Quantitative analysis of silybin A and B in dry extract of milk thistle (silybum marianum) by uplc / uv - vis method

Nội dung text: Quantitative analysis of silybin A and B in dry extract of milk thistle (silybum marianum) by uplc / uv - vis method

1. Journal of military pharmaco-medicine n o7-2017 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 (C 18 ; 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 has the effect of protecting the membrane Extract from the milk thistle seeds of liver cells and enhancing its function (Silybum marianum ) is being used as a and also protect the liver function from herbal therapy for hepatotoxicity and deterioration resulting from the invasion acute and chronic liver diseases [1, 2]. by deleterious substances [4]. Thus, silybin The pharmacological active ingredient is is considered as active compound of milk the flavonoid complex called silymarin, thistle extract. Silybin can be resolved into the main constituent accounts for about two 1:1 diastereoisomers, silybin A and 80% of the extract [3]. Silymarin is a silybin B. The concentration of silybin in complex of at least seven flavonolignans the main pharmaceutical products contain that are the most common class of silymarin present in the US and other compounds present in milk thistle extract countries ranging from 20% to 40% [5]. and one flavonoid, taxifolin. The relative Up to now, Vietnamese Pharmacopoeia abundance of each compound may vary hasn’t contained any monograph for depending on the sources of botanical quality control of dry extract of milk thistle. materials, suppliers and extraction In this study, we have developed and processes. Silybin represents about 50% validated a short UPLC/UV-Vis method to 70% of the silymarin extract. Silybin for the analysis of silybin A and B in dry (figure 1), a stabilizer of liver cell membrane, 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 5
2. Journal of military pharmaco-medicine N o7-2017 Figure 1: Chemical structure of silybin A (1a) and B (1b). MATERIALS AND METHODS - Equipment: Acquity UPLC H-Class 1. Materials and equipments. system including Acquity Autosampler, Quaternary solvent Manager, column oven - Materials: Dry extract of milk thistle and Empower sofware), the Acquity UPLC was purchased from Dongtai Kangning BEH column (C 18 ; 1.7 µm, 2.1 x 100 mm) Vegetable Extraction Co., LTD (China), in was used for the separation; Elmasonic December 2016. Sultrasonic (Elma), Mettler Toledo - Solvents: acetonitrile (ACN) (Merck, MS205DU. HPLC grade), 0.1% aqueous formic acid 2. Methods. (Merck, analytical grade); methanol - Chromatographic conditions: (MeOH) (Merck, HPLC grade); double- distilled water was prepared using + Detector PDA: detection wavelength Hamilton water still system (Hamilton was set at 280 nm. Laboratory Glass Ltd, UK). + Flow rate: 0.3 mL/minute. - Chemical: Silybin standard (racemic + Injection volume: 5 µL. o mixture of silybin A and B with 93.5% + Column temperature: 25 C. purity) was purchased from National Drug + A multistep gradient program of Quality Control. mobile phase as shown in table 1. 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 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 0 80 20 6
3. Journal of military pharmaco-medicine n o7-2017 - 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 1. System suitability. For evaluating system suitability, a standard solution was injected 6 times repeatedly into the UPLC system [6]. Results are shown in table 2 and chromatograms of standard and sample solutions were shown in figure 2. Table 2: Results of system suitability for analysis of silybin. Parameters Silybin A Silybin B Peak area (µV*s) S = 380,156 S = 386,267 RSD = 0.92% RSD = 0.90% Retention time (min) tR= 12.05 tR = 12.99 RSD = 0.10 % RSD = 0.09% Tailing factor 0.98 1.06 Resolution 1.60 1.60 Column efficiency (plate number) > 11,952 > 10,923 Figure 2: Chromagrams of standard silybin (a) and dry extract of milk thistle sample (b); 1: silybin A; 2: silybin B. 7
