QAMS For Determination Of Lactones In Andrographis Paniculata(Burm.f.)&Antitumor Activities Of Components Compatibility

Objective:To develop a quantitive analysis multi-components by single marker (QAMS) method for simultaneous determination Lactones and measure the content of total flavonoid in andrographis extract. We found out the effective components and optimal combination of active components from effective components and compound compatibility of the vitro anti-tumor activity.Method:To establish HPLC method in analysis on andrographis extract for the simultaneous determination of6main constituents (A,GDA, DOA, NA, DA and DDA). Using DDA as an indicator, a relative correction factor (RCF) between other components was established, respectively then to calculate the amount of each component. At the same time, the external reference method and regression equation method were used to determine the amounts of the above6constituents, to compare the difference between the calculated and validate the correctness and adaptability of QAMS. The RCF, ATR and RTR targeted accuracy were explored on different bands of chromatographic column and instrument. Meanwhile NaNO2-Al(NO3)3-NaOH was used as the colour system. UV visible spectrophotometry method was employed for the determination of flavonoid in andrographis extracts. The extracts and effective components of andrographis in4T-1and LLC rats tumor cell lines was measured by MTT assay to find out effective components, we discovered the active ingredient of antitumor by comparing andrographis ethanol extracts and effective components. A component-Knockout approach was applied to discover active components of effective components of AP for the synergistic effects on its antitumor activity. The optimal combination of active component from effective components of AP was studied by a simplex lattice design. The IC50level was used to evaluate the antitumor effect of component mixture.Results:1.The RCF of A, GDA, DOA, NA and DA had a good reproducibility (RSD<5%), the values of RCF were1.392,0.308,0.337,1.175,1.757. It is accurately determined peak position of target components combined RTR with internal standard△TR and peak shape. The analysis results have no statistically significant differences (p=95%, n=6) between QAMS and external method of six groups of the ethanol extract of andrographis. The A、GDA、DOA、NA、DA and DDA content of Andrographis paniculata were2.15%,0.21%,0.32%,0.72%,0.26%and0.77%; The A, GDA, DOA, NA, DA and DDA content of andrographis ethanol extract were5.81%,0.85%,0.86%,2.91%,1.46%and2.92%; The A, NA, DA and DDA content of andrographis effective components were45.38%,3.62%,8.08%and16.66%.2.The total flavonoids in andrographis extracts were determined by spectrophotometry, the regression equation is A=10.9110C+0.0064, r=0.9995, test range:17.00-82.10μg/mL, The average recovery was101.14%with the RSD of3.16%. The total flavonoid content of andrographis, ethanol extract and effective components were1.32%,15.05%and1.55%.3.The IC50of andrographis effective components are26.91±3.33μg/mL,44.16±4.28μg/mL in4T-1and LLC rats tumor cell lines. The IC50of andrographis ethanol extract are more than100μg/mL.The results in4T-1rats tumor cell lines of a component-knockout approach indicated that the IC50of andrographis effective components, all the compatibility group and A group are26.91±3.33μg/mL,46.64±6.24μg/mL and29.38±3.65μg/mL; The IC50of the group of compatibility Ⅱ, Ⅲ and IV are52.66±5.90μg/mL,43.22±2.52μg/mL and49.84±4.88μg/mL. The results in LLC rats tumor cell lines of a component-knockout approach indicated that the IC50of andrographis effective components, all the compatibility group and A group are44.16±4.28μg/mL,51.46±5.21μg/mL and40.80±1.00μg/mL; The IC50of the group of compatibility II and III are50.59±1.26μg/mL and63.64±2.46μg/mL. The optimal combination of A and DDA(12:1) was study by a simplex lattice design. The IC50of the optimize combination group was32.69±2.07.Conclusion:The method for simultaneously evaluating the six Lactones of A, GDA, DOA, NA, DA and DDA in andrographis extract is reliable and accurate, which could be used to control the quality of andrographis extract; the method is simple, reproducible and suitable for the determination of total flavones. The antitumor effect of andrographis effective components was remarkable which Andrographis diterpenes is the main active ingredients. The results a of component-knockout approach showed that andrographolide was the effective components of AP. The compatibility of andrographolide and dehydroandrographolide (12:1) will generate better efficacy of the other compatibility groups. The study resulted the major effective substances in effective components of AP towards LLC and4T-1anti-tumor effects.