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Construction method for screening active components of phytoestrogen

A technology of phytoestrogens and active ingredients, which is applied in the field of screening active ingredients of Chinese herbal medicines, and can solve the problems of large drug consumption, difficulty in high-throughput screening, and high cost

Inactive Publication Date: 2012-01-11
XINJIANG TECHN INST OF PHYSICS & CHEM CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The method of the invention is simple, sensitive, specific, and low in dosage, and can be used for high-throughput screening of phytoestrogens active ingredients, effectively solving the problem of large drug consumption, high cost, and high cost of existing phytoestrogens active ingredients screening methods. Complicated operation, poor reproducibility of results, difficulty in meeting high-throughput screening, etc.

Method used

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  • Construction method for screening active components of phytoestrogen
  • Construction method for screening active components of phytoestrogen
  • Construction method for screening active components of phytoestrogen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1: The present invention is based on the construction of co-transfection-based phytoestrogen active ingredient screening method (using estrogen receptor agonist 17β-estradiol (17β-E2) and inhibitor tamoxifen (TAM) verify)

[0026] Construction of pERα expression vector

[0027] Take routinely cultured human breast cancer MCF-7 cells (density 1 × 10 7 cells / ml) 1ml (take 3 parts in parallel), and extract total RNA with a total RNA extraction kit (TRNzol Regent) purchased from China Tiagen Company ( image 3 ). Design upstream and downstream primers according to the full-length human estrogen receptor gene (hERcDNAα) sequence (GI: 18860919), and the upstream primer is 5'-ATTC GAATTC ATGTCCAGCCAGGTGGTGGGCATTG-3', the downstream primer is 5'-ATTC CTCGAG TCAGTCCATGGCCTCGAGCAT-3'. EcoR I and Xho I restriction enzyme sites were added to both ends of the primers, and 4 protective bases were added. The reverse transcription kit (Reverse Transcriptase M-MLV (Rn...

Embodiment 2

[0043] Embodiment 2 measures the estrogen effect of several phytoestrogens

[0044] Using 17β-E2 as a positive control, the estrogenic effects of several phytoestrogens such as formononetin, chickpea germanin A, and genistein were determined.

[0045] Co-transfect MCF-7 cells according to the ratio (mass ratio) of 10:1 by pERE-luc and pERα in Example 1. After 24 hours of transfection, phytoestrogens to be tested (1×10 -12 -1×10 -4 mol L -1 ) to treat the cells, and detect the activity of luciferase for 24 hours. The results are shown in Table 3:

[0046] Table 3. Luciferase activity assay of several phytoestrogens compounds

[0047]

[0048] As shown in Table 3: each phytoestrogen can cause the expression of luciferase, and there is a dose-effect relationship. Compared with the solvent control, formononetin, biochanin A, genistein and 17β-E2 The highest increase was 3.23±0.04 times (P<0.05), 4.81±0.15 times (P<0.05), 7.36±0.05 times (P<0.01) and 9.85±0.17 (P<0.05), res...

Embodiment 3

[0050] For the estrogenic effect of several phytoestrogens measured in Example 2, the estrogen receptor inhibitor ICI 182,780 was used to inhibit the verification of luciferase activity activated by phytoestrogens:

[0051] MCF-7 cells were co-transfected with pERE-luc and pERα in Example 1 at a ratio (mass ratio) of 10:1. After 24 hours of transfection, the maximum effect of 17β-E2, formononetin and genistein was given. At the same time, the specific inhibitor of estrogen receptor was given 1×10 -6 mol L -1 The ICI was 182,780, and the activity of luciferase was detected after 24 hours. The results are shown in Table 4:

[0052] Table 4. Effect of ICI 182,780 on phytoestrogen luciferase activity

[0053]

[0054] As shown in Table 4: the estrogen receptor inhibitor ICI 182,780 can inhibit the luciferase activity of phytoestrogens formononetin and genistein, further illustrating the specificity and sensitivity of this method for phytoestrogens screening sex.

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Abstract

The invention relates to a method for screening active components of phytoestrogen. In the method, cotransfection is taken as a base to transfect human breast cancer MCF-7 cells by an expression vector having an exogenous human estrogen receptor gene and a luciferase reporter gene vector having an estrogen response sequence (3*ERE (Estrogen Reaction Element)) according to a ratio; then a phytoestrogen sample to be detected is added to detect the expression of the luciferase and the expression of the luciferase is regulated and controlled by the ERE, thus the active components of the phytoestrogen can be screened. The method has higher specificity and sensibility on screening the active components of the phytoestrogen and can be widely used as a model for screening the active components of the phytoestrogen. The method provided by the invention is simple and flexible, is good in specificity and low in drug dosage, can be used for screening high flux of the active components of the phytoestrogen and effectively solving the problems that the drug dosage is great, the cost is low, the operation is complicated, the result repeatability is poor, the high-flux screening is difficult to satisfy and the like in the traditional method for screening the active components of the phytoestrogen.

Description

technical field [0001] The invention relates to a method for screening active ingredients of phytoestrogens, which is suitable for screening active ingredients of Chinese herbal medicines. Background technique [0002] Phytoestrogens are a class of plant-derived non-steroidal polyphenolic compounds that can bind to estrogen receptors to exert weak estrogenic effects. In the past ten years, studies have found that phytoestrogens have a significant effect on the prevention and treatment of estrogen-related diseases such as breast cancer, prostate cancer, cardiovascular disease, osteoporosis, and menopausal syndrome. Endometrial cancer and breast cancer caused by Xifen, Renoxifene, etc. Since 1999, when the NIH of the United States included artificially synthesized estrogens on the carcinogenic "blacklist", screening and extracting phytoestrogens active ingredients from plants has become the most popular demand. [0003] At present, the screening of phytoestrogens active ingr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/66C12Q1/02
Inventor 马海蓉魏华波曹旭阿吉艾克拜尔·艾萨
Owner XINJIANG TECHN INST OF PHYSICS & CHEM CHINESE ACAD OF SCI
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