Objectives: To establish an UPLC/UV-Vis method that is suited with current conditions of the laboratory to quantify silybin A and B in dry extract of milk thistle. Materials and methods: Parameters of the method are validated.
Journal of military pharmaco-medicine no7-2017 QUANTITATIVE ANALYSIS OF SILYBIN A AND B IN DRY EXTRACT OF MILK THISTLE (Silybum marianum) BY UPLC/UV-VIS METHOD Dang Truong Giang*; Vu Tuan Anh* Hoang Kim Vuong Nam**; Chu Van Men* SUMMARY Objectives: To establish an UPLC/UV-Vis method that is suited with current conditions of the laboratory to quantify silybin A and B in dry extract of milk thistle Materials and methods: Parameters of the method are validated including: linear range, precision, accuracy Results: Chromatographic conditions of the method: using Cortecs UPLC BEH column (C18; 1.6 µm, 2.1 x 50 mm), acetonitrile and 0.1% aqueous formic acid as mobile phase following a multistep gradient program, monitored at 320 nm The established method has good precision (RSD = 2.37%) and accuracy (recovery ratio = 97.08 - 103.76%, average = 100.38%, RSD = 2.84%) Conclusions: UPLC/UV-Vis method for quantifying silybin A and B in dry extract of milk thistle has been developed and validated * Keywords: Milk thistle; Silybin A, B; HPLC INTRODUCTION Extract from the milk thistle seeds (Silybum marianum) is being used as a herbal therapy for hepatotoxicity and acute and chronic liver diseases [1, 2] The pharmacological active ingredient is the flavonoid complex called silymarin, the main constituent accounts for about 80% of the extract [3] Silymarin is a complex of at least seven flavonolignans that are the most common class of compounds present in milk thistle extract and one flavonoid, taxifolin The relative abundance of each compound may vary depending on the sources of botanical materials, suppliers and extraction processes Silybin represents about 50% to 70% of the silymarin extract Silybin (figure 1), a stabilizer of liver cell membrane, has the effect of protecting the membrane of liver cells and enhancing its function and also protect the liver function from deterioration resulting from the invasion by deleterious substances [4] Thus, silybin is considered as active compound of milk thistle extract Silybin can be resolved into two 1:1 diastereoisomers, silybin A and silybin B The concentration of silybin in the main pharmaceutical products contain silymarin present in the US and other countries ranging from 20% to 40% [5] Up to now, Vietnamese Pharmacopoeia hasn’t contained any monograph for quality control of dry extract of milk thistle In this study, we have developed and validated a short UPLC/UV-Vis method for the analysis of silybin A and B in dry extract of milk thistle * Vietnam Military Medical University ** College of Pharmacy, Lac Hong University Corresponding author: Chu Van Men (chuvanmen@gmail.com) Date received: 30/06/2017 Date accepted: 10/08/2017 Journal of military pharmaco-medicine No7-2017 Figure 1: Chemical structure of silybin A (1a) and B (1b) MATERIALS AND METHODS Materials and equipments - Materials: Dry extract of milk thistle was purchased from Dongtai Kangning Vegetable Extraction Co., LTD (China), in December 2016 - Solvents: acetonitrile (ACN) (Merck, HPLC grade), 0.1% aqueous formic acid (Merck, analytical grade); methanol (MeOH) (Merck, HPLC grade); doubledistilled water was prepared using Hamilton water still system (Hamilton Laboratory Glass Ltd, UK) - Chemical: Silybin standard (racemic mixture of silybin A and B with 93.5% purity) was purchased from National Drug Quality Control - Equipment: Acquity UPLC H-Class system including Acquity Autosampler, Quaternary solvent Manager, column oven and Empower sofware), the Acquity UPLC BEH column (C18; 1.7 µm, 2.1 x 100 mm) was used for the