Euphorbia helioscopia L is widespread in China and has a large number of flavonoids. Quercetin glycosides, having useful biological activities, are abundant in this plant, and no validated analytical method has so far been developed for their determination. We, therefore, standardized a reversedphase highperformance liquid chromatography (RPHPLC) assay for quercetin detection. For this, the plant was locally procured and identification was confirmed based on its morphohistological characteristics. Ethyl acetate extracts of leaves, stems, and roots were analyzed by RPHPLC using Agilent 1120 HPLC TCC18 column (250 9 4.6 mm; 5 lm) with UVdetector system. The mobile phase of methanol0.2% phosphoric acid (65:35) solution was used with the flow rate of 1.0 ml min1 at 30C, and the detection was performed at 360 nm wavelength. Our data show that the linear range of quercetin was 0.025–0.150 mg.ml1 (r = 0.9995; n = 6) with the recovery rate of 97.50–103.30% (average 100.40%; RSD = 2.28%). The target component was baseline separated during only the period of 9 min. The repeatability of RPHPLC analysis was demonstrated with an RSD of 1.77% (n = 6), and the highest quercetin content (average 1.42 mg g1dryweight) was present in leaves. It was, therefore, concluded that RPHPLC is a simple, rapid, accurate, and sensitive method for the detection of quercetin from Euphorbia helioscopia L.
Cell Biochem Biophys (2011) 61:59–64 DOI 10.1007/s12013-011-9161-0 ORIGINAL PAPER Quantitative Analysis of Quercetin in Euphorbia helioscopia L by RP-HPLC Hai Peng Liu • Xiao Feng Shi • You Cheng Zhang Zhong Xin Li • Lin Zhang • Zhe Yuan Wang • Published online: 17 February 2011 Ó Springer Science+Business Media, LLC 2011 Abstract Euphorbia helioscopia L is widespread in China and has a large number of flavonoids Quercetin glycosides, having useful biological activities, are abundant in this plant, and no validated analytical method has so far been developed for their determination We, therefore, standardized a reversed-phase high-performance liquid chromatography (RP-HPLC) assay for quercetin detection For this, the plant was locally procured and identification was confirmed based on its morpho-histological characteristics Ethyl acetate extracts of leaves, stems, and roots were analyzed by RP-HPLC using Agilent 1120 HPLC TCC18 column (250 4.6 mm; lm) with UV-detector system The mobile phase of methanol-0.2% phosphoric acid (65:35) solution was used with the flow rate of 1.0 ml min-1 at 30°C, and the detection was performed at 360 nm wavelength Our data show that the linear range of quercetin was 0.025–0.150 mg.ml-1 (r = 0.9995; n = 6) with the recovery rate of 97.50–103.30% (average 100.40%; RSD = 2.28%) The target component was baseline separated during only the period of The repeatability of RP-HPLC analysis was demonstrated with an RSD of 1.77% (n = 6), and the highest quercetin content (average 1.42 mg g-1dry-weight) was present in leaves It was, therefore, concluded that RP-HPLC is a simple, rapid, accurate, and sensitive method for the detection of quercetin from Euphorbia helioscopia L H P Liu Á Y C Zhang (&) Á Z X Li Á L Zhang Á Z Y Wang Department of General Surgery, Lanzhou University Second Hospital, 82 Cuiyingmen, Chengguan District, Lanzhou 730030, Gansu, China e-mail: zhangychmd@yahoo.com.cn X F Shi Institute of Materia Medica, Gansu Academy of Medical Sciences, Lanzhou 730050, Gansu, China Keywords Chinese herb Á Euphorbia helioscopia L Á Quercetin Á RP-HPLC Introduction Euphorbia helioscopia L (Family Euphorbiaceae) is widely distributed in most parts of China [1] It has been traditionally used for the prevention, improvement, or cure of various diseases such as liver cancer, esophagus cancer, nasopharyngeal carcinoma, bronchitis, acute glomerulonephritis, and epidemic parotitis in China for centuries [2–7] Phytochemical studies reveal that Euphorbia helioscopia L contains a variety of secondary metabolites Terpenoids [8–15], flavonoids [16, 17], tannins [18, 19], steroids and lipids [9] were isolated from this plant during the twentieth century Some of the compounds were found to have antitumor [20–22], bacteriostatic [23], anti-allergic and antiasthmatic [24], insecticidal [25], and inhibitory effect on mushroom tyrosinase in vitro [26] The recent studies have shown that terpenoids and flavonoids are the main biologically active substances The flavonoids isolated from Euphorbia helioscopia L mainly include quercetin [17], kaempferol, and flavonoid glycosides such as quercetin-3D-galactosidase [17], quercetin 3-b-glucoside, quercetin 