Journal of Natural Medicines

, Volume 67, Issue 2, pp 390–398 | Cite as

Development of simultaneous analysis for marker constituents in Hwangryunhaedok-tang (黄連解毒湯) and its application in commercial herbal formulas

  • Se Gun Kim
  • Amrit Poudel
  • Yun-Kyung Kim
  • Hyung-Kwon Jo
  • Hyun-Ju JungEmail author


This study was performed to develop methods for the chromatographic determination of biomarkers in Hwangryunhaedok-tang (HHT) and the quantitative evaluation of commercial HHT. To develop an analytical method, an RP-amide column (2.7 μm, 4.6 × 100 mm, Halo: Supelco, Bellefonte, PA) was used with a gradient solvent system of mixed acetonitrile and 0.1 % phosphoric acid/water and an ultra performance liquid chromatography-diode array detector. The method was validated by specificity, linearity, accuracy (recovery) and precision tests (repeatability, intra and inter-day). The correlation coefficients (R 2) of biomarkers were calculated as 0.9998–1.000 and their ranges were as follows: geniposide (62.5–1,000.0 μg/ml), berberine (31.3–500.0 mg/ml), palmatine (31.3–500.0 μg/ml), baicalin (125.0–1,500.0 μg/ml), baicalein (15.6–250.0 μg/ml) and wogonin (5.2–125.0 μg/ml), respectively. The limit of detection was 0.34–4.01 μg/ml, and the limit of quantification was 1.02–12.16 μg/ml. The intra-day and inter-day precision of six components were revealed as 0.02–2.48 % as a relative standard deviation (RSD). The repeatability value of biomarkers in three different concentrations of HHT was 0.29–2.98 % (RSD value) and recovery was 95.72–104.90 %. Among several extraction methods tested, biomarker content was higher with the 20 times extraction (20TE) and mixture of extract powder (MEP) methods than with any other method, and some differences among diverse pharmaceutical medicines were revealed. The validation data indicated that the method developed is suited to the determination of six marker compounds in HHT. The content of biomarkers by simultaneous analysis was evaluated in 20TE, MEP, USA formula and Taiwan formula.


Biomarkers Hwangryunhaedok-tang UPLC-DAD Method validation 



This paper was supported by Wonkwang University in 2009.


