Application of Syzygium aromaticum leaf extract in the preparation of drugs for the prevention and / or treatment of atherosclerosis

Abstract

This invention relates to the field of natural product chemistry, specifically to the application of a *Syzygium brevicornum* leaf extract in the preparation of drugs for the prevention and / or treatment of atherosclerosis. The preparation method of the *Syzygium brevicornum* leaf extract of this invention includes the following steps: taking dried *Syzygium brevicornum* leaves, extracting with ethanol solution, filtering, combining the extracts, and concentrating under reduced pressure to obtain a total extract of *Syzygium brevicornum* leaf ethanol extract; extracting the total extract of *Syzygium brevicornum* leaf ethanol extract with ethyl acetate to obtain the *Syzygium brevicornum* leaf extract. Using the RAW264.7 foam cell model, this invention found that the *Syzygium brevicornum* leaf extract prepared by this invention can reduce lipid accumulation in RAW264.7 macrophage-derived foam cells, suggesting that the *Syzygium brevicornum* leaf extract prepared by this invention can be used to prepare drugs for the prevention and / or treatment of atherosclerosis.

Description

[0001] This application is a divisional application of the following application:

[0002] Application date: August 30, 2022

[0003] Application Number: 202211061589.7

[0004] Invention Title: A Short-Inflorescence Syzygium Leaf Extract, Its Preparation Method and Application Technical Field

[0005] This invention relates to the field of natural product chemistry, specifically to the use of a short-chain Syzygium aromaticum leaf extract in the preparation of drugs for the prevention and / or treatment of atherosclerosis. Background Technology

[0006] Atherosclerosis (AS) is a chronic, inflammatory cardiovascular disease that can lead to heart disease, stroke, and even death, and has become a serious public health problem threatening human health. In the 1960s, the presence of macrophages in atherosclerotic plaques was discovered, and researchers now increasingly recognize the crucial role of monocytes / macrophages in this chronic inflammatory disease (see Liu, Shoujia; Yang, Zhicheng, et al. Monocyte recruitment and foam cell formation in atherosclerosis [J]. Chinese Journal of Arteriosclerosis, 2009, 17(11):957). In the early stages of AS progression, foam cell formation is a key factor. Foam cell formation originates from macrophages. Due to excessive cholesterol accumulation, the imbalance between the influx, esterification, and efflux of oxidized low-density lipoprotein (ox-LDL) in macrophages leads to the gradual foaming of macrophages. Simultaneously, inflammation is another important factor affecting atherosclerosis. Various cytokines secreted by foam cells, including inflammatory chemokines, activate endothelial cells and further recruit more inflammatory cells into the intima. Meanwhile, monocytes are driven to differentiate into macrophages and absorb large amounts of ox-LDL to form foam cells, which gradually accumulate in blood vessels, further accelerating the formation of atherosclerosis.

[0007] *Syzygium brachythyrsum* Merr. et Perry is an evergreen shrub or small tree belonging to the genus *Syzygium* in the family Myrtaceae. It is a historically significant and important functional herbal tea and folk medicine in Lingnan and Southwest China. Its fruit and leaves are used medicinally under the name "wild holly fruit" (see *Yunnan Traditional Chinese Medicine*). According to the *Flora of China*, *Syzygium brachythyrsum* has lung-moistening and asthma-relieving effects, and is mainly used to treat allergic asthma, tuberculosis, and asthma caused by the common cold. The inventors previously confirmed that *Syzygium brachythyrsum* leaves contain a large number of compounds, including flavonoids, tannins, phenolic acids, anthocyanins, and terpenes (see Jiaqi Qiu, Xuelian Chen, Pulin Liang, et al. Integrating approach to discover novel bergenin derivatives and phenolics with antioxidant and anti-inflammatory activities from bio-active fraction of *Syzygium brachythyrsum* [J]. Arabian Journal of Chemistry, 2021, 15, 103507). Epidemiological studies have shown that the general population and patients at risk of cardiovascular disease can reduce cardiovascular events by consuming natural products rich in polyphenols and triterpenes from their diet. Therefore, the medicinal value of *Syzygium brachythyrsum* leaves in reducing cardiovascular disease warrants further investigation. Summary of the Invention

[0008] The first objective of this invention is to provide a method for preparing a short-arcted Syzygium leaf extract, the second objective of this invention is to provide a short-arcted Syzygium leaf extract obtained by the method, and the third objective of this invention is to provide the application of the short-arcted Syzygium leaf extract.

