1. Secondary metabolite mapping identifies Scutellaria inhibitors of human lung cancer cells Jiayu Gao, Huiying Zhao, Peter J. Hylands, Olivia Corcoran Journal of Pharmaceutical and Biomedical Analysis, In press, 2010

2. Introduction Scutellaria baicalensis Georgi root, used as a adjuvant in conventional chemotherapy. Quality of herbs, wide variability in quality amongst commercial products of identical S. baicalensis and claimed formulation. Functional biomarkers, chemical constituents shown to have in vitro activity relevant to therapeutic use. Baicalin, baicalein, wogonin, and oroxylin A, functional biomarkers of anti-cancer activities against a wide range of cancer cells except lung cancer. HPLC and NMR secondary metabolite model for functional biomarker identification.

3. Method Metabolic profile (chemical fingerprint), generated by HPLC and NMR spectroscopic data. Principal component analysis (PCA) and Partial least squares analysis (PLS) PCA, a tool for outlier detection and discerning patterns and trends in multidimensional data. PLS, a model that describes the correlation between the data matrix and the feature of interest.

4. Preparation of extracts and MTT test 25% EtOH45% EtOH70% EtOH 100% EtOH 100% MeOH Methylene Chloride Ethyl Acetate ButanolWater

5. Initial analysis of HPLC and NMR data ？ ？

6. HPLC-PCA map IC 50 >124ug/ml 58<IC 50 <124ug/ml IC 50 <58ug/ml

7. Loading and contribution plot

8. NMR-PCA map IC 50 >124ug/ml 58<IC 50 <124ug/ml IC 50 <58ug/ml

9. Contribution plot Baicalin Baicalein Wogonin

10. PLS prediction of inhibition by S. baicalensis crude extracts ExtractIC 50 measured value (µg/ml) IC 50 predicted by NMR model (µg/ml) IC 50 predicted by HPLC model (µg/ml) a: 25% aqueous ethanol249.47165.27199.03 b: 45% aqueous ethanol175.35199.67160.51 c: 70% aqueous ethanol82.46124.41100.21 d: ethanol57.1833.9895.79 e:dichloromethane42.2387.6843.87 f: ethyl acetate82.7660.3294.08 g: butanol107.11101.7545.34 h: water123.59165.70190.78

11. Validation of Scutellaria inhibitors A: baicalin B: baicalein C: wogonin

12. Conclusion Baicalin, baicalein and wogonin are determined as functional biomarkers for S. baicalensis inhibition on human lung cancer cells. Secondary metabolite mapping quickly identified functional compounds from crude plant extract. This approach can be used as an important first step to qualify herbal prescriptions. The potential of NMR/HPLC-PLS analysis as a tool for the prediction of growth inhibitory activities of plant extracts.