Study On The Second Metabolite Of Essential Oil From Common Chinese Materia Medica As Citri Reticulatae Pericarpium And Citri Reticulatae Pericarpium Viride From Citrus L.

The plants in Citrus L. genus of Rutaceae family have been used as Chinese Materia Medica (CMM) for a long history such as Citri Reticulatae Pericarpium (CRP), Citri Reticulatae Pericarpium Viride (CRPV), etc. The CMMs from this genus have common and differention in nature and function, chemical constituents, pharmacology effects, etc. The representative CMMs as CRP and CRPV can fully demonstrate this trait. CRP and CRPV both come from the peel of Citrus reticulata Blanco or its cultivars, they become two kinds of CMM because of different harvest time. CRP is the dried pericarp of ripe fruit which is usually collected from September to December in each year. CRPV is the dried pericarp of young or immature fruits. The fallen young fruit is collected in May and June known commonly as "Geqingpf, which is also called Fructus Citri Immaturus (FCI). The immature fruit is collected in July and August, cut longitudinally into four-valved but connected at the base, removed from the emergences completely and dried, and known commonly as "Sihuaqingpi". They both belong to the qi-regulating drug, but have different effects and indications. CRP is good at regulating the flow of qi and invigorating the spleen function, while CRPV is good at soothing the liver and disintegrating stagnated qi. The channels entered by CRP are mainly at middle-and upper-jiao, while the channels entered by CRPV are mainly at middle-and lower-jiao. They are the representative of "One material used as two kinds of CMM""One material used as two kinds of CMM" is referred to that two kinds of CMM are come from the same part in the identical plant, but become two kinds of CMM due to different harvest time. They can be used as the model drug to study the scientific essence of "One material used as two kinds of CMM" because of the same origin and synthetic route of second metabolites.The main bioactive constituents of CRP and CRPV consist of essential oil and flavonoid. In the present study, many reports on CRP and CRPV focus on flavonoid, but few on essential oil which has strong pharmacologic bioactivities. Previous studies show that volatile oil is the main component that acts the function of regulating qi. CRP and CRPV have the same property and flavor, while differ in the effect of regulating qi. So, the different function should be related to their dissimilar constituents in essential oils based on the correlation between clinical effects and chemical constituents. Chinese pharmacopoeia2010Edition only uses hesperidin to control the quality of CRP and CRPV, but doesn’t apply essential oil to determine their quality, which can’t fully reflect their quality. Additional, the essential oil from C. reticulata peel is thought to be the main fragrance for making food, beverage, and cosmetic because of good flavor character. On account of these, this paper has done a systematic, broadly, and deep study on the essential oils of CRP and CRPV. The results can expound the scientific essence of relationship in "variety-components-property and effect" and "One material used as two kinds of CMM" in some degree, and have important meaning to the broad use of C. reticulata peel, industrialization of peel oil, local economical development.The concrete research content and experimental outcome are listed as follows.1Study on the extraction and GC-MS analysis method for oils of CRP and CRPV1.1Study on extracted methodBased on the existed method founded by Lab before and through systematic analyzing literature, a better extraction program has been determined. Now, the main extraction method is pressing for fresh peel in world because which can ensure the fine aromatic quality largely. But due to CRP and CRPV origin from the dried peel, pressing method isn’t suit for them. And so, this study still uses the classical method as steam distillation. The concrete plan is listed below.The sample was weighted10-30g respectively and then smashed. The smashed samples were swollen with about10times volume (v/w)100-300mL of distilled water in a Clevenger-type apparatus for soaking1h at40℃before extracting volatile oil for3-4h. Then, the essential oils were prepared according to the procedure described in Chinese Pharmacopoeia. The essential oils were dried over anhydrous sodium sulphate for24h at5℃and then stored in separate screw-capped vials in a refrigerator until needed. The yields of essential oils (v/w) should be calculated. The result shows that this method is workable. The yield of Guang CRP (3.56-8.71%) is significantly higher than that of other CRP and CRPV. In order to ensure that there are sufficient oils (not less than0.1mL) for analysis, the sample’s weight should not less than10g to Guang CRP and25g to other CRP and CRPV.1.2Study on GC-MS analysis methodSimilarly, a better method was established by referring the method used before in Lab and systematic literature research. It is listed below.An Agilent7890A gas chromatograph (USA) matched with an Agilent5975C mass spectrometer with a GC-MS ChemStation data system was used for GC-MS analysis. The gas chromatograph was coupled with a fused silica capillary column HP-5MS5%Phenyl Methyl Siloxane (30m×0.25mm i.d..0.25μm film thickness). The oven temperature was programmed from60℃(3-min hold) to250℃at4℃min-1. The carrier gas was helium at a constant flow of1mL min-1. Injector and ion-source were remained at280and230℃, individually. Splitting ratio was20:1. Solvent delay was3min. Electron impact mass spectra were taken at70eV Scan at2.29scans s-1from m/z30to350amu. The injection volume was1μL of a10%n-hexane solution to obtain the appropriate peak intensity. The molecular weight352of detected compound Pentacosane (n-C2sH52) is beyond350, so the reasonable m/z range should be from30to400amu.2The investigation on qualitative method of essential oil from CMM as CRP and CRPV2.1To deconvolve the overlapped peaks in the total ion chromatogram of essential oil from CMMEssential oil of CMM is a very complicated system and contains hundreds of chemical components. The overlapped or embedded peaks extensively exist in the total ion chromatogram (TIC) from volatile oil, which would make the accurately quantitative and qualitative analyses become more difficult. For that reason, the comparative analysis among different samples may be a hard task and even is