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Four kinds of licorice in human breast cancer cell proliferation and induce apoptosis Comparative Study_847

By Troy Gordon,2014-10-30 17:59
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Four kinds of licorice in human breast cancer cell proliferation and induce apoptosis Comparative Study_847

    Four kinds of licorice in human breast cancer cell proliferation and induce apoptosis Comparative Study

     Abstract Objective To compare the active ingredient in Xinjiang four kinds of wild licorice on human breast cancer cell proliferation with a view to filter out the

    pharmacodynamics of the best types of licorice. Methods MTT assay in different samples of the BCAP 37 cell

    proliferation, Hoechst33258 fluorescence staining of the samples BCAP 37 cells induced apoptosis. The results of

    four kinds of active ingredients of licorice in human breast cancer BCAP 37 cells, has significant growth inhibition and induction of apoptosis, and there are significant differences between species and origin. Conclusion The best anti-cancer

    efficacy of Alar licorice licorice species.

     Key words Glycyrrhiza active ingredient of human breast cancer cells, BCAP 37

     Comparative Study on the Anti-proliferation and

    Apoptosis-inducing Effects of Active Components from Four Glycyrrhiza Species on Human Breast Cancer BCAP-37 Cell

     Abstract: ObjectiveTo select the optimal glycyrrhiza species for medicinal utilization by comparing the anti-

    proliferation and apoptosis-inducing effects of active

    components from four wild glycyrrhiza species in Xinjiang on

    human breast cancer BCAP 37 cell. MethodsThe anti-

    proliferation effects of different samples on BCAP 37 cell

    were measured by MTT assay. The cell apoptotic rate was detected by Hoechst 33258 stain.ResultsActive components of four Glycyrrhiza species inhibited the proliferation of BCAP

     37 cells and induced apoptosis significantly. What's more, there were significant interspecific and spatial variations in the anti-proliferation and apoptosis-inducing effects.

    ConclusionThe optimal Glycyrrhiza species for anticancer

    utilization is Glycyrrhiza alalensis L.

     Key words: Glycyrrhiza; Active Component; Human breast cancer BCAP 37 Cell

     Licorice is a very important traditional Chinese medicine in China, known as "10 square nine grass" in the world. In

    recent years, with the development and utilization of the continuous deepening of licorice, licorice is widely used in medicine, food, health care products, cosmetics and other fields [1], licorice sharp increase in market demand. China is

    the world's exporting countries licorice, of which 80% of the wild licorice, only 20% of cultivated licorice. Chinese licorice reserves dropped from the founding of New China 200 ~ 2.5 million tons down to 50 ~ 700 thousand tons [2]. The existing wild liquorice resources have been unable to meet the growing market demand, therefore, imperative that artificial cultivation of licorice, wild licorice varieties of high-

    quality screening is particularly urgent. Quality level of licorice for its efficacy is ultimately measured by their

    predecessors only active ingredient content as an indicator of the quality of research conducted licorice is one-sided [3 ~

    7].

     Licorice most important active ingredients of two major categories of licorice flavonoids and glycyrrhizic acid all

    have anti-inflammatory, anti-virus and anti-tumor effect

    [8,9], the anti-tumor activity of licorice, particularly the attention of scholars home and abroad. Breast cancer is the most common malignancy of women worldwide. Recent report, the

    U.S. breast cancer incidence rate has risen to 1 / 7, while in our country over the past 10 years, breast cancer incidence rose by 27%, breast cancer has become a threat to women's lives and health, the number one killer of [10] . Therefore, this article studied the Xinjiang Glycyrrhiza inflata Glycyrrhiza inflata Batal., Glycyrrhiza glabra G. glabra L., Arar licorice G. alalensis L. and yellow liquorice G. eurycarpa L. other four kinds of wild licorice water extract of liquorice and liquorice flavonoids BCAP 37 pairs of

    human breast cancer cell proliferation with a view to filter

out the best licorice cultivation types of efficacy for high-

    quality large-scale artificial cultivation of liquorice in laying the foundation for the development of new drugs against

    cancer licorice to provide scientific basis.

     1 Equipment

     1.1 The cell line of human breast cancer cell lines BCAP

     37 by the Chinese Academy of Sciences Shanghai Institute of Cell Biology, Cell Bank provides.

     1.2 experimental samples of the samples sampling sites

    and sample names in Table 1. All samples were Shihezi University, Mr. Li Xueyu identified as the real thing. Table 1 Sampling sites and sample name (abbreviated)

     1.3 Reagents and Instruments

     1.3.1 Reagents RPMI1640 medium, Gibco, Inc.; calf serum, Hangzhou Sijiqing Biological Engineering Materials Co., Ltd.; HEPES, China and the United States bio-engineering company;

    trypsin, Shanghai Science and Technology Development Co., Ltd. Blue quarter; 4 thiazolyl blue (MTT), 2 methyl sulfoxide

    (DMSO), Sigma Company; cis-platinum (DDP), Jiangsu Stockhausen Pharmaceutical Co., Ltd.; Hoechst33258 staining kit, Pik-day

    Biotechnology Institute.

