英文邀请函格式参考5种（精选2篇）
篇1：英文邀请函格式参考5种
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篇2：英文邀请函格式参考5种
Recently,chromatographic fingerprint technique,as a more meaningful formulation for controlling the quality of TCM,has emphasized on the systemic characterization of compositions of samples and focus on identifying the stability of the plants.Fingerprint technique has been introduced and accepted by WHO as a strategy for the assessment of herbal medicines[7].Because of its advantages and popularization,TLC and HPLC fingerprint analysis have been received as the first choice[8,9,10].
Up to now,most of analytical methods established for the quality control of the crude drug were involved in the qualitation or quantification of one or two of components from only one comprising herb[11,12].However,the biological activities of these single chemical compounds alone could not fully account for the potency and efficacy of parent TCM complex.It is possible that the therapeutic effects of TCM complex are ultimately the results of a concerted action of multiple chemicals on the corresponding cellular targets.Considering all these factors discussed above,we made simultaneous determination of two bioactive components from two different raw drugs by HPLC-UV,using bioactive Emodin[13]and Icariin[14]as marker compounds.Also,three kinds of raw drugs were identified by TLC with Psoralen[15],Astragaloside[16]and Berberine[17]being marker compounds,respectively.In this paper,the structures of these marker compounds were shown in Figure 1.
1 Experimental
1.1 Apparatus
ALC-10ATVPhigh-performanceliquid chromatography system including a VP-ODS(150×4.6 mm)chromatographic column and UV-2450spectrophotometer(Shimadzu,Japan)was used to acquire chromatograms.Ultrasonic extraction device(Kedao ultrasound Instrument Co.Ltd.,Shanghai,China)was used for sample extraction.
1.2 Materials
GKG was the gift given by Hunan Yuanhang Pharmaceutical Co.Ltd.(Batch 051001,051002,051101).All crude drugs were purchased from the city of Changsha,China,and identified by Dr.Tang Yijia at Hunan Provincial Institute for Drug Control.The five reference substances of Emodin(Lot 110756-200110),Icariin(Lot110737-200312),Psoralen(Lot110739-200512),Astragaloside(Lot 110781-200512)and Berberine(Lot 110713-200208)were purchased from the National Institute for the Control of Pharmaceutical and Biological Products,China.HPLC grade methanol and acetonitrile,from Tedia company(U.S.A),were used for preparation of mobile phase.Other reagents were of analytical grade.
1.3 Qualitative identification
1.3.1 Reference and sample solution preparation
Preparation of reference solution:Standard solutions of three medical materials were prepared respectively in ethanol at concentration of 1.500 mg/m L for Psoralen,0.500 mg/m L for Astragaloside and Berberine each.
Preparation of sample solution:For Ficus simplicissima Lour.root and Astragalus monghbolicus(Bge.)Hsiao,15.0 g of GKG(accurately weighed)were dissolved respectively in 100 m L of methanol for ultrasonic extraction for 20 min,filtered.Then the filtrate was evaporated to dryness in water bath,dissolved in 8 m L of water and flowed through D101macroporous resin column(2.5 cm of diameter,30 cm of height).After that,400 m L of water,40%ethanol and60%ethanol were used one after another to elute.At last,60%ethanol solution was collected,evaporated,dissolved in 2 m L of methanol for use.For Mahonia bealei(Fort)Carr,instead of 8 m L of water to dissolve,20 m L of 0.5 mol/L hydrochloric acid solution were used,then extracted twice with 40 m L of ethyl acetate each,abandoned ethyl acetate liquid,regulated with ammonia to make p H=(12±0.05).After that,30 m L of chloroform were used to extract twice,then evaporated the combined chloroform liquid.Finally,2 m L of methanol were utilized to dissolve the residue as a sample solution for the test.
Preparation of the positive control solution of medicinal herbs:According to the above-mentioned method,Ficus simplicissima Lour.root(2 g),Astragalus monghbolicus(Bge.)Hsiao(2 g)and Mahonia bealei(Fort)Carr(3 g)were respectively made to be the positive control solution.
Preparation of negative control solution:It contains7 herbs lack of Ficus simplicissima Lour.root or Astragalus monghbolicus(Bge.)Hsiao or Mahonia bealei(Fort)Carr.
