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___________________________________________________________________________________________________________ National Formosa University Department of Biotechnology Service Commission Project Report   Project Number: 104AF105 Project Name: Anti-cancer activity evaluation of concentrated powder from Prunus mume fruit Requester (Institute): YOU DE LTD Implementation period: 2015/ 12/ 1 ~ 2016/ 2/ 28 Project Moderator: Dr. Yi-Ling Ye

Abstract

In this project, we evaluated the in vitro anti-colon cancer activity of P. mume samples provided by YOU DE LTD. We used two human adenocarcinoma WiDr and Caco-2 cell lines for research. First, diluted P. mume concentrated fruit powder into different concentrations (400 μg / ml, 800 μg / ml, 1200 μg / ml, 1600 μg / ml and 2000μg / ml) and used MTT assay to observe its cytotoxic effect on cancer cells. We found that higher concentration of P. mume concentrated fruit powder can kill more cancer cells. After data analysis, the IC₅₀ of P. mume for WiDr cell line is 1216.97 μg / ml while for Caco-2 cell line is 1243.39 μg / ml. The experimental results confirmed that P. mume concentrated fruit powder provided by YOU DE LTD has anti-cancer effect on WiDr and Caco-2 cells.   KEYWORDS: Anti-cancer activity, colon cancer, MTT assay, Prunus mume INTRODUCTION

I、Prunus mume：

Prunus mume (P. mume) is the deciduous trees of the rose family, sometimes also referred to as the fruit or flowers. P. mume originated in China, and it was later introduced to South Korea and Japan.[1] According to compendium of materia medica“Ben cao gang mu”, fruit of P. mume possesses various pharmacological activities. Wu et al., reported that a kind of ingredient named 5-hydroxymethylfurfural (HMF) in fruit of P. mume has anti–allergic effect,[2] other reports also showed that anti-oxidant activity [3] and anti-cancer activity by MK615[4,5] and C19H22O6[6] isolated from fruit of P. mume. It‟s indicate that P.mume not only has high ornamental value but also has effect on healthcare and pharmaceutical use.

1. Colon cancer：
2. Colon rectal cancer, also called colon cancer, is a cancer that originating in the colon or rectum (as part of the large intestine). Owing to the abnormal growth of cells in these tissue, it finally might infringe on or transferred to other areas of the body. Symptoms of colon cancer may include fecal blood, changes in bowel habits, weight loss, and fatigue. The majority etiology of colorectal cancer includes the lifestyle (diet, obesity, smoking, lack of exercise), aging, family history. Other risk factors include colon polyps and inflammatory bowel disease can also cause a high incidence of colorectal cancer.
3. MTT assay: value was measured spectrophotometrically. More live cells, more absorbance values obtained in final measurement.
4.            The MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay was the first homogeneous cell viability assay developed for a 96-well format that was suitable for high throughput screening (HTS) [7]. The MTT assay technology has been widely used for evaluation the viability of cells according to the mitochondria reductase activity. Through this enzyme activity, MTT (yellow color) is reduced to insoluble formazan (purple color) in living cells. After solubilize the insoluble formazan in DMSO, the O.D.
5. Specific aim of this study：Experimental design：The colonic cancer cell lines WiDr and Caco-2 were cultured with different concentration of concentrated P. mume fruit powder (400 μg/ml, 800 μg/ml, 1200 μg/ml, 1600 μg/ml and 2000 μg/ml). The inhibition of cell growth was measured by an MTT assay. The IC50 values of samples indicating 50% growth inhibition were estimated by an MTT assay. Data are expressed as the average of three analyses.

1. The colonic cancer cell lines WiDr and Caco-2 were cultured with different concentration of concentrated P. mume fruit powder (400 μg/ml, 800 μg/ml, 1200 μg/ml, 1600 μg/ml and 2000 μg/ml). The inhibition of cell growth was measured by an MTT assay. The IC₅₀ values of samples indicating 50% growth inhibition were estimated by an MTT assay. Data are expressed as the average of three analyses.
2. The concentrated P. mume fruit powder provided by YOU DE LTD was requested to evaluate the anti-colonic cancer activity in this project. Although many data showed that P. mume had killing effect on different cancer cell type, but, nobody did experiment about P. mume fruit killing colonic cancer cells at present. That‟s why we used the concentrated P. mume fruit powder to do this experiment.

