阅读说明：本技术 一种蓝莓花青素的应用 (Application of blueberry anthocyanin ) 是由 宋冬妍 温继龙 王宁 李殿俊 于航 于 2021-08-27 设计创作，主要内容包括：一种蓝莓花青素的应用,它涉及一种蓝莓花青素的应用。本发明提供一种蓝莓花青素的用途。一种蓝莓花青素的应用,莓花青素提取物在制备抗肿瘤药物中的用途。优点：由于GADD45A、GADD45B、DDIT4、CDKN1A、CHAC1ATF-4和EPHA2的表达在经蓝莓花青素处理后在HepG-2细胞中被更高地转录,因此蓝莓花青素能够通过相关途径诱导细胞凋亡；所以蓝莓花青素能够作为癌症预防的辅助成分用于制备抗肿瘤药物,尤其是对于舌鳞癌细胞系、非小细胞肺癌细胞系、肝癌细胞系和前列腺癌细胞系。(An application of blueberry anthocyanin relates to an application of blueberry anthocyanin. The invention provides application of blueberry anthocyanin. An application of blueberry anthocyanin, namely an application of a blueberry anthocyanin extract in preparation of antitumor drugs. The advantages are that: since the expression of GADD45A, GADD45B, DDIT4, CDKN1A, CHAC1ATF-4 and EPHA2 is more highly transcribed in HepG-2 cells after blueberry anthocyanin treatment, blueberry anthocyanin can induce apoptosis through related pathways; therefore, the blueberry anthocyanin can be used as an auxiliary component for preventing cancers and is used for preparing anti-tumor drugs, particularly for tongue squamous cancer cell lines, non-small cell lung cancer cell lines, liver cancer cell lines and prostate cancer cell lines.)

1. The application of the blueberry anthocyanin is characterized in that: the application of the blueberry anthocyanin extract in preparing anti-tumor drugs; the mass fraction of the blueberry anthocyanin in the blueberry anthocyanin extract reaches more than 95%.

2. The application of the blueberry anthocyanin according to claim 1, wherein the blueberry anthocyanin extract is used for preparing a medicine for resisting a head and neck squamous cell carcinoma cell line.

3. The use of blueberry anthocyanin according to claim 2, wherein the head and neck squamous cell line is tongue squamous cell SCC 15.

4. The application of the blueberry anthocyanin according to claim 1, wherein the blueberry anthocyanin extract is used for preparing anti-hepatoma cell line medicines.

5. The application of the blueberry anthocyanin as defined in claim 4, wherein the liver cancer cell line is liver cancer cell HuH-7 or liver cancer cell HLF.

6. The application of the blueberry anthocyanin according to claim 1, wherein the blueberry anthocyanin extract is used for preparing a prostate cancer cell line resistant medicament.

7. The application of blueberry anthocyanin according to claim 6, wherein the prostate cancer cell line is prostate cancer cell C4-2B.

8. The application of the blueberry anthocyanin according to claim 1, wherein the blueberry anthocyanin extract is used for preparing a non-small cell cancer cell line resistant medicament.

9. The application of the blueberry anthocyanin according to claim 1, wherein the non-small cell cancer cell line is non-small cell lung cancer cell H460.

Technical Field

The invention relates to application of blueberry anthocyanin.

Background

Blueberries are members of the ericaceae and bilberry genera and are known for their high content of flavonoids (anthocyanins, flavonols, procyanidins) and other phenolic compounds. The flavonoid is a three-membered ring compound and has the effect of protecting the heart, known that low-density lipoprotein is harmful to human bodies and is easy to cause coronary heart disease, and the flavonoid can inhibit the generation of the harmful low-density lipoprotein and has the effect of reducing thrombosis. The investigation proves that the mortality rate of coronary heart disease is lower for patients with high flavonoid intake. Anthocyanins belong to the flavonoid class of phenolic compounds, and are potent antioxidants that protect the human body from a harmful substance called free radicals. Anthocyanins can also enhance vascular elasticity, improve circulatory system and smoothness of skin, inhibit inflammation and allergy, and improve joint flexibility.

Disclosure of Invention

The invention provides application of blueberry anthocyanin.

An application of blueberry anthocyanin, an application of a blueberry anthocyanin extract in preparing an anti-tumor medicament; the mass fraction of the blueberry anthocyanin in the blueberry anthocyanin extract reaches more than 95%.

