Eliminate Liver Cancer Stem-Like Cells
B. 透過誘導分化治療癌症幹細胞
II. 鴉膽子 (Brucea javanica)
1. teroxirone 影響肝癌幹細胞型態
在不同肝癌細胞株 Huh7、HepG2 和 Hep3B 在含有特定生長因子 的培養基培養 7 天，當有 tumor sphere 形成時，分別加入 teroxirone (5、
10、20 或 30µM) ，之後利用顯微鏡觀察 0、24、48 或 72 小時 tumor sphere 的大小。Huh7 (Fig1A) 及 Hep3B (Fig3A)細胞株在不同時間點
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以及在處理不同 teroxirone 藥物濃度處理下 tumor sphere 大小、數量 有明顯減少的趨勢，而 HepG2 (Fig2A) 細胞株在不同時間點以及處理 不同 teroxirone 藥物濃度處理 tumor sphere 大小、數量則沒有明顯變 化。量化後也具統計意義(Fig1B, Fig2B and Fig3B)。
2.中藥夏枯草影響肝癌幹細胞型態
在不同肝癌細胞株 Huh7、HepG2 和 Hep3B 在含有特定生長因子 的培養基培養 7 天，當有 tumor sphere 形成時，加入夏枯草分別為(2、
4 或 6mg/mL)，利用顯微鏡觀察 0、24、48 或 72 小時 tumor sphere 的大小。在 Huh7 (Fig4A) 及 HepG2 (Fig5A) 細胞株處理夏枯草藥物 細胞大小及數量都有改變，在 Hep3B (Fig6A) 細胞株處理夏枯草不同 的時間點及藥物濃度中並沒有明顯變化，量化後也具統計意義(Fig4B, Fig5B and Fig6B)。
3.利用幹細胞標誌物 Nanog 鑑定肝癌類幹細胞
在不同肝癌細胞株在含有特定生長因子的培養基培養 7 天，當有 tumor sphere 形成時，Huh7、HepG2 和 Hep3B 加入夏枯草濃度為(2、
4 或 6mg/mL) ，再加入 Nanog 進一步利用活細胞顯微鏡觀察。(Fig8A, Fig8B and Fig8C)。Huh7、HepG2 和 Hep3B 分別加入 teroxirone 濃度 為 (5、10、20 或 30µM) ，再加入 Nanog 進一步利用活細胞顯微鏡觀
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察(Fig7A, Fig7B and Fig7C)，
4.利用螢光染劑確認肝癌幹細胞的細胞數量及自我更新能力
肝癌細胞株 Huh7 在含有特定生長因子的培養基培養 7 天，當有 tumor sphere 形成時， 分別加入不同中藥處理後加入 BrdU，再進一 步利用活細胞顯微鏡觀察 BrdU 的變化。在鴉膽子及夏枯草 BrdU 表 現量的變化及細胞數量有明顯下降(Fig9A, Fig9B, Fig9C, Fig9D)，而 苦蔘則沒有任何影響 (Fig9E, Fig9F)
陸、討論
1.teroxirone 抑制肝癌 tumor sphere 大小及數目
有研究指出 teroxirone 可以抑制非小細胞肺癌細胞(53)，所以本研 究推測 teroxirone 可以抑制肝癌 tumor sphere。而在我們實驗結果發現，
加入 teroxirone 處理下，Huh7 數量及大小都有隨著藥物處理濃度上升 而下降，另外有報導發現，夏枯草、鴉膽子及苦蔘對肝癌細胞有抑制 效果(47)。因此利用此三種中藥觀察對肝癌 tumor sphere 的影響，在 HepG2 的 tumor sphere 加入夏枯草處理後，數量及大小都有明顯下降，
Huh7 的 tumor sphere 加入鴉膽子處理後，數量及大小都有明顯下降。
在處理藥物的 tumor sphere 加入幹細胞標誌物 Nanog，雖然藥物會影 響 tumor sphere 大小，但是還是具有癌症幹細胞的功能。另外本研究 利用 BrdU 偵測藥物處理過後 spheroid 的自我更新能力，實驗發現
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Huh7 的 tumor sphere 在鴉膽子處理下，tumor sphere 的自我更新能力 有明顯的下降。結果發現藥物會影響肝癌類幹細胞的自我更新能力，
未來可以進一步利用西方墨點法觀察藥物是透過調控此特徵的相關 路徑例如: Wnt pathway，Hedgehog pathway…等等，來達到抑制肝癌 tumor sphere 生長。
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柒、參考文獻
1. Madka, V., and Rao, C. V. (2011) Cancer stem cell markers as potential targets for epithelial cancers. Indian J Exp Biol 49, 826-835
2. Al-Hajj, M., Wicha, M. S., Benito-Hernandez, A., Morrison, S. J., and Clarke, M. F. (2003) Prospective identification of tumorigenic breast cancer cells.
