and late stages. As a complex disease involving multiple mechanisms such as lipid metabolism disorders, chronic inflammation, and oxidative stress, single-target drugs are insufficient to effectively intervene in the multiple processes of its development.

[0003] Natural extracts possess multi-target synergistic effects, simultaneously regulating inflammatory pathways, scavenging free radicals, and improving endothelial function, thus better aligning with the systemic pathological characteristics of atherosclerosis. Furthermore, natural extracts exhibit high safety and few side effects, making them suitable for long-term prevention and control, particularly for patients intolerant to statins or requiring combination therapy. With their multi-dimensional regulatory advantages and sustainability, natural extracts offer important strategies for personalized treatment and preventative care of atherosclerosis, representing a key direction for filling current treatment gaps. Therefore, developing anti-atherosclerotic natural extracts is a significant societal need. Summary of the Invention

[0004] In view of this, the present invention provides the application of jujube bark extract in the preparation of anti-atherosclerotic drugs.

[0005] Furthermore, the preparation method of the *Ziziphus jujuba* peel extract includes the following steps:

[0006] After washing and drying, the peel is cut off from the jujube fruit and then dried to obtain dried jujube peel.

[0007] The dried jujube peel is pulverized into jujube peel powder, the jujube peel powder is mixed with an extraction solvent to form a mixture, and the mixture is subjected to ultrasonic extraction to obtain an extract.

[0008] The extract is centrifuged to obtain a supernatant, which is then rotary evaporated to obtain jujube peel powder. The jujube peel powder is then redissolved in distilled water to obtain jujube peel extract.

[0009] Furthermore, the extraction solvent is anhydrous methanol.

[0010] Furthermore, the mixing ratio of the jujube peel powder to the extraction solvent is 1:1 to 1:4.

[0011] Furthermore, the ultrasonic extraction time is 20 min to 40 min.

[0012] Furthermore, the centrifugation speed is 3000 rpm to 5000 rpm, and the centrifugation time is 8 min to 15 min.

[0013] Furthermore, the temperature of the rotary evaporation is 50°C to 70°C.

[0014] Furthermore, the mixing ratio of the dried jujube peel powder to the distilled water is 80 mg / ml to 120 mg / ml.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: Experiments show that the *Ziziphus jujuba* peel extract of this application can reduce inflammation levels, delay the formation of atherosclerotic plaques, reduce the number of monocytes / macrophages within plaques, and reduce the adhesion of endothelial cells to monocytes in mice with atherosclerosis. In other words, the *Ziziphus jujuba* peel extract of this application can effectively delay the occurrence of atherosclerosis. Attached Figure Description

[0016] Figure 1 The image shows the detection results of the Chinese jujube bark extract in Example 1 of this invention in reducing atherosclerosis-related inflammation.

[0017] Figure 2 The image shows the detection results of the reduction in atherosclerotic plaque size by the extract of jujube bark in Example 1 of the present invention. In the image, A represents the proportion of aortic tree plaque area, and B represents the proportion of aortic root plaque area.

[0018] Figure 3 The image shows the effect of the extract of jujube bark in Example 1 of the present invention in reducing the size of atherosclerotic plaques.

[0019] Figure 4 This is a diagram showing the detection results of the reduction of mononuclear / macrophage cells in atherosclerotic plaques by the extract of jujube bark in Example 1 of the present invention;

[0020] Figure 5 This is a diagram illustrating the effect of the extract of *Ziziphus jujuba* bark in Example 1 of the present invention in reducing the number of mononuclear / macrophage cells in atherosclerotic plaques.

[0021] Figure 6 The image shows the detection results of the reduction of monocyte adhesion and adhesion molecule expression by the extract of jujube bark in Example 1 of the present invention. In the image, A represents the number of monocytes adhering to endothelial cells, B represents the expression of VCAM1 in HUVEC cells, and C represents the expression of ICAM1 in HUVEC cells.

[0022] Figure 7The image shows the effect of the extract of Ziziphus jujuba bark in Example 1 of the present invention on reducing monocyte adhesion and improving the expression of adhesion molecules. In the image, A is a bright field image and B is a FITC fluorescence image.

[0023] The following detailed description, in conjunction with the accompanying drawings, will further illustrate the present invention. Detailed Implementation

[0024] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Several embodiments of the invention are illustrated in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

[0025] It should be noted that when a component is said to be "fixed to" another component, it can be directly on the other component or there may be an intervening component. When a component is said to be "connected to" another component, it can be directly connected to the other component or there may be an intervening component. The terms "vertical," "horizontal," "left," "right," and similar expressions used in this document are for illustrative purposes only.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0027] In a first aspect, Embodiment 1 of the present invention provides the application of *Ziziphus jujuba* peel extract in the preparation of anti-atherosclerotic drugs. Specifically, the preparation method of the *Ziziphus jujuba* peel extract includes the following steps:

[0028] After washing and drying, the peel is cut off from the jujube fruit and then dried to obtain dried jujube peel.