The invention has the advantages and the principle that:

the cause of apoptosis is as follows:

active oxygen is an apoptosis inducer for cells. Some of the agents that trigger apoptotic patterns are stimulators of cellular oxidative metabolism. Bufalin (Bufalin) has been reported to induce autophagy of cancer cells by producing reactive oxygen species in human colon cancer cells. Therefore, reactive oxygen species are considered to be essential mediators for monitoring cancer cell apoptosis. Cytochrome c is a key factor in the apoptotic process in this mechanism. When some factors induce disruption of the outer mitochondrial membrane, some mitochondrial apoptotic factors, such as cytochrome c, are released into the cytoplasm. Cytochrome c present in the cytoplasm may induce activation of caspase, resulting in apoptosis. It has been shown that cytoplasmic cytochrome c is involved in caspase-9 dependent activation of caspase-3, initiating the apoptotic program. Furthermore, it has been reported that a decrease in matrix metalloproteinases can trigger the opening of the mitochondrial pore, which in turn may release cytochrome c from the mitochondria into the cytoplasm.

② the Bax/Bcl-2 ratio is considered as the mitochondrion mediated apoptosis marker, the apoptosis of the cell is related to whether the Bax and Bcl-2 proteins are expressed in balance. Bax can increase permeability of the mitochondrial membrane, leading to entry of proteins in the inter-mitochondrial membrane space (cytochrome c) into the cytoplasm. Cytochrome c can initiate caspase activation and apoptosis. Bcl-2 belongs to an anti-apoptotic protein and can inhibit Bax-mediated mitochondrial membrane permeability.

③ the MAPK pathway is important for p38, JNK, NFKB and p53 to regulate cell proliferation and apoptosis. It is well known that the p53 protein plays an important role in growth arrest and apoptosis after cellular stress, including deoxyribonucleic acid damage, oxidative stress, and activation of oncogenes. The p53 protein is used as the transcription activator of p53 regulating gene and can induce cell cycle arrest, cell senescence or apoptosis.

DNA damage-inducing transcript 4(DDIT4) and growth arrest and DNA damage inducers alpha and beta (GADD 45A and GADD45B, respectively) are involved in the regulation of cell growth and apoptosis. GADD45A and GADD45B play important roles in negative growth control, including cell death and inhibition of cell growth. The proteins encoded by GADD45A and GADD45B were reported to be regulated by p53 tumor suppressor. The two proteins can also activate the p38/JNK kinase pathway and apoptosis through stress-responsive MTK1/MEKK4 kinase.

It is known that CHAC1 and ATF-4 are involved in intrinsic apoptotic signaling pathways in response to Endoplasmic Reticulum (ER) stress. ER stress-dependent apoptosis is mediated by activation of human caspases and pro-apoptotic members of the Bcl-2 family. Cyclin-dependent kinase inhibitor 1(CDKN1A) is involved in the positive regulation of reactive oxygen metabolic processes. CDKN1A (p21) can induce mitochondrial dysfunction and reactive oxygen species production through the series of signaling by GADD45-MAPK 14(p38 MAPK) -grb 2-TGFBR 2-TGFbeta. EPHA2 is an essential p 53-independent, caspase-8-dependent pro-apoptotic factor. The expressions of GADD45A, GADD45B, DDIT4, CDKN1A, CHAC1ATF-4 and EPHA2 are more highly transcribed in HepG-2 cells after being treated by blueberry anthocyanin, so that the blueberry anthocyanin can induce apoptosis through related pathways; therefore, the blueberry anthocyanin can be used as an auxiliary component for preventing cancers and is used for preparing anti-tumor drugs, particularly for tongue squamous cancer cell lines, non-small cell lung cancer cell lines, liver cancer cell lines and prostate cancer cell lines.

Detailed Description

The first embodiment is as follows: the embodiment is an application of blueberry anthocyanin, and the application of a blueberry anthocyanin extract in preparing an anti-tumor medicament; the mass fraction of the blueberry anthocyanin in the blueberry anthocyanin extract reaches more than 95%.

The second embodiment is as follows: the present embodiment differs from the present embodiment in that: the blueberry anthocyanin extract is used for preparing a medicine for resisting a head and neck squamous cell carcinoma cell line. The rest is the same as the first embodiment.

The third concrete implementation mode: the second embodiment differs from the first embodiment in that: the head and neck squamous carcinoma cell line is tongue squamous carcinoma cell SCC 15. The rest is the same as the second embodiment.