Proceedings of the National Academy of Sciences of the United States of America 100, 3983-3988
3. O'Brien, C. A., Pollett, A., Gallinger, S., and Dick, J. E. (2007) A human colon cancer cell capable of initiating tumour growth in immunodeficient mice.
Nature 445, 106-110
4. Ricci-Vitiani, L., Lombardi, D. G., Pilozzi, E., Biffoni, M., Todaro, M., Peschle, C., and De Maria, R. (2007) Identification and expansion of human colon-cancer-initiating cells. Nature 445, 111-115
5. Maenhaut, C., Dumont, J. E., Roger, P. P., and van Staveren, W. C. (2010) Cancer stem cells: a reality, a myth, a fuzzy concept or a misnomer? An analysis. Carcinogenesis 31, 149-158
6. Puglisi, M. A., Barba, M., Corbi, M., Errico, M. F., Giorda, E., Saulnier, N., Boninsegna, A., Piscaglia, A. C., Carsetti, R., Cittadini, A., Gasbarrini, A., and Sgambato, A. (2011) Identification of Endothelin-1 and NR4A2 as CD133-regulated genes in colon cancer cells. The Journal of pathology 225, 305-314
7. Boman, B. M., Fields, J. Z., Cavanaugh, K. L., Guetter, A., and Runquist, O. A.
(2008) How dysregulated colonic crypt dynamics cause stem cell overpopulation and initiate colon cancer. Cancer research 68, 3304-3313 8. Chiba, T., Kita, K., Zheng, Y. W., Yokosuka, O., Saisho, H., Iwama, A.,
Nakauchi, H., and Taniguchi, H. (2006) Side population purified from hepatocellular carcinoma cells harbors cancer stem cell-like properties.
Hepatology (Baltimore, Md.) 44, 240-251
9. Haraguchi, N., Ohkuma, M., Sakashita, H., Matsuzaki, S., Tanaka, F., Mimori, K., Kamohara, Y., Inoue, H., and Mori, M. (2008) CD133+CD44+ population efficiently enriches colon cancer initiating cells. Annals of surgical oncology 15, 2927-2933
10. Dalerba, P., Dylla, S. J., Park, I. K., Liu, R., Wang, X., Cho, R. W., Hoey, T., Gurney, A., Huang, E. H., Simeone, D. M., Shelton, A. A., Parmiani, G., Castelli, C., and Clarke, M. F. (2007) Phenotypic characterization of human colorectal cancer stem cells. Proceedings of the National Academy of Sciences of the United States of America 104, 10158-10163
64
11. Huang, E. H., Hynes, M. J., Zhang, T., Ginestier, C., Dontu, G., Appelman, H., Fields, J. Z., Wicha, M. S., and Boman, B. M. (2009) Aldehyde dehydrogenase 1 is a marker for normal and malignant human colonic stem cells (SC) and tracks SC overpopulation during colon tumorigenesis. Cancer research 69, 3382-3389
12. Ma, S., Chan, K. W., Lee, T. K., Tang, K. H., Wo, J. Y., Zheng, B. J., and Guan, X. Y. (2008) Aldehyde dehydrogenase discriminates the CD133 liver cancer stem cell populations. Molecular cancer research : MCR 6, 1146-1153
13. Boman, B. M., and Huang, E. (2008) Human colon cancer stem cells: a new paradigm in gastrointestinal oncology. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 26, 2828-2838
14. Huang, E. H., and Wicha, M. S. (2008) Colon cancer stem cells: implications for prevention and therapy. Trends in molecular medicine 14, 503-509
