Share This Article:

Development and Validation of an HPLC Method for Simultaneous Determination of Nine Active Components in ‘Da-Chai-Hu-Tang’

Abstract Full-Text HTML Download Download as PDF (Size:755KB) PP. 20-28
DOI: 10.4236/cm.2011.21004    4,745 Downloads   9,616 Views   Citations


In this study, a simple, reliable and accurate method for the simultaneous separation and determination of naringin, hesperidin, neohesperidin, baicalin, wogonoside, baicalein, wogonin, emodin and chrysophanol in ‘Da-Chai-Hu-Tang’ was developed by reverse-phase high-performance liquid chromatography (RP-HPLC). The chromatographic separation was performed on an Agilent ZORBAX C18 column (250 mm × 4.6 mm i.d., 5.0 μm), and the mobile phase composed of methanol and water containing 1% (v/v) acetic acid was used to elute the targets in a gradient elution mode. The flow rate and detection wavelength were set at 0.8 ml/min and 280 nm, respectively. All calibration curves of the nine components expressed good linearities (r2≥0.9992) within the tested ranges. The RSD values demonstrated the intra- and inter-day precisions were less than 2.89%, and the recoveries of the investigated compounds were between 96.22% and 105.28%. The proposed method is simple, precise, specific, sensitive, and successfully applied to determine the nine marker compounds in ‘Da-Chai-Hu-Tang’ for quality control.

Conflicts of Interest

The authors declare no conflicts of interest.

Cite this paper

L. Zheng and D. Dong, "Development and Validation of an HPLC Method for Simultaneous Determination of Nine Active Components in ‘Da-Chai-Hu-Tang’," Chinese Medicine, Vol. 2 No. 1, 2011, pp. 20-28. doi: 10.4236/cm.2011.21004.


