Radix Stemonae ( 百部 ) : From authentication and quality control to antitussive analysis

1. Radix Stemonae (百部) : From authentication and quality control to antitussive analysis Pang-Chui Shaw (邵鹏柱) Department of Biochemistry & Institute of Chinese Medicine The Chinese University of Hong Kong

2. Radix Stemonae (1) • A major antitussive in Chinese medicine • “Ben Cao Gang Mu” 本草綱目 • 主治咳嗽上氣 • PRC Pharmacopoeia 中華人民共和國藥典(2005) • moisten the lung and relieve cough • kill insects and worms Radix Stemonae (From Jing Shi Zheng Lei Ben Cao in Song dynasty)

3. Radix Stemonae (2) • Three Stemona species are listed in the PRC Pharmacopoeia Stemona japonica (Bl.) Miq. 蔓生百部 Stemona sessilifolia (Miq.) Miq. 直立百部 Stemona tuberosa Lour. 對葉百部

4. Challenges • It is difficult to identify Radix Stemonae when it is dried. • Substitutes and adulterants exist • Radix Stemonae has long been used for antitussive, however its active components were not defined.

5. A comprehensive approach to study Radix stemonae • Use DNA and chemical technologies to differentiate the three species used as Radix Stemonae, its substitute and its adulterant. • Purify and determine the chemicals in the Radix Stemonae • Study the antitussive properties of the purified chemicals

6. Sample List

7. Stemona authentication using DNA technology

8. DNA regions for Stemona authentication • trnL-F • Chloroplast gene spacer • Sequence more conserved • 5S rRNA • Genomic gene spacer • Highly variable among species of the same family

9. DNA extraction PCR DNASequencing Cloning Database Experimental procedures Sample collection

10. Comparison of trnL-F spacer sequences of Stemona

11. Comparison of 5S rDNA spacer sequences of Stemona

12. Summary • Radix Stemonae can be distinguished from the substitute of Asparagus by 5S rDNA spacer sequences and trnL-F sequences. • 5S rDNA spacer sequences can be further used to differentiate the four concerned Stemona species.

13. Stemona spp. authentication using Chemical technology

14. Comparison of HPLC-ELSD and HPLC-UV HPLC-ELSD totalalkaloids HPLC-UV total alkaloids ELSD is better than UV for the detection of Stemona alkaloids

15. Experimental procedure (1) Extraction HPLC-ELSD Purify chemicals

16. Experimental procedure (2) NMR MS 2D –NMR Chemical structure

17. Stemona alkaloids identified in Radix Stemonae (1) A) Alkaloid found in all three Stemona species B) Alkaloids found in S. japonica and S.sessilifolia

18. Stemona alkaloids identified in Radix Stemonae (2) C) Alkaloids found in S. tuberosa only New New

19. Stemona alkaloids identified in Radix Stemonae (3) D) Alkaloid found in S. japonica only E) Alkaloid found in S. sessilifolia only

20. HPLC-MS profiles of the three medicinal Stemona spp. Stemotinine S. tuberosa S. japonica S. tuberosa S. japonica S. tuberosa S. japonica Stemonidine S. sessilifolia S. tuberosa S. tuberosa S. sessilifolia S. tuberosa S. sessilifolia Three genuine Stemona can be differentiated by HPLC-MS analysis

21. 3 1 2 4 Type A Type B Type C Type D Four chemical-types were observed in HPLC-ELSD profile • Samples collected from: • Yunnan (3 samples) • Guangxi (2 samples) • Anhui (2 samples) • Purchased in Guangzhou (6 samples) Determine the identities of Compounds 1, 2, 3 & 4

22. 3 1 2 4 Type A Type B Type C Type D 4 tuberostemonine 3 neotuberostemonine 1 croomine 2 stemoninine

23. Study of the antitussive properties of Radix Stemonae • Do all four types of S. tuberosa have antitussive effect? • What chemicals in S. tuberosa have antitussive effect?

24. computer Instrument for anti-cough study ultrosonic nebulizer guinea pig microphone

25. 1st challenge 2nd challenge Antitussive effects of total alkaloids from different Stemona samples • Stemona samples at 100 mg/kg i.g. White columns represent first challenge with citric acid aerosol; black columns represent second challenge with citric acid aerosol after administration with test samples • (n=6) *** P<0.001 in A—C, • ** P<0.01 in D • Samples from • Yunnan • Anhui • Guangdong • Guangxi All four types of Baibu are active A B C D

26. Stemoninine (2) Croomine (1) ** ** ** ** Tuberostemonine (4) Neotuberostemonine (3) ** ** Antitussive effects of four alkaloids from S. tuberosa Antitussive effects (i.g.)* P<0.05, ** P<0.01 (n=6. paired-T test) : 1st challenge, : 2nd challenge

27. Antitussive mechanism • Remove the superior laryngeal nerve from the larynx of guinea pig, stimulate with electrical pulses and measure the contraction of abdomen muscle • Peripherally acting (inhibit airway nerves that evoke cough): • Neotuberostemonine • Tuberostemonine • Stemoninine • Centrally acting (act on the CNS) • Croomine

28. Summary • DNA sequences and chemical profiles were established for authentication. • Specific chemical markers were obtained. • S. tuberosa was found to have different chemical profiles and antitussive properties of total alkaloids were demonstrated. • The active components responsible for the antitussive properties were identified and their mechanism revealed.