[0009] This invention is achieved through the following technical solution:

[0010] According to a first aspect of the present invention, a method for preparing a short-arcted Syzygium leaf extract is provided, comprising the following steps:

[0011] Dried leaves of Syzygium brevicornum were extracted with ethanol solution, filtered, and the extracts were combined and concentrated under reduced pressure to obtain the total extract of Syzygium brevicornum leaf alcohol extract; the ethanol solution used was an aqueous solution of anhydrous ethanol with a volume fraction greater than 50%.

[0012] The total extract of Syzygium brevicornu leaf alcohol was extracted with ethyl acetate to obtain Syzygium brevicornu leaf extract.

[0013] In some embodiments, the leaves of *Syzygium brevicornu* are coarsely pulverized by passing them through a 4-mesh sieve before extraction with an ethanol solution.

[0014] In some embodiments, the ratio of short-stemmed Syzygium leaf to ethanol solution is 1g:5mL to 1g:15mL.

[0015] In some implementations, the extraction is performed by heating and reflux extraction, and the extraction is performed four times, each time for 1 hour.

[0016] In some embodiments, the extraction method is as follows: the total extract of Syzygium brevicornum leaf alcohol extract is extracted three times with ethyl acetate, the extracts are combined, and concentrated under reduced pressure to obtain Syzygium brevicornum leaf extract, wherein the ratio of ethyl acetate to total extract of Syzygium brevicornum leaf alcohol extract is 1 mL: 1 g.

[0017] According to a second aspect of the present invention, a short-arcted Syzygium leaf extract prepared by the above-described preparation method is provided.

[0018] According to a third aspect of the invention, the above-described short-arcted Syzygium leaf extract is provided for use in the preparation of pharmaceuticals or foods having anti-inflammatory and / or lipid-lowering effects.

[0019] According to a fourth aspect of the invention, the use of the above-described short-chain Syzygium leaf extract in the preparation of a medicament for the prevention and / or treatment of atherosclerosis is provided.

[0020] The beneficial effects of this invention include:

[0021] This invention, using an LPS / ox-LDL-induced RAW264.7 foam cell model, revealed that the *Syzygium aromaticum* leaf extract prepared according to this invention possesses significant anti-inflammatory and lipid-lowering effects, and can be used to prepare drugs or foods with anti-inflammatory and / or lipid-lowering effects. Furthermore, the *Syzygium aromaticum* leaf extract prepared according to this invention can reduce lipid accumulation in RAW264.7 macrophage-derived foam cells, suggesting that the *Syzygium aromaticum* leaf extract prepared according to this invention can be used to prepare drugs for the prevention and / or treatment of atherosclerosis. Attached Figure Description

[0022] Figure 1 The effect of short-chain Syzygium aromaticum leaf extract on the activity of RAW264.7 macrophages in cell experiments of this invention.

[0023] Figure 2 The effect of short-chain Syzygium leaf extract on LPS-induced inflammatory cytokine release in cell experiments of this invention.

[0024] Figure 3 The effect of Syzygium aromaticum leaf extract on ox-LDL-induced lipid accumulation in macrophages was detected by Oil Red O staining in the cell experiments of this invention.

[0025] Figure 4 The effect of Syzygium aromaticum leaf extract on Dil-ox-LDL fluorescence accumulation in macrophages was observed under a fluorescence microscope in the cell experiments of this invention.

[0026] Figure 5 The effect of short-chain Syzygium aromaticum leaf extract on macrophage lipid efflux rate was detected in the cell experiments of this invention using fluorescent NBD cholesterol detection. ##p<0.01 is compared with the blank control group, *p<0.05 and **p<0.01 are compared with the ox-LDL group.

[0027] Figure 6 This invention relates to the cell experiments conducted using RT-PCR to determine the effects of short-chain Syzygium aromaticum leaf extract on the mRNA expression of lipid uptake receptor CD36 and efflux receptors ABCG1 and SRB1 in macrophages. ##p<0.01 is compared to the blank control group, *p<0.05 and **p<0.01 are compared to the ox-LDL group. Detailed Implementation

[0028] The present invention will now be described in further detail with reference to the accompanying drawings. Unless otherwise specified, all reagents used in the following experiments are commercially available, and all experimental methods not specifically described are conventional methods known in the art.

[0029] Example 1

[0030] The preparation method of the short-chain Syzygium leaf extract in this embodiment includes the following steps:

[0031] (1) Alcohol extraction step: Weigh 10 kg of dried Syzygium brevicornum leaves, coarsely crush them through a 4-mesh sieve, and then extract them 4 times with 10 times the amount (100 L) of anhydrous ethanol, each time for 1 hour. After filtration, combine the extracts and concentrate them under reduced pressure to obtain about 500 g of total extract of Syzygium brevicornum leaf alcohol extract.

