TY - JOUR T1 - Optimization of a Reverse-Phase High Performance Liquid Chromatography (RP-HPLC) Method for Simultaneous Separation of Aloe-Emodin, Rhein, Emodin, Chrysophanol and Physcion TT - Optimization of a Reverse-Phase High Performance Liquid Chromatography (RP-HPLC) Method for Simultaneous Separation of Aloe-Emodin, Rhein, Emodin, Chrysophanol and Physcion AU - Yıldırım Baştemur, Gizem AU - Akpınar, Reyhan AU - Perçin Özkorucuklu, Sabriye AU - Kır, Esengül PY - 2021 DA - November DO - 10.31590/ejosat.987628 JF - Avrupa Bilim ve Teknoloji Dergisi JO - EJOSAT PB - Osman SAĞDIÇ WT - DergiPark SN - 2148-2683 SP - 972 EP - 984 IS - 27 LA - en AB - In this study, a reverse-phase high performance liquid chromatography (RP-HPLC) method with high separation efficiency, high detection sensitivity and excellent selectivity was successfully developed for the simultaneous quantitative determination of the aloe-emodin, rhein, emodin, chrysophanol, and physcion compounds. These optimum method conditions were determined by studying various columns, mobile phases and compositions, flow rates and column temperatures. The successful separation was carried out with a Supelcosil LC-18 column (250 × 4.6 mm, 5 µm) and a gradient program. The procedure was carried out at 20 °C with the flow rate of 1.0 mL/min and the injection volume of 20 μL utilizing an RP-HPLC method with DAD detector at 225 nm. Deionized water containing 0.5% orthophosphoric acid and methanol was used as the mobile phases A and B. This study showed an optimized analytical method can be effectively utilized to identify and measure anthraquinone compounds. KW - Reverse Phase High Performance Liquid Chromatography KW - Anthraquinon KW - Secondary Metabolites KW - Method Optimization N2 - In this study, a reverse-phase high performance liquid chromatography (RP-HPLC) method with high separation efficiency, high detection sensitivity and excellent selectivity was successfully developed for the simultaneous quantitative determination of the aloe-emodin, rhein, emodin, chrysophanol, and physcion compounds. These optimum method conditions were determined by studying various columns, mobile phases and compositions, flow rates and column temperatures. The successful separation was carried out with a Supelcosil LC-18 column (250 × 4.6 mm, 5 µm) and a gradient program. The procedure was carried out at 20 °C with the flow rate of 1.0 mL/min and the injection volume of 20 μL utilizing an RP-HPLC method with DAD detector at 225 nm. Deionized water containing 0.5% orthophosphoric acid and methanol was used as the mobile phases A and B. This study showed an optimized analytical method can be effectively utilized to identify and measure anthraquinone compounds. CR - Agarwal, S.K., Singh, S.S., Verma, S., & Kumar, S., (2000). Antifungal activity of anthraquinone derivatives from Rheum emodi. Journal of Ethnopharmacology, 72 (1-2), 43-46. doi: 10.1016/s0378-8741(00)00195-1 CR - Ahmad, W., Zaidi, S.M.A., Mujeeb, M., Ansari, S.H., & Ahmad, S., (2014). HPLC and HPTLC Methods by Design for Quantitative Characterization and in vitro Anti-oxidant Activity of Polyherbal Formulation Containing Rheum emodi. Journal of Chromatographic Science, 52 (8), 911-918. doi: 10.1093/chromsci/bmt123 CR - Aichner, D., & Ganzera, M., (2015). Analysis of anthraquinones in rhubarb (Rheum palmatum and Rheum officinale) by supercritical fluid chromatography. Talanta, 144, 1239-1244. doi: 10.1016/j.talanta.2015.08.011 CR - Chen, C., Fu, Z., Zhou, W., Chen, Q., Wang, C., Xu, L., Wang, Z. and Zhang, H., (2020). Ionic liquid-immobilized NaY zeolite-based matrix solid phase dispersion for the extraction of active constituents in Rheum palmatum L. Microchemical Journal, 152, 104245. doi: 10.1016/j.microc.2019.104245 CR - Chen, S.H., Lin, K.Y., Chang, C.C., Fang, C.L., & Lin, C.P., (2007). Aloe-emodin-induced apoptosis in human gastric carcinoma cells. Food and Chemical Toxicology, 45 (11), 2296-2303. doi: 10.1016/j.fct.2007.06.005 CR - Chien, S.C., Wu, Y.C., Chen, Z.W., & Yang, W.C., (2015). Naturally occurring anthraquinones: chemistry and therapeutic potential in autoimmune diabetes. Evidence-Based Complementary and Alternative Medicine, Article ID 357357. doi: 10.1155/2015/357357 CR - ElSohly, M.A., Gul, W., & Murphy, T.P., (2004). Analysis of the anthraquinones aloe-emodin and aloin by gas chromatography/mass spectrometry. International Immunopharmacology, 4 (14), 1739-1744. doi: 10.1016/j.intimp.2004.07.005 CR - Farooq, U., Pandith, S.A., Singh, Saggoo, M.I., & Lattoo, S.K., (2013). Altitudinal variability in anthraquinone constituents from novel cytotypes of Rumex nepalensis Spreng—a high value medicinal herb of North Western Himalayas. Industrial Crops and Products, 50, 112-117. doi: 10.1016/j.indcrop.2013.06.044 CR - Feng, S.X., Li, M.M., Zhao, D., Li, X.H., Zhang, L., Wang, Z., & Gao, N.N., (2017). Simultaneous Determination of 10 Anthraquinones in Rhubarb Based on HPLC-Q-HR/MS. Chinese Herbal Medicines, 9 (4), 388-395. doi: 10.1016/S1674-6384(17)60120-5 CR - Gao, X.Y., Jiang, Y., Lu, J., & Tu, P-.F., (2009). One single standard substance for the determination of multiple anthraquinone derivatives in rhubarb using high-performance liquid chromatography-diode array detection. Journal of Chromatography A, 1216 (11), 2118-2123. doi: 10.1016/j.chroma.2008.11.104 CR - Gautam, R., Srivastava, A., & Jachak, S.M., (2011). Simultaneous Determination of Naphthalene and Anthraquinone Derivatives in Rumex nepalensis Spreng. Roots by HPLC: Comparison of Different Extraction Methods and Validation. Phytochemical Analysis, 22 (2), 153-157. doi: 10.1002/pca.1261 CR - Guo, S., Feng, B., Zhu, R., Ma, J., & Wang, W., (2011). Preparative Isolation of Three Anthraquinones from Rumex japonicus by High-Speed Counter-Current Chromatography. Molecules, 16 (2), 1201–1210. doi: 10.3390/molecules16021201 CR - He, D., Chen, B., Tian, Q., & Yao, S., (2009). Simultaneous determination of five anthraquinones in medicinal plants and pharmaceutical preparations by HPLC with fluorescence detection. Journal of Pharmaceutical and Biomedical Analysis, 49 (4), 1123-1127. doi: 10.1016/j.jpba.2009.02.014 CR - Hu, S.S., Cao, W., Dai, H.B., Da, J.H., Ye, L.H., Cao, J., & Li, X.Y., (2014). Ionic-liquid-micelle-functionalized mesoporous Fe3O4 microspheres for ultraperformance liquid chromatography determination of anthraquinones in dietary supplements. Journal of Agricultural and Food Chemistry, 62 (35), 8822-8829. doi: 10.1021/jf502323f CR - Iizuka, A., Iijima, O.T., Kondo, K., Itakura, H., Yoshie, F., Miyamoto, H., Kubo, M., Higuchi, M., Takeda, H., & Matsumiya, R., (2004). Evaluation of Rhubarb using antioxidative activity as an index of pharmacological usefulness. Journal of Ethnopharmacology, 91 (1), 89-94. doi: 10.1016/j.jep.2003.11.021 CR - Koyama, J., Morita, I., & Kobayashi, N., (2007). Simultaneous determination of anthraquinones in rhubarb by high-performance liquid chromatography and capillary electrophoresis. Journal of