[1] W. Y. Jiang, “Therapeutic Wisdom in Traditional Chinese Medicine: A Perspective from Modern Science,” Trends in Pharmacological Sciences, Vol. 26, No. 11, 2005, pp. 558-563. doi:10.1016/
[2] B. T. J. Van, “Chemical Analysis of Ginkgo Biloba Leaves and Extracts,” Journal of Chromatography A, Vol. 967, No. 1, 2002, pp. 21-55. doi:10.1016/S0021-9673(02)00172-3
[3] R. L. J. Graham, C. Graham, S. M. Clean, T. B. Chen, M. O’Rourke, D. Hirst, D. Theakston and C. Shaw, “Identification and Functional Analysis of a Novel Bradykinin Inhibitory Peptide in the Venoms of New World Crotalinae Pit Vipers,” Biochemical and Biophysical Research Communications, Vol. 338, No. 3, 2005, pp. 1587-1592. doi:10.1016/j.bbrc.2005.10.130
[4] T. Wu, R. Abdulla, Y. X. Zhao, Y. Yang, J. Chen and H. A. Aisa, “Simultaneous Quantification of Seven Flavonoids in Flos Gossypii by LC,” Chromatographia, Vol. 68, No. 5, 2008, pp. 467-470. doi:10.1365/s10337-008-0723-2
[5] X. C. Ma, X. L. Xin, K. X. Liu, B. J. Zhang, F. Y. Li and D. A. Guo, “Simultaneous Determination of Nine Major Flavonoids in Sophora flavescens by RP-LC,” Chromatographia, Vol. 68, No. 5-6, 2008, pp. 471-474. doi:10.1365/s10337-008-0719-y
[6] X. W. Shi, J. L. Qi, Y. B. Wu, Y. Fu, Y. Z. Wang and D. Q. Zhang, “Simultaneous Quantification of Six Sesquiterpene Lactones in Inula Britannica L. by RP-LC,” Chromatographia, Vol. 68, No. 3, 2008, pp. 281-285. doi:10.1365/s10337-008-0698-z
[7] J. Kang, L. Zhou, J.B. Sun, J. Han and D. A. Guo, “Chromatographic Fingerprint Analysis and Characterization of Furocoumarins in the Roots of Angelica Dahurica by HPLC/DAD/ESI-MSN Technique,” Journal of Pharmaceutical and Biomedical Analysis, Vol. 47, No. 4-5, 2008, pp. 778-785. doi:10.1016/j.jpba.2008.03.010
[8] J. Su, P. Fu, Y. H. Shen, C. Zhang, M. J. Liang, R. H. Liu, H. L. Li and W. D. Zhang, “Simultaneous Analysis of Flavonoids from Hypericum Japonicum Thunb.ex Murray (Hypericaceae) by HPLC-DAD-ESI/MS,” Journal of Pharmaceutical and Biomedical Analysis, Vol. 46, No. 2, 2008, pp. 342-348. doi:10.1016/j.jpba.2007.10.032
[9] W. Li, Y. L. Deng, R. J. Dai, Y. H. Yu, M. K. Saeed, L. Li, W. W. Meng and X. S. Zhang, “Chromatographic Fingerprint Analysis of Cephalotaxus Sinensis from Various Sources by High-Performance Liquid Chroma- tography-Diodearray Detection-Electrospray Ionization- Tandem Mass Spectrometry,” Journal of Pharmaceutical and Biomedical Analysis, Vol. 45, No. 1, 2007, pp. 38-46. doi:10.1016/j.jpba.2007.05.027
[10] W. Jin, R. L. Ge, Q. J. Wei, T. Y. Bao, H. M. Shi and P. F. Tu, “Development of High-Performance Liquid Chromatographic Fingerprint for the Quality Control of Rheum Tanguticum Maxim.ex Balf.,” Journal of Chromatography A, Vol. 1132, No. 1-2, 2006, pp. 320-324. doi:10.1016/j.chroma.2006.08.022
[11] S. H. Zhou, “Thirty Cases of Chronic Cholecystitis Treated by Acupuncture and Oral Adiministration of Da Chai Hu Tang,” Journal of Traditional Chinese Medicine, Vol. 28, No. 2, 2008, pp. 173-174. doi:10.1016/S0254-6272(08)60039-4
[12] Y. Fumihiko, I. Akira, K. Yasuhiro, M. Akiyo, I. Hiroshige and K. Kazuo, “Effects of Dai-saiko-to (Da-Chai- Hu-Tang) on Plasma Lipids and Atherosclerotic Lesions in Female Heterozygous Heritable Kurosawa and Kusanagi Hyper Cholesterolemic (KHC) Rabbits,” Pharmacological Research, Vol. 50, No. 1-2, 2004, pp. 223- 230. doi:10.1016/j.phrs.2004.02.003
[13] I. Akira, T. I. Osamu, Y. Fumihiko, M. Bunsho, A. Sakae, K. Yasuhiro, K. Kazuo, M. Akiyo and I. Hiroshige, “Inhibitory Effects of Dai-saiko-to (Da-Chai-Hu-Tang) on the Progression of Atherosclerotic Lesions in Kurosawa and Kusanagi Hyper-Cholesterolemic Rabbits,” Journal of Ethnopharmacology, Vol. 63, No. 3, 1998, pp. 209-218. doi:10.1016/S0378-8741(98)00083-X
[14] Y. Masaki, S. Atsushi, J. Hiroko, Y. Naoki and H. Tadashi, “Glucosyl Hesperidin Prevents Endothelial Dysfunction and Oxidative Stress in Spontaneously Hypertensive rats,” Nutrition, Vol. 24, No. 5, 2008, pp. 470-476. doi:10.1016/j.nut.2008.01.010
[15] P. Kannampalli, H. P. Sang, C. K. Kyong, “Hesperidin a Flavanoglycone Protects against γ-irradiation Induced Hepatocellular Damage and Oxidative Stress in Sprague–Dawley Rats,” European Journal of Pharmacology, Vol. 587, No. 1, 2008, pp. 273-280. doi:10.1016/j.ejphar.2008.03.052
