莪术油口服吸收机理及制剂前研究-8

CHAPTER 2: ANALYTICAL METHODS OF ZEDOARY TURMERIC OIL 2.1. Materials2.1.1. CHEMICALSZedoary Turmeric Oil was purchased from Tianrei Pharmacy Limited Company, Zhejiang Province, China. Curdione, germacrone, furanodiene, curcumol were separated and purified from Institute of Chinese Medical Sciences, University of Macau (Macau, China) with the method described by F.Q.Yang (2005). The purity of above pure compounds was more than 98% and the structures were confirmed by comparing their UV, EI-MS and NMR data with literatures. Acetonitrile and methanol for HPLC analysis were purchased from Merck (Darmstadt, Germany). Water for HPLC analysis was prepared using a Millipore Milli-Q®Plus system (Millipore, Bedford, MA, USA). Other chemical reagents were used at least of analytical grades.2.1.2. INSTRUMENTSAgilent Series 1100 (Agilent Technologies, USA) liquid chromatograph, equipped with a vacuum degasser, a quaternary pump, an autosampler and a diode array detection (DAD) system. The HPLC column employed was an Agilent ZORBAX ODS column (250mm×4.6mm I.D., 5μm) with a ZORBAX ODS guard column (20mm×3.9mm I.D., 5μm).2.2. Methods2.2.1. ANALYTICAL METHODS OF ZRDOARY TURMERIC OIL AND ITS PURE COMPOUNDS2.2.1.1.HPLC ANALYSIS OF Z EDOARY T URME RIC O ILThe determination of Zedoary Turmeric Oil was conducted by HPLC-DAD. The mobile phase for HPLC analysis of Zedoary Turmeric Oil consisted of two solvent compositions, water (solvent A) and acetonitrile (solvent B). The flow rate of the mobile phase was set at 1.0ml/min with injection volume of 50μl and the column temperature at 25℃. The detective wavelength was 214nm. The HPLC condition used for Zedoary Turmeric Oil analysis was listed in Table 2.1.Table 2.1: Elution condition of Zedoary Turmeric OilSolvent A (%) 45 35 20 0 Solvent B (%) 55 65 80 100Figure 2.1a: HPLC chromatogram of Zedoary Turmeric OilFigure 2.1b: UV spectrum of curdione in Zedoary Turmeric OilFigure 2.1c: UV spectrum of germacrone in Zedoary Turmeric OilFigure 2.1d: UV spectrum of furanodiene in Zedoary Turmeric Oil2.2.1.2.HPLC ANALYSIS OF INDIVIDUAL PURE COMPONE NT OF Z EDOARYT URMERIC O ILThe chromatogram conditions of individual pure component in Zedoary Turmeric Oil were similar to the condition of the essential oil. The mobile phase consisted of water (solvent A) and acetonitrile (solvent B). Flow rate is at 1.0ml/min with injection volume of 50μl and column tempe rature at 25℃. The detective wavelength is 214nm with UV detection. The HPLC conditions used for analysis of individual pure component in Zedoary Turmeric Oil analysis were listed in Table 2.2.Table 2.2: Elution conditions of individual pure component in Zedoary Turmeric Oil Curdione 0 45 5515 35 65Furanodiene15 0 100Germacrone 0 45 55 15 35 65 25 20 80Curcumol 0 45 55 15 35 65 25 20 80Figure 2.2a: HPLC chromatogram of curdioneFigure 2.2b: HPLC chromatogram of furanodieneFigure 2.2c: HPLC chromatogram of germacroneFigure 2.2d: HPLC chromatogram of curcumolFigure 2.2e: UV spectrum of curdioneFigure 2.1f: UV spectrum of furanodieneFigure 2.1g: UV spectrum of germacroneFigure 2.1h: UV spectrum of curcumol2.2.2. PREFORMULATION STUDY OF ZEDOARY TURMERIC OIL Preformulation studies were carried out to choose appropriate experimental conditions for transport studies of Zedoary Turmeric Oil and its individual pure component.Phosphate buffered saline plus (PBS+) solution was used as the transport buffer in the transport studies using Caco-2 cell model. It was prepared by dissolving 1 of phosphate buffered saline tablet which containing 0.01M phosphate buffer, 0.0027M potassium chloride and 0.137M sodium chloride in 200ml water. Then calcium chloride and magnesium chloride were added to reach a final