CN117017968A - Application of hydroxytyrosol acetate in preparing antitumor products - Google Patents

Abstract

The invention provides application of hydroxytyrosol acetate in preparing antitumor products, wherein the hydroxytyrosol acetate is a metabolite derived from natural plants, has obvious effects of inhibiting tumor proliferation and promoting tumor cell apoptosis, is safe and reliable, and can be obtained by condensing hydroxytyrosol and acetic acid of natural plant species through biotransformation, so that the source of the product is rich. The hydroxytyrosol acetate has the antitumor effect discovered, provides a new medicine type for the discovery of antitumor medicines, and is hopeful to become a novel antitumor medicine through intensive research.

Description

Application of hydroxytyrosol acetate in preparing antitumor products

Technical Field

The invention belongs to the technical field of anti-tumor compound screening application, and particularly relates to application of hydroxytyrosol acetate in preparation of anti-tumor products.

Background

Hydroxytyrosol acetate (Hydroxytyrosol acetate, HTA) is a natural polyphenol compound in olive leaves, and the chemical structural formula of the hydroxytyrosol acetate is shown in figure 1. HTA is known to have antioxidant activity and to reduce MPP-induced oxidative damage in rats. The inventors have found in earlier work that HTA has antibacterial biological activity and Ghalandari et al demonstrated that HTA can inhibit the growth of Staphylococcus aureus and Staphylococcus epidermidis. In addition, HTA has been found to be associated with the prevention of cardiac and cerebral health. Daily intake of daily dietary levels of HTA can slightly improve cognitive abilities in alzheimer's disease mice. Gonz lez-Correa et al found that HTA had neuroprotective effects on rats, which provided a preliminary basis for further studies of polyphenols as potential neuroprotective compounds. HTA may prevent vascular endothelial cell necrosis by down-regulating HDAC 11-associated signaling pathways in atherosclerosis, and studies have found that HTA moiety exerts anti-inflammatory effects through TNFRSF1A/SIRT6/pkm 2-mediated signaling pathways. Dietary supplementation with HTA significantly reduces pro-inflammatory cytokines and prevents kidney damage by significantly blocking different inflammation-related pathways, which provides the basis for developing new diet strategies to prevent and manage systemic lupus erythematosus. Oral HTA inhibits rat platelet aggregation by decreasing thrombin synthesis and increasing nitric oxide production. However, there is no report of the anti-tumor effect of HTA.

Disclosure of Invention

The invention aims to provide application of hydroxytyrosol acetate in preparing anti-tumor products, so as to make up for the defects of the prior art.

The invention firstly provides an application of hydroxytyrosol acetate in preparing products for inhibiting tumor proliferation or promoting tumor cell apoptosis;

the tumor is liver cancer cell.

In yet another aspect, the invention provides a preparation for inhibiting tumor proliferation or promoting tumor cell apoptosis, said preparation comprising hydroxytyrosol acetate at a pharmacologically effective concentration.

The hydroxytyrosol acetate used in the invention is a metabolite derived from natural plants, has obvious effects of inhibiting tumor proliferation and promoting tumor cell apoptosis, is safe and reliable, and can be obtained by condensing hydroxytyrosol and acetic acid of natural plant species through bioconversion, so that the source of the product is rich. The hydroxytyrosol acetate has the antitumor effect discovered, provides a new medicine type for the discovery of antitumor medicines, and is hopeful to become a novel antitumor medicine through intensive research.

Drawings

Fig. 1: structure of hydroxytyrosol acetate.

Fig. 2: effect of HTA on BEL7402 cells, wherein panel a is a proliferation plot of HTA-inhibited BEL7402 cells, and panel B is a microscopic plot of HTA-treated BEL7402 cells, with I-VI being control, 20 μg/ml, 30 μg/ml,40 μg/m, 50 μg/ml, 60 μg/ml HTA-treated groups, respectively.

Fig. 3: HTA induced BEL7402 cell death data, FIG. A is a graph of cytotoxicity of HTA against BEL7402 cells, and FIG. B is a graph of the effect of HTA on genomic DNA, where lanes 1-5 are marker, control, 30 μg/ml,40 μg/ml, and HTA treated groups at 50 μg/ml concentrations, respectively.

