Search JIM Advanced Search

Journal of Integrative Medicine ›› 2014, Vol. 12 ›› Issue (5): 455-462.doi: 10.1016/S2095-4964(14)60037-6

• Research Article • Previous Articles     Next Articles

Simultaneous determination of five anthraquinones in a Chinese traditional preparation by RP-HPLC using an improved extraction procedure

Yan-bin Shia, Hui-li Lia, Hai-qin Wanga, Yan-biao Yangb, Xiao-yun Zhanga, Hui Wanga, Zong-jie Zhub, Zhi-ye Zhangb, Cheng-an Zhangb   

  1. School of Pharmacy, Lanzhou University, Lanzhou 730000, Gansu Province, China
    Department of Pharmaceutics, the First People’s Hospital of Lanzhou, Lanzhou 730000, Gansu Province, China
  • Received:2014-02-17 Accepted:2014-05-07 Online:2014-09-10 Published:2014-09-15


The stable quality of Chinese herbal medicines is a critical factor for their reliable clinical efficiency. An improved liquid-liquid extraction procedure and a liquid chromatographic method were developed to simultaneously analyze five anthraquinones (aloe-emodin, rhein, emodin, chrysophanol and physcion) in a Chinese traditional hospital preparation, Fuyankang mixture, in order to quantitatively control its quality in a more effective way. 


A more economical and repeatable extraction procedure based on conventional liquid-liquid extraction technique was developed and used to extract five marker components in Fuyankang mixture. These anthraquinones were separated in less than 20 min on a C18 column with methanol and 0.1% phosphoric acid (88:12, v/v) as mobile phase. The method was validated for specificity, precision, spiked recovery and stability. 


Compared to conventional liquid-liquid extraction, the improved liquid-liquid extraction was found to be more effective for simultaneous extraction of anthraquinones from an aqueous Chinese herbal preparation, especially for hydrophobic compounds. The improved extraction method was successfully applied to determine the content of five marker components in Fuyankang mixture by the means of reverse phase high-performance liquid chromatography. 


The improved extraction procedure may be suitable for routine quality control of Fuyankang mixture and other traditional preparations at city-level hospitals in China.

Key words: Improved liquid-liquid extraction, Fuyankang mixture, Drugs, Chinese herbal, Chromatography, High-performance liquid, Anthraquinones, Quality control

Figure 1

Chemical structure of analytes[8]Thus, the aim of the present investigation was to develop a convenient, effective, and reproducible extraction method based on conventional LLE technique for the analysis of anthraquinones in Fuyankang mixture using RP-HPLC. This method is necessary to establish routine quality control for this preparation or other TCM preparations at city-level hospitals in China."

Figure 2

The equipment of an improved liquid-liquid extraction"

Figure 3

Chromatograms of the five analytesA: Mixed standard solution (all concentrations of compounds 1 to 5 were 2.5 μg/mL); B: Fuyankang mixture sample spiked with reference substances (30 μg); C: Fuyankang mixture sample (Lot No. 20120207). Peaks: 1=aloe-emodin, 2=rhein, 3=emodin, 4=chrysophanol, 5=physcion. Analytical conditions: column, Diomonsil? C18; mobile phase, methanol-0.1% phosphoric acid (88:12, v/v); flow-rate, 0.8 mL/min; column temperature, 40 ℃; detection wavelength, 254 nm."