15. Sukowati, C. H., Rosso, N., Croce, L. S., and Tiribelli, C. (2010) Hepatic cancer stem cells and drug resistance: Relevance in targeted therapies for hepatocellular carcinoma. World journal of hepatology 2, 114-126
16. Watabe, T., and Miyazono, K. (2009) Roles of TGF-beta family signaling in stem cell renewal and differentiation. Cell research 19, 103-115
17. Amin, R., and Mishra, L. (2008) Liver stem cells and tgf-Beta in hepatic carcinogenesis. Gastrointestinal cancer research : GCR 2, S27-30
18. Beachy, P. A., Karhadkar, S. S., and Berman, D. M. (2004) Tissue repair and stem cell renewal in carcinogenesis. Nature 432, 324-331
19. Cheng, W. T., Xu, K., Tian, D. Y., Zhang, Z. G., Liu, L. J., and Chen, Y. (2009) Role of Hedgehog signaling pathway in proliferation and invasiveness of hepatocellular carcinoma cells. International journal of oncology 34, 829-836 20. Huang, S., He, J., Zhang, X., Bian, Y., Yang, L., Xie, G., Zhang, K., Tang, W.,
Stelter, A. A., Wang, Q., Zhang, H., and Xie, J. (2006) Activation of the hedgehog pathway in human hepatocellular carcinomas. Carcinogenesis 27, 1334-1340
21. Dean, M. (2006) Cancer stem cells: redefining the paradigm of cancer treatment strategies. Molecular interventions 6, 140-148
22. Reya, T., Duncan, A. W., Ailles, L., Domen, J., Scherer, D. C., Willert, K., Hintz, L., Nusse, R., and Weissman, I. L. (2003) A role for Wnt signalling in self-renewal of haematopoietic stem cells. Nature 423, 409-414
23. Yang, W., Yan, H. X., Chen, L., Liu, Q., He, Y. Q., Yu, L. X., Zhang, S. H., Huang, D. D., Tang, L., Kong, X. N., Chen, C., Liu, S. Q., Wu, M. C., and Wang, H. Y. (2008) Wnt/beta-catenin signaling contributes to activation of normal and tumorigenic liver progenitor cells. Cancer research 68, 4287-4295
65
24. Ali, I. U., Schriml, L. M., and Dean, M. (1999) Mutational spectra of PTEN/MMAC1 gene: a tumor suppressor with lipid phosphatase activity.
Journal of the National Cancer Institute 91, 1922-1932
25. Hu, T. H., Huang, C. C., Lin, P. R., Chang, H. W., Ger, L. P., Lin, Y. W., Changchien, C. S., Lee, C. M., and Tai, M. H. (2003) Expression and prognostic role of tumor suppressor gene PTEN/MMAC1/TEP1 in hepatocellular carcinoma. Cancer 97, 1929-1940
26. He, X. C., Yin, T., Grindley, J. C., Tian, Q., Sato, T., Tao, W. A., Dirisina, R., Porter-Westpfahl, K. S., Hembree, M., Johnson, T., Wiedemann, L. M., Barrett, T. A., Hood, L., Wu, H., and Li, L. (2007) PTEN-deficient intestinal stem cells initiate intestinal polyposis. Nature genetics 39, 189-198
27. Lee, T. K., Cheung, V. C., and Ng, I. O. (2013) Liver tumor-initiating cells as a therapeutic target for hepatocellular carcinoma. Cancer letters 338, 101-109 28. Luo, H., Hao, X., Ge, C., Zhao, F., Zhu, M., Chen, T., Yao, M., He, X., and Li,
J. (2010) TC21 promotes cell motility and metastasis by regulating the expression of E-cadherin and N-cadherin in hepatocellular carcinoma.