[0029] Preferably, the drying temperature is 50℃~80℃ and the drying time is 20h~25h. In this embodiment, the drying temperature is 60℃ and the drying time is 24h.

[0030] The dried jujube peel is pulverized into jujube peel powder, the jujube peel powder is mixed with an extraction solvent to form a mixture, and the mixture is subjected to ultrasonic extraction to obtain an extract.

[0031] Specifically, the jujube peel is pulverized to obtain initial powder, and the initial powder is sieved to obtain jujube peel powder. The sieve used for sieving 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.

[0032] The extract is centrifuged to obtain a supernatant, which is then rotary evaporated to obtain jujube peel powder. The jujube peel powder is then redissolved in distilled water to obtain jujube peel extract.

[0033] Preferably, the centrifugation speed is 3000 rpm to 5000 rpm, the centrifugation time is 8 min to 15 min, and the rotary evaporation temperature is 50℃ to 70℃. In this embodiment, the centrifugation speed is 4000 rpm, the centrifugation time is 10 min, and the rotary evaporation temperature is 60℃. After obtaining the jujube peel powder, the mixing ratio of the jujube peel powder to the distilled water is 80 mg / ml to 120 mg / ml. In this embodiment, the mixing ratio of the jujube peel powder to the distilled water is 100 mg / ml.

[0034] After obtaining the *Ziziphus jujuba* bark extract, an anti-atherosclerotic assay was performed on the extract.

[0035] Reduce inflammation associated with atherosclerosis:

[0036] Please see 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 was administered the *Ziziphus jujuba* bark extract (50 μL / 10 g) daily by gavage according to body weight, while the control group was administered distilled water (50 μL / 10 g) daily by gavage according to body weight. After 8 weeks, serum samples were collected to detect the level of the inflammation-related factor IL-6. The results showed that the *Ziziphus jujuba* bark extract in this embodiment could reduce the level of the inflammation-related factor IL-6.

[0037] Reduce the size of atherosclerotic plaques:

[0038] Please see 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 administered the *Ziziphus jujuba* bark extract (50 μL / 10 g) daily by gavage according to body weight, while the control group was administered distilled water (50 μL / 10 g) daily by gavage according to body weight. After 8 weeks, plaque size in sections of the aortic tree and aortic root was detected by Oil Red O staining. The results showed that the *Ziziphus jujuba* bark extract in this example can reduce plaque formation. It should be noted that... Figure 3 In the image, the red area represents the patchy area.

[0039] Reduce mononuclear / macrophage cells within atherosclerotic plaques:

[0040] Please see 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 administered the *Ziziphus jujuba* bark extract (50 μL / 10 g) daily by gavage according to body weight, while the control group was administered distilled water (50 μL / 10 g) daily by gavage 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 *Ziziphus jujuba* bark extract in this example could reduce the area of ​​monocytes / macrophages in plaques. It should be noted that... Figure 5 In this context, CD31 represents endothelial cell markers, MOMA-2 represents monocyte / macrophage markers, DAPI represents nuclear markers, and Merge represents additive effects.

[0041] Reduce monocyte adhesion and adhesion molecule expression:

[0042] Please see Figure 6 and Figure 7 Human umbilical vein endothelial cells (HUVECs) were seeded in 96-well cell culture plates at a density of 5000 cells / well. After cell adhesion, the extract of *Ziziphus jujuba* bark described in this example was added for pre-protection for 24 hours. The supernatant was discarded, and medium containing 20 μg / L TNF-α was added for stimulation for 6 hours. Human monocytic leukemia cell line (THP-1) was collected and cultured in BCECF working solution (10 μM) for 1 hour. Then, THP-1 cells were added to the 96-well plates seeded with HUVECs and cultured for 1 hour. The results were observed under a fluorescence microscope, and the number of mononuclear cells adhering to the endothelial cells was counted.

[0043] HUVECs were seeded in 96-well plates and cultured normally until the cells reached a suitable density. The old culture medium was discarded, and the cells were washed with 1×PBS. The treatment group was cultured with the *Ziziphus jujuba* bark extract described in this example for 24 h, while the control and model groups were cultured with an equal volume of pure water for 24 h. After culture, the model and treatment groups were cultured with a medium containing 20 μg / L TNF-α (TNF-α represents tumor necrosis factor α-induced inflammation), while the control group was cultured with an equal volume of 1×PBS. After 6 h, the expression levels of vascular cell adhesion molecule 1 (VCAM1) and intercellular adhesion molecule 1 (ICAM1) in the cells were detected.

[0044] It should be noted that, Figure 1 , Figure 2 , Figure 4 In the study of biological data, the number of replicates was n=6. * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, and P<0.05 indicates a statistically significant difference. Figure 6 In the biological studies, the replication factor was n=3. * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, and P<0.05 is considered statistically significant. Figure 6 In the text, - indicates no addition, + indicates addition, and percentage (%) indicates the volume percentage of the fruit 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 Ziziphus jujuba peel, and percentage (%) represents the volume percentage of the aqueous extract of peel in the cell culture medium.

