CUSTOMERS VENTURE SDN BHD Certificate No : AMC/nU1604/033 Lots.3A.010 & 3A.009 & 3A.011 & 3A.015, Sample Log Code : nu1604/022 4th Floor,Endah Parade, Sample Received Date : 19-Apr-2016 No.1,Jalan 1/149E,Taman Sri Endah, Issuance Date : 15-Jun-2016 Bandar Baru Sri Petaling, 57000 Kuala Lumpur. Tel: 03-9543 0392 Fax : Attention : Mr.Dickson Cheng Sample Description: Cure Liquefaction Silver 10ppm Water (14.04.2016) Test Summary: It is a cell-based colorimetric assay and a quantitative method to determine the cell viability. Yellow tetrazolium MTT is reduced to purple formazan by NADH released from mitochondrial activity in viable cells. Absorbance values of samples will be measured by ELISA reader. Objective: To measure the in-vitro cytotoxic effect of samples on breast cancer cell lines MCF-7 and MDA-MB-231. Page 1 of 8
Test procedures: Harvest the MCF-7 cells (Human Breast Adenocarcinoma) and MDA-MB-231 (Human Caucasian Breast Adenocarcinoma) Plate the cells in 96 well plate. Incubate overnight. Treatment of cells with water sample. Incubate the treated cells for 24 hours. Add MTT Reagent to each wells. Incubate for 4 hours. Remove media from each well. Resuspend the cells with DMSO. Obtain absorbance value from ELISA Reader. Result: MCF-7 Control Before Treated Page 2 of 8
MCF -7 After Treated with Silver Water Page 3 of 8
MDA Control Before Treated Page 4 of 8
MDA After Treated with Silver Water Page 5 of 8
Viability of cells (%) AMCEN LAB SDN BHD (1070971-W) Table : Viability of Cancer Cell (MDA-MB-231 & MCF-7) Versus Concentration of Silver Water 100 90 80 70 60 50 40 MDA-MB-231 MCF7 30 20 10 0 0 1 2 3 4 5 6 7 8 9 10 concentration (10ppm/ml) MDA OD1 OD2 OD3 OD average STDEV 10ppm 5.369128 6.617647059 5.594406 5.86039339 0.665403932 5ppm 10.73826 11.02941176 11.88811 11.2185929 0.597816712 2.5ppm 12.75168 16.91176471 14.68531 14.78291908 2.081760222 1.25ppm 18.79195 17.64705882 18.18182 18.2069411 0.572857058 0.625ppm 17.44966 16.91176471 18.18182 17.51441577 0.637497851 0.3125ppm 28.85906 40.44117647 36.36364 35.22129108 5.874952571 0.156ppm 85.90604 85.29411765 87.41259 86.20424844 1.090263557 0.078ppm 85.2349 88.23529412 86.01399 86.49472649 1.556896199 0.0390ppm 89.93289 97.79411765 91.60839 93.11179839 4.140641868 Control 100 100 100 100 0 Page 6 of 8
MDA OD average STDEV 10ppm 5.86039339 0.665404 5ppm 11.2185929 0.597817 2.5ppm 14.78291908 2.08176 1.25ppm 18.2069411 0.572857 0.625ppm 17.51441577 0.637498 0.3125ppm 35.22129108 5.874953 0.156ppm 86.20424844 1.090264 0.078ppm 86.49472649 1.556896 0.0390ppm 93.11179839 4.140642 MCF OD1 OD2 OD3 OD average STDEV 10ppm 0.779220779 1.123595506 1.137980085 1.01359879 0.203104697 5ppm 1.948051948 2.528089888 2.275960171 2.250700669 0.290842801 2.5ppm 2.337662338 2.668539326 2.560455192 2.522218952 0.168719895 1.25ppm 2.467532468 2.247191011 2.987197724 2.567307068 0.379958829 0.625ppm 2.727272727 3.230337079 3.556187767 3.171265858 0.417602793 0.3125ppm 10.51948052 7.865168539 7.254623044 8.546424034 1.735772269 0.156ppm 39.48051948 44.38202247 48.79089616 44.2178127 4.657359994 0.078ppm 53.50649351 55.33707865 49.50213371 52.78190196 2.984195083 0.0390ppm 71.94805195 74.71910112 74.25320057 73.64011788 1.483772614 Control 100 100 100 100 0 MCF OD average STDEV 0.0390ppm 73.64011788 1.483772614 0.078ppm 52.78190196 2.984195083 0.156ppm 44.2178127 4.657359994 0.3125ppm 8.546424034 1.735772269 0.625ppm 3.171265858 0.417602793 1.25ppm 2.567307068 0.379958829 2.5ppm 2.522218952 0.168719895 5ppm 2.250700669 0.290842801 10ppm 1.01359879 0.203104697 Page 7 of 8
Conclusion: The testing on silver water against two breast cancer cells i.e MDA-MB-231 and MCF7 have been successfully done and the solution inhibited both cells with IC50 values of IC 50= 0.3ppm (for MDA) and IC50=0.088 ppm (For MCF7). The results of the present study showed potent cytotoxic effects on MCF-7 cells and MDA-MB-231. The criteria of cytotoxicity for the crude extract, as established by the U.S. National Cancer Institute (NCI), is an IC 50 < 20 ppm in the preliminary assay. Yeow Liang Ming General Manager B. Sc., M.Sc., M.B.A. M1737/4047/2000 Page 8 of 8