The fourth concrete implementation mode: the present embodiment differs from the present embodiment in that: the blueberry anthocyanin extract is used for preparing anti-hepatoma cell line medicines. The rest is the same as the first embodiment.

The fifth concrete implementation mode: the fourth difference between the present embodiment and the specific embodiment is: the liver cancer cell line is liver cancer cell HuH-7 or liver cancer cell HLF. The rest is the same as the fourth embodiment.

The sixth specific implementation mode: the present embodiment differs from the present embodiment in that: the blueberry anthocyanin extract is used for preparing a medicine for resisting a prostate cancer cell line. The rest is the same as the first embodiment.

The seventh embodiment: the sixth difference from the present embodiment is: the prostate cancer cell line is prostate cancer cell C4-2B. The rest is the same as the sixth embodiment.

The specific implementation mode is eight: the present embodiment differs from the present embodiment in that: the blueberry anthocyanin extract is used for preparing a non-small cell cancer cell line resistant medicament. The rest is the same as the first embodiment.

The specific implementation method nine: eighth embodiment differs from the eighth embodiment in that: the non-small cell cancer cell line is a non-small cell lung cancer cell H460. The rest is the same as the embodiment eight.

The invention is not limited to the above embodiments, and one or a combination of several embodiments may also achieve the object of the invention.

The following experiments are adopted to verify the effect of the invention:

example 1: an application of blueberry anthocyanin, an application of a blueberry anthocyanin extract in preparing an anti-tumor medicament; the mass fraction of the blueberry anthocyanin in the blueberry anthocyanin extract is 99.3%; in particular to application of the blueberry anthocyanin extract in preparing medicines for resisting squamous cell carcinoma SCC15, liver cancer cell HuH-7, liver cancer cell HLF, prostate cancer cell C4-2B and non-small cell lung cancer cell H460.

And (3) cell viability detection:

reagent: cell counting Kit CCK8 Kit (same as Japan), DMEM high-sugar liquid Medium, RPMI Medium 1640basic, normal saline, pancreatin Cell digestive juice, special fetal calf serum, penicillin-streptomycin solution.

Consumable material: pasteur pipet, 25cm square culture bottle with inclined mouth, 15mL aseptic centrifuge tube, 96-hole cell culture plate.

Medicine preparation: the purity of the blueberry anthocyanin extract is 99.3 percent.

The experimental method comprises the following steps:

(1) cancer cell and media selection: selecting experimental cancer cells and a liquid culture medium as shown in table 1; secondly, adding bovine serum and a penicillin/streptomycin solution into the liquid culture medium to prepare a complete culture medium, wherein the volume ratio of the liquid culture medium to the bovine serum is 9: 1; the volume ratio of the liquid culture medium to the penicillin/streptomycin solution is 99:1, the concentration of penicillin G sodium salt in the penicillin/streptomycin solution is 10KU/mL, and the concentration of streptomycin sulfate is 10 mg/mL.

TABLE 1

(2) The cell culture method comprises the following steps: taking out the frozen tube from a refrigerator at minus 80 ℃, quickly placing the frozen tube in a water bath kettle at 37 ℃, incubating for 1-2 min, taking out, sucking out the experimental cancer cells (shown in table 1) into a 15mL centrifuge tube, adding 10mL of complete culture medium, uniformly mixing, centrifuging (1500rpm for 5min), discarding the supernatant after centrifugation, suspending the cells by using 4mL of complete culture medium, transferring the cells to an oblique-mouth cell culture bottle, placing the cells in a 37 ℃ C., and 5% CO₂Carrying out static culture in an incubator;

(3) cell proliferation and toxicity detection: firstly, discarding a culture medium in a bottle, adding 1mL of physiological saline to clean cells (when the physiological saline is added, placing a pipettor at the bottom of the bottle, not directly cleaning the pipettor by using a gun, slightly shaking the culture bottle), sucking out the physiological saline, adding 1mL of trypsin digestive juice, digesting the cells to a cell suspension (or semi-suspension) state, adding 4mL of complete culture medium, resuspending cells by using a Pasteur tube, centrifuging (1500rpm, 5min), discarding supernatant, and adding 4mL of complete culture medium to suspend the cells to obtain cell suspension;

(4) preparing 100 mu L of cell suspension in a 96-well plate; the plates were pre-incubated in an incubator for 24 hours (37 ℃ C., 5% CO)₂)。