[16] G. H. Xu, D. H. Liu, J. C. Chen, X. Q. Ye, Y. Q. Ma and J. Shi, “Juice Components and Antioxidant Capacity of Citrus Varieties Cultivated in China,” Food Chemistry, Vol. 106, No. 2, 2008, pp. 545-551. doi:10.1016/j.foodchem.2007.06.046
[17] P. F. Sebastian, W. Cristina, C. P. Alejandro and M. Mariel, “Synergistic Interaction between Hesperidin, a Natural Flavonoid, and Diazepam,” European Journal of Pharmacology, Vol. 512, No. 2-3, 2005, pp. 189-198. doi:10.1016/j.ejphar.2005.02.039
[18] K. Gaganjit, T. Naveen and C. Kanwaljit, “Beneficial Effect of Hesperidin on Lipopolysaccharide-induced Hepatotoxicity,” Toxicology, Vol. 226, No. 2-3, 2006, pp. 152-160. doi:10.1016/j.tox.2006.06.018
[19] L. L. Liu, L. K. Gong, H. Wang, Y. Xiao, X. F. Wu, Y. H. Zhang, X. Xue, X. M. Qi and J. Ren, “Baicalin Inhibits Macrophage Activation by Lipopolysaccharide and Protects Mice from Endotoxin Shock,” Biochemical Pharmacology, Vol. 75, No. 4, 2008, pp. 914-922. doi:10.1016/j.bcp.2007.10.009
[20] H. Y. Lin, S. C. Shen, C. W. Lin, L. Y. Yang and Y. C. Chen, “Baicalein Inhibition of Hydrogen Peroxide-induced Apoptosis via ROS-dependent Heme Oxygenase 1 Gene Expression,” Biochimica et Biophysica Acta, Vol. 1773, No. 7, 2007, pp. 1073-1086. doi:10.1016/j.bbamcr.2007.04.008
[21] K. J. Woo, J. H. Lim, S. Suh, Y. K. Kwon, S. W. Shin, S. C. Kim, Y. H. Choi, J. W. Park and T. K. Kwon, “Differential Inhibitory Effects of Baicalein and Baicalin on LPS-induced Cyclooxygenase-2 Expression through Inhibition of C/EBPβ DNA-binding Activity,” Immunobiology, Vol. 211, No. 5, 2006, pp. 359-368. doi:10.1016/j.imbio.2006.02.002
[22] H. Mika, O. Takashi, F. Harumi, T. Miho, Y. Shin-ichi, U. Sadaharu, N. Koji, Y. Hisako, T. Kachio and M. Akio, “Difference of Growth-inhibitory Effect of Scutellaria Baicalensis-producing Flavonoid Wogonin among Human Cancer Cells,” Cancer Letters, Vol. 245, No. 1-2, 2007, pp. 269-274. doi:10.1016/j.canlet.2006.01.011
[23] C. S. Chen, N. J. Chen, L. W. Lin, C. C. Hsieh, G. W. Chen and M. T. Hsieh, “Effects of Scutellariae Radix on Gene Expression in HEK 293 Cells Using cDNA Microarray,” Journal of Ethnopharmacology, Vol. 105, No. 3, 2006, pp. 346-351. doi:10.1016/j.jep.2005.11.012
[24] Y. Hu, Y. Yang, Q. D. You, W. Liu, H. Y. Gu, L. Zhao, K. Zhang, W. Wang, X. T. Wang and Q. L. Guo, “Oroxylin A Induced Apoptosis of Human Hepatocellular Carcinoma Cell Line HepG2 was Involved in Its Antitumor Activity,” Biochemical and Biophysical Research Communications, Vol. 351, No. 2, 2006, pp. 521-527. doi:10.1016/j.bbrc.2006.10.064
[25] Y. Ding, L. Zhao, H. Mei, S. L. Zhang, Z. H. Huang, Y. Y. Duan and P. Ye, “Exploration of Emodin to Treat Alpha-naphthylisothiocyanate-induced Cholestatic Hepatitis via Anti-inflammatory Pathway,” European Journal of Pharmacology, Vol. 590, No. 1-3, 2008, pp. 377-386. doi:10.1016/j.ejphar.2008.06.044
[26] J. M. Cherng, W. Chiang, J. H. Wang, C. M. Lin, C. Y. Lee, C. M. Shih and L. C. Chiang, “Anthraquinones of Edible Wild Vegetable Cassia Tora Stimulate Proliferation of Human CD4+ T Lymphocytes and Secretion of Interferon-gamma or Interleukin 10,” Food Chemistry, Vol. 107, No. 4, 2008, pp. 1576-1580. doi:10.1016/j.foodchem.2007.10.005
[27] X. Zhou, B. A. Song, L. H. Jin, D. Y. Hu, C. L. Diao, G. F. Xu, Z. H. Zou and S. Yang, “Isolation and Inhibitory Activity against ERK Phosphorylation of Hydroxyanthraquinones from Rhubarb,” Bioorganic & Medicinal Chemistry Letters, Vol. 16, No. 3, 2006, pp. 563-568. doi:10.1016/j.bmcl.2005.10.047
[28] I. Akira, T. I. Osamu, K. Kazuo, I. Hiroshige, Y. Fumihiko, M. Hiroko, K. Masayoshi, H. Masami, T. Hiroshi and M. Teruhiko, “Evaluation of Rhubarb Using Antioxidative Activity as an Index of Pharmacological Usefulness,” Journal of Ethnopharmacology, Vol. 91, No. 1, 2004, pp. 89-94. doi:10.1016/j.jep.2003.11.021
[29] Y. C. Chen, S. C. Shen, W. R. Lee, F. L. Hsu, H. Y. Lin, C. H. Ko and S. W. Tseng, “Emodin Induces Apoptosis in Human Promyeloleukemic HL-60 Cells Accompanied by Activation of Caspase 3 Cascade but Independent of Reactive Oxygen Species Production,” Biochemical Pharmacology, Vol. 64, No. 12, 2002, pp. 1713-1724.

comments powered by Disqus

Copyright © 2018 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.