3-b-galactosidase, quercetin 3-b-galactosidase-2-gallic acid [27], and quercetin-5, 3-2-D-galactosidase [16] Quercetin glycosides appear to predominate in Euphorbia helioscopia L Quercetin is known to have a variety of biological activities and may play an important role in the medicinal effects of Euphorbia helioscopia L To our knowledge, no validated analytical method has been so far reported for determining quercetin contents in Euphorbia helioscopia L Therefore, in this study, we developed a rapid, simple and selective reversed-phase high-performance 123 60 liquid chromatography (RP-HPLC) method for the detection of quercetin contents in Euphorbia helioscopia L Cell Biochem Biophys (2011) 61:59–64 and filtered with 0.45-lm Millipore membrane filter before use RP-HPLC Materials and Methods Samples The plant of Euphorbia helioscopia L was procured from Dingxi, Gansu Province in China during the months of June, July and August, 2008 The plant identification was authenticated based on its specific morphological and histological characteristics by the School of Life Sciences, Lanzhou University The plant was air-dried immediately after collection, and some of them were first separated into roots, stems and leaves and then preserved The samples were stored in bags and kept in a dry and cool place for later use Instrumentation Analytical balance (AE260, Mettle Company, Switzer¨ CHI AG, Switzerland), and land), Rotary Evaporators (BU Agilent 1120 High-Performance Liquid Chromatography system (Agilent Technologies, Santa Clara, CA, USA) were used in this study Chemicals and Reagents Standard quercetin was obtained from National Institute for the Control of Pharmaceutical and Biological Products (Batch number: 100081-200406) Methanol used was of HPLC-specific grade (Yu Wang-Chemical Industry Company, Shan Dong; China) Quercetin contents in Euphorbia helioscopia L were analyzed by using RP-HPLC At first, three different buffers including methanol buffer (buffer A), acetonitrile buffer (buffer B), and 0.4% phosphoric acid buffer (buffer C), were mixed at three different ratios such as 60:20:20; 65:20:15; and 70:15:15 It was found that the whole separation course required almost 21 and the peak of quercetin was obtained at the 15th minute and was asymmetric Thus, we opted to use 0.2% phosphoric acid buffer (buffer D) and mixed buffer A and buffer D at the ratios of 70:30; 60:40; and 65:35 The quercetin peak time now shifted to the fifth minute point and the whole separation course required while the peak obtained was symmetric Since quercetin is reported to have the maximum absorption at 250 and 360 nm wavelengths, we, therefore, compared the shapes of quercetin peaks at these wavelengths and found that the shape of the peak was more symmetric and higher at the detection wavelength of 360 nm We used Agilent 1120 system with TC-C18 column (250 4.6 mm, lm) The mobile phase was methanol-0.2% phosphoric acid (65:35) solution The flow rate was 1.0 ml min-1 The column temperature was 30°C and the detection wavelength was 360 nm The quercetin peaks were identified and quantified against the external reference standards Quercetin standard was purchased from the National Institute for the Control of Pharmaceutical and Biological Products, China The solvents used were of spectra analytical grade and were filtered through 0.45 lm filters before use Statistical Analysis Sample Preparation The pulverized plant material (about 60 mesh; g) was reflux-extracted twice, using 50 ml of methanol for h each time in a water bath at 80°C The extracts were filtered through filter paper (Whatman filter paper, 1003090/grade No 3) and collected in a flask The pooled extract was evaporated under vacuum to dryness The residue was dissolved in 30 ml of distilled water and extracted six times using 30 ml of petroleum ether each time The petroleum ether extraction liquid was discarded and 10 ml of HCL (10%) was added to water liquid extract which was extracted for 30 in a water bath at 90°C After rapid cool off, the liquid was extracted five times using 30 ml of ethyl acetate each time The ethyl acetate extraction was collected, evaporated to dryness, re-dissolved in methanol, transferred to a 25-ml measuring flask 123 The data were acquired and analyzed by Agilent 1120 HPLC software The standard curve of quercetin was obtained by plotting concentrations against peak area and the regression equation and correlation coefficient (r2) were determined The sample analyses were performed in triplicate and the mean values were calculated RSD \ 3% was considered