concentration of 0.9mM and 0.4mM respectively. The pH value of the buffer was attained at pH7.4.2.2.2.1.D ETE RMINATION OF PROPORTION OF E ACH CONSTITUENTSThe main constituents in Zedoary Turmeric Oil were curdione, curcumenol, germacrone, curzerene, furanodie ne and β-elemene etc according to their peak area in the HPLC chromagram of Zedoary Turmeric Oil. As the actual proportion of above pure compounds in Zedoary Turmeric Oil was not clear, it is necessary to evaluate their positive contents in Zedoary Turmeric Oil to determine their loading concentrations in the transport experiments.Zedoary Turmeric Oil and the active compounds including curdione, germacrone, furanodiene and curcumol were dissolved in methanol respectively to reach the sameconcentration of 100μg/ml. By HPLC-DAD analysis, area percentages of the same components in Zedoary Turmeric Oil against in pure compounds were calculated to confirm the proportion of each constituent mentioned above in Zedoary Turmeric Oil.2.2.2.2.S AMPLE PRE PARATION FOR AQUEOUS SOLUBILITY DETERMINATIONStock solutions of Zedoary Turmeric Oil and the pure compounds were prepared by dissolving each constituent into DMSO to reach the concentration of 10mg/ml. The working solutions of each constituent was obtained by dilution of the primary stock solution to required concentrations with phosphate buffered saline plus (PBS+) maintaining the content of DMSO constantly (0.5%).2.2.2.3.D ETE RMINATION OF SOLUBILITYSame amounts of Zedoary Turmeric Oil and pure compounds were dissolved in PBS+ buffer (pH7.4) and methanol respectively at difference concentrations to investigate the different solubility in PBS+buffer and methanol. It is considered that all constituents could be completely dissolved in methanol. The percentages of DMSO in all samples were maintained constantly at 1%. Samples were analyzed by HPLC-DAD. The amount percentage of each compound dissolved in PBS+ buffer was calculated against the contents in methanol to investigate the solubility profile in aqueous solution.Solubility of each component with different concentrations of DMSO (1% and 0.5%) was also investigated. These were also performed against PBS+ buffer and methanol and the amount percentages were calculated as mentioned above. The investigation was performed at different concentrations in parallel.2.2.2.4.D ETE RMINATION OF STABILITY2.2.2.4.1. Stability of samples for permeability studyStock solution of each constituent of Zedoary Turmeric Oil with a concentration of 10mg/ml was diluted with PBS+at pH7.4 respectively to reach the concentrations according to their solubility levels and determined DMSO contents. The prepared solutions were incubated in water bath at 37 for 2 hours. Samples were taken at different time intervals and the drug concentrations were measured by HPLC. The percentage of each components of Zedoary Turmeric Oil was plotted against time to gain stability profiles.The stability of germacrone was performed at both pH7.4 and pH6.0 in PBS+to investigate the pH dependent stability profile of germacrone in Zedoary Turmeric Oil.2.2.2.4.2. Stability of samples for HPLC analysisWorking solutions in PBS+for permeability study were diluted by methanol to improve their solubility and stability for HPLC analysis. The stability of solutions with methanol was conducted by determining their amounts decrease within 24 hours at 25 and the dilution ratio was also optimized by comparison of different methanol proportions.2.2.3. VALIDATION OF HPLC METHODSHPLC methods validation was conducted by determining the linear range, intra-day and inter-day precision, accuracy and sample stability of Zedoary Turmeric Oil and pure compounds. The precision and stability assay was defined as