International journal of oncology 37, 853-859
29. Ji, J., Yamashita, T., Budhu, A., Forgues, M., Jia, H. L., Li, C., Deng, C., Wauthier, E., Reid, L. M., Ye, Q. H., Qin, L. X., Yang, W., Wang, H. Y., Tang, Z. Y., Croce, C. M., and Wang, X. W. (2009) Identification of microRNA-181 by genome-wide screening as a critical player in EpCAM-positive hepatic cancer stem cells. Hepatology (Baltimore, Md.) 50, 472-480
30. Nowak, D., Stewart, D., and Koeffler, H. P. (2009) Differentiation therapy of leukemia: 3 decades of development. Blood 113, 3655-3665
31. Vicente Lopez, M. A., Vazquez Garcia, M. N., Entrena, A., Olmedillas Lopez, S., Garcia-Arranz, M., Garcia-Olmo, D., and Zapata, A. (2011) Low doses of bone morphogenetic protein 4 increase the survival of human adipose-derived stem cells maintaining their stemness and multipotency. Stem cells and development 20, 1011-1019
32. Zhang, L., Sun, H., Zhao, F., Lu, P., Ge, C., Li, H., Hou, H., Yan, M., Chen, T., Jiang, G., Xie, H., Cui, Y., Huang, X., Fan, J., Yao, M., and Li, J. (2012) BMP4 administration induces differentiation of CD133+ hepatic cancer stem cells, blocking their contributions to hepatocellular carcinoma. Cancer research 72, 4276-4285
33. Pardal, R., Clarke, M. F., and Morrison, S. J. (2003) Applying the principles of stem-cell biology to cancer. Nature reviews. Cancer 3, 895-902
34. Reya, T., Morrison, S. J., Clarke, M. F., and Weissman, I. L. (2001) Stem cells, cancer, and cancer stem cells. Nature 414, 105-111
66
35. Van Den Berg, D. J., Sharma, A. K., Bruno, E., and Hoffman, R. (1998) Role of members of the Wnt gene family in human hematopoiesis. Blood 92, 3189-3202
36. Singh, S. K., Hawkins, C., Clarke, I. D., Squire, J. A., Bayani, J., Hide, T., Henkelman, R. M., Cusimano, M. D., and Dirks, P. B. (2004) Identification of human brain tumour initiating cells. Nature 432, 396-401
37. Singh, S. K., Clarke, I. D., Terasaki, M., Bonn, V. E., Hawkins, C., Squire, J., and Dirks, P. B. (2003) Identification of a cancer stem cell in human brain tumors. Cancer research 63, 5821-5828
38. Shimano, K., Satake, M., Okaya, A., Kitanaka, J., Kitanaka, N., Takemura, M., Sakagami, M., Terada, N., and Tsujimura, T. (2003) Hepatic oval cells have the side population phenotype defined by expression of ATP-binding cassette transporter ABCG2/BCRP1. The American journal of pathology 163, 3-9 39. Chiasson, B. J., Tropepe, V., Morshead, C. M., and van der Kooy, D. (1999)
Adult mammalian forebrain ependymal and subependymal cells demonstrate proliferative potential, but only subependymal cells have neural stem cell characteristics. The Journal of neuroscience : the official journal of the Society for Neuroscience 19, 4462-4471
40. Wang, M. L., Chiou, S. H., and Wu, C. W. (2013) Targeting cancer stem cells:
emerging role of Nanog transcription factor. OncoTargets and therapy 6, 1207-1220
41. Stacey, D. W., and Hitomi, M. (2008) Cell cycle studies based upon quantitative image analysis. Cytometry. Part A : the journal of the International Society for Analytical Cytology 73, 270-278
42. Pardal, R., Molofsky, A. V., He, S., and Morrison, S. J. (2005) Stem cell self-renewal and cancer cell proliferation are regulated by common networks that balance the activation of proto-oncogenes and tumor suppressors. Cold Spring Harbor symposia on quantitative biology 70, 177-185
43. Treasure, J. (2005) Herbal medicine and cancer: an introductory overview.
Semin Oncol Nurs 21, 177-183
44. Ruan, W. J., Lai, M. D., and Zhou, J. G. (2006) Anticancer effects of Chinese herbal medicine, science or myth? Journal of Zhejiang University. Science. B 7, 1006-1014
45. Hsu, H. Y., Yang, J. J., and Lin, C. C. (1997) Effects of oleanolic acid and ursolic acid on inhibiting tumor growth and enhancing the recovery of hematopoietic system postirradiation in mice. Cancer letters 111, 7-13
46. Tsai, S. J., and Yin, M. C. (2008) Antioxidative and anti-inflammatory protection of oleanolic acid and ursolic acid in PC12 cells. Journal of food
67
science 73, H174-178
47. Kim, S. H., Huang, C. Y., Tsai, C. Y., Lu, S. Y., Chiu, C. C., and Fang, K.
(2012) The aqueous extract of Prunella vulgaris suppresses cell invasion and migration in human liver cancer cells by attenuating matrix metalloproteinases.