separation; Elmasonic Sultrasonic (Elma), Mettler Toledo MS205DU Methods - Chromatographic conditions: + Detector PDA: detection wavelength was set at 280 nm + Flow rate: 0.3 mL/minute + Injection volume: µL + Column temperature: 25oC + A multistep gradient program of mobile phase as shown in table Table 1: Gradient condition for analysis of silybin in dry extract of milk thistle Time (min) Flow rate (mL/min) Water (%) Methanol (%) 0.1% aqueous acetic acid (%) 0.3 50 30 20 0.5 0.3 45 35 20 2.5 0.3 35 45 20 6.0 0.3 35 45 20 10.0 0.3 30 50 20 15.0 0.3 20 60 20 20.0 0.3 80 20 Journal of military pharmaco-medicine no7-2017 - Sample preparation: 10 mg of dry extract of milk thistle was ultrasonically extracted with about 25 mL of methanol in a volumetric flask of 25 mL for 15 minutes Methanol was added to volumetric mark After that, this solution was filtered through a 0.45 µm filter membrane and injected into the UPLC system for analysis - Standard solution preparation: Dissolving and diluting an accurate amount of silybin standard in methanol by using volumetric flasks and Eppendorf micropipets to obtain a suitable range of working standard solutions - Method validation: The analysis method was fully validated in accordance to ICH guideline [6] RESULTS AND DISCUSSIONS System suitability For evaluating system suitability, a standard solution was injected times repeatedly into the UPLC system [6] Results are shown in table and chromatograms of standard and sample solutions were shown in figure Table 2: Results of system suitability for analysis of silybin Parameters Silybin A Silybin B Peak area (µV*s) S = 380,156 RSD = 0.92% Retention time (min) tR= 12.05 RSD = 0.10 % tR = 12.99 RSD = 0.09% Tailing factor 0.98 1.06 Resolution 1.60 1.60 > 11,952 > 10,923 Column efficiency (plate number) S = 386,267 RSD = 0.90% Figure 2: Chromagrams of standard silybin (a) and dry extract of milk thistle sample (b); 1: silybin A; 2: silybin B Journal of military pharmaco-medicine No7-2017 The results in table show that the repeatability of peak areas and retention times are good (both RSD < 2%) Peaks of silybin A and B are sharp and proportional (tailing factor for sylibin A and B were 0.98 and 1.06, respectively) Theoretic plate number for sylibin A and B are 11,952 and 10,923, respectively Therefore, the chromatographic system is suitable for analyzing samples Specificity Retention times of the peak in the chromatogram of sample solution and in the chromatogram of standard solution are coincident At retention times of each peak of silybin A and B in standard and sample and correlation coefficient of silybin A and B UV spectra in standard and sample were more than 0.999 These results prove that the established method has good specificity Linear range and calibration curves For evaluating the linear range of the established method, concentrations of silybin A and B were analyzed [6] Results are showed in table and figure Table 3: Correlation between peak areas and concentrations of silybin A and B Peak area (µV*s) No Concentration (µg/mL) 2.34 4.68 70839 78019 9.35 142756 156514 23.38 353853 391459 37.40 566159 623065 46.75 702105 787906 Regression equation Y = 15042.7X + 1248.35 Y = 16.786.1X - 449.78 correlation coefficient R2 0.9999 0.9999 Silybin A Silybin B 35379 39931 The results of figure show linear correlation between peak areas and concentrations of silybin A and B following the regression equation Y = 15042.7 X + 1248.35 and Y = 16786.1X - 449.78, respectively with correlation coefficient of 0.9999 for both compounds Figure 3: Calibration curves of silybin A (a) and B (b) Journal of military pharmaco-medicine no7-2017 Precision Precision of the established method is evaluated based on the repeatability of individual experiments quantifying silybin A and B in the raw