1.3.2 Chromatography of TLC
According to TLC test(see appendix VIB,Chinese Pharmacopoeia,2005),Ficus simplicissima Lour.,Astragalus monghbolicus(Bge.)Hsiao and Mahonia bealei(Fort)Carr were identified in their optimal reagent systems.20μL of sample solution,20μL of negative control solution,4μL of reference solution and 4μL of positive control solution were applied onto the same silicon G thin-layer plate.The plate was dried in a current of air and developed with a mobile phase of n-hexane-ethyl acetate(4∶1,v/v)for Ficus simplicissima Lour.,chloroform-ethyl acetate-methanol-water(20∶40∶22∶10,v/v/v/v)for Astragalus monghbolicus(Bge.)Hsiao and benzene-ethyl acetate-isopropyl alcohol-methanol-concentrated ammonia(6∶3∶1.5∶1.5∶0.5,v/v/v/v/v)for Mahonia bealei(Fort)Carr under laboratory condition[temperature(25±3)℃].All spots were visualized in UV detection at 254 nm for Ficus simplicissima Lour.and 365 nm for Mahonia bealei(Fort)Carr.As for Astragalus monghbolicus(Bge.)Hsiao,the developed plate was immersed in freshly prepared sulphuric acid alcohol solution,and the resultant plate was then heated at 105℃for 5 min until the spots were clear to see.
1.4 Quantitative determination
1.4.1 Referenceandsample
solution preparation 1.20 mg Emodin reference substance and2.00 mg Icariin reference substance were transferred separately into 10 m L measuring flasks and mixed with methanol.The content of each flask was completed to volume with the mobile phase to get the concentrations of 0.120 mg/m L of Emodin or 0.200 mg/m L of Icariin,then filtered through 0.45μm filters.
For the Emodin,2.0 g of GKG were used to prepare the sample solution.It was dissolved in 25mL methanol,extracted three times with chloroform(30,20 and 15m L)and dried,then the residue was dissolved in 10mL of methanol,filtered by 0.45μm filter.As for the Icariin,4.0g of GKG were dissolved in 50 m L of methanol,filtrated and dried.The residue was dissolved in 25 m L of methanol,filtrated with 0.45μm filter and used for HPLC analysis.
We also prepared negative control solution which contains 7 herbs lack of Rumex japonicus Houtt root or Epimedium brevicornum Maxim.
1.4.2 Chromatographic conditions of HPLC
The samples were then chromatographed under the following chromatographic conditions:Chromatographic column:VP-ODS 150 mm×4.6 mm analytical column(Shimadzu).Mobile phase:methanol-0.25%phosphoric acid(80∶20,v/v)for Emodin,acetonitrile-water(26∶74,v/v)for Icariin.The final p H-value was adjusted to(3.0±0.07)with o-phosphoric acid by a p H-meter.Flow rate:1.1 m L/min for Emodin and 1.0m L/min for Icariin.Column temperature:45℃for Emodin and 30℃for Icariin,with UV detection at 436nm for Emodin and 270 nm for Icariin.
1.4.3 Analytical data treatment
Reference solution,sample solution and negative sample solution were injected precisely into liquid chromatography for determination.The relative peak area ratios were then plotted versus the corresponding concentrations of Emodin or Icariin to get the calibration graph and compute the corresponding regression equation.Then concentrations of Emodin and Icariin were calculated from the linear regressions.
2 Results and discussion
2.1 Identification by TLC
The sample tracks were scanned at UV light.It was clearly evident that no interfering compound eluted in the sample tracks to affect the quantitation of the targeted marker.As the results were showed in Figure 2,the spots of the sample solution showed significantly the same as the reference substance,the negative samples in the corresponding location displayed no interference.
To identify the herbs,the chief active components or index ingredients should be choosed,such as Ficus simplicissima Lour.Root,Astragalus monghbolicus(Bge.)Hsiao and Mahonia bealei(Fort)Carr in our study.For Ficus simplicissima Lour.,GKG was originally resolved in methanol for ultrasonic extraction and filtered through 0.45μm membrane.Then the filtrate was extracted by ethyl acetate three times,condensed and spotted the plate,using N-hexane-ethyl acetate as developing agent.The results showed that there appeared orange spot rather than blue white in the corresponding place of reference substance,it was evident that there was interference.Therefore,macroporous adsorptive resins were used for purification,and the spots were clear to see.For Astragalus monghbolicus(Bge.)Hsiao and Mahonia bealei(Fort)Carr,official methods in Chinese Pharmacopiea were used and refined.The suitable amount of spotting for Astragalus monghbolicus(Bge.)Hsiao was 25～30μL.