as significant for the precision (repeatability) and the accuracy (recovery) of the method used Results RP-HPLC Optimization Conditions The best mobile phase was methanol-0.2% phosphoric acid (65:35) solution, the flow rate was 1.0 ml min-1, the Cell Biochem Biophys (2011) 61:59–64 61 Fig Standard peak of quercetin Quercetin content in Euphorbia helioscopia L was determined by RP-HPLC using Agilent 1120 system with TC-C18 column (250 4.6 mm, lm) and UV detector system The assay was optimized using the mobile phase of methanol0.2% phosphoric acid (65:35) solution and a flow rate of 1.0 ml min-1at the column temperature of 30°C and the detection wavelength of 360 nm The standard solutions of quercetin were prepared at the concentrations of 0.025, 0.050, 0.075, 0.100, 0.125 and 0.150 mg ml-1 The least-square regression analysis of quercetin calibration graph was: y = 6.00460 108 x ? 41710.9 (r = 0.9995) where, y represents peak area, x represents concentration in mg ml-1 and r represents correlation coefficient Fig Sample peak of quercetin Quercetin content in Euphorbia helioscopia L were determined by RP-HPLC as described before Quercetin sample peaks were identified and quantified against the external reference standards The assay was performed in triplicate and average quercetin content (mg g-1) was calculated column temperature was 30°C, and the detection wavelength was 360 nm The standard (reference) and sample peaks of quercetin are shown in Figs and 2, respectively Recovery Rate Validation Six samples from the same source were reflux-extracted, processed in parallel, analyzed by RP-HPLC, and compared The relative standard deviation (RSD) was 1.77% By adding different concentrations of standard quercetin (e.g., 4.05, 2.70, and 1.35 mg g-1) into samples with known (2.69 mg g-1) quercetin content, the recovery rate of the method was determined at the reaction conditions used As shown in Table 1, the recovery rate ranged from 97.50 to 103.30% (average 100.40%; RSD 2.28%; n = 6) 123 62 Cell Biochem Biophys (2011) 61:59–64 Table Recovery rates (n = 6) Background (mg g-1) Added (mg g-1) Detected (mg g-1) 2.69 4.05 6.63 2.69 4.05 6.71 99.30 2.69 2.70 5.43 101.50 2.69 2.70 5.48 103.30 2.69 1.35 4.07 102.20 2.69 1.35 4.02 98.50 Recovery rate (%) Average recovery rate (%) RSD (%) 100.40 2.28 97.50 RSD represents relative standard deviation Linearity Discussion The linearity of the method was found to be from 0.025 to 0.150 mg ml-1 The standard solutions of quercetin were prepared at the concentrations of 0.025, 0.050, 0.075, 0.100, 0.125 and 0.150 mg ml-1 The least-square regression analysis of quercetin calibration graph was: y = 6.00460 108 x ? 41710.9; r = 0.9995 where y represents peak area, x represents concentration in mg ml-1, and r represents correlation coefficient The average quercetin contents of leaves, stems, and roots were 1.42 mg g-1 (RSD = 1.38%), 0.02 mg g-1 (RSD = 1.42%) and 0.04 mg g-1 (RSD = 1.87%), respectively The results are shown in Table HPLC method is gaining importance for the analysis of plant extracts Although TLC is also a simple method, it sometimes produces doubtful results RP-HPLC has been previously used for the analysis of flavonoids in plants e.g to distinguish species based on quantitative variation of flavonoids [28] and for quantitative analysis of flavonoid aglycones [29] The analysis of quercetin content in Euphorbia helioscopia L using RP-HPLC has not been previously reported, and in this study, we standardized the RP-HPLC method for the detection of quercetin in Euphorbia helioscopia L During optimization of RP-HPLC, we initially used different mobile phases comprising three different buffers mixed at three different ratios Since the quercetin peak obtained was asymmetric, we re-adjusted the mobile phase and found that methanol buffer mixed with 0.2% phosphoric acid buffer at the ratio of 65:35 yielded a highly symmetric quercetin peak at the flow rate of 1.0 ml min-1 at 30°C Since the previous studies [30, 31] indicate that quercetin has the maximum absorption at 250 and 360 nm, we tested and found that the shape of the peak was more symmetric at the detection UV wavelength of 360 nm using Agilent 1120 system with TC-C18 column (250 4.6 mm, Table Quercetin contents of whole plant at different times Table Quercetin contents of roots, stems and leaves Quercetin Contents of Whole Plant at Different Times The average quercetin contents in the whole plant were 3.57 mg g-1 (RSD = 1.22%), 2.69 mg g-1 (RSD = 1.49%), and 1.48 mg g-1 (RSD = 1.17%) in June, July, and August, respectively The