AJAC  Vol.3 No.10 , October 2012
Utilization of Dispersive Liquid-Liquid Microextraction Coupled with HPLC-UV as a Sensitive and Efficient Method for the Extraction and Determination of Oleanolic Acid and Ursolic Acid in Chinese Medicinal Herbs
Abstract: Isomeric triterpenic acids of oleanolic acid (OA) and ursolic acid (UA) both have very low ultraviolet absorption and always exist in the same plant, so the separation and simultaneous determination of them have been a difficult task. In this study, a sensitive method combining dispersive liquid-liquid microextraction (DLLME) with HPLC-UV was developed for the extraction and determination of OA and UA in traditional Chinese medicinal herbs (CMHs). Variables influencing DLLME such as type and volume of extraction solvent, volume of dispersive solvent, ionic strength, aqueous phase pH, extraction time, centrifugation speed and time, and sample volume were investigated and optimized. Under the optimum conditions, both OA and UA attained favorable extraction efficiencies with enrichment factors 1378 and 933, respectively. The linear dynamic ranges of 0.07 - 30.4 μg?mL–1 for OA and 0.08 - 33.6 μg?mL–1 for UA were obtained with square correlation coefficients of 0.9963. The detection limits of OA and UA were both 0.02 μg?mL–1. The method recoveries ranged between 88.2% - 116.2% for OA and 85.7% - 108.2% for UA with the RSDs (n = 5) lower than 8.6%. The proposed method was successfully applied to concentrate and simultaneously determine these two triterpenic acids in Hedyotis diffusa and Eriobotrya japonica samples.
Cite this paper: Y. Hao, X. Chen, S. Hu, X. Bai and D. Gu, "Utilization of Dispersive Liquid-Liquid Microextraction Coupled with HPLC-UV as a Sensitive and Efficient Method for the Extraction and Determination of Oleanolic Acid and Ursolic Acid in Chinese Medicinal Herbs," American Journal of Analytical Chemistry, Vol. 3 No. 10, 2012, pp. 675-682. doi: 10.4236/ajac.2012.310089.

[1]   J. Liu, “Pharmacology of Oleanolic Acid and Ursolic Acid,” Journal of Ethnopharmacology, Vol. 49, No. 2, 1995, pp. 57-68. doi:10.1016/0378-8741(95)90032-2

[2]   V. Mandal and S. C. Mandal, “Design and Performance Evaluation of a Microwave Based Low Carbon Yield- ingextraction Technique for Naturally Occurring Bioactive Triterpenoid: Oleanolic Acid,” Biochemical Engineering Journal, Vol. 50, No. 1-2, 2010, pp. 63-70. doi:10.1016/j.bej.2010.03.005

[3]   A. T. Dinkova-Kostova, K. T. Liby, K. K. Stephenson, W. D. Holtzclaw, X. Q. Gao, N. Suh, C. Williarrli, R. Risingsong, T. Honda, G. W. Gribble, M. B. Sporn and P. Talalay, “Extremely Potent Triterpenoid Inducers of the Phase 2 Response: Correlations of Protection against Oxidant and In?ammatory Stress,” Proceedings of the National Academy of Sciences of the United States of America, Vol. 102, No. 12, 2005, pp. 4584-4589. doi:10.1073/pnas.0500815102

[4]   S. Schwaiger, I. Zeller, P. P?lzelbauer, S. Frotschnig, G. Laufer, B. Messner, V. Pieri, H. Stuppner and D. Bernhard, “Identi?cation and Pharmacological Characterization of the Anti-In?ammatory Principal of the Leaves of Dwarf Elder (Sambucus ebulus L.),” Journal of Ethnopharmacology, Vol. 133, No. 2, 2011, pp. 704-709. doi:10.1016/j.jep.2010.10.049

[5]   J. P. Benincá, J. B. Dalmarco, M. G. Pizzolatti and T. S. Fr?de, “Analysis of the Anti-Inflammatory Properties of Rosmarinus Officinalis L. in Mice,” Food Chemistry, Vol. 124, No. 2, 2011, pp. 468-475.

[6]   K. Mazumder, E. Siwu, S. Nozaki, Y. Watanabe and K. Tanaka, “Ursolic Acid Derivatives from Bangladeshi Medicinal Plant, Saurauja Roxburghii: Isolation and Cytotoxic Activity against A431 and C6 Glioma Cell Lines,” Phytochemistry Letters, Vol. 4, No. 3, 2011, pp. 287-291. doi:10.1016/j.phytol.2011.04.019

[7]   S. L. Yan, C. Y. Huang, S. T. Wu and M. C. Yin, “Oleanolic acid and Ursolic Acid Induce Apoptosis in Four Human Liver Cancer Cell Lines,” Toxicology in Vitro, Vol. 24, No. 3, 2010, pp. 842-848. doi:1016/j.tiv.2009.12.008

