莪術(shù)油口服吸收機(jī)理及制劑前研究-8

1、CHAPTER 2: ANALYTICAL METHODS OF ZEDOARY TURMERIC OIL2.1. Materials. 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, Univer

2、sity 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 (Darmsta

3、dt, 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. . INSTRUMENTSAgilent Series 1100 (Agilent Technologies, USA) liquid chromatograph, equipped with a vacuum degasser,

4、 a quaternary pump, an autosampler and a diode array detection (DAD) system. The HPLC column employed was an Agilent ZORBAX ODS column (250mm I.D., 5m) with a ZORBAX ODS guard column (20mm I.D., 5m).2.2. Methods. ANALYTICAL METHODS OF ZRDOARY TURMERIC OIL AND ITS PURE COMPOUNDS.1. HPLC ANALYSIS OF Z

5、EDOARY TURMERIC OILThe 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). T of 50l and the column temperature at 25. The detective wavelengt

6、h was 214nm. The HPLC condition used for Zedoary Turmeric Oil analysis was listed in Table 2.1.Table : Elution condition of Zedoary Turmeric OilTime (minutes)0152545Solvent A (%)4535200Solvent B (%)556580100min0510152025303540mAU020406080100120140Figure a: HPLC chromatogram of Zedoary Turmeric Oilnm

7、200225250275300325350375mAU0100200300400Figure : UV spectrum of curdione in Zedoary Turmeric Oilnm200225250275300325350375mAU0100200300400500600Figure : UV spectrum of germacrone in Zedoary Turmeric Oil200225250275300325350375mAU0100200300400500600700800nmFigure : UV spectrum of furanodiene in Zedoa

8、ry Turmeric Oil.2. HPLC ANALYSIS OF INDIVIDUAL PURE COMPONENT OF ZEDOARY TURMERIC OILThe 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. The d

9、etective 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 : Elution conditions of individual pure component in Zedoary Turmeric OilCompoundsTime (minutes)Solvent A (%)Solvent B (%

10、)04555Curdionuranodiene15010004555153565Germacrone25208004555153565Curcumol252080min02468101214mAU020406080100120140160Figure : HPLC chromatogram of curdionemin02468101214mAU020406080100120140160Figure : HPLC chromatogram of furanodienemin05101520mAU050100150200250300350Figure : HPLC ch

11、romatogram of germacronemin05101520mAU012345Figure : HPLC chromatogram of curcumolnm200225250275300325350375mAU050100150200250300350Figure : UV spectrum of curdionenm200220240260280300320340360380mAU0255075100125150175Figure : UV spectrum of furanodienenm200225250275300325350375mAU0100200300400500Fi

12、gure : UV spectrum of germacronenm200225250275300325350375mAU2468101214Figure : UV spectrum of curcumol. PREFORMULATION STUDY OF ZEDOARY TURMERIC OILPreformulation studies were carried out to choose appropriate experimental conditions for transport studies of Zedoary Turmeric Oil and its individual

13、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 phosphate buffer, potassium chloride and sodium chloride in 200ml wate

14、r. Then calcium chloride and magnesium chloride were added to reach a final concentration of and respectively. The pH value of the buffer was attained at pH7.4.1. DETERMINATION OF PROPORTION OF EACH CONSTITUENTSThe main constituents in Zedoary Turmeric Oil were curdione, curcumenol, germacrone, curz

15、erene, furanodiene and -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 load

16、ing 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 same concentration of 100g/ml. By HPLC-DAD analysis, area percentages of the same components in

17、Zedoary Turmeric Oil against in pure compounds were calculated to confirm the proportion of each constituent mentioned above in Zedoary Turmeric Oil.2. SAMPLE PREPARATION FOR AQUEOUS SOLUBILITY DETERMINATIONStock solutions of Zedoary Turmeric Oil and the pure compounds were prepared by dissolving ea

18、ch 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%).3. DETERMINATION OF

19、 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

20、 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

21、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.4. DETERMINAT

22、ION OF STABILITY.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 p

23、repared 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 germacron

24、e was performed at both pH7.4 and pH6.0 in PBS+ to investigate the pH dependent stability profile of germacrone in Zedoary Turmeric Oil.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 H

25、PLC 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. VALIDATION OF HPLC METHODSHPLC methods validation was conducted by determining t

26、he 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 concentrati

27、ons from nominal values and evaluated by the formula of Difference% = (determined-nominal)/nominal 100.2.3. Results. PREFORMULATION STUDIES AND SAMPLE TREATMENT OF ZEDOARY TURMERIC OIL.1. PROPORTION OF EACH CONSTITUENTS IN ZEDOARY TURMERIC OILThe results were show in Table 2.3. The content of curcum

28、ol in Zedoary Turmeric Oil was too low to be quantified by HPLC analysis.Table : Proportion of individual constituents in Zedoary Turmeric OilCompoundcurdionegermacronefurnodienecurcumolProportion (%)N/DN/D: not detectable.2. SOLUBILITY OF CONSTITUENTS OF ZEDOARY TURMERIC OIL.2.1. Solubility in aque

29、ous 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 conce

30、ntrations 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%. How

31、ever, 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 : Solubility in aqueous buffer with 1% DMSOPure compoundcurdione (g/ml)6030Percent (%)*germacrone (g/ml)3520Percent (%)furan

32、odiene (g/ml)4525Percent (%)curcumol (g/ml)10020Percent (%)Zedoary Turmeric Oilcurdione (g/ml)200100Percent (%)germacrone (g/ml)200100Percent (%)furanodiene (g/ml)200100Percent (%)* Percent (%) stands for the ratio of peak area of individual component in PBS+ against peak area of individual componen