(5) Dissolving the blueberry anthocyanin extract with complete culture medium according to the blueberry anthocyanin concentrations of 2mg/mL, 4mg/mL, 8mg/mL, 12mg/mL, 16mg/mL, 20mg/mL and 24mg/mL respectively to prepare a blueberry anthocyanin-containing culture medium, and filtering with a 0.22-micrometer filter membrane;

(6) grouping according to an additive group, an additive group (0) (namely, no additive group) and a blank group, wherein the additive group comprises the following components in parts by weight: adding 100 mu L of blueberry anthocyanin-containing culture medium with different blueberry anthocyanin concentrations into each hole of the culture plate of the dosing group, and adding the culture plate of the dosing group; 0, dosing group: adding 100 mu L of culture medium into each hole of the culture plate of the drug adding group 0 to obtain the drug adding group 0 culture plate; blank group: each hole in the blank group of culture plates is a hollow hole, so that a blank group of culture plates is obtained;

(7) and (4) incubating the medicated group culture plate, the 0 medicated group culture plate and the blank group culture plate obtained in the step (6) in an incubator for 24 h.

(8) The Cell counting Kit CCK8 Kit was diluted with liquid medium at a ratio of 1:10 to obtain a diluted CCK-8 solution.

(9) Adding 100 mu L of diluted CCK-8 solution into each hole of the culture plate added with the medicine in the step (7) to obtain a culture plate added with the medicine CCK-8; in the step (7), 100 mu L of diluted CCK-8 solution is added into each hole of the 0-medicine-adding group culture plate to obtain a 0-medicine-adding group CCK-8 culture plate; adding 100 mu L of liquid culture medium into each hole of the hollow white tissue culture plate in the step (7) to obtain a blank control culture plate; (Care not to generate bubbles in the well, which would affect the OD reading).

(10) And incubating the drug-adding group CCK-8 culture plate, the 0 drug-adding group CCK-8 culture plate and the blank control culture plate in an incubator for 4 hours.

(11) And measuring the absorbance of each hole of the drug adding group CCK-8 culture plate, the 0 drug adding group CCK-8 culture plate and the blank control culture plate at 450nm by using an enzyme-labeling instrument.

The Kit is used for detecting the Cell proliferation-toxicity of a head and neck squamous Cell carcinoma Cell line by using a Cell Counting Kit-8, wherein the Cell Counting Kit-8 is called CCK-8 for short, and is a rapid high-sensitivity detection Kit which is based on WST-8 (chemical name: 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfobenzene) -2H-tetrazole monosodium salt) and is widely applied to Cell proliferation and cytotoxicity. The working principle is as follows: in the presence of an electron coupling agent, it is reduced by mitochondrial dehydrogenases to produce a highly water-soluble orange-yellow formazan product (formazan). The shade of color is proportional to the proliferation of cells and inversely proportional to cytotoxicity. OD value was measured at a wavelength of 450nm using a microplate reader, indirectly reflecting the number of living cells.

The activity was calculated, and the calculation results are shown in table 2:

cell viability (%) ([ a (dosed) -a (blank) ]/[ a (0 dosed) -a (blank) ] × 100

A (dosing): absorbance of the drug adding group hole; a (blank): absorbance of blank set wells; a (0 dosing): 0 absorbance of the wells of the kit.

TABLE 2

As can be seen from Table 2, the survival rates of the tonsil cancer cell lines, the non-small cell lung cancer cell lines and the liver cancer cell lines are gradually reduced along with the increase of the concentration of the blueberry anthocyanin, and the inhibition effect on the cell growth is gradually obvious, so that the growth of the tonsil cancer cell lines, the non-small cell lung cancer cell lines and the liver cancer cell lines is obviously influenced by the blueberry anthocyanin, and the inhibition effect and the concentration of the blueberry anthocyanin form a dose-dependent relationship. In contrast, prostate cancer cell lines are more sensitive to blueberry anthocyanin extract, and prostate cancer cells show high inhibition rate in low dose of blueberry anthocyanin pure extract and do not change with the change of anthocyanin dose.

And (4) conclusion: according to the apoptosis mechanism of HepG-2 cells, the blueberry anthocyanin extract has obvious cancer cell apoptosis inducing effect on tongue squamous cancer cell lines, non-small cell lung cancer cell lines, liver cancer cell lines and prostate cancer cell lines.