2.2 Assaying by HPLC
The results showed that the peak of Emodin or I-cariin in the sample was well-resolved peak,and the resolution with its adjacent chromatographic peaks was more than 1.5.The calculated theoretical plate number was more than 3,000 according to the peak of Emodin or Icariin.At the same time,the negative chromatography lacks of a corresponding peak in the corresponding position of the peak of Emodin or Icariin,which explained that there was no negative interference.See Figure 3 and Figure 4.
2.3 Method validation for HPLC
Validation of quantitative method includes evaluation of the following performance parameters,such as specificity,reproducibility,linearity,limits of sensitivity,precision,stability,recovery and robustness,according to the guidelines of ICH[18]and International Union of Pure and Applied Chemistry(IUPAC)[19].
2.3.1 Specificity and reproducibility
10μL of reference solution,10μL of sample solution and 10μL of negative sample solution were injected precisely into liquid chromatography for determination under the optimum condition mentioned above.The sample solution has a corresponding peak in the corresponding position of the peak of reference solution with the same retention time,7.5 min for Emodin and 22 min for Icariin respectively.Moreover,there was no negative interference.See Figure 3 and Figure 4.
For intraday and inter-day reproducibility test,six portions of sample solution for Emodin or Icariin were each analyzed to determine the intraday and inter-day reproducibility of the peak areas under the optimum conditions in this experiment.R.S.D.of the peak areas was both quite low(Table 1).
2.3.2 Linearity and detection limit
A stock solution of 0.12 mg/m L of Emodin or 0.20 mg/m L of Icariin was prepared in a 50 m L volumetric flask by dissolving 6 mg of Emodin or 10 mg of Icariin in mobile phase.Appropriate amounts of the stock solution were injected to obtain solutions containing 120.0,480.0,960.0,1 440.0,1920.0 and 2 400.0 ng of Emodin respectively and 200.0,800.0,1 600.0,2 400.0,3 200.0 and 4 000.0 ng of I-cariin respectively.Each solution was injection in triplicate into the chromatograph.The series of the standard solution of Emodin or Icariin were tested to determine the linearity between the standard concentrations and the peak areas.The calibration curve was prepared with the absolute amount(ng)as independent variable(X)and the peak area of Emodin or Icariin as dependent variable(Y).The corresponding regression equations were computed and found to be:Y=2 375.1x+75 229(r=0.9996)for Emodin and Y=2 105.2x+46 846(r=0.9994)for Icariin.
Serial amounts of Emodin or Icariin in mobile phase were performed.A linear relationship was obtained in the range of 120～2 400 ng for Emodin or200～4 000 ng for Icariin.
2.3.3 Precision and stability
Repeatability was calculated by analyzing 6 samples.The precisions expressed as the relative standard deviation(R.S.D.)for peak area were determined for Emodin or Icariin standards by repeated analysis.The precision results obtained from Table 1 showed that the R.S.D.for peak area was both quite low.
For stability test,the sample solution(ethanol extract)was analyzed every 2 h within 10 h(n=6).R.S.D.=2.57%for Emodin and R.S.D.=3.18%for Icariin(n=6).
2.3.4 Sample analysis,recovery and robustness
The contents of the Emodin or Icariin were calculated from the linear regression.The average contents in three lots of GKG are 0.869 mg/g(CV=2.3%)for Emodin and1.014 mg/g(CV=3.1%)for Icariin respectively.According to 80%average value of the total number,the value is 0.695 mg/g for Emodin and 0.811 mg/g for Icariin respectively.Considering the source of medicinal herbs and the actual process of production and storage,we set a limit that at least 0.60 mg/g of Emodin and 0.80 mg/g of Icariin should be contained in the finished drug.
Note:R.S.D.Relative standard deviation;Data are means±R.S.D.of six replicates
The recovery experiment was performed for six times by adding Emodin or Icariin to the sample solution according to the procedure described above.The amounts of Emodin or Icariin recovered in relation to the results obtained in the intermediate precision study were calculated.The mean recoveries were 98.75%for E-modin and 98.83%for Icariin(Table 1).
Robustness was examined by changing slightly in the chromatography conditions,such as the proportion of mobile phase,flow rate and column temperature.In all the deliberately varied chromatographic conditions,the chromatogram of sample solution showed satisfactory resolution(Table 2).
Note：覮All the values of R.S.D.<5%
3 Conclusions
TLC is a glob ally accepted rational and practical solution to characterize the crude plant drug,pharmacologically active constituent enriched standardized extracts and their formulations.This standardized TLC procedure may be used effectively for screening the Ficus simplicissima Lour.root,Astragalus monghbolicus(Bge.)Hsiao and Mahonia bealei(Fort)Carr.as well as quality evaluation of the plant.