[8]   Y. Kashiwada, H. K. Wang, T. Nagao, S. Kitanaka, I. Yasuda, T. Fujioka, T. Yamagishi, L. M. Cosentino, M. Kozuka, H. Okabe, Y. Ikeshiro, C. Q. Hu, E. Yeh and K. H. Lee, “Anti-AIDS Agents. 30 Anti-HIV Activity of Oleanolic Acid, Pomolic Acid, and Structurally Related Triterpenoids,” The Journal of Natural Products, Vol. 61, No. 9, 1998, pp. 1090-1095. doi:10.1021/np9800710

[9]   J. F. Rivero-Cruz, M. Zhu, A. D. Kinghorn and C. D. Wu, “Antimi-crobial Constituents of Thompson Seedless Raisins (Vitis vinif-era) against Selected Oral Pathogens,” Phytochemistry Letters, Vol. 1, No. 3, 2008, pp. 151-154. doi:10.1016/j.phytol.2008.07.007

[10]   K. Horiuchi, S. Shiota, T. Hatano, T. Toshita, T. Kuroda and T. Tsuchiya, “Antimicrobial Activity of Oleanolic acid from Salvia Officinalis and Related Compounds on Vancomycin-Resistant Enterococci (VRE),” Biological and Pharmaceutical Bulletin, Vol. 30, No. 6, 2007, pp. 1147-1149. doi:10.1248/bpb.30.1147

[11]   J. A. Rodriguez, L. Astudillo and G. Schmeda-Hirsch- mann, “Oleanolic Acid Promotes Healing of Acetic Acid- Induced Chronic Gastric Lesions in Rats,” Pharmacological Research, Vol. 48, No. 3, 2003, pp. 291-294. doi:10.1016/S1043-6618(03)00155-5

[12]   C. L. Meloa, M. Queiroza, S. Fonsecaa, A. Bizerrab, T. Lemosb, T. Meloc, F. Santosc and V. Raoc, “Oleanolic Acid, a Natural Triterpenoid Improves Blood Glucose Tolerance in Norma Mice and Ameliorates Visceral Obesity in Mice Fed a High-Fat Diet,” Chemico- Bio- logical Interactions, Vol. 185, No. 1, 2010, pp. 59-65. doi:10.1016/j.cbi.2010.02.028

[13]   I. Kazmi, M. Rahman, M. Afzal, A. G. Gupta, S. Saleem, O. Afzal, M. A. Shaharyar, U. Nautiyal, S. Ahmed and F. Anwar, “Anti-Diabetic Potential of Ursolic Acid Stearoyl Glucoside: A New Triterpenic Gycosidic Ester from Lantana Camara,” Fitoterapia, Vol. 83, No. 1, 2012, pp. 142- 146.

[14]   H. X. Liu, Y. H. Shi, D. X. Wang, G. L. Yang, A. M. Yu and H. Q. Zhang, “MECC Determination of Oleanolic Acid and Ursolic Acid Isomers in Ligustrum lucidum Ait,” Journal of Pharmaceutical and Biomedical Analysis, Vol. 32, No. 3, 2003, pp. 479-485.

[15]   P. Yang, Y. Q. Li, X. Liu and S. X. Jiang, “Determination of Free Isomeric Oleanolic Acid and Ursolic Acid in Pterocephalus Hookeri by Capillary Zone Electrophoresis,” Journal of Pharmaceutical and Biomedical Analysis, Vol. 43, No. 4, 2007, pp. 1331-1334.

[16]   X. H. Xu, Q. Su and Z. H. Zang, “Simultaneous Determination of Oleanolic Acid and Ursolic Acid by RP- HPLC in the Leave of Eriobotrya japonica Lindl,” Journal of Pharmaceutical Analysis, Vol. 2, 2012, pp. 238- 240. doi:10.1016/j.jpha.2012.01.006

[17]   H. H. Wang, Z. Z. Wang and W. B. Guo, “Comparative Determination of Ursolic Acid and Oleanolic Acid of Macrocarpium of?cinalis (Sieb. et Zucc.) Nakai by RP- HPLC,” Industrial Crops and Products, Vol. 28, No. 3, 2008, pp. 328-332. doi:10.1016/j.indcrop.2008.03.004

[18]   G. L. Li, X. L. Zhang, J. M. You, C. H. Song, Z. W. Sun, L. Xia and Y. R. Suo, “Highly Sensitive and Selective Pre-Column Derivatization High Performance Liquid Chromatography Approach for Rapid Determination of Triterpenes Oleanolic and Ursolic Acids and Application to Swertia Species: Optimization of Triterpenic Acids Extraction and Pre-Column Derivatization Using Response Surface Methodology,” Analytica Chimica Acta, Vol. 688, No. 2, 2011, pp. 208-218. doi:10.1016/j.aca.2011.01.010

[19]   M. Wójciak-Kosior, “Separation and Determination of Closely Related Triterpenic Acids by High Performance Thin-Layer Chromatography after Iodine Derivatization,” Journal of Pharmaceutical and Biomedical Analysis, Vol. 45, No. 2, 2007, pp. 337-340.