29. Patents applied and papers published Papers published • Isolation and chemotaxonomic significance of tuberostemospironine-type alkaloids from Stemona tuberosa, Phytochemistry, 2006, 67, 52-57. • New alkaloids and chemical diversity of Stemona tuberosa. J. Nat. Prod. 2006, 69, 749 – 754. • Analyses of Stemona alkaloids in Stemona tuberosa by liquid chromatography/tandem mass spectrometry, Rapid Commun. Mass Spect. 2006, 20, 1030-1038. • Antitussive effects of Stemona tuberosa with different chemical profiles, J.Ethnopharmacol. 2006, 108, 46-53. Patents applied • Method of making antitussive medicine and relieving cough • 制备止咳药及减轻咳嗽的方法

30. Institute of Chinese Medicine CUHK Hong Kong Jockey Club Institute of Chinese Medicine Dept. of Biochemistry CUHK • Prof. P. C. Shaw • Mr. K. L. Wong • Dr. Y.T. Xu • Mr. P.M. Hon • Dr. R.W. Jiang • Dr. S.L. Li • Miss L. Cheng • Mr. Y.M. Chan • Miss. M.F. He • Mr. Y. P. Chan • Mr. M. Li • Ms. K. Y. Law • Dr. H. X. Xu Chengdu Institute of Biology, The Chinese Academy of Sciences Dept. of Biology, CUHK • Dr. Y. Zhou • Dr. L.S. Ding • Prof. Paul. P.H. But Institute of Botany University of Vienna Dept. of Chemistry, CUHK • Prof. T. W. Chan • Prof. H. Greger Financial support from HKJCICM

31. Thank you for your attention

32. Morphology of S. tuberosa Leaves opposite or whorled 對生葉片或輪生葉片 Vine with branched stem Flower: perianth (花被) /stamen (雄蕊) is obvious peduncle axillary (腋生花)

33. Morphology of S. japonica Whorled leaves with petiole 輪生葉,有明顯葉柄 Vine (攀爬植物) Flower: perianth (花被) / stamen (雄蕊) is obvious Peduncle borne on leaf midvein 花梗由葉的中央葉脈長出

34. Morphology of S. sessilifolia Subshrub (亞灌木) Whorled leaves with short petiole or subsessile (輪生葉, 葉柄短或近無葉柄) Inflorescences borne at base of stem (幼穗由莖底長出) Flower: perianth (花被) / stamen (雄蕊) is obvious

35. Substitute & adulterant of Radix Stemonae Asparagus filicinus 羊齒天門冬 (Adulterant) Stemona parviflora 細花百部 (Substitute) Herbs, stems erect, unarmed Leaf spur short, not spinescent (非針刺狀) • Vines with branched stem, base woody • Leaves alternate (葉互生); petiole sometimes curved • peduncle axillary (花序腋生)

36. S. tuberosa Smooth outer surface of epidermal cells (Phase contrast mode) Cell Characteristic fibers in the cortex

37. S. japonica Cortex Epidermis No fiber in the cortex Thicken strips on outer surface of epidermal cells (Phase contrast mode)

38. S. sessilifolia Cortex Thicken strips on outer surface of epidermal cells (Phase contrast mode) No fiber in the cortex

39. Advantages of Molecular Techniques • Not limited to the sample’s physical form • Less affected by the age and growth condition of the sample • Minute amount is sufficient • May be adopted to screen many samples in a relatively short period of time • Even individual genetic difference can be identified

40. Advantages of Chemical Techniques • The efficacy of medicinal herbs are directly contributed by its chemicals contained • Chemical profile and marker may be used to authenticate a highly processed product • The efficacy of medicinal herbs can be qualitatively and quantitativelyevaluated by determining the bioactive chemicals in the materials

41. Cough responses in the control group after operation during 60 min (n=6) Reproducible cough responses can be induced

42. Cough responses after neotuberostemonine treatment in 60 min (n=6)

43. Cough responses after tuberostemonine treatment in 60 min (n=6)

44. Cough responses after stemoninine treatment in 60 min (n=6)

45. Cough responses and respiratory movements after croomine treatment (n=6)

46. Comparison of the three genuine species of Radix Stemonae by LC-MS • HPLC • C18 column (250 x 4.6mm, 5um) • Gradient elution • H2O (A) and CH3CN containing 0.1% Et3N • Mass spectra • TSQ 7000 ESI/MS detector

47. Correlation between chemistry andpharmacology of S. tuberosa

48. Chemical study on S. tuberosa • Different chemical patterns among S. tuberosa samples • Four chemical-types were observed

49. Action mechanisms of antitussive Stemona alkaloids

50. Mechanism of cough--- laryngeal nerve model (1)