the relative standard deviation (RSD) and the accuracy was defined of the percentage difference of the determined concentrations from nominal values and evaluated by the formula of Difference% = [(determined-nominal)/nomin al] ×100.2.3. Results2.3.1. PREFORMULATION STUDIES AND SAMPLE TREATMENT OF ZEDOARY TURMERIC OIL2.3.1.1.P ROPORTION OF EACH CONSTITUENTS IN Z EDOARY T URMERIC O ILThe results were show in Table 2.3. The content of curcumol in Zedoary Turmeric Oil was too low to be quantified by HPLC analysis.Table 2.3: Proportion of individual constituents in Zedoary Turmeric OilProportion (%) 14.88 8.85 11.39 N/DN/D: not detectable2.3.1.2.S OLUBILITY OF CONSTITUENTS OF Z EDOARY T URME RIC O IL2.3.1.2.1. Solubility in aqueous bufferZedoary Turmeric Oil is an essential oil extracted from the Zedoary Turmeric with high lipophilicity and poor aqueous solubility. These experiments were carried out to determine the amount of constituents in Zedoary Turmeric Oil that could be dissolved into the PBS+buffer. Different concentrations of individual component in PBS+ solutions and methanol solutions with 1% DMSO were analyzed by HPLC-DAD. The result was shown in Table 2.4.Among four constituents, only curdione and curcumol could be well dissolved in PBS+buffer and their dissolved percentages were approached to 100%. However, germacrone and furanodiene were not well dissolved in the aqueous solution at the tested concentrations either as single compound or as those in the essential oil.Table 2.4: Solubility in aqueous buffer with 1% DMSOPure compoundcurdione (μg/ml)60 30Percent (%)* 97.93 100.77 germacrone (μg/ml)35 20 Percent (%) 59.52 60.89Percent (%) 70.75 70.38 curcumol (μg/ml)100 20 Percent (%) 99.77 103.62curdione (μg/ml)200 100Percent (%) 103.63 105.65 germacrone (μg/ml)200 100 Percent (%) 71.65 79.01 furanodiene (μg/ml)200 100 Percent (%) 61.03 72.40* Percent (%) stands for the ratio of peak area of individual component in PBS+ against peak area of individual component in methanol.2.3.1.2.2. Solubility at different content of DMSOAqueous solubility of Zedoary Turmeric Oil in PBS+ with different concentrations of DMSO was also investigated. The results were shown in Table 2.5. The results indicate that solubility of most constituents could not be increased greatly by increasing the concentration of DMSO from 0.5% to 1%. As it has been reported previously that certain amount of DMSO will affect the integrity and may bring toxicity problem to the Caco-2 monolayer, so the principle of selecting the concentration of DMSO used in transport experiments should bee as low as possible. In the present study, 0.5% DMSO was used in the following permeability study.Moreover, the effect of 0.5% DMSO on the integrity of Caco-2 cell monolayer was also investigated and will be described in Chapter 3.Table 2.5: Solubility of constituents of Zedoary Turmeric Oil at the DMSO content of 0.5% and 1%Percent (%)* Concentration (μg/ml) 0.5% DMSO 1% DMSOgermacrone10 40.36 58.6320 63.46 41.07furanodiene12 18.83 23.9725 41.43 45.65germacrone in Zedoary Turmeric Oil 100 73.84 74.34 200 75.02 83.57furanodiene in Zedoary Turmeric Oil 100 64.61 72.89 200 69.82 81.58* Percent (%) stands for the ratio of peak area of individual component in PBS+ against peak area of individual component in methanol.2.3.1.3.S TABILITY OF CONSTITUENTS OF Z EDOARY T URMERIC O IL2.3.1.3.1. Stability of samples for permeability studyThe stability of Zedoary Turmeric Oil and the pure compounds in PBS+ buffer (pH7.4) at 37℃were studied. The results were shown in Figure 2.3. These results indicated that curdione either in Zedoary Turmeric Oil or in pure compounds were quite stable after 2 hours incubation in PBS+ buffer (pH7.4) at 37℃. However, other