柑橘属常用中药材陈皮、青皮次生代谢产物之挥发油成分研究
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摘要
芸香科Rutaceae柑橘属Citrus L植物药用历史悠久,许多常见中药材如陈皮、青皮、枳实、枳壳、化橘红、橘红、橘叶、橘络、橘核、佛手等均来自该属植物。源于该属的药材在中药性效、化学成分、药理作用等方面既存在共性,又各具特征。代表性药材陈皮Citri Reticulatae Pericarpium (CRP)、青皮Citri Reticulatae Pericarpium Viride (CRPV)即充分体现了这一特点。陈皮、青皮同来源于柑橘属柑橘Citrus reticulata Blanco种及其变种,因不同的采收期而成为两味中药。陈皮为9-12月果实成熟时采摘的果皮；青皮为5-6月收集自落的幼果(个青皮),或7-8月采收的未成熟果实的果皮(四花青皮)。陈皮、青皮均为理气药,但临床应用和作用特点各有不同。陈皮的功效为理气健脾,燥湿化痰；青皮的功效为疏肝破气,消积化滞。陈皮长于理气健脾,青皮长于疏肝破气。陈皮作用部位主要在中上焦,青皮主要在中下焦。二者来源相同,是体现中药“一体二用”的代表。“一体二用”是指来源于同一基原植物和同一入药部位,因不同的生长期采收而成为功效、作用特点、临床应用不同的两味中药。陈皮和青皮因有相同的遗传背景和次生代谢产物合成途径,可将二者作为模式药物进行中药“一体二用”的研究。
陈皮、青皮的活性成分主要包括挥发油、黄酮成分。目前,关于陈皮、青皮的研究报道主要集中于黄酮类成分,而对具有较强生物活性的挥发油关注较少。已有的研究表明,挥发油是陈皮、青皮产生理气作用的主要部位。陈皮、青皮具有相同的性味,但其调气作用有强弱之别,陈皮为理气健脾,青皮为疏肝破气。基于此,从临床作用和化学成分的相关性考虑,理气作用的强弱应与其挥发油中所含主要成分含量有关；《中国药典》2010版仅以橙皮苷一个成分作为陈皮、青皮药材的质量控制指标,并未将挥发油成分纳入品质评价体系,这不能全面的反映陈皮、青皮品质；此外,由于柑橘挥发油优良的芳香品质及丰富的资源,它被认为是制作食品、饮料、化妆品的主要香料来源。鉴于此,本文对陈皮、青皮挥发油成分做了一较系统、全面、深入的研究。研究结果为揭示阐明“品种-成分-性效”相关性,中药“一体二用”的科学内涵奠定了一定的基础；并对柑橘果皮的综合利用,橘皮精油的产业化建设,促进地方经济发展具有重要意义。
具体研究内容及实验结果如下：
1.陈皮、青皮挥发油提取、GC-MS分析方法学研究
1.1提取方法学研究
在实验室已建立方法的基础上,通过系统查阅文献及实际操作,确定了一较好的提取方案。目前国际上,对于新鲜柑橘果皮精油的提取主要采用压榨法,因为这能充分地保障橘皮精油优良的芳香品质。但由于陈皮、青皮是以干燥的橘皮入药,因此压榨法并不适用于两药挥发油的提取。基于此,本研究仍采用经典的水蒸汽蒸馏提取法,具体方案如下：取粉碎后的样品约10-30g与约10倍量(V/W)蒸馏水(mL)于40℃时在一挥发油提取器(Clevenger-type apparatus)中溶胀1h,随后提取挥发油3-4h,得无色或淡黄色澄明油状液体。提取完毕后,即让挥发油在提取装置中静置1h,后读取挥发油的量(mL),并收集。挥发油于约5℃时通过无水硫酸钠脱水24h,后装于具塞小瓶,置于冰箱中,以备气相色谱-质谱(Gas Chromatograph-Mass Spectrometer, GC-MS)分析所需。计算挥发油的收率(v/w)。结果表明该方法切实可行,提取效果较好。从各样品挥发油得率看：广陈皮的挥发油得率(3.56-8.71%)明显高于其他陈皮、青皮(<0.7-4.2%)。从实验结果来看,为了确保有足够的挥发油(≥0.1mL)供分析用,应当确保样品的量为：广陈皮不少于10g(贮藏年限较长的广陈皮价格较高),其他陈皮、青皮不少于25g,提取溶剂用量约为样品量的10倍(V/W)。
1.2GC-MS分析方法学研究
同样地,在实验室已有分析方法的基础上,通过系统查阅文献及实际操作,确定本研究中采取的方法为：Agilent7890A/5975C GC-MS气相色谱一质谱联用仪(美国),匹配一化学工作站MSD ChemStation E.02.01.1177,质谱图匹配的谱库为NIST (National Institute of Standards and Technology,美国国家标准技术研究所)08MS database。色谱柱HP-5MS5%Phenyl Methyl Siloxane (30m×0.25mm×0.25μm)。