33、t in methanol.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 i

34、ncreasing 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 lo

35、w 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 : Solubility of constituents of Zedoary Turmeric Oil at the DMSO con

36、tent of 0.5% and 1%Percent (%)*Concentration (g/ml)0.5% DMSO1% DMSO10germacrone2012furanodiene25100germacrone in Zedoary Turmeric Oil200100furanodiene in Zedoary Turmeric Oil200* Percent (%) stands for the ratio of peak area of individual component in PBS+ against peak area of individual component i

37、n methanol.3. STABILITY OF CONSTITUENTS OF ZEDOARY TURMERIC OIL.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 i

38、n 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 i

39、ncubation 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 by adjusting pH to 6.0. The following permeability studies were performed in PBS+ buffer at pH7.4.Table

40、 : Stability of Zedoary Turmeric Oil in PBS+ (pH7.4) after 150 minutes incubationCompoundcurdionegermacronefuranodienePercent (%)Table : Stability of pure compounds in PBS+ (pH7.4) after 120 minutes incubationCompoundcurdionegermacronefuranodienecurcumolPercent (%)Table : Stability of germacrone in

41、PBS+ after 120 minutes incubationgermacronePercent (%)Stability of Zedoary Turmeric Oil in PBS+ (pH7.4)5060708090100110120050100150200Time (minutes)Remaining percentage (%)curdionegermacronefuranodieneStability of pure compounds in PBS+ (pH7.4)80859095100105050100150Time (minutes)Remaining percentag

42、e (%)curdionegermacronefuranodienecurcumolStability of germacrone in PBS+5060708090100110120020406080100120140Time (minutes)Remaining percentage (%)pH 7.4pH 6.0Figure : Stability of constituents of Zedoary Turmeric Oil in PBS+ at 37.3.2. Stability of samples for HPLC analysisAll samples attained aft

43、er 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 dil

44、ution 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 : Stability of Zedoary Turm

45、eric Oil for HPLC analysis within 21 hoursCompoundcurdionegermacronePercent (%)Table : Stability of pure compounds for HPLC analysis within 21 hoursCompoundcurdionegermacronecurcumolPercent (%)Table : Stability of furanodiene for HPLC analysis within 24 hoursCompoundfuranodienePercent (%)Stability o

46、f Zedoary Turmeric Oil for HPLC analysis50607080901001101200510152025Time (hours)Remaining percentage (%)curdionegermacroneStability of pure compounds for HPLC analysis50607080901001100510152025Time (hours)Remaining percentage (%)curdionegermacronecurcumolStability of furanodiene for HPLC analysis50

47、60708090100110051015202530Time (hours)Remianing percentage (%)furanodieneFigure : Stability of constituents of Zedoary Turmeric Oil for HPLC analysis. 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

48、 calibration curves of Zedoary Turmeric Oil and the pure compounds were linear over the studies concentrations (R20.999). 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 t

49、hat the developed HPLC methods were suitable for the sample analysis in the present study.1. CALIBRATION CURVEWorking stock solutions containing Zedoary Turmeric Oil and the active compounds were prepared in PBS+ and diluted with methanol for construction of calibration curves. The calibration curve

50、s 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 : Linear regression data of Zedoary Turmeric Oil

51、 and the individual tested componentLinear regression dataAnalytesRegression equationr2Linear range/gml-1LOQ/gml-1Zedoary Turmeric OilcurdioneA=3.1225germacroneA=furanodieneA=1.5625Pure compoundcurdioneA=0.7825germacroneA=furanodieneA=0.3712curcumolA=6.25100Curdione in Zedoary Turmeric Oily = 2.981x

52、 + 0.7913R2 = 1020406080051015202530Concentration (g/ml)Peak area Germacrone in Zedoary Turmeric Oily = 5.7829x + 0.25R2 = 0.999701020304001234567Concentration (g/ml)Peak area Furanodiene in Zedoary Turmeric Oily = 5.3709x + 0.7292R2 = 0.9999050100150051015202530Concentration (g/ml)Peak area Curdion

53、e of pure compoundy = 21.199x + 4.8826R2 = 0.99990100200300400500600051015202530Concentration (g/ml)Peak area Germacrone of pure compoundy = 58.39x + 2.9753R2 = 0.9997010020030040001234567Concentration (g/ml)Peak area Furanodiene of pure compoundy = 20.405x + 1.3567R2 = 0.999905010015020025030002468

54、101214Concentration (g/ml)Peak area Curcumol of pure compoundy = 8.4499x - 0.0542R2 = 0.999902004006008001000020406080100120Concentration (g/ml)Peak areaFigure : Standard curves of Zedoary Turmeric Oil and pure compounds.2. PRECISIONSame solutions of Zedoary Turmeric Oil and pure compounds were inje

55、cted 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 : Intra-day precision of investigated compoundsZedoary Turmeric OilPure compoun

56、dsInjection Timecurdionegermacronefuranodienecurdionegermacronefuranodienecurcumol1st742nd8093rd6241054th107655th56th686RSD (%)Table : Inter-day precision of investigated compoundsZedoary Turmeric Oil (g/ml)Pure compounds (g/ml)Daycurdionegermacronefuranodienecurdionegermacronefuranodienecurcumol1st

57、2nd3rdRSD (%).3. ACCURACYIn 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 : Accuracy of develpoed HPLC methodsAnalytesConcentration

58、 (g/ml)Determined concentration (g/ml)Accuracy (%)2550curdione in Zedoary Turmeric Oil1002550germacrone in Zedoary Turmeric Oil1002550furanodiene in Zedoary Turmeric Oil100110curdione as pure compound202germacrone as pure compound2015furanodiene as pure compound10510curcumol as pure compound202.4. Discussion. SOLUBILITY OF ZEDO