CN116642988A - Method for measuring content of multiple components in myrobalan and constructing characteristic fingerprint - Google Patents
Method for measuring content of multiple components in myrobalan and constructing characteristic fingerprint Download PDFInfo
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Abstract
Description
技术领域Technical Field
本发明涉及中药分析领域,尤其涉及一种诃子中多成分的含量测定及特征指纹图谱构建方法。The invention relates to the field of traditional Chinese medicine analysis, and in particular to a method for determining the contents of multiple components in terminalia chebula and constructing a characteristic fingerprint spectrum.
背景技术Background Art
中药诃子是使君子科植物诃子Terminalia chebula Retz.或绒毛诃子Terminalia chebula Retz.var.tomentella Kurt.的干燥成熟果实,具有涩肠止泻,敛肺止咳,降火利咽的功效,主要用于久泻久痢,便血脱肛,肺虚喘咳,久嗽不止,咽痛音哑。The Chinese medicine Terminalia chebula Retz is the dried mature fruit of Terminalia chebula Retz. or Terminalia chebula Retz.var.tomentella Kurt. of the Combretaceae family. It has the effects of astringing the intestines and stopping diarrhea, astringing the lungs and stopping coughs, reducing fire and relieving sore throats. It is mainly used for long-term diarrhea and dysentery, rectal prolapse due to bloody stools, lung deficiency, wheezing and coughing, long-term coughs, and sore throats and hoarseness.
诃子中含有大量生物活性成分,包括多酚类、黄酮类、多糖类、萜类等,其中含量最多的是鞣质类化合物,以可水解鞣质为主,如没食子酸、安石榴苷、柯里拉京、鞣花酸等。诃子中化学物质的解析和含量测定及特征指纹图谱研究有助于药材质量评价、活性成分发现、作用机理研究。但现有分析检测方法只能定量检测诃子中的一种或少数几种鞣质类化合物,不足以更全面反映其鞣质类成分的质量波动情况。Terminalia chebula contains a large number of bioactive ingredients, including polyphenols, flavonoids, polysaccharides, terpenes, etc. Among them, the most abundant are tannin compounds, mainly hydrolyzable tannins, such as gallic acid, punicalagin, corilagin, ellagic acid, etc. The analysis and content determination of chemical substances in Terminalia chebula and the study of characteristic fingerprints are helpful for the quality evaluation of medicinal materials, the discovery of active ingredients, and the study of mechanism of action. However, the existing analytical detection methods can only quantitatively detect one or a few tannin compounds in Terminalia chebula, which is not enough to more comprehensively reflect the quality fluctuation of its tannin components.
发明内容Summary of the invention
针对以上技术问题,本发明提供一种诃子中多成分的含量测定及特征指纹图谱构建方法和应用。本发明提供的含量测定方法可同时准确检测诃子中十余种鞣质类化合物,灵敏度高且精密度、重复性、稳定性均良好,不仅可用于构建诃子特征指纹图谱,还能用于构建与诃子有共同成分的西青果的特征指纹图谱。In view of the above technical problems, the present invention provides a method and application for determining the content of multiple components in Terminalia chebula and constructing a characteristic fingerprint. The content determination method provided by the present invention can accurately detect more than ten tannin compounds in Terminalia chebula at the same time, with high sensitivity and good precision, repeatability and stability. It can be used not only to construct a characteristic fingerprint of Terminalia chebula, but also to construct a characteristic fingerprint of Cibotium barometz which has common components with Terminalia chebula.