柱箱升温程序：初始温度60℃,保持3min,以4℃/min的速率升至250℃。载气为He,恒定流速1mL/min。进样口(气化温度),离子源的温度分别为280,230℃。分流比20：1。溶剂延迟：3.00min。电离方式：El (Electron impact)源；电子能量：70eV；质量范围(m/z):30～350amu;扫描间隔：2.29scans s-1。进样前,样品被稀释为10%的n-hexane(正己烷)溶液。进样量1μL。
通过本研究发现,以上参数还可进一步的优化,具体有：由于在药材FCI-1-b, FCI-2-b, FCI-3-b挥发油中鉴定的化合物Pentacosane(正二十五烷)的分子量为352,超出了350的上限,因此恰当的质量范围(m/z)应为：30-400amu。
2中药挥发油成分定性方法学研究——以陈皮、青皮为例
2.1中药挥发油总离子流谱图中重叠峰的拆分
中药挥发油是一个十分复杂的体系,含有数以百计的化学成分,且许多成分性质相近,这导致应用GC-MS分析得到的总离子流谱图(Total ion chromatogram, TIC)中存在着大量的重叠、甚至内嵌峰。如何对这些重叠峰进行拆分,并定性,将对挥发油成分的正确分析起到至关重要的作用。本研究应用气-质化学工作站(ChemStation)匹配的／AMDIS (Automated Mass Spectral Deconvolution&Identification System,自动质谱退卷积定性系统)拆分中药挥发油TIC中的重叠峰：并首次系统、深入地阐述了如何应用该程序拆分中药挥发油TIC中的重叠峰(以陈皮、青皮为例)。该研究具有较好的创新性和实用性,对于解决色谱分析中色谱峰重叠问题具有很好的启示作用。研究结果表明,AMDIS能初步分析TIC中含有多少化合物。而后,通过对各待鉴定化合物对应扫描点提纯谱图及该扫描点原始谱图与参考谱库中各匹配化合物的比对、分析,确定其结构；或否定某处提纯谱图及其对应的化合物(即此提纯谱图并不合理,其对应的化合物实际并不存在)。进一步,通过选择各化合物的特征性碎片离子峰,并分析它们的变化规律,可确认已有的鉴定结果。尤其,当被鉴定物浓度较低,且受到干扰时,AMDIS分析方法具有较大优势。它能有效去除干扰并获得“纯净”的谱图信息,从而增大目标化合物定性的可靠性。不过,当2个化合物的保留时间十分接近时,AMDIS有可能会漏检。总之,AMDIS的提纯质谱图的功能,可有效地对重叠峰中混合质谱图进行拆分,进而准确的对各物质定性。
2.2Kovdts保留指数在中药挥发油成分定性中的作用
随着GC-MS的普及,现在,对于中药挥发油成分的定性主要通过MS完成。但是仅仅依靠MS对化合物定性是不够的,因为许多萜烯类化合物有着基本相同的质谱。因此对于补充质谱数据而言,一些关于保留行为的知识是非常需要的。这其中最著名的就是Kovats于1958年提出的保留指数(Retention index, RI)概念。本研究应用Van den Dool与Kratz提出的公式计算程序升温保留指数(temperature-programmed retention indices,IP)。
研究结果表明,保留指数在中药挥发油成分定性中发挥着重要作用,具有很大的参考价值。对于中药挥发油成分的鉴定,应将MS与保留指数结合起来,保留指数和其它手段综合定性是气相色谱发展的重要趋势之一。
3中药挥发油成分定量方法学研究——以陈皮、青皮为例
应用2.2中的方法对TIC中的谱峰定性,定性后的色谱峰采用总体积积分和归一化法进行定量,并假定所有化合物的响应因子均为1。尽管面积归一化法是一较粗略的定量方法,但由于其操作简便,因此本研究中仍采用此方法定量。对于TIC中的重叠峰,应用AMDIS拆分定性后,通过各定性化合物的特征性碎片离子峰对各物质进行一大致的峰面积定量。研究结果表明，此方法切实可行,数据可靠。
4同来源陈皮、青皮挥发油成分比较研究
由于陈皮、青皮的来源复杂,为了确保一个真正的比较研究,实验中的药材均首次采用了定种、定产地、定植株(1棵或相邻的几颗)的方法进行收集。同时,实验中各批次药材的提取方法也相同。因此遗传因素,生态环境,及技术参数的影响均被去除,一个真正的比较分析实验得以进行。共对来源于2个变种：大红袍(产地：重庆市璧山县、四川夹江马村)、椪柑(产地：四川蒲江)总计19批药材进行了研究。
研究结果表明不同采收时间的陈皮、青皮(大红袍、椪柑)挥发油成分在性质、含量上均有一定的差异。并且源于不同变种的陈皮、青皮在挥发油大类成分上均呈现一致的变化规律：即随着采收时间的延长,单萜烯类成分的含量呈现先升高,再降低的趋势；含氧化合物含量呈现先降低,再升高的趋势。以挥发油成分含量为变量进行聚类分析,结果将不同采收期橘皮分为3类：早期采收的橘皮、中期采收的橘皮、晚期采收的橘皮,这与按采收期不同将橘皮分为个青皮、青皮、陈皮3类一致。