[1] Yang HX, Yang YB, Zeng YY . Preparation and application of Fule mixture(Fuyankang mixture)[J]. Zhongguo Yi Yuan Yao Xue Za Zhi, 2001,21(12):733
[2] Wang J, Zhao H, Kong W, Jin C, Zhao Y, Qu Y, Xiao X . Microcalorimetric assay on the antimicrobial property of five hydroxyanthraquinone derivatives in rhubarb(Rheum palmatum L.) to Bifidobacterium adolescentis[J]. Phytomedicine, 2010,17(8-9):684-689
doi: 10.1016/j.phymed.2009.10.009
[3] Wu YW, Gao WY, Xiao XH, Liu Y . Calorimetric investigation of the effect of hydroxyanthraquinones in Rheum officinale Baill on Staphylococcus aureus growth[J]. Thermochim Acta, 2005,429(2):167-170
doi: 10.1016/j.tca.2005.03.008
[4] Chueh WH, Lin JY . Protective effect of isoquinoline alkaloid berberine on spontaneous inflammation in the spleen, liver and kidney of non-obese diabetic mice through downregulating gene expression ratios of pro-/anti-inflammatory and Th1/Th2 cytokines[J]. Food Chem, 2012,131(4):1263-1271
doi: 10.1016/j.foodchem.2011.09.116
[5] Kuo CL, Chi CW, Liu TY . The anti-inflammatory potential of berberine in vitro and in vivo[J]. Cancer Lett, 2004,203(2):127-137
doi: 10.1016/j.canlet.2003.09.002
[6] Chagas-Paula DA , Oliveira RB, da Silva VC, Gobbo-Neto L, Gasparoto TH, Campanelli AP, Faccioli LH, Da Costa FB.Chlorogenic acids from Tithonia diversifolia demonstrate better anti-inflammatory effect than indomethacin and its sesquiterpene lactones[J]. J Ethnopharmacol, 2011,136(2):355-362
doi: 10.1016/j.jep.2011.04.067
[7] Yun N, Kang JW, Lee SM . Protective effects of chlorogenic acid against ischemia/reperfusion injury in rat liver: molecular evidence of its antioxidant and anti-inflammatory properties[J]. J Nutr Biochem, 2012,23(10):1249-1255
doi: 10.1016/j.jnutbio.2011.06.018
[8] Genovese S, Tammaro F, Menghini L, Carlucci G, Epifano F, Locatelli M . Comparison of three different extraction methods and HPLC determination of the anthraquinones aloe-emodine, emodine, rheine, chrysophanol and physcion in the bark of Rhamnus alpinus L.(Rhamnaceae)[J]. Phytochem Anal, 2010,21(3):261-267
doi: 10.1002/pca.v21:3
[9] Ren P, Qin F, Huang X, Zhu ZY . Simultaneous LC analysis of aloe-emodin, rhein, emodin and chrysophanol in Rhizoma rhei-type preparations[J]. Chromatographia, 2009,70(9-10):1515-1517
doi: 10.1365/s10337-009-1341-3
[10] Wang JB, Li HF, Jin C, Qua Y, Xiao XH . Development and validation of a UPLC method for quality control of rhubarb-based medicine: fast simultaneous determination of five anthraquinone derivatives[J]. J Pharm Biomed Anal, 2008,47(4-5):765-770
doi: 10.1016/j.jpba.2008.03.011
[11] Tian K, Zhang HG, Chen XG, Hua ZD . Determination of five anthraquinones in medicinal plants by capillary zone electrophoresis with β-cyclodextrin addition[J]. J Chromatogr A, 2006,1123(1):134-137
doi: 10.1016/j.chroma.2006.04.021
[12] Lü HX, Wang JB, Wang XC . Rapid separation and determination of structurally related anthraquinones in Rhubarb by pressurized capillary electrochromatography[J]. J Pharm Biomed Anal, 2007,43(1):352-357
doi: 10.1016/j.jpba.2006.06.023
[13] Koyama J, Morita I, Kobayashi N . Simultaneous determination of anthraquinones in rhubarb by high-performance liquid chromatography and capillary electrophoresis[J]. J Chromatogr A, 2007,1145(1-2):183-189
doi: 10.1016/j.chroma.2007.01.076
[14] Sun SW, Yeh PC . Analysis of rhubarb anthraquinones and bianthrones by microemulsion electrokinetic chromatography[J]. J Pharm Biomed Anal, 2005,36(5):995-1001
doi: 10.1016/j.jpba.2004.08.039
[15] Xu FG, Liu Y, Zhang ZJ, Song R, Dong H, Tian Y . Rapid simultaneous quantification of five active constituents in rat plasma by high-performance liquid chromatography/tandem mass spectrometry after oral administration of Da-Cheng-Qi decoction[J]. J Pharm Biomed Anal, 2008,47(3):586-595