materials [6] Results are showed in table Table 4: Precision of the analytical method for silybin A and B No Weight of materials (mg) Silybin A (mg/g) Silybin B (mg/g) 10.02 32.69 63.35 10.15 32.92 63.78 10.11 30.92 63.09 10.12 32.33 62.51 10.05 31.93 61.89 10.24 32.05 60.65 Mean ± SD 32.14 ± 0.71 62.55 ± 1.14 RSD (%) 2.19 1.82 The results in table show the established method has good repeatability with all RSDs less than 2.19% The content of silybin A and B in dry extract of milk thistle are 32.14 mg/g and 62.55 mg/g, respectively Accuracy Accuracy of the established method is evaluated by spiking an exact amount of silybin A and B into the dry extract sample of milk thistle Then, determine recovery ratio by comparing and recovery amount to the spiked amount of silybin A and B [6] Samples were injected times repeatedly into the UPLC system Results are showed in table Table 5: Accuracy of the analytical method for silybin A and B No Silybin A Added (µg) Recovered (µg) 46.75 44.92 46.75 Silybin B Added (µg) Recovered (µg) Recovery (%) 96.09 46.75 45.36 97.03 45.98 98.35 46.75 44.78 95.79 46.75 46.17 98.76 46.75 46.43 99.32 46.75 46.12 98.65 46.75 46.38 99.21 46.75 46.44 99.34 46.75 46.70 99.89 46.75 Recovery (%) 45.58 97.50 46.75 45.84 98.05 Mean 45.87 98.11 Mean 45.92 98.21 RSD (%) 1.18 1.18 RSD (%) 1.60 1.60 The results in table reveal that the established method has good accuracy with recovery ratio = 96.09 - 99.89%, RSDs less than 1.60% Journal of military pharmaco-medicine No7-2017 LOD and LOQ By diluting standard solutions of silybin A and B and analyzing with the UPLC system, LOD and LOQ of the established method were determined based on signal to noise [6] LOD was about 0.31 µg/mL and LOQ was about 0.93 µg/mL CONCLUSION A suitable method had been developed and validated for quantifying silybin A and B in dry extract of milk thistle The established method has good precision and accuracy This study also provided valuable information for the content of silybin A and B in our dry extract of milk thistle being about 32.14 mg/g and 62.55 mg/g, respectively ACKNOWLEDGEMENT This research was supported by “National Program KC.10/16-20” under grant number KHCN-KC.10.12/16-20 REFERENCES Flora K, Hahn M., Rosen H, Benner K Milk thistle (Silybum marianum) for the 10 therapy of liver disease Am J Gastroenterol 1998, 93, pp.139-143 Gazak R, Walterova D, Kren V Silybin and silymarin-new and emerging applications in medicine Curr Med Chem 2007, 14, pp.315-338 Weyhenmeyer R, Mascher H, Birkmayer J Study on dose-linearity of the pharmacokinetics of silibinin diastereomers using a new stereospecific assay Int J Clin Pharmacol Ther Toxicol 1992, 30, pp.134-138 Valenzuela A, Guerra R, Garrido A Silybin dihemisuccinate protects rat erythrocytes against phenylhydrazine-induced lipid peroxidation and hemolysis Planta Med 1987, 53, pp.402-405 Lee J.I, Narayan M, Barrett J.S Analysis and comparison of active constituents in commercial standardized silymarin extracts by liquid chromatography-electrospray ionization mass spectrometry J Chromatogr B Analyt Technol Biomed Life Sci 2007, 845, pp.95-103 ICH guidelines Q2 (R1) validation of analytical procedures: text and methodology 1996 ... times of the peak in the chromatogram of sample solution and in the chromatogram of standard solution are coincident At retention times of each peak of silybin A and B in standard and sample and. .. results in table show that the repeatability of peak areas and retention times are good (both RSD < 2%) Peaks of silybin A and B are sharp and proportional (tailing factor for sylibin A and B were... preparation: Dissolving and diluting an accurate amount of silybin standard in methanol by using volumetric flasks and Eppendorf micropipets to obtain a suitable range of working standard solutions