5不同贮藏年限广陈皮(新会陈皮)挥发油成分对比研究
研究结果表明源于茶枝柑的不同贮藏年限的广陈皮(新会陈皮)拥有几乎相同的物质基础。直观及聚类分析均表明：贮藏期为4年的新会陈皮挥发油成分与其他5批贮藏年限更长的新会陈皮有较显著性差异,后5批之间的差异性并不显著。与4年新会陈皮相比,后5批广陈皮总单萜烯类化合物含量有所升高,而总含氧化合物含量则降低。这表明,就挥发油类成分而言,陈皮确实需要“陈”,但这种“陈”也是有限度的,“陈”到一定程度即达到最佳。具体的变化趋势和内在规律还需要在今后的研究中做进一步探讨。传统认为,陈皮在存放过程中,挥发性成分多已挥发,使得其他成分能更好地发挥药理作用。但本研究发现,随着贮藏年限的延长,挥发油的得率并未降低,这说明挥发油类成分仍是新会陈皮发挥药效的重要物质基础。
6陈皮、青皮、橘皮挥发油化学型分析
Lota等根据化合物柠檬烯(Limonene)与γ-萜品烯(y-Terpinene)的含量,将来源于不同柑橘变种果皮挥发油的化学型分为两类：柠檬烯(83.8～96.2%)型与柠檬烯/γ-萜品烯(52.2～81.3%/11.2～36.7%)型。受Lota的启发,并在Lota的基础上,本研究首次系统分析了源于不同变种、不同产地陈皮、青皮(橘皮)挥发油的化学型。已有的研究结果表明,区分陈皮、青皮挥发油的化学型主要看4个物质的含量高低,即：柠檬烯、γ-萜品烯、芳樟醇(Linalool)、2-甲氨基苯甲酸甲酯(Benzoic acid,2-(methylamino)-, methyl ester)。据此,本研究中来源于大红袍、椪柑的陈皮、青皮(橘皮)挥发油归属于柠檬烯/芳樟醇型；源于茶枝柑的广陈皮挥发油归属于柠檬烯/γ-萜品烯/2-甲氨基苯甲酸甲酯化学型。
7挥发油成分在陈皮、青皮品质评价中的作用
本研究表明,挥发油成分在陈皮、青皮品质评价系统中具有重要作用。首先,同来源的陈皮、青皮挥发油成分具有显著性差异,并且这种差异的总体特征在来源于不同变种、不同产地的陈皮、青皮均一致。直观分析及聚类分析均表明：这种变化趋势与传统的按采收时间将橘皮分为个青皮、青皮、陈皮3种类型也是一致的。其次,就挥发油成分而言,陈皮中来源于茶枝柑的道地药材“广陈皮”与其他陈皮、青皮具有显著性差异。从化学型上来看,广陈皮具有一特殊的化学型；另从挥发油在药材中的含量来看,广陈皮也明显高于其他陈皮、青皮。因此可以推断,广陈皮之所以成为道地药材,并显现比其他陈皮更好的药效,挥发油成分发挥了重要的作用。据此,建议《中国药典》在今后的版本中将挥发油成分纳入陈皮、青皮品质评价体系,具体如下：化学型概念,得率,籍此将广陈皮与其他陈皮区分；挥发油成分中两大主成分(总单萜烯、总含氧化合物)的含量比例关系,籍此区分个青皮、青皮、陈皮。此结论与课题组之前对陈皮、青皮次生代谢产物之黄酮化合物研究结果一致,即：在品质评价体系的构建中,建议将广陈皮与陈皮区分,个青皮与四花青皮区分。
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 CRPV
1.1Study on extracted method
Based 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 method
Similarly, 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 CRPV
2.1To deconvolve the overlapped peaks in the total ion chromatogram of essential oil from CMM
Essential 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 CMM
The 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 CRPV
According 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 origin
A 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 years
There 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 CRP
Lota 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 CRPV
This 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.