doi: 10.1016/j.jpba.2008.02.005
[16] Zuo Y, Wang C, Lin Y, Guo J, Deng Y . Simultaneous determination of anthraquinones in Radix Polygoni Multiflori by capillary gas chromatography coupled with flame ionization and mass spectrometric detection[J]. J Chromatogr A, 2008,1200(1):43-48
doi: 10.1016/j.chroma.2008.01.058
[17] Singh NP, Gupta AP, Sinha AK, Ahuja PS . High-performance thin layer chromatography method for quantitative determination of four major anthraquinone derivatives in Rheum emodi[J]. J Chromatogr A, 2005,1077(2):202-206
doi: 10.1016/j.chroma.2005.03.130
[18] Que NN, Ren YJ, Li SL, Wang YP, Li H . Determination of rheum anthraquinones in Jiangzhi Yihao by thin-layer chromatography scanner[J]. Jie Fang Jun Yao Xue Xue Bao, 2001,17(4):211-214
[19] Wei Y, Zhang T, Ito Y . Preparative separation of rhein from Chinese traditional herb by repeated high-speed counter-current chromatography[J]. J Chromatogr A, 2003,1017(1-2):125-130
doi: 10.1016/j.chroma.2003.08.015
[20] Poole CF . New trends in solid-phase extraction[J]. Trends Anal Chem, 2003,22(6):362-373
doi: 10.1016/S0165-9936(03)00605-8
[21] Catalina BO, Fuensanta SR . Separation and preconcentration by dispersive liquid-liquid microextraction procedure: a review[J]. Chromatographia, 2009,69(11-12):1149-1159
doi: 10.1365/s10337-009-1104-1
[22] Shi ZH, Jiang HX, Yan JJ, Zhang HY . Determination of anthraquinone derivatives in Chang-Qing tea by using cloud-point extraction and high-performance liquid chromatography[J]. Food Anal Methods, 2012,5(4):659-663
doi: 10.1007/s12161-011-9299-9
[23] Jeannot MA, Przyjazny A, Kokosa JM . Single drop microextraction — development, applications and future trends[J]. J Chromatogr A, 2010,1217(16):2326-2336
doi: 10.1016/j.chroma.2009.10.089
[24] Ye CW, Gao J, Yang C, Liu XJ, Li XJ, Pan SY . Development and application of an SPME/GC method for the determination of trace phthalates in beer using a calix[6]arene fiber[J]. Anal Chim Acta, 2009,641(1-2):64-74
doi: 10.1016/j.aca.2009.02.052
[25] Jin DH, Liang FH, Li JJ, Zhang HQ . Determination of anthraquinones in Semen Cassiae by microwave assistant extraction-high-performance liquid chromatography[J]. Fen Zi Ke Xue Xue Bao, 2007,23(6):429-433
[26] Zou JL, Xie ZY, Jiang XF, Chen XX, Yao MC . Determination of anthraquinones in different Rhubarb and Coptis Xiexin Decoction by HPLC[J]. Shi Jie Ke Xue Ji Shu Zhong Yi Yao Xian Dai Hua, 2008,10(4):61-65
[27] Zhang HF, Shi YP . Temperature-assisted ionic liquid dispersive liquid-liquid microextraction combined with high performance liquid chromatography for the determination of anthraquinones in Radix et Rhizoma Rhei samples[J]. Talanta, 2010,82(3):1010-1016
doi: 10.1016/j.talanta.2010.06.008
[28] He D, Chen B, Tian Q, Yao S . Simultaneous determination of five anthraquinones in medicinal plants and pharmaceutical preparations by HPLC with fluorescence detection[J]. J Pharmaceut Biomed Anal, 2009,49(4):1123-1127
doi: 10.1016/j.jpba.2009.02.014
[29] Liu JP, Li G. Pharmaceutics and pharmacokinetics[M]. Beijing: People’s Medical Publishing House, 2011: 16
[1] Shu-yi Chen, Qun-wei Chen, Liu-mei Shou, Hong Pan, Shan-ming Ruan, Zhe-hao Liang, Qi-jin Shu. Stevens-Johnson syndrome/toxic epidermal necrolysis successfully treated with Chinese herbal medicine Pi-Yan-Ning: A case report. Journal of Integrative Medicine, 2021, 19(6): 555-560.
[2] Xin Yin, Shu-bin Cai, Lan-ting Tao, Lu-ming Chen, Zhong-de Zhang, Su-hong Xiao, Arthur Yin Fan, Xu Zou. Recovery of a patient with severe COVID-19 by acupuncture and Chinese herbal medicine adjuvant to standard care. Journal of Integrative Medicine, 2021, 19(5): 460-466.