[0045] Example 2 of this invention provides the application of jujube bark extract in the preparation of anti-atherosclerotic drugs, which differs from the application described in Example 1 in that...

[0046] In the preparation of the jujube peel extract, the mixing ratio of the jujube peel powder to the extraction solvent is 1:1.

[0047] Example 3 of this invention provides the application of jujube bark extract in the preparation of anti-atherosclerotic drugs, which differs from the application described in Example 1 in that...

[0048] In the preparation of the jujube peel extract, the mixing ratio of the jujube peel powder to the extraction solvent is 1:4.

[0049] Example 4 of this invention provides the application of *Ziziphus jujuba* bark extract in the preparation of anti-atherosclerotic drugs, which differs from the application described in Example 1 in that...

[0050] In the preparation of the jujube bark extract, the ultrasonic extraction time is 20 minutes.

[0051] Example 5 of this invention provides the application of jujube bark extract in the preparation of anti-atherosclerotic drugs, which differs from the application described in Example 1 in that...

[0052] In the preparation of the jujube bark extract, the ultrasonic extraction time is 40 minutes.

[0053] Comparative Example 1 of this invention provides the application of Ziziphus jujuba bark extract in the preparation of anti-atherosclerotic drugs, which differs from the application described in Example 1 in that:

[0054] In the preparation of the jujube peel extract, the mixing ratio of the jujube peel powder to the extraction solvent is 1:0.5.

[0055] Comparative Example 2 of this invention provides the application of Ziziphus jujuba bark extract in the preparation of anti-atherosclerotic drugs, which differs from the application described in Example 1 in that:

[0056] In the preparation of the jujube peel extract, the mixing ratio of the jujube peel powder to the extraction solvent is 1:8.

[0057] Comparative Example 3 of this invention provides the application of Ziziphus jujuba bark extract in the preparation of anti-atherosclerotic drugs, which differs from the application described in Example 1 in that:

[0058] In the preparation of the jujube bark extract, the ultrasonic extraction time is 5 minutes.

[0059] Comparative Example 4 of this invention provides the application of Ziziphus jujuba bark extract in the preparation of anti-atherosclerotic drugs, which differs from the application described in Example 1 in that:

[0060] In the preparation of the jujube bark extract, the ultrasonic extraction time is 60 minutes.

[0061] Based on the aforementioned experimental methods, the specific effects of the *Ziziphus jujuba* peel extract described in Examples 1 to 5 and Comparative Examples 1 to 4 of this application on relevant indicators of anti-atherosclerosis were tested. The test results are shown in Table 1 below:

[0062] Table 1

[0063] ,

[0064] As shown in the table above, by setting the preparation parameters in the preparation method of the *Ziziphus jujuba* peel extract in this application, the effect of the *Ziziphus jujuba* peel extract on improving atherosclerosis can be significantly enhanced. Experiments show that the *Ziziphus jujuba* peel extract in this application can reduce inflammation levels, delay the formation of atherosclerotic plaques, reduce the number of monocytes / macrophages within plaques, and reduce the adhesion of endothelial cells to monocytes in mice with atherosclerosis. In other words, the *Ziziphus jujuba* peel extract in this application can effectively delay the occurrence of atherosclerosis.

[0065] In the description of this specification, references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions 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 one or more embodiments or examples.

[0066] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. The application of *Ziziphus jujuba* bark extract in the preparation of drugs for treating atherosclerosis, wherein the preparation method of the *Ziziphus jujuba* bark extract includes the following steps: After washing and drying, the peel is cut off from the jujube fruit and then dried to obtain dried jujube peel. The dried jujube peel is pulverized into jujube peel powder, and the jujube peel powder is mixed with an extraction solvent to form a mixture. The extraction solvent is anhydrous methanol, and the mixing ratio of the jujube peel powder to the extraction solvent is 1:1 to 1:

4. The mixture is subjected to ultrasonic extraction to obtain an extract. The ultrasonic extraction time is 20 min to 40 min. The extract is centrifuged to obtain a supernatant, which is then rotary evaporated to obtain jujube peel powder. The jujube peel powder is then redissolved in distilled water to obtain jujube peel extract.

2. The application according to claim 1, characterized in that, The ultrasonic extraction time is 30 minutes.

3. The application according to claim 1, characterized in that, The centrifugation speed is 3000 rpm to 5000 rpm, and the centrifugation time is 8 min to 15 min.

4. The application according to claim 1, characterized in that, The rotary evaporation temperature is 50℃~70℃.

5. The application according to claim 4, characterized in that, The rotary evaporation temperature is 60°C.

6. The application according to claim 1, characterized in that, The jujube peel powder, after being redissolved in distilled water, has a concentration of 80 mg / ml to 120 mg / ml.

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