Chromatography A, 1145 (1-2), 183-189. doi: 10.1016/j.chroma.2007.01.076 CR - Koyama, J., Morita, I., Tagahara, K., Nobukuni, Y., Mukainaka, T., Kuchide, M., Tokuda, H., & Nishino, H., (2002). Chemopreventive effects of emodin and cassiamin B in mouse skin carcinogenesis. Cancer Letters, 182 (2), 135-139. doi: 10.1016/S0304-3835(02)00100-3 CR - Kuo, C.H., & Sun, S.W., (2003). Analysis of nine rhubarb anthraquinones and bianthrones by micellar electrokinetic chromatography using experimental design. Analytica Chimica Acta, 482 (1), 47-58. doi: 10.1016/S0003-2670(03)00169-7 CR - Liu, S.Y., Sporer, F., Wink, M., Jourdane, J., Henning, R., Li, Y.L., & Ruppel, A., (1997). Anthraquinones in Rheum palmatum and Rumex dentatus (Polygonaceae), and phorbol esters in Jatropha curcas (Euphorbiaceae) with molluscicidal activity against the schistosome vector snails Oncomelania, Biomphalaria, and Bulinus. Tropical Medicine and International Health, 2 (2): 179-188. doi: 10.1046/j.1365-3156.1997.d01-242.x CR - Li-Weber M., (2013). Targeting apoptosis pathways in cancer by Chinese medicine. Cancer Letters, 332 (2), 304-312. doi: 10.1016/j.canlet.2010.07.015 CR - Locatelli M., (2011). Anthraquinones: analytical techniques as a novel tool to investigate on the triggering of biological targets. Current Drug Targets, 12 (3) 366-380. doi: 10.2174/138945011794815338 CR - Lü, H., Wang, J., Wang, X., Lin, X., Wu, X., & Xie Z., (2007). Rapid separation and determination of structurally related anthraquinones in Rhubarb by pressurized capillary electrochromatography. Journal of Pharmaceutical and Biomedical Analysis, 43 (1), 352-357. doi: 10.1016/j.jpba.2006.06.023 CR - Mandrioli, R., Mercolini, L., Ferranti, A., Fanali, S., & Raggi, M.A., (2011). Determination of aloe emodin in Aloe vera extracts and commercial formulations by HPLC with tandem UV absorption and fluorescence detection. Food Chemistry, 126 (1), 387-393. doi: 10.1016/j.foodchem.2010.10.112 CR - Mehta, J., (2012). Separation and characterization of anthraquinone derivatives from Cassia fistula using chromatographic and spectral techniques. International Journal of Chemical Sciences, 10(1), 306-316. CR - Rafaelly, L., Heron, S., Nowik, W., & Tchapla, A., (2008). Optimisation of ESI-MS detection for the HPLC of anthraquinone dyes. Dyes and Pigments, 77 (1), 191-203. doi: 10.1016/j.dyepig.2007.05.007 CR - Reynolds T., (2004). Aloes: The genus Aloe (Medicinal and Aromatic Plants − Industrial Profiles). CRC Press. Rong, F., Xi, H., Yang, W., Ping, R., Feng, Q., Chunhu, Z., Lan, F., Lichen, G., Zhaoqian, L., & Honghao, Z., (2011). Determination of anthraquinones by UPLC method in patient with craniocerebral injuries after oral administration of rhubarb. Word Science and Technology, 13, 676-680. CR - Shang, X., & Yuan, Z., (2003). Determination of hydroxyanthraquinoids in Rhubarb by cyclodextrin-modified micellar electrokinetic chromatography using a mixed micellar system of sodium dodecyl sulfate and sodium cholate. Journal of Pharmaceutical and Biomedical Analysis, 31 (1), 75-81. doi: 10.1016/s0731-7085(02)00596-4 CR - Sharma, N., Kumar, R., Sinha, A.K., Reddy, P.B., Nayeem, S.M., & Deep, S., (2012). Anthraquinone derivatives based natural dye from Rheum emodi as a probe for thermal stability of proteins: Spectroscopic and chromatographic studies. Journal of Pharmaceutical and Biomedical Analysis, 62, 96-104. doi: 10.1016/j.jpba.2011.12.017 CR - Shi, Y.B., Li, H.L., Wang, H.Q., Yang, Y.B., Zhang, X.Y., Wang, H., Zhu, Z.J., Zhang, Z.Y., & Zhang, C.A., (2014). Simultaneous