components such as germacrone, furanodiene and curcumol in Zedoary Turmeric Oil or in pure compounds were relatively unstable, the remaining percentages after 2 hours incubation were still above 85%.The stability of germacrone at pH6.0 was also investigated as shown in Figure 2.3. This result indicated that the stability of germacrone did not obviously increased byadjusting pH to 6.0. The following permeability studies were performed in PBS+ buffer at pH7.4.Table 2.6a: Stability of Zedoary Turmeric Oil in PBS+ (pH7.4) after 150 minutes incubationPercent (%) 100.89 92.83 86.45 Table 2.6b: Stability of pure compounds in PBS+ (pH7.4) after 120 minutes incubationPercent (%) 100.01 85.64 88.88 88.52 Table 2.6c: Stability of germacrone in PBS+ after 120 minutes incubationFigure 2.3: Stability of constituents of Zedoary Turmeric Oil in PBS+ at 37℃2.3.1.3.2. Stability of samples for HPLC analysisAll samples attained after permeability studies were diluted with methanol for HPLC analysis. The dilution ratios of all constituents were optimized and their stabilities after dilution were investigated at room temperature. The results indicated that curdione, germacrone and curcumol were stable within 24 hours at the dilution ratio of 1:1 (PBS+: methanol, v/v) either in Zedoary Turmeric Oil or in pure compounds. However, furanodiene was only stable within 24 hours with high content of methanol such as dilution ratio of 1:4 (PBS+: methanol, v/v). The results were shown in Figure 2.4.Table 2.7a: Stability of Zedoary Turmeric Oil for HPLC analysis within 21 hours Compound curdione germacronePercent (%) 112.71 102.14Table 2.7b: Stability of pure compounds for HPLC analysis within 21 hoursTable 2.7c: Stability of furanodiene for HPLC analysis within 24 hoursPercent (%) 91.76Figure 2.4: Stability of constituents of Zedoary Turmeric Oil for HPLC analysis2.3.2. VALIDATION OF HPLC METHODSThe solutions for HPLC validation were prepared by the instrument of item 2.3.1.3.2. The validation results showed that the calibration curves of Zedoary Turmeric Oil and the pure compounds were linear over the studies concentrations (R2>0.999). The RSD of the intra-day, inter-day precision were within 5% and the accuracy of the constituents at high, middle, low concentrations were all from 95% to 105%. These indicated that the developed HPLC methods were suitable for the sample analysis in the present study.2.3.2.1.C ALIBRATION CURVEWorking stock solutions containing Zedoary Turmeric O il and the active compounds were prepared in PBS+and diluted with methanol for construction of calibration curves. The calibration curves were constructed by plotting the peak areas (A) versus the concentration (C) of each analyte. The results indicated that the investigated compounds had good linearity within the tested ranges. The results were shown in Table 2.8 and Figure 2.5.Table 2.8: Linear regression data of Zedoary Turmeric Oil and the individual tested componentAnalytes LOQ/μg·ml-12-1Zedoary Turmeric Oilcurdione A=2.981C+0.7913 1.0000 3.12~25 3.12 germacrone A=5.7829C+0.250.9997 1.56~6.25 1.56 furanodiene A=5.3709C+0.72920.9999 1.56~25 1.56Pure compoundcurdione A=21.199C+4.88260.99990.78~250.78 germacrone A=58.39C+2.97530.99970.098~6.250.098 furanodiene A=20.405C+1.35670.99990.37~120.37 curcumol A=8.4499C-0.05420.9999 6.25~100 3.12Figure 2.5: Standard curves of Zedoary Turmeric Oil and pure compounds2.3.2.2.P RECISIONSame solutions of Zedoary Turmeric Oil and pure compounds were injected to HPLC-DAD for 6 times. The performance was repeated on the next day and the third day. The precisions for intra-day and inter-day of investigated compounds were shown in Table 2.9 and Table 2.10 respectively.Table 2.9: Intra-day precision