为达到上述发明目的,本发明提供了如下的技术方案:In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
一种诃子中多成分的含量测定方法,用甲醇水溶液对诃子进行提取,用超高效液相色谱法(UPLC)对待测成分进行含量测定,所述待测成分包括诃子次酸、没食子酸、4-没食子酰莽草酸、5-没食子酰莽草酸、安石榴苷(安石榴苷A&B)、柯里拉京、1,3,6-三-O-没食子酰基-β-D-葡萄糖、诃子宁、尿石素M5、诃子鞣酸、诃子林鞣酸和鞣花酸;所述超高效液相色谱法的色谱条件包括:A method for determining the contents of multiple components in terminalia chebula comprises extracting terminalia chebula with a methanol aqueous solution, and determining the contents of the components to be determined by ultra-high performance liquid chromatography (UPLC), wherein the components to be determined include terminalia chebula acid, gallic acid, 4-galloylshikimic acid, 5-galloylshikimic acid, punicalagin (punicalagin A & B), corilagin, 1,3,6-tri-O-galloyl-β-D-glucose, terminalia chebula, urolithin M5, terminalia tannic acid, terminalia chebula tannic acid and ellagic acid; and the chromatographic conditions of the ultra-high performance liquid chromatography include:
色谱柱:五溴苯基高效液相色谱柱;Chromatographic column: Pentabromophenyl HPLC column;
流动相:流动相A为0.095%~0.105%v/v甲酸水溶液,流动相B为甲醇,梯度洗脱,所述梯度洗脱的程序如下:Mobile phase: Mobile phase A is 0.095% to 0.105% v/v formic acid aqueous solution, mobile phase B is methanol, gradient elution, the gradient elution procedure is as follows:
流速为:0.28~0.32ml/min;Flow rate: 0.28~0.32ml/min;
检测波长为:250~290nm;Detection wavelength: 250~290nm;
上述色谱条件能够检测出诃子中的多种鞣质成分,包括诃子次酸、没食子酸、4-没食子酰莽草酸、5-没食子酰莽草酸、安石榴苷(安石榴苷A&B)、柯里拉京、1,3,6-三-O-没食子酰基-β-D-葡萄糖、诃子宁、尿石素M5、诃子鞣酸、诃子林鞣酸和鞣花酸,灵敏度高且精密度、重复性、稳定性均良好,可用于诃子的质量评价、诃子提取物或提取液中鞣质成分的含量检测、诃子特征指纹图谱构建。该含量测定方法还可以用于与诃子含有类似鞣质类化合物的西青果中鞣质类化合物的含量测定及特征指纹图谱构建。The above chromatographic conditions can detect a variety of tannin components in myrobalan, including myrobalan acid, gallic acid, 4-galloylshikimic acid, 5-galloylshikimic acid, punicalagin (punicalagin A & B), corilagin, 1,3,6-tri-O-galloyl-β-D-glucose, myrobalanine, urolithin M5, myrobalan tannic acid, myrobalan tannic acid and ellagic acid, with high sensitivity and good precision, repeatability and stability, and can be used for the quality evaluation of myrobalan, the content detection of tannin components in myrobalan extracts or extracts, and the construction of characteristic fingerprints of myrobalan. The content determination method can also be used for the content determination of tannin compounds in the fruit of the citrus aurantium containing similar tannin compounds as myrobalan and the construction of characteristic fingerprints.
结合第一方面,所述甲醇水溶液的体积百分浓度为40%~100%。进一步优选为甲醇,即100%v/v甲醇。In combination with the first aspect, the volume percentage concentration of the methanol aqueous solution is 40% to 100%, and more preferably methanol, that is, 100% v/v methanol.
优选地,提取的方式为超声提取,提取的温度为30~50℃,提取的时间为10~30min。Preferably, the extraction method is ultrasonic extraction, the extraction temperature is 30 to 50° C., and the extraction time is 10 to 30 minutes.
结合第一方面,所述五溴苯基高效液相色谱柱为Cosmosil PBr Packed Column(2.1×100mm,2.6μm)。该色谱柱不仅可在反相条件下分离大极性化合物,可在100%水条件下应用,适用于极性化合物分离;且对于环状化合物或胺类化合物有明显优势。对于诃子中的待测成分来说,该色谱柱分离效果较好。In combination with the first aspect, the pentabromophenyl high performance liquid chromatography column is a Cosmosil PBr Packed Column (2.1×100 mm, 2.6 μm). The chromatographic column can not only separate highly polar compounds under reverse phase conditions, but also can be used under 100% water conditions, and is suitable for the separation of polar compounds; and has obvious advantages for cyclic compounds or amine compounds. For the components to be tested in Terminalia chebula, the chromatographic column has a good separation effect.
结合第一方面,流动相A优选采用0.10%v/v甲酸水溶液。In combination with the first aspect, the mobile phase A is preferably a 0.10% v/v formic acid aqueous solution.
结合第一方面,所述流速为0.3ml/min。In combination with the first aspect, the flow rate is 0.3 ml/min.
结合第一方面,所述色谱条件还包括柱温为30~60℃;优选为30℃。In combination with the first aspect, the chromatographic conditions also include a column temperature of 30-60°C, preferably 30°C.
结合第一方面,所述诃子与所述甲醇水溶液的质量体积比为1:50~500(g:ml)。优选采用1:250~500(g:ml)。In combination with the first aspect, the mass volume ratio of the terminalia chebula to the methanol aqueous solution is 1:50-500 (g:ml), preferably 1:250-500 (g:ml).