Experimental materials：

1. Experimental equipment：Thermostatic circulator                                 (Digisystem,Germany)CO₂ Incubators                                      (Heal force,Hong Kong)
2. UV/VIS Spectrophotometer                                       (Metertech)
3. Microscope                                                 (Nikon,Japan)
4. Centrifuge                               (5922 Microprocessor,Kubota, Japan)
5. Consumable material：10ml pipette                                                 (Falcon,USA)III、Experimental Drugs：Antibiotic                                       (Biological Industries,Israel)Dimethyl sulfoxide (DMSO)                                     (Sigma,USA)Glucose                                            (Riedel-de Haën,Seelze)Human transferrin                                             (Sigma,USA)Minimum Essential Medium (MEM)                               (Sigma,USA)Soudium Bicarbonate                                   (Riedel-de Haën,Seelze)Trypan blue                                       (Biological Industries,Israel)
6. Concentrated P. mume fruit powder                   (provided by YOU DE LTD)
7. Soudium Pyruvate                                 (Biological Industries,Israel)
8. Non-essential amino acid (NEAA)                     (Biological Industries,Israel)
9. L-glutamin                                       (Biological Industries,Israel)
10. HEPES                                           (PanreacAppliChem,USA)
11. Fetal bovine serum (FBS)                           (Biological Industries,Israel)
12. Dulbecco‟s Modified Eagle‟s Medium(DMEM)                     (Sigma,USA)
13.           3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)           (Sigma,USA)
14. 15、50ml Centrifuge tube                                      (Falcon,USA)
15. Cryogenic Vial                                           (Corning® ,Mexico) hemocytometer                                        (Marienfeld,Germany) 96well                                                 (Nunc^Tm,Denmark)

Methods：

The human colon cancer cell lines was purchased from Food Industry Research and Development Institute (FIRDI) in Hsinchu, Taiwan. Caco-2 cells (BCRC Number: 60182) and WiDr cells (BCRC Number 60157) are human colorectal adenocarcinoma cell lines. WiDr cell is the goblet cells HT-29 cell-derived cells. Both of these two cells are adherent cells. WiDr cell and Caco-2 cell were cultured in MEM and D-MEM medium, supplemented with 10% fetal bovine serum. The medium was changed once two days. Concentrated P. mume fruit powder stock solution (5000 μg / ml) was diluted in medium ranged from concentration 400 μg / ml, 800 μg / ml, 1200 μg / ml, 1600 μg / ml to 2000 μg / ml. After 100 μL of each cell culture was placed in a 96 well plate at a concentration of 2×10⁴ cells/mL, cells were cultivated in a 5% CO₂ incubator at 37 °C for 24 h. Then the medium was aspirated, and the cells were co-cultured with different concentration of concentrated P. mume fruit powder for 24 hours. After 24 hours, medium was removed, MTT reagent was added. After 4 hours incubation, insoluble crystallizes dissolved in DMSO. The absorbance values (O.D.) of each well was measured.

Results

Fig 1. The cell cytotoxicity effect of concentrated P. mume fruit powder on WiDr cells MTT assay was performed on WiDr cells after 24 h treatment with increasing concentrations of concentrated P. mume fruit powder solution. Using the medium treated only as 100% cell viability, the cell survival % by different concentration of concentrated P. mume fruit powder was calculated. Experiments were performed three times Fig 2. The cell cytotoxicity effect of concentrated P. mume fruit powder on WiDr cells The cell cytotoxicity percentage by different concentration of concentrated P. mume fruit powder solution was calculated. Data are presented as the average of three independent experiments. *P<0.05 and ***P<0.01 for comparisons between the medium treated control group (CTL) versus that in the presence of different concentration of concentrated P. mume fruit powder solution. Fig 3. The cell cytotoxicity effect of concentrated P. mume fruit powder on Caco-2 cells MTT assay was performed on Caco-2 cells after 24 h treatment with increasing concentrations of P. mume fruit powder solution. Using the medium treated only as 100% cell viability, the cell survival % by different concentration of concentrated P. mume fruit powder was calculated. Experiments were performed three times.   Fig 4. The cell cytotoxicity effect of concentrated P. mume fruit powder on Caco-2 cells The cell cytotoxicity percentage by different concentration of concentrated P. mume fruit powder solution was calculated. Data are presented as the average of three independent experiments. *P<0.05 and ***P<0.01 for comparisons between the medium treated control group (CTL) versus that in the presence of different concentration of concentrated P. mume fruit powder solution.