陈皮、青皮的活性成分主要包括挥发油、黄酮成分。目前,关于陈皮、青皮的研究报道主要集中于黄酮类成分,而对具有较强生物活性的挥发油关注较少。已有的研究表明,挥发油是陈皮、青皮产生理气作用的主要部位。陈皮、青皮具有相同的性味,但其调气作用有强弱之别,陈皮为理气健脾,青皮为疏肝破气。基于此,从临床作用和化学成分的相关性考虑,理气作用的强弱应与其挥发油中所含主要成分含量有关；《中国药典》2010版仅以橙皮苷一个成分作为陈皮、青皮药材的质量控制指标,并未将挥发油成分纳入品质评价体系,这不能全面的反映陈皮、青皮品质；此外,由于柑橘挥发油优良的芳香品质及丰富的资源,它被认为是制作食品、饮料、化妆品的主要香料来源。鉴于此,本文对陈皮、青皮挥发油成分做了一较系统、全面、深入的研究。研究结果为揭示阐明“品种-成分-性效”相关性,中药“一体二用”的科学内涵奠定了一定的基础；并对柑橘果皮的综合利用,橘皮精油的产业化建设,促进地方经济发展具有重要意义。
具体研究内容及实验结果如下：
1.陈皮、青皮挥发油提取、GC-MS分析方法学研究
1.1提取方法学研究
在实验室已建立方法的基础上,通过系统查阅文献及实际操作,确定了一较好的提取方案。目前国际上,对于新鲜柑橘果皮精油的提取主要采用压榨法,因为这能充分地保障橘皮精油优良的芳香品质。但由于陈皮、青皮是以干燥的橘皮入药,因此压榨法并不适用于两药挥发油的提取。基于此,本研究仍采用经典的水蒸汽蒸馏提取法,具体方案如下：取粉碎后的样品约10-30g与约10倍量(V/W)蒸馏水(mL)于40℃时在一挥发油提取器(Clevenger-type apparatus)中溶胀1h,随后提取挥发油3-4h,得无色或淡黄色澄明油状液体。提取完毕后,即让挥发油在提取装置中静置1h,后读取挥发油的量(mL),并收集。挥发油于约5℃时通过无水硫酸钠脱水24h,后装于具塞小瓶,置于冰箱中,以备气相色谱-质谱(Gas Chromatograph-Mass Spectrometer, GC-MS)分析所需。计算挥发油的收率(v/w)。结果表明该方法切实可行,提取效果较好。从各样品挥发油得率看：广陈皮的挥发油得率(3.56-8.71%)明显高于其他陈皮、青皮(<0.7-4.2%)。从实验结果来看,为了确保有足够的挥发油(≥0.1mL)供分析用,应当确保样品的量为：广陈皮不少于10g(贮藏年限较长的广陈皮价格较高),其他陈皮、青皮不少于25g,提取溶剂用量约为样品量的10倍(V/W)。
1.2GC-MS分析方法学研究
同样地,在实验室已有分析方法的基础上,通过系统查阅文献及实际操作,确定本研究中采取的方法为：Agilent7890A/5975C GC-MS气相色谱一质谱联用仪(美国),匹配一化学工作站MSD ChemStation E.02.01.1177,质谱图匹配的谱库为NIST (National Institute of Standards and Technology,美国国家标准技术研究所)08MS database。色谱柱HP-5MS5%Phenyl Methyl Siloxane (30m×0.25mm×0.25μm)。
柱箱升温程序：初始温度60℃,保持3min,以4℃/min的速率升至250℃。载气为He,恒定流速1mL/min。进样口(气化温度),离子源的温度分别为280,230℃。分流比20：1。溶剂延迟：3.00min。电离方式：El (Electron impact)源；电子能量：70eV；质量范围(m/z):30～350amu;扫描间隔：2.29scans s-1。进样前,样品被稀释为10%的n-hexane(正己烷)溶液。进样量1μL。
通过本研究发现,以上参数还可进一步的优化,具体有：由于在药材FCI-1-b, FCI-2-b, FCI-3-b挥发油中鉴定的化合物Pentacosane(正二十五烷)的分子量为352,超出了350的上限,因此恰当的质量范围(m/z)应为：30-400amu。
2中药挥发油成分定性方法学研究——以陈皮、青皮为例
2.1中药挥发油总离子流谱图中重叠峰的拆分