Fig. 4: BEL7402 cell Mitochondrial Membrane Potential (MMP) and cytochrome C level map BEL7402 cells were treated with HTA (0. Mu.g/ml (I), 30. Mu.g/ml (II), 40. Mu.g/ml (III), 50. Mu.g/ml (IV)), incubated with mitochondrial dye rhodamine 123 (2.0. Mu.M) for 10min at 37℃and washed with PBS. FIG. A is a graph of the effect of HTA on mitochondrial function observed under a fluorescence microscope, and FIG. B is a graph of the change in the MMP of BEL7402 cells after HTA treatment detected by a fluorescence spectrophotometer; panel C is a red-green fluorescence ratio chart, and panel D is a Western blot analysis chart of cytochrome C expression.

Fig. 5: graph of HTA effect on BEL7402 cell Bax and Bcl-2 expression.

Detailed Description

According to the invention, through researching the influence of HTA on proliferation and apoptosis of liver cancer BEL7402 cells, the HTA can inhibit proliferation of BEL7402 cells and can induce apoptosis of BEL7402 cells.

The hydroxytyrosol acetate (HTA, > 97%) used in the examples of the present invention may be hydroxytyrosol acetate isolated from olive leaves (Olea Europeae L.), but may also be commercially available hydroxytyrosol acetate. RPMI1640 medium and fetal bovine serum were purchased from HyClone, BCA protein assay kit from Pierce, cell culture plates from Corning, primary antibody from Santa Cruz, secondary antibody from Solarbio, primary anti-dilution from Biyuntian, protein electrophoresis apparatus from Beijing Liujia instruments.

BEL7402 human liver cancer cell line was supplied from the national academy of sciences pharmacology laboratory in RPMI1640 medium supplemented with 10% FBS+penicillin and streptomycin antibiotics (Sigma) in 5% CO ₂ Culturing at 37 ℃.

The present invention will be described in detail with reference to the following examples and the accompanying drawings.

Example 1: influence of hydroxytyrosol acetate HTA on BEL7402 human hepatoma cells

1. BEL7402 cell proliferation assay/MTT assay

Cells were plated in 96-well plates (about 5X 10) ³ 200 mu L/well) were inoculated and grown for 24 hours, and the cells were plated in 96-well plates @About 1X 10 ⁵ Mu L/well) was inoculated with growth for 24h, and the control group was treated with 1% alcohol. After 48h incubation, 40. Mu.L MTT solution (5 mg/mL) was added to each well. After incubation for a further 3h, the supernatant was taken, formaldehyde was dissolved in 150. Mu.L of DMSO and mixed on a shaker for 30min. Absorbance at 570nm was measured using ELx808 TM. The cell viability ratio was calculated as (%) = (1- (OD control-treated OD)/OD control) ×100%.

Weighing a certain amount of HTA, adding absolute ethyl alcohol, preparing mother liquor of 3, 4, 5, 6 and 7mg/mL, comparing absolute ethyl alcohol, adding HTA solutions with different concentrations according to the proportion of 1% (v/v), obtaining different groups with final concentrations of 30, 40, 50, 60 and 70 mug/mL, and comparing absolute ethyl alcohol. The growth of BEL7402 human hepatoma cells at different HTA concentrations was observed.

The results show that HTA significantly inhibited the proliferation of BEL7402 cells and that cell viability decreased with increasing concentration (fig. 2). BEL7402 cell viability after HTA treatment was 87.67% at 30. Mu.g/ml. Thus, HTA can be used as a component of liver cancer treatment to develop drugs.

2. LDH assay

The leakage rate of BEL7402 cells after HTA treatment was measured. Cells were plated in 96-well plates (about 1X 10) ⁵ Well) were inoculated and cultured for 24 hours, then treated with HTA (0, 30, 40, 50, 60, 70. Mu.g/mL) at a given concentration, and the control group was treated with 1% alcohol. After 24h incubation, cells were detected with LDH detection kit. The absorbance at 490nm was measured with ELx 808. TM. And the LDH content was calculated, and the LDH leakage rate was calculated as LDH leakage rate=extracellular LDH concentration/(extracellular LDH content+intracellular LDH content) ×100%.

The DNA fragments were analyzed by agarose gel electrophoresis. BEL7402 cells (about 1X 10) ⁵ Well) was inoculated on a 6-well plate for 24 hours, incubated with HTA (0, 30, 40, 50. Mu.g/mL) for 48 hours, and collected by centrifugation. Total DNA was purified using the general genomic DNA extraction kit (TAKARA Biotechnology Co., ltd., china) according to the manufacturer's instructions. After the DNA was dissolved on a 2% agarose gel, it was stained with ethidium bromide and irradiated with ultraviolet light (us Bio Rad Chemi Doc XRS imaging system).