[0032] (2) Extraction steps: The total extract of brachythyrsum leaf alcohol extract obtained in step (1) was extracted three times with an equal volume (about 500 mL) of ethyl acetate. The extracts were combined and concentrated under reduced pressure to obtain 200 g of ethyl acetate fraction, which is brachythyrsum leaf extract (BE).

[0033] Below, cell experiments were conducted using the short-chain Syzygium leaf extract (BE) prepared in Example 1 to investigate the pharmacological effects of the short-chain Syzygium leaf extract.

[0034] 1. Activity assay of Syzygium aromaticum leaf extract on RAW264.7 macrophages

[0035] The extract of *Syzygium brevicornu* leaves was dissolved in dimethyl sulfoxide (DMSO) and then diluted to working concentrations using DMEM medium. The density was 2 × 10⁻⁶. 4 RAW264.7 cells were seeded at 100 cells / well in 96-well plates. After 24 hours, the culture medium was discarded, and the cells were incubated with various concentrations of Syzygium aromaticum leaf extract for another 24 hours. Cytotoxicity was then assessed using an MTT assay. All experiments were performed three times, and the data were calculated as mean ± SD.

[0036] The results are as follows Figure 1 As shown, the extract of Syzygium aromaticum leaf at concentrations below 25 μg / mL was not toxic to RAW264.7 cells. Therefore, concentrations of 6.25, 12.5, and 25 μg / mL were selected for subsequent experiments.

[0037] 2. Experiment on the effect of Syzygium aromaticum leaf extract on LPS-induced inflammatory response in RAW264.7 cells.

[0038] According to 2×10 5 RAW264.7 cells were seeded in 24-well plates and cultured at 37°C for 24 h. The drug-treated groups were then incubated with medium containing different concentrations of *Syzygium brevicornu* leaf extract, the positive control group was incubated with 1 μg / mL dexamethasone, and the blank control group was incubated with an equal volume of fresh complete cell culture medium. Except for the blank control group, both the drug-treated and positive control groups were incubated with 2 μg / mL LPS. After 24 h, the supernatant was collected, and the NO content was determined using the Griess method. All experiments were performed three times, and the data were calculated as mean ± SD.

[0039] The results are as follows Figure 2 As shown, extracts of *Syzygium brevicornu* leaves at concentrations of 6.25, 12.5, and 25 μg / mL reduced NO release from foam cells in a dose-dependent manner.

[0040] 3. Experiment on the effect of Syzygium brevicornu leaf extract on lipid accumulation in foam cells

[0041] (1) With 1.2×10 5 RAW264.7 macrophages were added to 24-well plates at a density of cells / mL and cultured in a cell culture incubator at 37°C and 5% CO2 for 24 h. Gradual concentrations of drugs were added to each treatment group, and except for the control group, ox-LDL was added to each well at a final concentration of 80 μg / mL. Cells were then cultured for another 24 h. Cells were fixed with paraformaldehyde for 30 min, stained with prepared Oil Red staining solution for 20-30 min, the staining solution was discarded, and the cells were washed several times with PBS. Lipid accumulation was observed and photographed under an inverted microscope.

[0042] The results are as follows Figure 3As shown, extracts of Syzygium aromaticum leaves (6.25, 12.5, 25 μg / mL) reduced red lipid droplets in cells to varying degrees.

[0043] (2) RAW264.7 cells were divided into groups of 1×10⁻⁶. 5 Cells were seeded at a density of [number] cells / well in 24-well plates. After 24 hours, the drug-treated groups were treated with different concentrations of *Syzygium brevicornu* leaf extract and 20 μg / mL Dil-ox-LDL, the model group was treated with 20 μg / mL Dil-ox-LDL, and the control group was treated with an equal volume of fresh complete cell culture medium. After 3 hours of culture, the culture medium was discarded, and the cells were washed three times with PBS and observed under a fluorescence microscope. Following the same procedure, after washing three times with PBS, the cells were collected in the dark and the fluorescence intensity of Dil-ox-LDL was detected by flow cytometry.

[0044] The results are as follows Figure 4 As shown, the Dil-ox-LDL experiment more clearly demonstrates that the extracts of Syzygium aromaticum leaves (6.25, 12.5, 25 μg / mL) can reduce the uptake of ox-LDL in RAW264.7 cells.

[0045] Therefore, it can be seen that the extract of Syzygium brevicornu leaves can reduce the uptake of ox-LDL by macrophages and inhibit the formation of foam cells by macrophages.