结合第一方面,所述含量测定方法包括以下步骤:In combination with the first aspect, the content determination method comprises the following steps:
步骤a、用甲醇水溶液对诃子进行提取,制成供试品溶液;Step a, extracting Terminalia chebula with methanol-water solution to prepare a test solution;
步骤b、配制所述待测成分的对照品溶液;Step b, preparing a reference solution of the component to be tested;
步骤c、用所述超高效液相色谱法对所述对照品溶液和供试品溶液进行测定,用标准曲线法计算所述供试品中所述待测指标成分的含量。Step c, using the ultra-high performance liquid chromatography method to measure the reference solution and the test solution, and using the standard curve method to calculate the content of the index component to be measured in the test solution.
第二方面,本发明还提供了一种诃子的特征指纹图谱构建方法,包括以下步骤:In a second aspect, the present invention also provides a method for constructing a characteristic fingerprint of Terminalia chebula, comprising the following steps:
S1、配制指标成分的对照品溶液;所述指标成分包括诃子次酸、没食子酸、5-没食子酰莽草酸、安石榴苷(安石榴苷A&B)、柯里拉京、1,3,6-三-O-没食子酰基-β-D-葡萄糖、诃子宁、尿石素M5、诃子鞣酸、诃子林鞣酸和鞣花酸;S1. Prepare a reference solution of the index components; the index components include chebulic acid, gallic acid, 5-galloylshikimic acid, punicalagin (punicalagin A & B), corilagin, 1,3,6-tri-O-galloyl-β-D-glucose, chebulic acid, urolithin M5, chebulic acid, chebulic acid and ellagic acid;
S2、取不同批次诃子,分别用甲醇进行提取,得到不同批次诃子的供试品溶液,用上述含量测定方法中的超高效液相色谱法对所述对照品溶液和供试品溶液进行测定,记录指纹图谱,将所述指纹图谱全部导入中药色谱指纹图谱相似度评价系统软件,以一批诃子的图谱为参照图谱,选择时间宽度0.1min,采用中位数法以Mark峰进行峰匹配,对其进行模式识别获得指纹图谱及对照指纹图谱,选择吸收信号较强、分离度与稳定性较好的峰作为特征峰,计算各特征峰的相对保留时间、相对峰面积。S2. Take different batches of Terminalia chebula and extract them with methanol respectively to obtain test solutions of different batches of Terminalia chebula, measure the reference solution and the test solution with the ultra high performance liquid chromatography in the above-mentioned content determination method, record the fingerprint, import all the fingerprints into the Chinese medicine chromatographic fingerprint similarity evaluation system software, take the spectrum of a batch of Terminalia chebula as the reference spectrum, select the time width of 0.1min, use the median method to perform peak matching with Mark peak, perform pattern recognition on it to obtain fingerprint spectrum and reference fingerprint spectrum, select peaks with strong absorption signal, good separation and stability as characteristic peaks, and calculate the relative retention time and relative peak area of each characteristic peak.
以本发明提供的诃子的特征指纹图谱构建方法构建得到的特征指纹图谱能够获得上述12种鞣质类化合物的图谱信息,可用于对诃子中的鞣质类化合物进行快速评价,为诃子的质量研究提供数据支持。The characteristic fingerprint constructed by the characteristic fingerprint construction method of Terminalia chebula provided by the present invention can obtain the spectrum information of the above-mentioned 12 tannin compounds, which can be used for rapid evaluation of the tannin compounds in Terminalia chebula and provide data support for the quality research of Terminalia chebula.
第三方面,本发明还提供了一种诃子特征指纹图谱,所述诃子特征指纹图谱包含12个特征指纹峰:依次为诃子次酸、没食子酸、5-没食子酰莽草酸、安石榴苷A、安石榴苷B、柯里拉京、1,3,6-三-O-没食子酰基-β-D-葡萄糖、诃子宁、诃子鞣酸、诃子林鞣酸、鞣花酸和4-O-(3”,4”-Di-O-galloyl-α-L-rhamnopyra nosyl)ellagic acid。In the third aspect, the present invention also provides a terminalia chebula characteristic fingerprint, which comprises 12 characteristic fingerprint peaks: terminalia chebula acid, gallic acid, 5-galloylshikimic acid, punicalagin A, punicalagin B, corilagin, 1,3,6-tri-O-galloyl-β-D-glucose, terminalia chebula, terminalia tannic acid, terminalia chebula tannic acid, ellagic acid and 4-O-(3",4"-Di-O-galloyl-α-L-rhamnopyra nosyl)ellagic acid.