of investigated compoundsInjection TimeZedoary Turmeric Oil Pure compoundscurdione germacrone f uranodiene curdione germacrone f uranodiene curcumolst2nd102.5457 78.2007 82.6575 19.5204 2.3692 5.1680 27.7789 3rd102.6127 79.0506 79.9142 19.5014 2.3310 5.1741 27.4835 4th101.6412 78.1810 82.2547 19.4966 2.3293 5.2536 28.0625 5th102.1102 78.5169 80.9501 19.5252 2.2977 5.2352 28.5115 6th102.5457 78.9715 81.9285 19.5109 2.2096 5.2108 28.4406 RSD (%) 0.68 0.98 1.00 0.01 0.05 0.04 0.56Table 2.10: Inter-day precision of investigated compoundsDayZedoary Turmeric Oil (μg/ml) Pure compounds (μg/ml)curdione germacrone f uranodiene curdione germacrone f uranodiene curcumol 1st102.0599 78.2139 81.4841 19.5117 2.3058 5.1965 28.2180 2nd101.4011 79.5993 80.6368 19.6157 2.3224 5.1924 28.4938 3rd99.7373 76.8206 77.8966 19.6284 2.2800 5.0977 27.7592 RSD (%) 1.20 0.94 1.88 0.06 0.02 0.06 0.372.3.2.3.A CCURACYIn order to validate the accuracy of developed HPLC methods, solutions of Zedoary Turmeric Oil and the active compounds were analyzed at high, middle and low concentrations. The results were shown in Table 2.11.Table 2.11: Accuracy of develpoed HPLC methodsAnalytes Concentration(μg/ml)Determined concentration(μg/ml)Accuracy (%)curdione in Zedoary Turmeric Oil 25 24.4578 101.91 50 51.6596 103.32 100 105.5321 105.53germacrone in Zedoary Turmeric Oil 25 26.0048 104.02 50 47.0789 94.16 100 101.6914 101.69furanodiene in Zedoary Turmeric Oil 25 24.6563 98.63 50 49.1452 98.29 100 99.0449 99.04curdione as pure compound1 0.9570 95.70 10 9.7236 97.24 20 19.9562 99.78germacrone as pure compound 0.2 0.1916 95.81 2 2.0924 104.62 20 20.9318 104.66furanodiene as pure compound1 1.0356 103.56 5 4.7509 95.02 10 9.6502 96.50curcumol as pure compound5 4.9976 99.95 10 10.0312 100.31 20 20.7247 103.622.4.1. SOLUBILITY OF ZEDOARY TURMERIC OIL AND PURE COMPOUNDSThe investigation of the aqueous solubility of tested components indicated that the constituents of curdione and curcumol both in pure compounds and in Zedoary Turmeric Oil could be completely dissolved in the PBS+buffer at tested concentrations. But germacrone and furanodiene were not well dissolved in PBS+ whatever in the form of single compound or in the essential oil. The dissolved percentages of above components were maintained at about 60%~70% and did not increase greatly by increasing the concentration of DMSO from 0.5% to 1%. Thus, in the following permeability studies, the concentrations of germacrone, furanodiene and Zedoary Turmeric Oil were reduce as low as possible to minimize the dissolved components in PBS+ buffer but also satisfy the quantification requirement of HPLC.2.4.2. STABILITY OF ZEDOARY TURMERIC OIL AND PURE COMPOUNDSThe stability of constituents in Zedoary Turmeric Oil in PBS+ was not consentaneous. Except curdione which was stable in PBS+, other components including germacrone, furanodiene and curcumol were unstable in tested aqueous system and above 85% of the tested components are remained after 2 hours incubation. It is expected that recovery of above components after permeability study may be slightly reduced. However, the reason that leads to the stability problem for the investigated components remains unclear.In this chapter, the precise and accurate HPLC analytical methods were d eveloped and validated. Results showed that these developed analytical methods are suitable for the determination of the concentrations of Zedoary Turmeric Oil and the individual component. Moreover, some preformulation studies, e.g. stability and solubility studies, were also conducted to optimize the conditions that may be used in the following permeability studies investigate the absorption mechanisms of selected components in Zedoary Turmeric Oil.。