  1. 1.
    Zhang J, Wider B, Shang H, Li X, Ernst E (2012) Quality of herbal medicines: challenges and solutions. Complement Ther Med 20:100–106PubMedCrossRefGoogle Scholar
  2. 2.
    Bartosińska JP, Pietrzak A, Szepietowski J, Dreiher J, Maciejewski R, Chodorowska G (2011) Traditional Chinese medicine herbs are they safe for psoriatic patients? Folia Histochem Cytobiol 49:201–205PubMedGoogle Scholar
  3. 3.
    Jiang WY (2005) Therapeutic wisdom in traditional Chinese medicine: a perspective from modern science. Trends Pharmacol Sci 26:558–563PubMedCrossRefGoogle Scholar
  4. 4.
    Kinsel JF, Straus SE (2003) Complementary and alternative therapeutics: rigorous research is needed to support claims. Annu Rev Pharmacol Toxicol 43:463–484PubMedCrossRefGoogle Scholar
  5. 5.
    Tse TW (2003) Use of common Chinese herbs in the treatment of psoriasis. Clin Exp Dermatol 28:469–475PubMedCrossRefGoogle Scholar
  6. 6.
    Cheng CW, Bian ZX, Li YP, Moher D, Wu TX, Dagenais S, Li J, Li TQ (2008) Transparently reporting adverse effects of traditional Chinese medicine interventions in randomized controlled trials. Zhong Xi Yi Jie He Xue Bao 6:881–886PubMedCrossRefGoogle Scholar
  7. 7.
    Koo J, Arain S (1998) Traditional Chinese medicine for the treatment of dermatologic disorders. Arch Dermatol 134:1388–1393PubMedCrossRefGoogle Scholar
  8. 8.
    Hwang YS, Shin CY, Huh Y, Ryu JH (2002) Hwangryun-Hae-Dok-tang (Huanglian-Jie-Du-Tang) extract and its constituents reduce ischemia-reperfusion brain injury and neutrophil infiltration in rats. Life Sci 71:2105–2117PubMedCrossRefGoogle Scholar
  9. 9.
    Suenaga T, Shirakawa T, Harada N, Tao C, Yamasaki M, Komatsu H, Matsumoto N, Kida M, Kawamoto Y, Murakami Y (1991) Mechanism of protective effects of ouren-gedoku-to and san’ou-syashin-to on the gastric mucosa. Nippon Yakurigaku Zasshi 98:319–325PubMedCrossRefGoogle Scholar
  10. 10.
    Arakawa K, Saruta T, Abe K, Iimura O, Ishii M, Ogihara T, Hiwada K, Fukiyama K, Fujishima M, Mizuno Y, Kikuchi T, Takaori S (2006) Improvement of accessory symptoms of hypertension by TSUMURA Orengedokuto Extract, a four herbal drugs containing Kampo-Medicine Granules for ethical use: a double-blind, placebo-controlled study. Phytomedicine 13:1–10PubMedCrossRefGoogle Scholar
  11. 11.
    International conference of harmonization (1995) Q2A: Text on validation of analytical procedures. US FDA Federal Register 11260–11266Google Scholar
  12. 12.
    International conference of harmonization (1997) Q2B: Validation of analytical procedures—methodology. US FDA federal register 27463–27467Google Scholar
  13. 13.
    Korea Food and Drug Administration (2010) Guideline for chemical profile determination of herbal medicine. 9–23Google Scholar
  14. 14.
    Ouyang E, Zhang C, Li X (2011) Simultaneous determination of geniposide, chlorogenic acid, crocin1, and rutin in crude and processed Fructus Gardeniae extracts by high performance liquid chromatography. Pharmacogn Mag 7:267–270PubMedCrossRefGoogle Scholar
  15. 15.
    Koo HJ, Lim KH, Jung HJ, Park EH (2006) Anti-inflammatory evaluation of gardenia extract, geniposide and genipin. J Ethnopharmacol 103:496–500PubMedCrossRefGoogle Scholar
  16. 16.
    Jang MH, Kim HY, Kang KS, Yokozawa T, Park JH (2009) Hydroxyl radical scavenging activities of isoquinoline alkaloids isolated from Coptis chinensis. Arch Pharm Res 32:341–345PubMedCrossRefGoogle Scholar
  17. 17.
    Yang Q, Zhang F, Gao SH, Sun LN, Chen WS (2010) Determination of bioactive compounds in Cortex Phellodendri by high-performance liquid chromatography. J AOAC Int 93:855–861PubMedGoogle Scholar
  18. 18.
    Gao J, Sanchez-Medina A, Pendry BA, Hughes MJ, Webb GP, Corcoran O (2008) Validation of a HPLC method for flavonoid biomarkers in skullcap (Scutellaria) and its use to illustrate wide variability in the quality of commercial tinctures. J Pharm Pharm Sci 11:77–87PubMedGoogle Scholar
  19. 19.
    Meng W, Xiaoliang R, Xiumei G, Vincieri FF, Bilia AR (2009) Stability of active ingredients of traditional Chinese medicine (TCM). Nat Prod Commun 4:1761–1776PubMedGoogle Scholar

Copyright information

© The Japanese Society of Pharmacognosy and Springer 2012

Authors and Affiliations

  • Se Gun Kim
    • 1
  • Amrit Poudel
    • 1
  • Yun-Kyung Kim
    • 1
  • Hyung-Kwon Jo
    • 2
  • Hyun-Ju Jung
    • 1
    Email author
  1. 1.Department of Oriental Pharmacy and Wonkwang-Oriental Medicines Research InstituteWonkwang UniversityIksan-siRepublic of Korea
  2. 2.Hanpoong Pharm and FoodsJeonju-siRepublic of Korea

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