[3] Abdulhakim Abubakar, Abdullahi Balarabe Nazifi, Idris Mohammed Maje, Yusuf Tanko, Joseph Akpojo Anuka, Ezzeldin Mukthar Abdurahman. Antihyperglycaemic activity of ethylacetate extract of Chlorophytum alismifolium in type 2 diabetes: The involvement of peroxisome proliferator-activated receptor-γ and dipeptidyl peptidase-4. Journal of Integrative Medicine, 2021, 19(1): 78-84.
[4] Qing-lan Wang, Yan-yan Tao, Hong-dong Xie, Cheng-hai Liu, Ping Liu. Fuzheng Huayu recipe, a traditional Chinese compound herbal medicine, attenuates renal interstitial fibrosis via targeting the miR-21/PTEN/AKT axis. Journal of Integrative Medicine, 2020, 18(6): 505-513.
[5] Ming-sheng Lyu, De-ying Li, Shao-zhong Zhou, Cheng-jun Ban, Jun Yan. Adult-onset Still's disease successfully treated with Chinese herbal medicine: A case report with 15-month follow-up. Journal of Integrative Medicine, 2020, 18(6): 530-534.
[6] Arthur Yin Fan, Sherman Gu, Sarah Faggert Alemi, Research Group for Evidence-based Chinese Medicine. Chinese herbal medicine for COVID-19: Current evidence with systematic review and meta-analysis. Journal of Integrative Medicine, 2020, 18(5): 385-394.
[7] Bin Lan, Jin-wen Ge, Shao-wu Cheng, Xi-long Zheng, Jun Liao, Chao He, Zheng-qing Rao, Guo-zuo Wang. Extract of Naotaifang, a compound Chinese herbal medicine, protects neuron ferroptosis induced by acute cerebral ischemia in rats. Journal of Integrative Medicine, 2020, 18(4): 344-350.
[8] Deng-hai Zhang, Kun-lun Wu, Xue Zhang, Sheng-qiong Deng, Bin Peng. In silico screening of Chinese herbal medicines with the potential to directly inhibit 2019 novel coronavirus. Journal of Integrative Medicine, 2020, 18(2): 152-158.
[9] Rabie Kachkoul, Tarik Squalli Housseini, Mohamed Mohim, RadouaneEl Habbani, Youssef Miyah, Anissa Lahrichi. Chemical compounds as well as antioxidant and litholytic activities of Arbutus unedo L. leaves against calcium oxalate stones. Journal of Integrative Medicine, 2019, 17(6): 430-437.
[10] Asmae Assaidi, Ikram Dib, Monique Tits, Luc Angenot, Said Bellahcen, Nourelhouda Bouanani, Abdelkhaleq Legssyer, Mohammed Aziz, Hassane Mekhfi, Mohammed Bnouham, Michel Frederich, Abderrahim Ziyyat. Chenopodium ambrosioides induces an endothelium-dependent relaxation of rat isolated aorta. Journal of Integrative Medicine, 2019, 17(2): 115-124.
[11] Rohitash Jamwal. Bioavailable curcumin formulations: A review of pharmacokinetic studies in healthy volunteers. Journal of Integrative Medicine, 2018, 16(6): 367-374.
[12] Shohini Chakraborty, Nashra Afaq, Neelam Singh, Sukanta Majumdar. Antimicrobial activity of Cannabis sativaThuja orientalis and Psidium guajava leaf extracts against methicillin-resistant Staphylococcus aureus. Journal of Integrative Medicine, 2018, 16(5): 350-357.
[13] Carola Analía Torres, Cristina Marisel Pérez Zamora, María Beatriz Nuñez, Ana María Gonzalez. In vitro antioxidant, antilipoxygenase and antimicrobial activities of extracts from seven climbing plants belonging to the Bignoniaceae. Journal of Integrative Medicine, 2018, 16(4): 255-262.
[14] Sandeep Kumar Singh, Arjun Patra. Evaluation of phenolic composition, antioxidant, anti-inflammatory and anticancer activities of Polygonatum verticillatum (L.). Journal of Integrative Medicine, 2018, 16(4): 273-282.
[15] Nanaware Sadhana, Sathiyanarayanan Lohidasan, Kakasaheb Ramoo Mahadik. Marker-based standardization and investigation of nutraceutical potential of Indian propolis. Journal of Integrative Medicine, 2017, 15(6): 483-494.
Full text