中药挥发油是一个十分复杂的体系,含有数以百计的化学成分,且许多成分性质相近,这导致应用GC-MS分析得到的总离子流谱图(Total ion chromatogram, TIC)中存在着大量的重叠、甚至内嵌峰。如何对这些重叠峰进行拆分,并定性,将对挥发油成分的正确分析起到至关重要的作用。本研究应用气-质化学工作站(ChemStation)匹配的／AMDIS (Automated Mass Spectral Deconvolution&Identification System,自动质谱退卷积定性系统)拆分中药挥发油TIC中的重叠峰：并首次系统、深入地阐述了如何应用该程序拆分中药挥发油TIC中的重叠峰(以陈皮、青皮为例)。该研究具有较好的创新性和实用性,对于解决色谱分析中色谱峰重叠问题具有很好的启示作用。研究结果表明,AMDIS能初步分析TIC中含有多少化合物。而后,通过对各待鉴定化合物对应扫描点提纯谱图及该扫描点原始谱图与参考谱库中各匹配化合物的比对、分析,确定其结构；或否定某处提纯谱图及其对应的化合物(即此提纯谱图并不合理,其对应的化合物实际并不存在)。进一步,通过选择各化合物的特征性碎片离子峰,并分析它们的变化规律,可确认已有的鉴定结果。尤其,当被鉴定物浓度较低,且受到干扰时,AMDIS分析方法具有较大优势。它能有效去除干扰并获得“纯净”的谱图信息,从而增大目标化合物定性的可靠性。不过,当2个化合物的保留时间十分接近时,AMDIS有可能会漏检。总之,AMDIS的提纯质谱图的功能,可有效地对重叠峰中混合质谱图进行拆分,进而准确的对各物质定性。
2.2Kovdts保留指数在中药挥发油成分定性中的作用
随着GC-MS的普及,现在,对于中药挥发油成分的定性主要通过MS完成。但是仅仅依靠MS对化合物定性是不够的,因为许多萜烯类化合物有着基本相同的质谱。因此对于补充质谱数据而言,一些关于保留行为的知识是非常需要的。这其中最著名的就是Kovats于1958年提出的保留指数(Retention index, RI)概念。本研究应用Van den Dool与Kratz提出的公式计算程序升温保留指数(temperature-programmed retention indices,IP)。
研究结果表明,保留指数在中药挥发油成分定性中发挥着重要作用,具有很大的参考价值。对于中药挥发油成分的鉴定,应将MS与保留指数结合起来,保留指数和其它手段综合定性是气相色谱发展的重要趋势之一。
3中药挥发油成分定量方法学研究——以陈皮、青皮为例
应用2.2中的方法对TIC中的谱峰定性,定性后的色谱峰采用总体积积分和归一化法进行定量,并假定所有化合物的响应因子均为1。尽管面积归一化法是一较粗略的定量方法,但由于其操作简便,因此本研究中仍采用此方法定量。对于TIC中的重叠峰,应用AMDIS拆分定性后,通过各定性化合物的特征性碎片离子峰对各物质进行一大致的峰面积定量。研究结果表明，此方法切实可行,数据可靠。
4同来源陈皮、青皮挥发油成分比较研究
由于陈皮、青皮的来源复杂,为了确保一个真正的比较研究,实验中的药材均首次采用了定种、定产地、定植株(1棵或相邻的几颗)的方法进行收集。同时,实验中各批次药材的提取方法也相同。因此遗传因素,生态环境,及技术参数的影响均被去除,一个真正的比较分析实验得以进行。共对来源于2个变种：大红袍(产地：重庆市璧山县、四川夹江马村)、椪柑(产地：四川蒲江)总计19批药材进行了研究。
研究结果表明不同采收时间的陈皮、青皮(大红袍、椪柑)挥发油成分在性质、含量上均有一定的差异。并且源于不同变种的陈皮、青皮在挥发油大类成分上均呈现一致的变化规律：即随着采收时间的延长,单萜烯类成分的含量呈现先升高,再降低的趋势；含氧化合物含量呈现先降低,再升高的趋势。以挥发油成分含量为变量进行聚类分析,结果将不同采收期橘皮分为3类：早期采收的橘皮、中期采收的橘皮、晚期采收的橘皮,这与按采收期不同将橘皮分为个青皮、青皮、陈皮3类一致。
5不同贮藏年限广陈皮(新会陈皮)挥发油成分对比研究
研究结果表明源于茶枝柑的不同贮藏年限的广陈皮(新会陈皮)拥有几乎相同的物质基础。直观及聚类分析均表明：贮藏期为4年的新会陈皮挥发油成分与其他5批贮藏年限更长的新会陈皮有较显著性差异,后5批之间的差异性并不显著。与4年新会陈皮相比,后5批广陈皮总单萜烯类化合物含量有所升高,而总含氧化合物含量则降低。这表明,就挥发油类成分而言,陈皮确实需要“陈”,但这种“陈”也是有限度的,“陈”到一定程度即达到最佳。具体的变化趋势和内在规律还需要在今后的研究中做进一步探讨。传统认为,陈皮在存放过程中,挥发性成分多已挥发,使得其他成分能更好地发挥药理作用。但本研究发现,随着贮藏年限的延长,挥发油的得率并未降低,这说明挥发油类成分仍是新会陈皮发挥药效的重要物质基础。