结合第三方面,以柯里拉京的峰为参照峰(H6),各共有峰与参照峰的相对保留时间在规定值的±5%的范围之内;规定值为:H1峰(诃子次酸)为0.2220–0.2453,H2峰(没食子酸)为0.4239–0.4685,H3峰(5-没食子酸酰莽草酸)为0.6435–0.7112,H4峰(安石榴苷A)为0.7405–0.8185,H5峰(安石榴苷B)为0.8456–0.9346,H6峰(柯里拉京)为1.000,H7峰(1,3,6-三-O-没食子酰基-β-D-葡萄糖)为1.056–1.167,H8峰(诃子宁)为1.167–1.290,H9峰(诃子鞣酸)为1.306–1.444,H10峰(诃子林鞣酸)为1.501–1.659,H11峰(鞣花酸)为1.532–1.693,H12峰(4-O-(3”,4”-Di-O-galloyl-α-L-rhamnopyranosyl)el lagic acid)为1.728–1.910。In combination with the third aspect, the peak of corilagin is used as the reference peak (H6), and the relative retention time of each common peak and the reference peak is within the range of ±5% of the specified value; the specified value is: H1 peak (chebulic acid) is 0.2220–0.2453, H2 peak (gallic acid) is 0.4239–0.4685, H3 peak (5-gallic acid ylshikimic acid) is 0.6435–0.7112, H4 peak (punicalagin A) is 0.7405–0.8185, H5 peak (punicalagin B) is 0.8456–0.9346, H6 peak (corilagin The peak values of H1 (H2) and H3 (H4) were 1.000, H7 (1,3,6-tri-O-galloyl-β-D-glucose) were 1.056–1.167, H8 (chebulic acid) were 1.167–1.290, H9 (chebulic acid) were 1.306–1.444, H10 (chebulic acid) were 1.501–1.659, H11 (ellagic acid) were 1.532–1.693, and H12 (4-O-(3”,4”-Di-O-galloyl-α-L-rhamnopyranosyl)el lagic acid) were 1.728–1.910.
第四方面,本发明还提供了上述的含量测定方法在西青果鞣质类成分含量测定及特征指纹图谱构建中的应用。In a fourth aspect, the present invention also provides the application of the above-mentioned content determination method in the determination of tannin components in western green fruit and the construction of characteristic fingerprints.
利用本发明提供的诃子的上述含量测定方法能够测定西青果鞣质类成分含量,构建西青果的特征指纹图谱,快速地反映西青果质量情况。The method for determining the content of terminalia chebula provided by the present invention can be used to determine the content of tannin components in terminalia chebula, construct a characteristic fingerprint of terminalia chebula, and quickly reflect the quality of terminalia chebula.
本发明的有益效果在于:The beneficial effects of the present invention are:
本发明建立的诃子中多成分的含量测定方法能够同时测定诃子中的12种鞣质类化合物,经方法学验证,该含量测定方法的灵敏度高且精密度、重复性、稳定性均良好,符合方法学要求。本发明还建立诃子的特征指纹图谱构建方法,并获得了诃子的12个特征指纹峰。本发明选择柯里拉京为参照峰,确定了诃子特征指纹图谱共有峰的相对保留时间。本发明提供的含量测定方法可以用于与诃子含有相似鞣质类化合物的西青果中鞣质类化合物的含量测定以及特征指纹图谱的构建,可以实现诃子、西青果质量评价及样品检测。The method for determining the content of multiple components in terminalia chebula established by the present invention can simultaneously determine 12 tannin compounds in terminalia chebula. After methodological verification, the method for determining the content is highly sensitive and has good precision, repeatability and stability, and meets the methodological requirements. The present invention also establishes a method for constructing a characteristic fingerprint of terminalia chebula, and obtains 12 characteristic fingerprint peaks of terminalia chebula. The present invention selects corilagin as a reference peak, and determines the relative retention time of the common peaks of the terminalia chebula characteristic fingerprint. The method for determining the content of tannin compounds in citrus aurantium containing similar tannin compounds to terminalia chebula and the construction of characteristic fingerprints can realize the quality evaluation of terminalia chebula and citrus aurantium and sample detection.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1为实施例3中不同药材供试品溶液的液相色谱图;FIG1 is a liquid chromatogram of different medicinal material test sample solutions in Example 3;
图2为实施例3中不同药材供试品溶液的TIC质谱图;FIG2 is a TIC mass spectrogram of different medicinal material test solutions in Example 3;
图3为实施例4中35批诃子样品特征指纹图谱和对照指纹图谱;FIG3 is a characteristic fingerprint of 35 batches of Terminalia chebula samples and a control fingerprint in Example 4;
图4为实施例5中28批西青果样品特征指纹图谱和对照指纹图谱;Figure 4 is the characteristic fingerprints and control fingerprints of 28 batches of Xiqing fruit samples in Example 5;
图5为实施例1、6、7、8中对照品溶液的液相色谱图;Fig. 5 is a liquid chromatogram of the reference substance solution in Examples 1, 6, 7, and 8;
图6为对比例1中使用不同色谱柱得到的液相色谱图。FIG6 is a liquid chromatogram obtained using different chromatographic columns in Comparative Example 1.