determination of five anthraquinones in a Chinese traditional preparation by RP-HPLC using an improved extraction procedure. Journal of Integrative Medicine 12 (5), 455-462. doi: 10.1016/S2095-4964(14)60037-6 CR - Singh, N.P., Gupta, A.P., Sinha, A.K., & Ahuja, P.S., (2005). High-performance thin layer chromatography method for quantitative determination of four major anthraquinone derivatives in Rheum emodi. Journal of Chromatography A, 1077 (2), 202-206. doi: 10.1016/j.chroma.2005.03.130 CR - Smolarz, H.D., Swatko-Ossor, M., Ginalska, G., & Medynska, E., (2013). Antimycobacterial effect of extract and its components from Rheum rhaponticum. Journal of AOAC International, 96 (1), 155-160. doi: 10.5740/jaoacint.12-010 CR - Su, Y.T., Chang, H.L., Shyue, S.K., & Hsu, S.L., ( 2005). Emodin induces apoptosis in human lung adenocarcinoma cells through a reactive oxygen species-dependent mitochondrial signaling pathway. Biochemical Pharmacology, 70 (2), 229-241. doi: 10.1016/j.bcp.2005.04.026 CR - Sun, S.W., & Yeh, P.C., (2005). Analysis of rhubarb anthraquinones and bianthrones by microemulsion electrokinetic chromatography. Journal of Pharmaceutical and Biomedical Analysis 36, (5), 995-1001. doi: 10.1016/j.jpba.2004.08.039 CR - Tian, J., Chen, X. and Bai, X., (2012). Comparison of dispersive liquid-liquid microextraction based on organic solvent and ionic liquid combined with high-performance liquid chromatography for the analysis of emodin and its metabolites in urine samples. Journal of Separation Science, 35 (1), 145-152. doi: 10.1002/jssc.201100729 CR - Tian, K., Wang, Y., Chen, Y., Chen, X., & Hu, Z., (2007). Application of 1-alkyl-3-methylimidazolium-based ionic liquids as background electrolyte in capillary zone electrophoresis for the simultaneous determination of five anthraquinones in Rhubarb. Talanta, 72 (2), 587-593. doi: 10.1016/j.talanta.2006.11.027 CR - Uzun, M., & Demirezer, L.O., (2019). Anti-aging power of Rumex crispus L.: Matrixmetalloproteinases inhibitor, sun protective and antioxidant. South African Journal of Botany, 124, 364-371. doi: 10.1016/j.sajb.2019.05.028 CR - VanMen, C., Jang, Y.S., Zhu, H.M., Lee, J.H., Trung, T.N., Ngoc, T.M., Kim, Y.K., & Kang, J.K., (2012). Chemical-based species classification of rhubarb using simultaneous determination of five bioactive substances by HPLC and LDA analysis. Phytochemical Analysis, 23 (4), 359-364. doi: 10.1002/pca.1365 CR - Wang, A., Zhou, Y., Wu, F., He, P., & Fang, Y., (2004). Determination of active ingredients in Huangdan Yinchen Keliby CZE with amperometric detection. Journal of Pharmaceutical and Biomedical Analysis, 35, 959–964. doi:10.1016/j.jpba.2004.02.031 CR - Wang, G.Y., & Shi, Y.P., (2014). Simultaneous Determination of Anthraquinone Derivatives in Radix et Rhizoma Rhei-based Medicines by Ultra-Performance LC-ESCI-MS/MS Multiple Reaction Monitoring. Acta Chromatographica, 26 (2), 229-242. doi: 10.1556/AChrom.26.2014.2.3 CR - Wang, Y., Xiong, H., Zhang, X., & Wang, S., (2010). Electrochemical study of Aloe-emodin on an ionic liquid-type carbon paste electrode. Microchimica Acta, 169, 255-260. doi: 10.1007/s00604-010-0348-7 CR - Wang, Z., Hu, J., Du, H., He, S., Li, Q., & Zhang, H., (2016). Microwave-assisted ionic liquid homogeneous liquid–liquid microextraction coupled with high performance liquid chromatography for the determination of anthraquinones in Rheum palmatum L. Journal of Pharmaceutical and Biomedical Analysis, 125, 178-185. doi: 