[1] Wei-xiong Liang. Problems-solving strategies in clinical treatment guideline for traditional Chinese medicine and integrative medicine. Journal of Chinese Integrative Medicine, 2008, 6(1): 1-4
[2] Zhao-guo Li. Discussion on English translation of commonly used sentences in traditional Chinese medicine: part one. Journal of Chinese Integrative Medicine, 2008, 6(1): 107-110
[3] Jun Hu, Jian-ping Liu. Non-invasive physical treatments for chronic/recurrent headache. Journal of Chinese Integrative Medicine, 2008, 6(1): 31
[4] Xue-mei Liu, Qi-fu Huang, Yun-ling Zhang, Jin-li Lou, Hong-sheng Liu, Hong Zheng. Effects of Tribulus terrestris L. saponion on apoptosis of cortical neurons induced by hypoxia-reoxygenation in rats. Journal of Chinese Integrative Medicine, 2008, 6(1): 45-50
[5] . Uniform requirements for manuscripts submitted to biomedical journals: Writing and editing for biomedical publication (Chinese version, part two). Journal of Chinese Integrative Medicine, 2010, 8(11): 1001-1005
[6] Daniel Weber, Janelle M Wheat, Geoffrey M Currie. Inflammation and cancer: Tumor initiation, progression and metastasis,and Chinese botanical medicines. Journal of Chinese Integrative Medicine, 2010, 8(11): 1006-1013
[7] Hong Liu , Guo-liang Zhang, Li Shen , Zhen Zeng, Bao-luo Zhou, Cheng-hai Liu, Guang Nie . Application and evaluation of a pseudotyped virus assay for screening herbs for anti-H5Nl avian influenza virus. Journal of Chinese Integrative Medicine, 2010, 8(11): 1036-1040
[8] Zhao-guo Li . A discussion of English translation of 1995 and 1997 Chinese National Standards of Traditional Chinese Medical Terminologies for Clinical Diagnosis and Treatment. Journal of Chinese Integrative Medicine, 2010, 8(11): 1090-1096
[9] Rui Jin, Bing Zhang. A complexity analysis of Chinese herbal property theory: the multiple formations of herbal property (Part 1). Journal of Chinese Integrative Medicine, 2012, 10(11): 1198-1205
[10] Hui-min Liu, Xian-bo Wang, Yu-juan Chang, Li-li Gu. Systematic review and meta-analysis of randomized controlled trials of integrative medicine therapy for treatment of chronic severe hepatitis. Journal of Chinese Integrative Medicine, 2012, 10(11): 1211-1228