1. School of Pharmaceutical Science, Peking University ( 北京大学药学院 )

1. Studies on Chromatography Fingerprint and Quality Evaluation of Radix Astragali and its Germplasm Resources (黄芪及其种质资源的指纹图谱与质量评价研究) 1. School of Pharmaceutical Science, Peking University (北京大学药学院) 2. School of Chinese Medicine, Hong Kong Baptist University (香港浸会大学中医药学院) Liu Jing (刘靖)1, Zhao Yuying (赵玉英)1, Chen Hubiao (陈虎彪)1,2

2. Introduction • Radix Astragali, knows as “Huangqi” in China, is one of the most popular herbal medicines known worldwide to reinforce “qi”. • Radix Astragali is prepared from the dried roots of Astragalus membranaceus (Fisch.) Bge and Astragalus membranaceus (Fisch.) Bge var. mongholicus (Bge) Hsiao recorded in China Pharmacopoeia (2005 edit). • Radix Astragali has been proved to be an immunostimulant, hepatoprotective, diuretic, antidiabetic, analgesic, expectorant, and sedative drug. • The constituents most often associated with the activity of Radix Astragali are isoflavonoids, saponins, polysaccharides, γ-aminobutyric acid (GABA), and various trace elements. Astragaloside Ⅳ is normally used as a marker for quality control.

3. Our Work • Germplasm Resource Investigation and Collection of Radix Astragali. • Established Chromatography Fingerprint of flavonoids in Radix Astragali by HPLC-UV. • Quality Evaluation : Determined four isoflavonoids and Astragaloside Ⅳ in Radix Astragali by HPLC of 37 batches of A. membranaceus var. mongholicus and 12 batches of A. membranaceus（Fisch.）Bge. from different cultivated places. • Made a chemical comparison with different harvest time, different age of cultivated sources, different commercial specification, and xylem& phloem of A. membranaceus var. mongholicusby using the method above.

4. Our Work • Germplasm Resource Investigation and Collection of Radix Astragali. • Established Chromatography Fingerprint of flavonoids in Radix Astragali by HPLC-UV. • Quality Evaluation : Determined four isoflavonoids and Astragaloside Ⅳ in Radix Astragali by HPLC of 37 batches of Astragulusmembranaceus var. mongholicus and 12 batches of A. membranaceus from different cultivated places. • Made a chemical comparison with different harvest time, different age of cultivated sources, different commercial specification, and xylem& phloem of A. membranaceus var. mongholicusby using the method above.

5. Germplasm Resource Investigation and Collection • From July, 2005 to September, 2005, we made the germplasm resource investigation of Radix Astragali. • Collected 94 batches of A. membranaceus var. mongholicus and 17 batches of A. membranaceus（Fisch.）Bge. from different places in China.

6. Astragulus membranaceus var. mongholicus Astragulus membranaceus

7. Collection of Astragulus membranaceus var. mongholicus

8. Collection of Astragulus membranaceus var. mongholicus

9. Collection of Astragulus membranaceus var. mongholicus

10. Collection ofAstragulus membranaceus var. mongholicus

11. Collection ofAstragulus membranaceus var. mongholicus

12. Collection ofAstragulus membranaceus var. mongholicus

13. Collection of Astragulus membranaceus

14. Collection of Astragulus membranaceus

15. Germplasm Resource Investigation and Collection • Astragulusmembranaceus var. mongholicus was widely cultivated mainly in Neimenggu, Shanxi and Gansu Province. • A. membranaceus was distributed in Heilongjiang Province and northeast of Neimenggu Province. • The main current of “Huangqi” is A. membranaceus var. mongholicusin these few years in the herb market in China.

16. Astragulus membranaceus var. mongholicus Cultivated on the plain in Neimenggu Cultivated on the mountain in Shanxi Cultivated on the plain in Shanxi Cultivated on the plain in Gansu

17. Astragulus membranaceus Cultivated on the plain in Neimenggu Cultivated on the plain in Shanxi(陕西) Wild on the mountain in Heilongjiang Wild on the mountain in Heilongjiang

18. Our Work • Germplasm Resource Investigation and Collection of Radix Astragali. • Established Chromatography Fingerprint of flavonoids in Radix Astragali by HPLC-UV. • Quality Evaluation : Determined four isoflavonoids and Astragaloside Ⅳ in Radix Astragali by HPLC of 37 batches of A. membranaceus var. mongholicus and 12 batches of A. membranaceus（Fisch.）Bge. from different cultivated places. • Made a chemical comparison with different harvest time, different age of cultivated sources, different commercial specification, and xylem& phloem of A. membranaceus var. mongholicus by using the method above.

19. Established Chromatography Fingerprint of flavonoids in Radix Astragali by HPLC-UV Detected 21 batches of A. membranaceus var. mongholicus which are cultivated in different places and analyzed the similarity of samples by using the software of Computer Aided Similarity Evaluation.