results are shown in Table Quercetin Contents of Leaves, Stems and Roots Month No Quercetin contents (mg g-1) Average (mg g-1) RSD (%) Parts June 3.57 1.22 Leaves 1.3954 1.4308 1.4274 July 3.54 3.62 3.55 2.73 2.65 2.69 August 1.46 1.49 1.49 2.69 1.48 RSD represents relative standard deviation 123 1.49 1.17 Stems Roots No Quercetin contents (mg g-1) 0.0211 0.0208 0.0214 0.0441 0.0428 0.0443 Average (mg g-1) RSD (%) 1.4179 1.38 0.0211 1.42 0.0437 1.87 RSD represents relative standard deviation Cell Biochem Biophys (2011) 61:59–64 lm) The quercetin peaks were identified and quantified against the external reference standards For validation of analytical methods, the guidelines from the International Conference on the Harmonization of Technical Requirements for the Human Use (ICH) [32] and USP 24 [33] recommend that the tests of linearity, sensitivity, precision, specificity, and accuracy of the method be performed The type of method and its use determine the parameters to be evaluated, particularly when the samples are complex biologic matrices such as extractive solutions from plants In this study, the linearity of the HPLC method for quercetin was found to be in the range 0.025–0.150 mg ml-1 at six concentration levels and the representative linear equation for quercetin was: y = 6.00460 108 x ? 41710.9 (n = 6; r = 0.9995) The method was found to be highly sensitive as the limit of detection (LOD) values were within the range 0.025–0.150 mg ml-1 Since the target component was baseline separated during the period of merely min, the analytical method also proved to be appreciably rapid For further validation, we also determined precision (repeatability) and accuracy (recovery) of the method The repeatability of RP-HPLC analysis was demonstrated with an RSD of 1.77% (n = 6) The accuracy of HPLC method for quercetin analysis, based on percent recovery, was determined by adding different concentrations of standard quercetin (e.g., 4.05, 2.70, 1.35 mg g-1) into samples with known quercetin content (i.e., the background of 2.69 mg g-1) The recovery rate for quercetin was found to be 97.50–103.30% (average 100.40%; RSD = 2.28%), indicating that the analyses performed were highly accurate We found that quercetin content in the plant were the highest with an average of 3.57 mg g-1 dry weight in the month of June, followed by July, and August samples with the average contents of 2.69 and 1.48 mg g-1 dry weight, respectively This indicates that June is the best harvest time for optimal quercetin yield from Euphorbia helioscopia L More importantly, leaves had the highest quercetin content with an average of 1.42 mg g-1 dry weight while stems and roots had lower quercetin contents (0.02 and 0.04 mg g-1, respectively) Thus, we conclude that quercetin is mainly distributed in leaves of Euphorbia helioscopia L HPLC with diode array detector (DAD) and electrospray ionization mass spectrometry (ESI–MS) was previously used to detect 13 bioactive compounds in Flos Lonicerae In this study [31], the optimal chromatographic conditions were obtained on a C18 column (250 4.6 mm; lm) at 30°C The mobile phase comprises acetic acid aqueous (0.4% v/v) and acetonitrile with a gradient elution at the flow rate of ml min-1 at 360 nm The method provided satisfactory precision and accuracy In another study [34], a capillary zone electrophoresis method was established for analysis of aglycone quercetin in mulberry (Morus alba L.) 63 leaves Baseline separation of all compounds was obtained within 16.5 under the following conditions: 150 mM boric acid (pH 10.0) using a fused-silica capillary with an effective length of 42.5 cm (50 lm inner diameter) at 32°C and the voltage of 15 kV Although all the above methods provide satisfactory precision and accuracy in determination of flavonoids, there are also some caveats involved As for example, the capillary zone electrophoresis results are influenced by too many factors such as electrolyte concentrations and pH, surfactant concentrations, organic solvents, temperature, and the voltage used We used HPLC for determination of quercetin content because it is a relatively simple method as compared with HPLC combined with ESI–MS whereas the efficiency of the latter approach may be slightly better In this study, however, we have determined only one constituent of Euphorbia helioscopia L; therefore, more studies will be required to further evaluate sensitivity, precision and accuracy of this method for simultaneous determination of different flavonoids in the plant 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