10.1016/j.jpba.2016.03.046 CR - Wang, Z., Ma, P., Xu, L., He, C., & Peng, Y., (2013). Evaluation of the content variation of anthraquinone glycosides in rhubarb by UPLC-PDA. Chemistry Central Journal, 7 (1), 170. doi: 10.1186/1752-153X-7-170 CR - Wei, S.Y., Yao, W.X., Ji, W.Y., Wei, J.Q., & Peng, S.Q., (2013). Qualitative and quantitative analysis of anthraquinones in rhubarbs by high performance liquid chromatography with diode array detector and mass spectrometry. Food Chemistry, 141 (3), 1710-1715. doi: 10.1016/j.foodchem.2013.04.074 CR - Wijesekara, I, Zhang, C., Van-Ta, Q., Vo, T.S., Li, Y.X., & Kim, S.K., (2014). Physcion from marine-derived fungus Microsporum sp. induces apoptosis in human cervical carcinoma HeLa cells. Microbiological Research, 169 (4), 255-261. doi: 10.1016/j.micres.2013.09.001 CR - Wolfender, J.L., Marti, G., Thomas, A., & Bertrand, S., (2015.) Current approaches and challenges for the metabolite profiling of complex natural extracts. Journal of Chromatography A, 1382, 136-164. doi: 10.1016/j.chroma.2014.10.091 CR - Xu, F., Liu, Y., Zhang, Z., Song, R., Dong, H., & Tian, Y., (2008). 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. Journal of Pharmaceutical and Biomedical Analysis, 47 (3), 586-595. doi: 10.1016/j.jpba.2008.02.005 CR - Yanwen, W., Wenyuan, G., Xiaohe, X., & Yi, L., (2005). Calorimetric investigation of the effect of hydroxyanthraquinones in Rheum officinale Baill on Staphylococcus aureus growth. Thermochimica Acta, 429, 167–170. doi:10.1016/j.tca.2005.03.008 CR - Yao, X.S., Wu, L.J., & Wu, J.Z., (2004). Natural Medicine Chemistry. People Health Press;Beijing, China; pp. 150. CR - Yen, G.C., Duh, P.D., & Chuang, D.Y., (2000). Antioxidant activity of anthraquinones and anthrone. Food Chemistry, 70 (4), 437-441. doi: 10.1016/s0308-8146(00)00108-4 CR - You, X,, Feng, S., Luo, S., Cong, D., Yu, Z., Yang, Z., & Zhang, J., (2013). Studies on a rhein-producing endophytic fungus isolated from Rheum palmatum L. Fitoterapia, 85 (1), 161-168. doi: 10.1016/j.fitote.2012.12.010 CR - Zhan, H., Fang, J., Wu, H.W., Yang, H.J., Li, H., Wang, Z.J., Yang, B., Tang, L.Y., Fu, M.H. Rapid determination of total content of five major anthraquinones in Rhei Radix et Rhizoma by NIR spectroscopy. Chinese Herbal Medicines, 2017, vol. 9, no.3, p. 250, 10.1016/S1674-6384(17)60101-1. CR - Zhang, H.F., & Shi, Y.P., (2010). 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. Talanta, 82 (3), 1010-1016. doi: 10.1016/j.talanta.2010.06.008 CR - Zhang, L.S., Hu, S., Chen, X., Bai, X.H., & Li, Q.S., (2013). A new ionic liquid–water–organic solvent three phase microextraction for simultaneous preconcentration flavonoids and anthraquinones from traditional Chinese prescription. Journal of Pharmaceutical and Biomedical Analysis, 86, 36-39. doi: 10.1016/j.jpba.2013.07.007 CR - Zou, J.L., Xie, Z.Y., Jiang, X.F., Chen, X.X., & Yao, M.C., (2008). Determination of Anthraquinones in Different Compatibility of Dahuang Huanglian Xiexin Decoction by HPLC. World Science and Technology, 10 (4), 61-64. doi: 10.1016/S1876-3553(09)60021-1 CR - Zuo, Y., Wang, C., Lin, Y., Guo, J., & Deng, Y., (2008). Simultaneous determination of anthraquinones in radix Polygoni multiflori by capillary gas chromatography coupled with flame ionization and mass spectrometric detection. Journal of Chromatography A, 1200 (1), 43-48. doi: 10.1016/j.chroma.2008.01.058 UR - https://doi.org/10.31590/ejosat.987628 L1 - https://dergipark.org.tr/tr/download/article-file/1944060 ER -