LDH leakage test results showed that there was no significant difference in cytotoxicity of low concentration HTA compared to control group, and high concentration HTA showed significant cytotoxicity (fig. 3A). Genomic DNA was then assayed and the results showed that HTA induced DNA damage, exhibiting a distinct DNA ladder (fig. 3B). These results indicate that HTA can induce cell death by inducing apoptosis in BEL7402 cells.

3. Mitochondrial membrane potential detection

Mitochondrial Membrane Potential (MMP) was measured by mitochondrial potential sensor JC-1 (Molecular Probes, USA). Cells treated with HTA at a concentration (about 1X 105 Cells/mL) were incubated with JC-1 dye (10. Mu.g/mL) for 10min, washed with JC-1 buffer, and analyzed by fluorescence microscopy and fluorescence spectrophotometer (fluorescent red, excitation 525nm, emission 590nm; fluorescent green, excitation 490nm, emission 530 nm).

As shown in fig. 4, HTA can induce mitochondrial dysfunction, which in turn leads to the release of cytochrome C. After HTA treatment, the red fluorescence intensity gradually decreased and the green fluorescence intensity gradually increased as the HTA concentration increased (fig. 4A). Quantitative detection was performed using a fluorescence spectrophotometer, and the result showed that the ratio of red-green fluorescence intensity was continuously decreased (fig. 4B). The ratio of red to green fluorescence intensities was approximately 3.41 in untreated BEL7402 cells, and decreased to 2.49, 1.71 and 0.39 in 30, 40 and 50 μg/mL treated cells, respectively (FIG. 4C). To further confirm this result, cytochrome C was detected, and the result showed that HTA treatment could cause release of cytochrome C (fig. 4D). Thus, enhancement of apoptosis by HTA may be mediated through the mitochondrial pathway.

4. Western Blot analysis

To determine the effect of HTA on anti/pro-apoptotic proteins, western blot detection was performed. After 24h treatment with HTA (0, 30, 40 and 50. Mu.g/mL) at a concentration, cells were washed 2 times with frozen PBS and cell pellet was lysed with RIPA buffer containing fresh protease inhibitor cocktail (50. Mu.g/mL aprotinin, 0.5mM phenylmethanesulfonyl fluoride (PMSF), 1mM sodium orthovanadate, 10mM sodium fluoride and 10mM beta-glycerophosphate). Protein concentration was determined using BCA protein assay (Biocolor BioScience & Technology, china). Protein samples were solubilized on a 10-15% SDS-PAGE gel, transferred to nitrocellulose membrane, and probed with protein specific antibodies and enzyme-labeled secondary antibodies.

Expression detection of HTA-treated BEL7402 apoptosis-related proteins, wherein Bcl and Bax are both important apoptosis-related proteins in the apoptotic pathway. The results indicated that Bcl-2 expression was reduced and Bax expression was significantly increased in HTA treated cells (fig. 5). The results indicate that HTA can alter Bcl and Bax expression.

In conclusion, HTA not only inhibits proliferation of BEL7402 cells, but also induces apoptosis of BEL7402 cells; therefore, HTA has the application potential of inhibiting tumor cell proliferation and promoting tumor cell apoptosis. In addition, the naturally-occurring HTA mainly exists in the olive leaves, the olive leaves belong to byproducts in the olive tree planting process, and by researching the HTA anti-tumor effect, the HTA is hopeful to be developed into a novel anti-tumor drug, so that the HTA has positive significance for tumor treatment on one hand, on the other hand, the development of the olive leaves can be increased, the high-valued utilization degree of the olive leaves is improved, and the high-quality development of the olive industry is promoted.

Claims (5)

1. An application of hydroxytyrosol acetate is characterized in that the application is the application in preparing a product for inhibiting tumor proliferation or promoting tumor cell apoptosis.

2. The use according to claim 1, wherein the tumor cell is a liver cancer tumor cell.

3. A preparation for inhibiting tumor proliferation or promoting tumor cell apoptosis, wherein the preparation comprises hydroxytyrosol acetate with a pharmacologically effective concentration.

4. The article of claim 3, wherein the article is a functional food or pharmaceutical product.

5. The article of claim 3, wherein the article is a liquid article.