The American journal of Chinese medicine 40, 643-656
48. Chen, M., Chen, R., Wang, S., Tan, W., Hu, Y., Peng, X., and Wang, Y. (2013) Chemical components, pharmacological properties, and nanoparticulate delivery systems of Brucea javanica. International journal of nanomedicine 8, 85-92
49. Pu, L. P., Chen, H. P., Cao, M. A., Zhang, X. L., Gao, Q. X., Yuan, C. S., and Wang, C. M. (2013) The antiangiogenic activity of Kushecarpin D, a novel flavonoid isolated from Sophora flavescens Ait. Life sciences 93, 791-797 50. Neidhart, J. A., Derocher, D., Grever, M. R., Kraut, E. H., and Malspeis, L.
(1984) Phase I trial of teroxirone. Cancer treatment reports 68, 1115-1119 51. Atassi, G., Spreafico, F., Dumont, P., Nayer, P., and Klastersky, J. (1980)
Antitumoral effect in mice of a new triepoxyde derivative: 1, 3, 5-triglycidyl-s-triazinetrione (NSC 296934). Eur J Cancer 16, 1561-1567 52. Dombernowsky, P., Lund, B., and Hansen, H. H. (1983) Phase-I study of
alpha-1,3,5-triglycidyl-s-triazinetrione (NSC 296934). Cancer Chemother Pharmacol 11, 59-61
53. Wang, J. P., Lin, K. H., Liu, C. Y., Yu, Y. C., Wu, P. T., Chiu, C. C., Su, C. L., Chen, K. M., and Fang, K. (2013) Teroxirone inhibited growth of human non-small cell lung cancer cells by activating p53. Toxicology and applied pharmacology 273, 110-120
59. National Institutes of Health
http://stemcells.nih.gov/staticresources/info/basics/StemCellBasics.pdf
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捌、圖示
Fig.1 不同濃度 teroxirone 對 Huh7 tumor sphere 的影響
Huh7 培養在 ultra-low attachment dish 七天，加入 teroxirone 處理 0、5、10、
20 μM，利用顯微鏡觀察 0、24、48 或 72 小時 tumor sphere 的大小及數量。A.
隨著時間及藥物濃度上升，tumor sphere 大小有 dose-dependent 下降。(scale bar, 100μm.)B. 觀察大於 60μm 的 tumor sphere 數量，隨著時間及藥物濃度上升，tumor sphere 數量有 dose-dependent 下降。
A
B
N=2
0 10 20 30 μM
μm
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Fig.2 不同濃度 teroxirone 對 HepG2 tumor sphere 的影響
HepG2 培養在 ultra-low attachment dish 七天，加入 teroxirone 處理 0、5、10、
20 μM，利用顯微鏡觀察 0、24、48 或 72 小時 tumor sphere 的大小及數量。A.
隨著時間及藥物濃度上升，tumor sphere 大小沒有變化。(scale bar, 100 μm.) B. 觀 察大於60μm 的 tumor sphere 數量，隨著時間及藥物濃度上升，tumor sphere 數 量沒有差異。
A
B
N=2
70
Fig.3 不同濃度 teroxirone 對 Hep3B tumor sphere 的影響
Hep3B 培養在 ultra-low attachment dish 七天，加入 teroxirone 處理 0、5、10、
20 μM，利用顯微鏡觀察 0、24、48 或 72 小時 tumor sphere 的大小及數量。A.