DISCUSSION：

In our study, higher the concentration of concentrated P. mume fruit powder solution can kill more cancer cells. After data analysis, IC₅₀ of concentrated P. mume fruit powder provided by YOU DE LTD on WiDr cell lines is 1216.97 μg / ml while IC₅₀ on the Caco-2 cell lines is 1243.39 μg / ml. Sunaga et al., reported that C₁₉H₂₂O₆ (2-hydroxy-1- [(7-hydroxy-2-oxo- 2H-chromen -6-yl) methyl]- 2-methylpropyl- (2Z)-3-methyl-but- e-enoate: prunate) is a compound extracted from P. mume fruit. It can kill four kinds of cancer cell, HEP-2, SKOV3, SW-156 and HEC-1-B. Experimental results showed C₁₉H₂₂O₆ inhibit 81-96% of cancer cells by concentration of 100 μg / ml , its IC₅₀ ranged between 39 to 58 μg / ml/.[4] Concentrated P. mume fruit powder used in our study is a mixture, further analyze the anti-cancer active components within it and mechanism for anti-cancer effect is needed in the future.

References：

[1].Ma Jiachi,Chen Quan,Gu Yuanhui, Li Yiping, Fang Wei, Liu Meiling,Chen Xiaochang, Guo Qingjin, Ma Shixun,(2012) Effect of colon cancer cell-derived IL-1α on the migration and proliferation of vascular endothelial cells. Zhonghua Zhong Liu Za Zhi. 2015 Nov;37(11):810-5. [2].Wu SF, Chen YH, Lin CL, Yang JC, Du YC, Lee JJ, Wu YC and Chang FR, (2011) Qualitative and Quantitative Analyses of the Anti-Allergic Constituent of Commercial Prunus mume Products in Taiwan. Journal of Food and Drug Analysis, 19, 66-72. [3].Jiayi Shi,Jinyan Gong,Ji‟er Liu,Xiaoqin Wu,Ying Zhang, (2009) Antioxidant capacity of extract from endible flowers of Prunus mume in China and its active compenents, LWT-Food Science and Technology, 42, 477-482. [4].Sunaga N, Hiraishi K, Ishizuka T, Kaira K, Iwasaki Y, (2011) MK615, A Compound Extract from the Japanese Apricot “Prunus mume” Inhibits In vitro Cell Growth and Interleukin-8 Expression in Non-small Cell Lung Cancer Cells. J Cancer Sci Ther S11:002. doi:10.4172/1948-5956.S11-002 [5].Tada KI, Kawahara KI, Matsushita S, Hashiguchi T, Maruyama I, Kanekura T, (2012) MK615, a Prunus mume Steb. Et Zucc („Ume‟) Extract, Attenuates the Growth of A375 Melanoma Cells by Inhibiting the ERK1/2-Id-1 Pathway. Phytother Res. 26, 833-8. [6].Jeong JT, Moon JH, Park KH, Shin CS, (2006) Isolation and Characterization of a New Compound from Prunus mume Fruit that Inhibits Cancer Cells, J.Agric. Food Chem. 54, 2123-2128 [7] Riss TL, Moravec RA, Niles AL, Benink HA, Worzella TJ, and Minor L, (2013) Cell Viability Assays Assay Guidance Manual; Last Update: June 29, 2015.