Study On The Inhibitory Effect Of β, β-Dimethacryloyl Shikonin On The Expression Of EGFR In Lithospermum Erythrorhizon L. Based On Capillary Electrophoresis Microfilter

Objective:This study was based on the previous experiment of using enzyme micro-reactor screen effective compounds from Chinese medicine in our laboratory. The human epidermal growth factor receptor (EGFR) was immobilizeded on the inner of the capillary wall as the molecular target. The capillary electrophoresis EGFR micro-reactor was prepared to study the inhibition of Arnebia euchroma extractive β,β’-dimethylacrylshikonin for EGFR autophosphorylation. It was dedicated to develop a rapid method to research the active ingredient in anticancer herb.Method:1. An EGFR micro-reactor was prepared by immobilizing the enzyme on the capillary wall with covalent bonding method. To verify the effect of enzyme immobilization, ATP was selected as the substrate and bare tube as the blank.2. The impact of different incubation time on yield of the enzyme was tested to determine the best incubation time.3. To determine the correlation between the concentration and the peak area, ATP and ADP standard sample were selected as the substrate and product.4. The positive drug Gefitinib was selected as the positive control group. The IC50 of Gefitinib to EGFR was calculated to test the accuracy of this method.5. Five different concentrations of ATP and two different concentrations of Arnebia euchroma extractive β, β’-dimethylacrylshikonin were selected and mixed respectively. The background buffer replaced β, β’-dimethylacrylshikonin was used as controll. According to the results and the Michaelis-Menten equation, the Michaelis-constant(Km) was calculated and the inhibition type was determined by using the double reciprocal plot.6. Five different concentrations of Arnebia euchroma extractive β, β’-dimethylacrylshikonin were selected and mixed with ATP as the five samples. Then each inhibition rate was measured three times. The inhibition rate curve was fitted and the IC50 was calculated based on the experimental results.7. Gefitinib was mixed with β, β’-dimethylacrylshikonin and ATP, then injected into the capillary enzyme micro-reactor to obvious the effect of combining Gefitinib with β, β’-dimethylacrylshikonin.Result:1. An active EGFR micro-reactor was built.2. lmin was chosen as the experimentally incubation time.3. When ATP concentration between 0.01mmol/L to 1mmol/L,the linear relation between concentration and peak area of ATP were good. Y=1.2E+5x+1956.8, R2=0.9967. ADP has good linear relationship between 0.12mmol/L-1.17mmol/L. Y=3.7E+5x-7428.7, R2=0.9985.4. Comparing the positive drug Gefitinib group with blank group. According to the inhibition rate of different concentrations of inhibitors, The S curve was fitted by Graphpad. From the curve, we obtained that the half maximal inhibitory concentration of Gefitinib was 39.80μmol/L.5. Two different concentrations of β, β’-dimethylacrylshikonin with five ATP concentrations were mixed each other. While replacing β, p’-dimethylacrylshikonin with background buffer to mixed with ATP as the control group. According to the experimental results of three groups, the Km of control group was calculated to be 0.4366mmol/L by using the double reciprocal plot. Other two groups which add inhibitor have similar Km and smaller Vmax. So β, β’-dimethylacrylshikonin may be a noncompetitive inhibitor.6. According to the inhibition rate results of different concentration of inhibitor, inhibition ratio S curve was fitted. We obtained the IC50 of β, β’-dimethylacrylshikoninis 77.08μmol/L.7. Gefitinib was mixed with β, β’-dimethylacrylshikonin and ATP, then introduced them into the enzyme micro-reactor. The results showed that, compared with no addition of β, β’-dimethylacrylshikonin, the consumption of Gefitinib was reduced and the inhibition was not weakened.Conclusion:A CE EGFR enzyme micro-reactor method was established to study the inhibition of Herbal Extracts β, β’-dimethylacrylshikonin for EGFR.It is simple and reliable, and can be widely used in study active component of medicine herbal extractive.