     1.3.2 Instrument CO2 incubator, the United States (Forma); Metertech Σ960 automated microplate reader;

    fluorescence microscope, OlympusBX51; Clean Benches, Shanghai purification equipment plant; blood cell count boards, Shanghai Medical Optical Instrument Factory.

     2 Methods

     2.1 The acquisition and processing experimental samples

    collected strains of the same age (the same root thickness) of four kinds of licorice roots material, immediately placed in 80 ? oven drying to constant weight, and then use herbs grinder be crushed into powder, over 100 mesh sieve, spare.

     2.2 Preparation of active ingredient Licorice

     2.2.1 licorice root aqueous extract accurate and that

    taking 1.0 g Licorice root powder plus 10 ml distilled water at room temperature for 48 h, 12 000 r / min centrifugation 10 min, 0.22 μm microporous membrane filter sterilization, plus sample before use cell culture medium According to the literature [11] preparation of different samples containing glycyrrhizic acid for 1 200 μg / ml of the tested samples.

     2.2.2 Licorice Flavonoids Weigh accurately 1.0 g licorice

    root powder, with 250 ml absolute ethanol plus 5 drops of concentrated hydrochloric acid solution as the extracting agent in the Soxhlet extractor in a water bath heated at 95 ?

    under the conditions of extraction 6 h, concentrated to 30 ml,

     0.22 μm microporous membrane filter sterilization, plus samples of cell culture medium according to previous literature [11] with different sample preparation of total flavonoids of 50 μg / ml of the tested samples.

     2.3 Cell culture BCAP-37 cells containing 10% inactivated fetal calf serum RPMI1640 medium, by adding NaHCO3 2 g / L, glutamine 0.24 g / L, Hepes 2.4 g / L, penicillin 100 U / ml, chain mycin 100 mg / ml, placed in 37 ?, 5% CO2 incubator box

    training. Experimental cells were in logarithmic growth phase, conventional Taiwan phenol blue counting of living cells is greater than 95%.

     2.4 cell proliferation inhibition assay (MTT) will be in the logarithmic phase of the BCAP 37 cells were treated

    with trypsin digestion, made of 2.0 × 105A/ ml of cell

    suspension, and then 100 μl per well to 96 well plates

    inoculated , in 37 ?, 5% CO2 incubator box replaced after 24 h in culture medium, the experimental group of subjects with different samples of 100 μl, final concentrations were as

    follows: water extract of Glycyrrhiza glycyrrhizin concentration of 600 μg / ml, Licorice Flavonoids 25 μg /

    ml, each sample are equipped with eight parallel holes, the positive control group to join with cisplatin 100 μl: the

    final concentration of 50 μg / ml, the blank control group,

    adding an equal volume of growth medium. To continue to develop 24 h, 48 h and 72 h, each hole by adding 20 μl MTT

    solution (concentration of 5 mg / ml), light-vibration culture

    plate, into the incubator in a further 4 h incubation, the

    exhaustion supernatant, for each hole plus DMSO 150 μl,

    oscillator oscillation 10 min, with the microplate reader to detect the hole absorbance (OD) determination of the

wavelength λ = 570 nm. Inhibition rate calculated as follows:

    inhibition rate (%) = (OD value of the control group - OD

    value of experimental group) / OD value of the control group

    × 100%.

     2.5 apoptosis rate experiment to take ordinary clean coverslip immersed in 70% ethanol 20 min, 0.9% NaCl solution, washed three times, and then washed a second cell culture medium. The coverslip placed in six-well plates, the species

    into the cells, cultured overnight, so that about 50% ~ 80% full. Add licorice water extract and Licorice Flavonoids and continue to train 48 h, exhaustion culture medium by adding 0.5 ml fixative and fixed 10 min. To the stationary phase, washed with 0.9% NaCl twice, 3 min / times, exhaust liquid, washing with the shaker. Add 0.5 ml Hoechst33258 Staining, dyeing and 5 min, also used the shaker. Washed with 0.9% NaCl

    twice, 3 min / times. Anti-drip drop of liquid on the

    fluorescence quenching Fengpian glass slide, covered with coverslip affixed cells try to avoid air bubbles. Fluorescence microscope (Ex350nm, Em460nm) randomly selected five of its

    field of vision pictures of apoptotic cells counted after the normal cells, apoptosis rate = apoptotic cells / (apoptotic

    cells + normal cells) × 100%.

     2.6 Statistical analysis All data were obtained by the experimental software processing SPSS12.0 between treatment

    group and one-way ANOVA, P

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