20. 21 batches of Astragulus membranaceus var. mongholicus

21. 21 batches of Astragulus membranaceus var. mongholicus

22. Method • Chromatographic Condition Jasco-High Performance Liquid Phase System Model (Jasco, Japan) HPLC column: YMC-Pack ODS-A 250×4.60mm Mobile Phase: UV detection: 210nm

23. Chromatography Fingerprint of flavonoids in Radix Astragali by HPLC-UV

24. Chromatography Fingerprint of flavonoids in Radix Astragali by HPLC-UV • Analyzed the similarity of 21 samples by using the software of Computer Aided Similarity Evaluation

25. The similarity of 21 samples

26. Our Work • Germplasm Resource Investigation and Collection of Radix Astragali. • Established Chromatography Fingerprint of flavonoids in Radix Astragali by HPLC-UV. • Quality Evaluation : Determined four isoflavonoids and Astragaloside Ⅳ in Radix Astragali by HPLC of 37 batches of A. membranaceus var. mongholicus and 12 batches of A. membranaceus（Fisch.）Bge. from different cultivated places. • Made a chemical comparison with different harvest time, different age of cultivated sources, different commercial specification, and xylem& phloem of A. membranaceus var. mongholicus by using the method above.

27. Quality Evaluation Determined four isoflavonoids and Astragaloside Ⅳ in Radix Astragali by HPLC-UV and HPLC-ELSD of 37 batches of A. membranaceus var. mongholicus and 12 batches of A. membranaceus（Fisch.）Bge. from different cultivated places.

28. Simultaneous determination of four isoflavonoids Chromatographic Condition Jasco-High Performance Liquid Phase System Model (Jasco, Japan) HPLC column: YMC-Pack ODS-A 250×4.60mm UV detection: 248nm Method

29. Simultaneous determination of four isoflavonoids Method Mobile Phase:

30. Four Isoflavonoids for quantification Calycosin-7-O-β-D-glucoside (1) Ononin (2) Calycosin (3) Formononetin (4)

31. HPLC-UV Chromatograms

32. Determination of Astragaloside Ⅳ by HPLC-ELSD Chromatographic Condition Shimadzu-High Performance Liquid Phase System Model (Shimadzu, Japan) HPLC column: YMC-Pack ODS-A 250×4.60mm Mobile Phase:Acetonitrile : Water = 35 : 65 Flow rate: 0.8ml/min Detection condition: Drift Tube Temperature: 102.0℃ Gas Flow rate: 2.8L/min

33. HPLC-ELSD Chromatograms

34. Quality Evaluation of Astragulus membranaceus var. mongholicus

35. Quality Evaluation of Astraulus membranaceus var. mongholicus

36. Quality Evaluation of Astragulus membranaceus var. mongholicus *: Can be detected, but cannot be quantified.

37. Quality Evaluation of Astragulus membranaceus *: Can be detected, but cannot be quantified.

38. Conclusion of Quality Evaluation A. membranaceus var. mongholicus > A. membranaceus

39. Our Work • Germplasm Resource Investigation and Collection of Radix Astragali. • Established Chromatography Fingerprint of flavonoids in Radix Astragali by HPLC-UV. • Quality Evaluation : Determined four isoflavonoids and Astragaloside Ⅳ in Radix Astragali by HPLC of 37 batches of A. membranaceus var. mongholicus and 12 batches of A. membranaceus（Fisch.）Bge. from different cultivated places. • Made a chemical comparison with different harvest time, different age of cultivated sources, different commercial specification, and xylem& phloem of A. membranaceus var. mongholicus by using the method above.

40. Different harvest time *: Can be detected, but cannot be quantified.

41. Conclusion: Spring > Autumn

42. Different age of cultivated sources *: Can be detected, but cannot be quantified.

43. Different age of cultivated sources

44. Conclusion: One-year > Two-year

45. Different commercial specification

46. Conclusion: Three-class ≌ Two-class > First-class

47. Comparison of Xylem and Phloem

48. Conclusion: Isoflavonoids : Xylem > Phloem Astragaloside Ⅳ : Phloem > Xylem

49. Conclusion of Chemical Comparison • The contents of isoflavonoids and Astragaloside Ⅳ in Radix Astragali harvested in Autumn are higher than harvested in Spring. The reason may be the plant would accumulate these chemical constituents in the soil in Winter. • The contents of isoflavonoids and Astragaloside Ⅳ in one-year cultivated plant are higher than those in two-year cultivated plant. We would pay attention to shortening the growth time limit of Radix Astragali.

50. Conclusion of Chemical Comparison • The contents of isoflavonoids and Astragaloside Ⅳ in Radix Astragali don’t decrease with the commercial specification on the Chinese Traditional Medicine market, on the contrary, the contents in the First-class of Radix Astragali are the lowest. It is not significant to classify the commercial specification based on the diameter of the rhizome of Radix Astragali. • The contents of isoflavonoids in the xylem of Radix Astragali are higher than those in the phloem, while the content of Astragaloside Ⅳ in the xylem is extremely lower than that in the phloem of Radix Astragali.