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Objectives: Establishing a HPLC method that was suitable with current conditions of the laboratory to quantification of trans-resveratrol in Rhizoma reynoutriae japonicae. Material and methods: Parameters of the established method are validated including - linear range, precision, accuracy.
JOURNAL OF MILITARY PHARMACO-MEDICINE No7-2015 QUANTIFICATION OF TRANS-RESVERATROL IN Rhizoma reynoutriae japonicae BY HPLC Hoang Van Tan*; Trinh Nam Trung**; Hoang Viet Dung** Nguyen Van Bach**; Nguyen Trong Diep** SUMMARY Objectives: Establishing a HPLC method that was suitable with current conditions of the laboratory to quantification of trans-resveratrol in Rhizoma reynoutriae japonicae Material and methods: Parameters of the established method are validated including: linear range, precision, accuracy Results: Chromatographic conditions of the method: using Sunfire RP-18 column, acetonitrile and 0.1% formic acid as mobile phase following a multi-step gradient program, monitored at 305 nm The established method had the high precision (RSD = 1.65%) and high accuracy (recovery ratio = 97.32 - 102.47%, average = 99.13%, RSD = 1.14%) Conclusion: A suitable HPLC method for quantifying trans-resveratrol in Rhizoma reynoutriae japonicae was validated * Key words: Rhizoma reynoutriae japonicae; Trans-resveratrol; HPLC INTRODUCTION Reynoutria japonica Hautt was used to treat diseases based on traditional experience, including: treating pain of arthritis by rheumatism, swell and pain by trauma, persistant pain… [6] Information of articles indicated trans-resveratrol in Rhizoma reynoutriae japonicae possesses some interesting bioactivities, specially, the effect of modulating blood lipid status [1] So, we selected this compound as a bioactive marker for controlling quality of medicinal plants as well as semi-products or endproducts afterwards According to references, there were some researches of establishing HPLC methods for determinating transresveratrol together with other compounds [2, 3, 5] However, these results were not in accordance with our research target being only to quantify the content of transresveratrol Besides, our laboratory conditions were different from the published ones Therefore, this research was carried out to establish HPLC method for determinating trans-resveratrol in Rhizoma reynoutriae japonicae that was suitable for our laboratory conditions and research target MATERIALS AND METHODS Materials and equipment - Materials: Rhizoma reynoutriae japonicae was harvested in Sapa district, Laocai province in 01 - 2015 - Solvents: acetonitrile (ACN) (Merck, HPLC grade), 0.1% formic acid (Merck, analytical grade) * Hospital of Military Region ** Vietnam Military Medical University Corresponding author: Hoang Viet Dung (vietdungk85@yahoo.com) JOURNAL OF MILITARY PHARMACO-MEDICINE No7-2015 - Chemical: trans-resveratrol (content 98%, provided by Centre of Pharmaceutical Research and Training, Vietnam Military Medical University) - Equipment: analyzing by waters HPLC system (Sunfire RP-18 column; autosamples 2695; deetector PDA 2998; empower sofware), extraction by Elmasonic machine, analysis balance of Mettler Toledo… RESULTS AND DISCUSSION System suitability For evaluating system suitability, carrying out to inject a standard solution times repeatedly into the HPLC system [4] Results were showed in table Table 1: Results of evaluating system suitability Methods - Chromatographic conditions: CONTENT RESULT Peak area (µV*s) S = 338,479 RSD = 0.90% Retention time (minute) tR = 16.46 RSD = 0.05% + Detector UV: 305 nm + Injection volume: 20 µL + Column temperature: 280C + A multistep gradient program of mobile phase: - 18 min, CAN - 0.1% formic acid (25:75, v/v); 18 - 19 from 20 to 80% ACN; 19 - 29 min, ACN - 0.1% formic acid (80:20, v/v); 29 - 30 from 80 to 25% ACN; 30 - 40 min, CAN - 0.1% formic acid (80:20, v/v) Tailing factor 1.09 Plate count > 10,000 0.010 0.008 0.006 0.004 AU + Flow rate: 1.0 mL/minute 0.002 0.000 -0.002 - Sample solution preparation: -0.004 - Standard solution preparation: 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 Minutes 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 Figure 1: Chromatogram of the blank sample res v eratrol - 16.466 0.020 0.018 0.016 0.014 0.012 0.010 AU 0.5 g pulverized Rhizoma reynoutriae japonicae was ultrasonically extracted with about 45 mL of methanol for 60 (repeatedly times) All of extraction solutions were collected in a 200 mL volumetric flask This solution is filtered through a 0.45 μm filter membrane The filtrate (20 μL) was injected into the HPLC system for analysis 0.008 0.006 0.004 0.002 6.2 mg trans-resveratrol is dissolved by methanol in a 50 mL volumetric flask (stock standard solution) From stock standard solution, carrying out to dilute using solvent of methanol and volumetric flasks to obtain working standard solutions 0.000 -0.002 -0.004 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 Minutes 10.00 11.00 12.00 13.00 14.00 15.00 Figure 2: Chromatogram of a standard solution 16.00 17.00 18.00 JOURNAL OF MILITARY PHARMACO-MEDICINE No7-2015 concentrations of trans-resveratrol and analyze these