4. Journal of military pharmaco-medicine N o7-2017 The results in table 2 show that the the chromatogram of standard solution repeatability of peak areas and retention are coincident. At retention times of each times are good (both RSD < 2%). Peaks peak of silybin A and B in standard and of silybin A and B are sharp and sample and correlation coefficient of proportional (tailing factor for sylibin A and silybin A and B UV spectra in standard B were 0.98 and 1.06, respectively). and sample were more than 0.999. These Theoretic plate number for sylibin A and B results prove that the established method are 11,952 and 10,923, respectively. has good specificity. Therefore, the chromatographic system is 3. Linear range and calibration curves. suitable for analyzing samples. For evaluating the linear range of the 2. Specificity. established method, 6 concentrations of Retention times of the peak in the silybin A and B were analyzed [6]. Results chromatogram of sample solution and in are showed in table 3 and figure 3. Table 3: Correlation between peak areas and concentrations of silybin A and B. Peak area (µV*s) No Concentration (µg/mL) Silybin A Silybin B 1 2.34 35379 39931 2 4.68 70839 78019 3 9.35 142756 156514 4 23.38 353853 391459 5 37.40 566159 623065 6 46.75 702105 787906 Regression equation Y = 15042.7X + 1248.35 Y = 16.786.1X - 449.78 correlation coefficient R 2 0.9999 0.9999 The results of figure 3 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). 8
5. Journal of military pharmaco-medicine n o7-2017 4. Precision. Precision of the established method is evaluated based on the repeatability of 6 individual experiments quantifying silybin A and B in the raw materials [6]. Results are showed in table 4. 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) 1 10.02 32.69 63.35 2 10.15 32.92 63.78 3 10.11 30.92 63.09 4 10.12 32.33 62.51 5 10.05 31.93 61.89 6 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 4 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. 5. 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 6 times repeatedly into the UPLC system. Results are showed in table 5. Table 5: Accuracy of the analytical method for silybin A and B. Silybin A Silybin B No Added (µg) Recovered (µg) Recovery (%) Added (µg) Recovered (µg) Recovery (%) 1 46.75 44.92 96.09 46.75 45.36 97.03 2 46.75 45.98 98.35 46.75 44.78 95.79 3 46.75 46.17 98.76 46.75 46.43 99.32 4 46.75 46.12 98.65 46.75 46.38 99.21 5 46.75 46.44 99.34 46.75 46.70 99.89 6 46.75 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 5 reveal that the established method has good accuracy with recovery ratio = 96.09 - 99.89%, RSDs less than 1.60%. 9
6. Journal of military pharmaco-medicine N o7-2017 6. LOD and LOQ. therapy of liver disease. Am J Gastroenterol. By diluting standard solutions of silybin 1998, 93, pp.139-143. A and B and analyzing with the UPLC 2. Gazak R, Walterova D, Kren V . Silybin system, LOD and LOQ of the established and silymarin-new and emerging applications method were determined based on signal in medicine. Curr Med Chem. 2007, 14, to noise [6]. LOD was about 0.31 µg/mL pp.315-338. and LOQ was about 0.93 µg/mL. 3. Weyhenmeyer R, Mascher H, Birkmayer CONCLUSION J. Study on dose-linearity of the pharmacokinetics of silibinin diastereomers using a new A suitable method had been developed stereospecific assay. Int. J Clin Pharmacol. and validated for quantifying silybin A and Ther Toxicol. 1992, 30, pp.134-138. B in dry extract of milk thistle. The established method has good precision 4. Valenzuela A, Guerra R, Garrido A. and accuracy. This study also provided Silybin dihemisuccinate protects rat erythrocytes valuable information for the content of against phenylhydrazine-induced lipid silybin A and B in our dry extract of milk peroxidation and hemolysis. Planta Med. thistle being about 32.14 mg/g and 62.55 1987, 53, pp.402-405. mg/g, respectively. 5. Lee J.I, Narayan M, Barrett J.S. Analysis ACKNOWLEDGEMENT and comparison of active constituents in commercial standardized silymarin extracts by This research was supported by liquid chromatography-electrospray ionization “National Program KC.10/16-20” under mass spectrometry. J Chromatogr B Analyt grant number KHCN-KC.10.12/16-20. Technol Biomed Life Sci. 2007, 845, pp.95-103. REFERENCES 6. ICH guidelines . Q2 (R1) validation of 1. Flora K, Hahn M., Rosen H, Benner K. analytical procedures: text and methodology. Milk thistle ( Silybum marianum ) for the 1996. 10