[20]   C. B. Ojeda, F. S. Rojas and J. M. C. Pavón, “Determination of Cobalt in Food, Environmental and Water Samples with Preconcentration by Dispersive Liquid-Liquid Micro Extraction,” American Journal of Analytical Chem- istry, Vol. 3, 2012, pp. 125-130. doi:10.4236/ajac.2012.32018

[21]   S. Berijani, Y. Assadi, M. Anbia, M. R. M. Hosseini and E. Aghaee, “Dispersive Liq-uid-Liquid Microextraction Combined with Gas Chromatog-raphy-Flame Photometric Detection: Very Simple, Rapid and Sensitive Method for the Determination of Organ Phosphorus Pesticides in Water,” Journal of Chromatography A, Vol. 1123, No. 1, 2006, pp. 1-9.

[22]   M. I. Leong, C. C. Chang, M. R. Fuh and S. D. Huanga, “Low Toxic Dispersive Liquid-Liquid Microextraction Using Halosolvents for Extraction of Polycyclic Aromatic Hydrocarbons in Water Samples,” Journal of Chroma-tography A, Vol. 1217, No. 34, 2010, pp. 5455-5461. doi:10.1016/j.chroma.2010.06.056

[23]   L. J. He, X. L. Luo, H. X. Xie, C. J. Wang, X. Jiang and K. Lu, “Ionic Liquid-Based Dispersive Liquid-Liquid Microextraction Followed High-Performance Liquid Chro- matography for the Determina-tion of Organophosphorus Pesticides in Water Sample,” Analytica Chimica Acta, Vol. 655, No. 1-2, 2009, pp. 52-59.

[24]   S. Q. Gao, J. Y. You, X. Zheng, Y. Wang, R. B. Ren, R. Zhang, Y. P. Bai and H. Q. Zhang, “Determination of Phenylurea and Triazine Herbicides in Milk by Microwave Assisted Ionic Liquid Microextraction High-Performance Liquid Chromatography,” Talanta, Vol. 82, No. 4, 2010, pp. 1371-1377.

[25]   E. Z. Jahromi, A. Bidari, Y. Assadi, M. R. M. Hosseini and M. R. Jamali, “Dispersive Liquid-Liquid Microex- traction Combined with Graphite Furnace Atomic Absorption Spectrometry: Ultra Trace Determination of Cadmium in Water Samples,” Analytica Chimica Acta, Vol. 585, No. 2, 2007, pp. 305-311.

[26]   H. F. Zhang and Y. P. Shi, “Temperature-Assisted Ionic Liquid Dispersive Liquid-Liquid Microextraction Com- bined with High Performance Liquid Chromatography for the Determination of Anthraquinones in Radix et Rhizoma Rhei Samples,” Talanta, Vol. 82, No. 3, 2010, pp. 1010-1016. doi:10.1016/j.talanta.2010.06.008

[27]   A. Daneshfar, T. Khezeli and H. J. Lotfi, “Determination of Cholesterol in Food Samples Using Dispersive Liquid-Liquid Microextraction followed by HPLC-UV,” Journal of Chromatography B, Vol. 877, No. 4, 2009, pp. 456-460. doi:10.1016/j.jchromb.2008.12.050

[28]   M. Bashiri-Juybari, A. Mehdinia, A. Jabbari and Y. Yamini, “Determination of Amitraz in the Honey Samples by Dispersive Liquid-Liquid Microextraction Followed by Gas Chromatography-Flame Ionization Detection C,” American Journal of Analytical Chemistry, Vol. 2, 2011, pp. 632-637.

[29]   M. Xiong, J. L. Ruan, Y. L. Cai and Y. Tang, “Extraction and Determination of Some Psychotropic Drugs in Urine Samples using Dispersive Liquid-Liquid Microextraction Followed by High-Performance Liquid Chromatography,” Journal of Pharmaceutical and Bio-medical Analysis, Vol. 49, No. 2, 2009, pp. 572-578.

[30]   J. Tian, X. Chen and X. H. Bai, “Comparison of Dispersive Liquid-Liquid Microextraction Based on Organic Solvent and Ionic Liquid Combined with High-Performance Liquid Chromatography for Analysis of Emodin and Its Metabolites in Urine Samples,” Journal of Separation Science, Vol. 35, No. 1, 2012, pp. 145- 152.

[31]   L. H. Li, H. F. Zhang, S. Hu and X. H. Bai, “Dispersive Liquid-Liquid Microextraction Coupled with High-Performance Liquid Chromatography for Determination of Coumarin Compounds in Radix Angelicae Dahuricae,” Chromatographia, Vol. 75, No. 3, 2012, pp. 131-137. doi:10.1007/s10337-011-2177-1