6陈皮、青皮、橘皮挥发油化学型分析
Lota等根据化合物柠檬烯(Limonene)与γ-萜品烯(y-Terpinene)的含量,将来源于不同柑橘变种果皮挥发油的化学型分为两类：柠檬烯(83.8～96.2%)型与柠檬烯/γ-萜品烯(52.2～81.3%/11.2～36.7%)型。受Lota的启发,并在Lota的基础上,本研究首次系统分析了源于不同变种、不同产地陈皮、青皮(橘皮)挥发油的化学型。已有的研究结果表明,区分陈皮、青皮挥发油的化学型主要看4个物质的含量高低,即：柠檬烯、γ-萜品烯、芳樟醇(Linalool)、2-甲氨基苯甲酸甲酯(Benzoic acid,2-(methylamino)-, methyl ester)。据此,本研究中来源于大红袍、椪柑的陈皮、青皮(橘皮)挥发油归属于柠檬烯/芳樟醇型；源于茶枝柑的广陈皮挥发油归属于柠檬烯/γ-萜品烯/2-甲氨基苯甲酸甲酯化学型。
7挥发油成分在陈皮、青皮品质评价中的作用
本研究表明,挥发油成分在陈皮、青皮品质评价系统中具有重要作用。首先,同来源的陈皮、青皮挥发油成分具有显著性差异,并且这种差异的总体特征在来源于不同变种、不同产地的陈皮、青皮均一致。直观分析及聚类分析均表明：这种变化趋势与传统的按采收时间将橘皮分为个青皮、青皮、陈皮3种类型也是一致的。其次,就挥发油成分而言,陈皮中来源于茶枝柑的道地药材“广陈皮”与其他陈皮、青皮具有显著性差异。从化学型上来看,广陈皮具有一特殊的化学型；另从挥发油在药材中的含量来看,广陈皮也明显高于其他陈皮、青皮。因此可以推断,广陈皮之所以成为道地药材,并显现比其他陈皮更好的药效,挥发油成分发挥了重要的作用。据此,建议《中国药典》在今后的版本中将挥发油成分纳入陈皮、青皮品质评价体系,具体如下：化学型概念,得率,籍此将广陈皮与其他陈皮区分；挥发油成分中两大主成分(总单萜烯、总含氧化合物)的含量比例关系,籍此区分个青皮、青皮、陈皮。此结论与课题组之前对陈皮、青皮次生代谢产物之黄酮化合物研究结果一致,即：在品质评价体系的构建中,建议将广陈皮与陈皮区分,个青皮与四花青皮区分。
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 CRPV
1.1Study on extracted method
Based 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 method
Similarly, 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 CRPV
2.1To deconvolve the overlapped peaks in the total ion chromatogram of essential oil from CMM
Essential 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 CMM
The 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 CRPV
According 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 origin
A 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 years
There 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 CRP
Lota 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 CRPV
This 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.
引文
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