隨著時間及藥物濃度上升，tumor sphere 大小有 dose-dependent 下降。(scale bar, 100 μm.) B. 觀察大於 60μm 的 tumor sphere 數量，隨著時間及藥物濃度上升，
tumor sphere 數量有 dose-dependent 下降。
B A
N=2
71
Fig.4 不同濃度夏枯草(PV)對 Huh7 tumor sphere 的影響
Huh7 培養在 ultra- low attachment dish 七天，加入夏枯草濃度 2、4 或 6 mg/mL，
利用顯微鏡觀察 0、24、48 或 72 小時 tumor sphere 的大小及數量。A.6 mg/mL 的夏枯草處理 48 小時，tumor sphere 變小。(scale bar, 100 μm.) B. 觀察大於 60μm 的 tumor sphere 數量，隨著時間及藥物濃度上升，tumor sphere 數量有
dose-dependent 下降。
A
B
N=2
72
Fig.5 不同濃度夏枯草(PV)對 HepG2 tumor sphere 的影響
HepG2 培養在 ultra-low attachment dish 七天，加入夏枯草濃度 2、4 或 6 mg/mL，
利用顯微鏡觀察 0、24、48 或 72 小時 tumor sphere 的大小及數量。A. 6 mg/mL 的夏枯草處理 24 小時，tumor sphere 變小。(scale bar, 100 μm.) B.觀察大於 60μm 的 tumor sphere 數量，隨著時間及藥物濃度上升，tumor sphere 數量有
dose-dependent 下降。
A
B
N=2
73
Fig.6 不同濃度夏枯草(PV) 對 Hep3B tumor sphere 的影響
Hep3B 培養在 ultra-low attachment dish 七天，加入夏枯草濃度 2、4 或 6 mg/mL，
利用顯微鏡觀察 0、24、48 或 72 小時 tumor sphere 的大小及數量。A. 隨著藥物 濃度及時間增加，tumor sphere 沒有變化。(scale bar, 100 μm.) B. 觀察大於 60μm 的 tumor sphere 數量，隨著時間及藥物濃度上升，tumor sphere 數量沒有變化。
A
B
N=1
74
A B
C
DAPI Nanog Merge DAPI Nanog Merge
DAPI Nanog Merge
Fig.7 利用幹細胞標誌物 Nanog 鑑定不同肝 癌類幹細胞對 teroxirone 的影響
Huh7、HepG2 和 Hep3B 培養在 ultra-low attachment dish 七天，teroxirone 濃度 0、5、10、20 μM，處理 48 小時。利用活細胞顯微鏡觀察 tumor sphere 的 Nanog 表現。(blue:DAPI,green: Nanog, scale bar, 100 μm.) A. Huh7 類幹細胞，處理不同濃度的 teroxirone，都有 Nanog 表現 B. HepG2 類幹細胞，
加入不同濃度的 teroxirone，都有 Nanog 表現 C.
Hep3B 類幹細胞，加入不同濃度的 teroxirone，都有 Nanog 表現。
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Fig.8 利用幹細胞標誌物 Nanog 鑑定不同 濃度夏枯草對肝癌類幹細胞的影響
Huh7、HepG2 和 Hep3B 培養在 ultra-low attachment dish 七天，加入夏枯草濃度 2、4 或 6 mg/mL，處理 48 小時。利用活細胞顯微鏡觀察 tumor sphere 的 Nanog 表現。(blue: Hoechst 33342,green: Nanog, scale bar, 100 μm.) A.在 Huh7 的類幹細胞，處理不同濃度的夏枯草，都有 Nanog 表現 B.在 HepG2 的類幹細胞，加入不同濃度的夏 枯草，都有 Nanog 表現 C.在 Hep3B 的類幹細胞，
加入不同濃度的夏枯草，都有 Nanog 表現。
C
A B
Hoechst 33342 Nanog Merge Hoechst 33342 Nanog Merge
Hoechst 33342 Nanog Merge
mg/mL
76
mg/mL
mg/mL
A
C
E
B
D
F
N=1
N=1
N=1
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Fig.9 利用螢光染劑確認不同中藥對Huh7的細胞存活及自我更新能力 的影響
Huh7 培養在 ultra-low attachment dish 七天，分別加入夏枯草、鴉膽子及苦
Huh7 培養在 ultra-low attachment dish 七天，分別加入夏枯草、鴉膽子及苦