| Objective Ambergris is a dried product of intestinal secretions form Physeter catodon Linnaeus,which is one of the species of Physeteridae animal.It is one of the four greatest spices in China(sandalwood,incense,ambergris,musk),and also has been used as medical material for a long time due to its various medicinal effects.However,the description of Ambergris is always mysterious.Even now,it is hard to find a specific way to distinguish it.Therefore,the identification method and the quality control improvement of Ambergris highlight their significance.Based on the literature summaries,taking certified products and suspected products as examining subjects,traditional and morden Chinese medicine identification methods as well as technologies would be used to study each subject,including morphologic characteristics,microscopic characteristics,PE extract componentsas well as relative ratio.Extracting,isolating and purifying the main characteristic component of Ambergris-amberin to create a standard product,which would be used to determine the content of ambrein and cholestanol in Amergris.A TLC method would be constructed to identify the authenticity of Ambergris quickly and simply.Thereout,this paper would provide scientific basis for qulity control of Ambergris from different levels and aspects,as well as providing reference to improve its quality standard.Methods1.Morphological character and microscopic character identification of certified and suspected subjects Using Chinese traditional morphological and microscopic identification methods-compare the features of shape,color,smell,texture,taste,fire phenomenon and color of ethanol extract between two different subjects to find out the differences.2.The optimization of selecting of Ambergris`s extracting conditions and GC-MS analyzing.Gas chromatography-mass spectrometry(GC-MS)would be used to analyze and identify the components of certified subject.By changing extract solvents,time for extraction or solid-liquid ratio,the results from GC-MS would helped to support the analysis.The same process would be used on suspected subjects.By comparing the different results between two different subjects to enhance identifications.3.Preparation of amberin`s standard product Taking petroleum ether(PE)as solvent and certified subjects as solute,samples would be extracted by going through ultrasonic.Next,silica gel column chromatography with gradient elution would be used to elute the samples to get raw amberin,taking n-hexane-Et OAc as eluents.Then,the monomer of amberin would be purified by washing with acetone and preparative HPLC.Its structure would be identified by NMR spectral data and other methods.4.Content determination of the main components in Ambergris-ambrein and cholestanol In order to set up a content determination for anbrein and cholestenol,HPLC-ELSD would be used.Me OH-MTBE would be regarded as the mobile phase for Cosmosil Cholester column to eluting with a gradient program.5.TLC identification of Ambergris According to TLC method,extracted samples in PE would be used with developing solvent(hexane-chloroform,volume ratio 1:5)to applied on a silica G plate.By comparing the different point positions of standard products of both ambrein and cholestenol with the point positions of certified subject,the standard TLC would be set up.Then,it would be used to compared with suspected subjects.Results1.The main differences between the certified ambergris and its suspected samples could be listed out as follow:(1)The color of certified ambergris could be yellowish-brown,kelly green,medium brown or variegated.The shapes of its small pieces usually could be kidney shaped,lump,while the bulks generally being like layer banded.The surface of Ambergris is often blocked with squid beaks.As for suspected samples,those often have various colors in the appearance and the textures are always grease or powder,without squid beaks.(2)Certified ambergris would turn into oil gelatinous when it been melted.It also could shows filamentous when picking up.The smoke rises with a straight line,along with specific fragrance when it is burning,leaving the black ash.The ethanol extraction of it is yellow,partly golden or yellowish-pink.The suspected samples would be melted completely when heating up.Smoke woule be decentralized.Its ethanol extracts could be transparent.As for microscopic differences,it shows that both subjects have a large amount of oil droplets and yellowish-brown lumps.However,the certified products sometimes show cut in beaks,while the suspected samples are common featherlike crystals.2.The results of Ambergris`s component extraction show that extracting samples by ultrasonic for 40 min with the solid-liquid ratio of PE is 1:60(g/m L)at room temperature is appropriate,which lead the extraction rate to nearly 97.3%.GC-MS results show that the main component for Ambergris is ambrein,followed by cholestanone and cholestenol.In addition,dihydro-γ-ionone and ambrox can be detected from all samples,some of them can be detected out other characteristic compounds like: sclareolide,ambrial and ambreinolide.However,none of the suspected samples have the characteristic components above.Hence,those components metioned above can be judged as counterfeit basing on the results.3.The results of preparation of ambrein`s standard product illustrated that the extract rate is88.2% by ultrasonic extract samples with using PE repeatedly.Taking n-hexane-Et OAc as eluents,the concentrate is isolated through silica gel chromatography column at atmospheric pressure.The raw amberin could be gained at the eluents volume ratio of 10:1.Washing with acetone when obtained.After structural identification,taking 100% methanol as mobile phase for preparation HPLC to purify ambrein product.4.The results of contents determination of ambrein and cholestanol displayed that it is feasible to establish the contents of ambrein and cholestanol by HPLC-ELSD.The experimental results are as follow: the content of ambrein is above 29%,up to 60%,while the content of cholestanol is about 3.7%~16%.5.TLC of Ambergris illustrated that the effect of using n-hexane-chloroform as developing agent is appropriate and the volume ratio is 1:5.Ambrein and cholestanol are the main spots in TLC,so it could determine the TLC spectrum of authentic ambergris.Therefore,suspected samples can be quickly identified through this method.Conclusion This study illustrates that Ambergris collected in different areas and countries have great similarity in the characters,microscopic characters,chemical compositions.Because of the length of time discharged from the bodies of sperm whale and the differences in external force,there may have some distinctions in appearance,but the burning phenomena and microscopic characteristics are basically same.As for the results from GC-MS analysis,the chemical components of genuine exist great similarities,with ambrein as the main component,followed by sterols components,while the adulterants without the components above.The experiment took the properties of amberin into consider,and established the measure of extracting,isolating and purifying amberin,which was feasible and simple. Besides,the experiment also changes the situation of the amberin standard is invisible today.Futhermore,it provides the necessary condition and basic guarantee on researching the authenticity identification of ambergris.The determination content of ambrein and cholestanol has changed the situation that the characteristic components in Ambergris cannot be verified and without experimental data to support according to the current papers.Therefore,this method provides the reference for quality evaluation of Ambergris.However,TLC can provide a rapid method on identification the authenticity of Ambergris.Testing samples base on the aspects that mentioned,which can provide different levels and angles of the scientific basis for improving the quality standard of ambergris.In addition,to identify the authenticity of Ambergris by GC-MS or TLC is an accurate and quick way. |
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