solutions [4] Results were showed in table and figure 0.12 0.10 resveratrol - 16.441 AU 0.08 0.06 0.04 0.02 Table 2: Correlation between peak areas and concetrations of trans-resveratrol CONCENTRATION (µg/ml) PEAK AREA (µV*s) 1.22 116,445 Figure 3: Chromatogram of a sample solution 3.04 339,568 6.08 708,856 The result in table showed the repeatibility of peak areas and the retention times were good (both RSD < 1%) Peak of trans-resveratrol is sharp and proportional with tailing factor = 1.09 Plate count was more than 10,000 Therefore, the chromatography system is suitable for analyzing samples 9.11 1,082.241 12.2 1,436.228 0.00 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 Minutes 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 S (μV*s) Specificity Retention times of the peak in the chromatogram of sample solution and in the chromatogram of standard solution are coincident At this retention time, there was no peak in the chromatogram of blank sample Match coefficients, determinated at points in peak of a sample solution by comparison between the UV-Vis geometry at these points and that of standard trans-resveratrol using PDA detector, were from 0.992 to 0.997 These results proved that the established method has the high specificity Linear range For evaluating the linear range of the established method, carrying out to prepare C (μg/mL) Figure 4: Graph of linear correlation between peak areas and concentrations of trans-resveratrol The result of figure showed that there was linear correlation between peak areas and concentrations of trans-resveratrol following the regression equation y = 120603x - 26752 with correlation coefficient R2 = 0.9998 JOURNAL OF MILITARY PHARMACO-MEDICINE No7-2015 Precision Table 4: Results of evaluating accuracy Precision of the established method was evaluated based on determinating repeatibility of individual experiments by extration and quantifying trans-resveratrol in the materials times repeatedly [4] Results were showed in table N0 ADDITION AMOUNT (µg) RECOVERY AMOUNT (µg) RECOVERY 486 472.99 97.32 486 498.00 102.47 486 474.62 97.66 486 477.44 98.24 486 476.95 98.14 486 490.54 100.93 RATIO (%) Table 3: Results of evaluating precision N0 PEAK AREA (µV*s) CONTENT OF TRANSRESVERATROL IN MATERIALS (%) 258727 0.0862 258329 0.0860 267112 0.0887 268729 0.0892 260163 0.0866 267198 0.0887 Statistic Average content = 0.0876 (%) RSD = 1.65% The result in table showed the established method had the high repeatibility with RSD = 1.65% The content of transresveratrol in Rhizoma reynoutriae japonicae harvested in Sapa district was 0.0876% Statistic Average recovery ratio = 99.13% RSD = 1.14% The result in table showed the established method has the high accuracy with recovery ratio = 97.32 - 102.47%, RSD = 1.14% LOD and LOQ By diluting standard solutions of transresveratrol and analyzing with the HPLC system, LOD and LOQ of the established method were determinated based on signal to noise [4] LOD was about 0.03 μg/mL and LOQ was about 0.1 μg/mL Accuracy Accuracy of the established method was evaluated by adding an exact amount of trans-resveratrol into the sample of Rhizoma reynoutriae japonicae Then, determinating recovery ratio by comparing an recovery amount to an addition amount of trans-resveratrol [4] Samples were injected times repeatedly into the HPLC system Results were showed in table CONCLUSION A suitable method had been developed and validated for quantifying trans-resveratrol in Rhizoma reynoutriae japonicae The established method had precision and accuracy This study also provided valuable information for the content of trans-resveratrol in Rhizoma reynoutriae japonicae harvested in Sapa district being about 0.0876% JOURNAL OF MILITARY PHARMACO-MEDICINE No7-2015 REFERENCES Arichi H, Kimura Y, Okuda H et al Effects of stilbene components of the roots of Polygonum cuspidatum Sieb et Zucc on lipid metabolism Chem Pharm Bull 1982, 30 (5), pp.1766-1770 Chinese Pharmacopoeia 2005, 1454, pp.180-181 Dionex Determination of anthraquinones and stilbenes in Giant Knotweed Rhizome by HPLC with UV detection Application note 232 ICH guidelines Q2 (R1) validation of analytical procedures: text and methodology 1996 Laszlo Mark, Martin S Pour Nikfardjam, Peter Avar et al A validated HPLC method for the quantitative analysis of trans-resveratrol and trans-piceid in Hungarian wines Journal of Chromatographic Science 2005, Vol 43, pp.445-449 National Institute of Medicinal Materials Medicinal plants and animals in Vietnam Publishing house of Science and Technology Hanoi 2004, pp.529-531 ... evaluated by adding an exact amount of trans-resveratrol into the sample of Rhizoma reynoutriae japonicae Then, determinating recovery ratio by comparing an recovery amount to an addition amount of trans-resveratrol. .. trans-piceid in Hungarian wines Journal of Chromatographic Science 2005, Vol 43, pp.445-449 National Institute of Medicinal Materials Medicinal plants and animals in Vietnam Publishing house of Science... were injected times repeatedly into the HPLC system Results were showed in table CONCLUSION A suitable method had been developed and validated for quantifying trans-resveratrol in Rhizoma reynoutriae