sometimes impossible. However, the GC-MS ChemStation is always compiled with Automated Mass Spectral Deconvolution&Identification System (AMDIS), which is excellent in solving the above problem. This paper first expounds how to deconvolve the overlapped peaks in TIC by AMDIS systematically and deeply. The research has well innovation, practical utility, and enlightening role to deconvolve the overlapped peaks in chromatographic analysis. First, AMDIS can detect there are how many components in TIC through the run function. Then, by analyzing the extracted spectrum of corresponding scan point of detected component and the original spectrum of this scan point, and their counterparts’spectra in the referred MS Library, researchers can ascertain the component’s structure accurately or deny some compounds, which don’t exist in nature. Furthermore, through examining the changeability of characteristic fragment ion peaks of identified compounds, the previous outcome can be affirmed again. The result demonstrates that AMDIS can efficiently deconvolve the overlapped peaks in TIC by taking out the spectrum of matching scan point of discerned component. And then, it can exactly identify the component’s structure.2.2Study on the role of Kovats Retention index for the identification of components in essential oil from CMMThe separation and identification of monoterpenes and sesquiterpenes in plant or CMM essential oils relies heavily on GC. Even where combined GC-MS is used for the analysis, assignments often cannot be made on the basis of mass spectrometric data only, as many terpenes have essentially identical mass spectra. Hence some knowledge of retention characteristics is often required to complement mass spectral data. The most notable one is Retention index (RI) proposed by Kovats in1958. This study used the formula advocated by Van den Dool and Kratz to calculate temperature-programmed Retention indices (ITp). The result demonstrates that RI plays an important role in oil components identification. The combined utilization of MS and RI can greatly enhance the accuracy of identification.3Study on the quantitation method for CMM oil as CRP and CRPVAccording to the deconvolved chromatogram and mass spectra, the quantitative analysis of each component can be directly calculated by the overall volume integration method (OVI). They are proportionate to the content of the peak as integration based on TIC.4Comparative studies on the essential oils of CRP and CRPV from the same originA really contrastive study of the constitutions in oils of CRPV and CRP could be implemented because they are from the same origin such as the cultivar and environment. Likewise, extraction conditions were identical for all samples. So, the impacts of hereditary, environmental, and technological factors on the chemical components of oils were thought inappreciable. Nineteen samples from2varieties as C. reticulata’Dahongpao’(produced in Chongqing Bishan county and Sichuan Jiajiang Ma country) and C. poonensis Tanaka (produced in Sichuan Pujiang) were analyzed.The outcome shows that oils from CRPV and CRP varied both qualitatively and quantitatively to some degree. Chemical changeability of CRPV and CRP oil presented a rhythm as follows:The content of monoterpenes first go up, then go down; while the content of oxygenated compounds first drop then go up with the later of harvest time. Cluster analyses show that the peel oil can be classified as three types, which is identical to the classification of FCI, CRPV, and CRP according to their different collecting time point.5Chemical variability of essential oils in Xinhui CRP stored in different yearsThere is little difference in the chemical components between different samples, but a great variation in quantitation between the Xinhui CRP stored in4years and the other5samples stored in more than4years. This study found that the oil yield doesn’t drop as the extending of stored years, which demonstrates that oil is still the important active components of Xinhui CRP.6The chemotypes of essential oils in CRPV and CRPLota et al differentiate the chemotypes of peel oils from different cultivars of C. reticulata by the contents of Limonene and γ-Terpinene. In this way, they distinguish two chemotypes as Limonene and Limonene/γ-Terpinene. Enlightened by Lota, this paper first analyzes the chemotype of CRP and CRPV oils. In fact, all oils consist of Limonene as a primary component, and most oils comprise γ-Terpinene as the second high amount component. So, all oils can be classified as the chemotype of Limonene. Then, according to the content of y-Terpinene, it can be continually sub classified as the chemotype of Limonene or Limonene/y-Terpinene. Whereas except for these two important components, another two prominent component as Linalool and Benzoic acid,2-(methylamino)-, methyl ester should be noticed. So the oils of CRPV and CRP from the cultivar of C. Reticulata ’Dahongpao’ in this case can be successively sub classified as the chemotype of Limonene/Linalool. The essential oils of CRPV and CRP from the cultivar of C. Reticulata ’Chachi’ produced in Guangdong can be classified as the chemotype of Limonene/γ-Terpinene/Benzoic acid,2-(methylamino)-, methyl ester.7The role of essential oil in the quality evaluation of CRP and CRPVThis study demonstrates that oil plays an important role in the quality evaluation of CRP and CRPV. First, the oils of CRP and CRPV from the same origin differ with each other to some extent, and such difference presents the conformation in different varieties such as C. Reticulata’Dahongpao’and C. poonensis Tanaka. Visual and cluster analysis show that the peel oils from the same origin can be classified as three types, which is identical to the classification of FCI, CRPV, and CRP according to their different collecting time point. Second, Guang CRP sharply differs with other CRP which has a special chemotype and higher yield. So, the Guang CRP become a genuine CMM partly because the contribution of oil. So, this paper suggests Chinese Pharmacopoeia to adopt essential oil to evaluate the quality of CRP and CRPV, which include the chemotype, yield, and the chemical changeability such as monoterpenes and oxygenated compounds. Through this criterion, Guang CRP can be distinguished from other CRP, CRP can be differed with CRPV, and FCI can be disparted with CRPV. The conclusion is identical with the outcome concluded by our research team before to the second metabolite as flavonoid.