[0046] 4. Experiment on the effect of Syzygium brevicornu leaf extract on cholesterol efflux from foam cells

[0047] RAW264.7 cells were divided into groups of 5 × 10⁻⁶. 5 Cells were seeded at a density of [number] cells / well in 12-well plates. After 24 hours, 80 μg / mL ox-LDL and 5 μg / mL 25-NBD fluorescent cholesterol were added and incubated for another 24 hours. Then, according to the drug administration groups, different concentrations of *Syzygium spicata* leaf extract and 50 μg / mL HDL were added to phenol red-free medium and the cells were cultured for another 24 hours. The supernatant was aspirated, and the cells were washed three times with PBS. Cells were lysed using RIPA, and the cell lysates were collected. The fluorescence intensity of the supernatant and lysates was detected at an excitation wavelength of 469 nm and an emission wavelength of 538 nm. The NBD-cholesterol efflux rate was calculated as: FI supernatant / (FI supernatant + FI cell lysate) × 100%. All experiments were performed three times, and the data were calculated as mean ± SD.

[0048] The results are as follows Figure 5 As shown, extracts of Syzygium aromaticum leaves (6.25, 12.5, 25 μg / mL) can promote the efflux of cholesterol from foam cells.

[0049] 5. RT-PCR determination of the effect of short-chain Syzygium aromaticum leaf extract on the mRNA expression of lipid uptake receptor CD36 and efflux receptors ABCG1 and SRB1 in macrophages.

[0050] RAW264.7 cells were divided into groups of 1×10⁻⁶. 6 RAW264.7 cells in logarithmic growth phase were seeded into 6-well plates and cultured at 37°C for 24 h. Afterward, cells were administered according to their assigned groups. 24 h later, total RNA was extracted from macrophages using TRIzol reagent. After determining RNA purity and concentration, cDNA was synthesized using a Roche cDNA reverse transcription kit. PCR experiments were then performed using a Roche FastStart Universal SYBR Green Master real-time PCR System 7500 (Applied Biosystems) (primer sequences are shown in Table 1). The expression of CD36, SRB1, and ABCG1 in each group of cells was detected. All experiments were performed three times, and the data were calculated as mean ± SD.

[0051] Table 1 Primer sequences of the target gene

[0052]

[0053] The results are as follows Figure 6 As shown, the extracts of *Syzygium brevicornu* leaves (6.25, 12.5, 25 μg / mL) reduced ox-LDL-induced CD36 mRNA expression and promoted SRB1 / ABCG1 mRNA expression, indicating that *Syzygium brevicornu* leaf extract can reduce cellular lipid uptake by inhibiting CD36 mRNA expression and promote cholesterol efflux from foam cells by promoting ABCG1 / SRB1 mRNA expression.

[0054] In summary, this invention, using an LPS / ox-LDL-induced RAW264.7 foam cell model, revealed that the *Syzygium aromaticum* leaf extract prepared in this invention possesses significant anti-inflammatory and lipid-lowering effects, and can be used to prepare drugs or foods with anti-inflammatory and / or lipid-lowering effects. Furthermore, the *Syzygium aromaticum* leaf extract prepared in this invention can reduce lipid accumulation in RAW264.7 macrophage-derived foam cells, suggesting that the *Syzygium aromaticum* leaf extract prepared in this invention can be used to prepare drugs for the prevention and / or treatment of atherosclerosis.

[0055] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. The application of *Syzygium brevicornu* leaf extract in the preparation of drugs for the prevention and / or treatment of atherosclerosis, wherein, The preparation method of extract from Syzygium brevicornu leaves includes the following steps: Dried leaves of Syzygium brevicornum were extracted with anhydrous ethanol four times for one hour each time. The extracts were then combined after filtration and concentrated under reduced pressure to obtain the total extract of Syzygium brevicornum leaf alcohol. The total extract of Syzygium brevicornu leaf alcohol was extracted with ethyl acetate to obtain Syzygium brevicornu leaf extract.

2. The application according to claim 1, characterized in that, Before extraction with anhydrous ethanol, the leaves of *Syzygium brevicornum* were coarsely crushed by passing them through a 4-mesh sieve.

3. The application according to claim 1 or 2, characterized in that, The ratio of *Syzygium brevicornu* leaves to anhydrous ethanol is 1g:5mL to 1g:15mL.

4. The application according to claim 1 or 2, characterized in that, The extraction method described is reflux extraction.

5. The application according to claim 1 or 2, characterized in that, The extraction method is as follows: the total extract of Syzygium aromaticum leaf alcohol extract is extracted three times with ethyl acetate, the extracts are combined, and concentrated under reduced pressure to obtain Syzygium aromaticum leaf extract. The ratio of ethyl acetate to total extract of Syzygium aromaticum leaf alcohol extract is 1 mL: 1 g.

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