具体实施方式DETAILED DESCRIPTION
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.
诃子中含有大量生物活性成分,其中含量最多的是鞣质类化合物,以可水解鞣质为主,如没食子酸、安石榴苷、柯里拉京、鞣花酸等。现有分析检测方法只能定量检测诃子中的一种或少数几种鞣质类化合物,不足以更全面反映其鞣质类成分的质量波动情况。针对该问题,本发明实施例提供了一种诃子中多成分的含量测定方法以及诃子的特征指纹图谱构建方法,并提供了基于该含量测定方法的西青果特征指纹图谱构建方法。Terminalia chebula contains a large number of biologically active ingredients, among which the most abundant are tannin compounds, mainly hydrolyzable tannins, such as gallic acid, punicalagin, corilagin, ellagic acid, etc. Existing analytical detection methods can only quantitatively detect one or a few tannin compounds in Terminalia chebula, which is not enough to more comprehensively reflect the quality fluctuation of its tannin components. In view of this problem, the embodiment of the present invention provides a method for determining the content of multiple components in Terminalia chebula and a method for constructing a characteristic fingerprint of Terminalia chebula, and provides a method for constructing a characteristic fingerprint of Cibotium barometz based on the content determination method.
下面分多个实施例对本发明进行进一步的说明。The present invention is further described below with reference to a number of embodiments.
以下实施例中所采用的主要仪器:The main instruments used in the following examples are:
超高效液相色谱仪(ACQUITYH class plus,美国Waters公司);AgilentInfinitiy II 1260超高效液相色谱仪串联6550Q-TOF高分辨率质谱仪(美国Agilent公司);万分之一天平(ME204,瑞士METTLER公司);十万分之一天平(New Classic MS,瑞士METTLER公司);智能超声波清洗器(DL-720B,上海之信仪器有限公司);高速冷冻离心机(5430R,德国Eppendorf公司);Ultra-high performance liquid chromatography (ACQUITY H class plus, Waters, USA); Agilent Infinitiy II 1260 ultra-high performance liquid chromatograph connected in series with 6550Q-TOF high-resolution mass spectrometer (Agilent, USA); 1/10,000 balance (ME204, METTLER, Switzerland); 1/100,000 balance (New Classic MS, METTLER, Switzerland); intelligent ultrasonic cleaner (DL-720B, Shanghai Zhixin Instrument Co., Ltd.); high-speed refrigerated centrifuge (5430R, Eppendorf, Germany);
以下实施例所用的试剂为:The reagents used in the following examples are:
甲醇(色谱纯,Sigma-Aldrich贸易有限公司);无水乙醇(分析纯,天津市康科德科技有限公司);甲酸(色谱纯,上海阿拉丁生化科技股份有限公司);纯净水(广州屈臣氏食品饮料有限公司);二甲基亚砜(分析纯,天津市大茂化学试剂厂)。Methanol (chromatographic grade, Sigma-Aldrich Trading Co., Ltd.); anhydrous ethanol (analytical grade, Tianjin Concord Technology Co., Ltd.); formic acid (chromatographic grade, Shanghai Aladdin Biochemical Technology Co., Ltd.); purified water (Guangzhou Watsons Food and Beverage Co., Ltd.); dimethyl sulfoxide (analytical grade, Tianjin Damao Chemical Reagent Factory).
以下实施例中所采用的试药:The reagents used in the following examples are:
诃子(北京同仁堂天津河西大药房有限公司);没食子酸(J05GB153704)、安石榴苷(J03HB186918)、柯里拉京(A02GB143893)、诃子鞣酸(M29GB143373)、诃子林鞣酸(J21HB174660)、1,3,6-三-O-没食子酰基-β-D-葡萄糖(J11GB154490)、鞣花酸(S24D11G135548),均购于上海源叶生物科技有限公司;诃子次酸、4-没食子酰莽草酸、5-没食子酰莽草酸、诃子宁、尿石素M5,均由本实验室自制(纯度大于98%)。Terminalia chebula (Beijing Tongrentang Tianjin Hexi Pharmacy Co., Ltd.); gallic acid (J05GB153704), punicalagin (J03HB186918), corilagin (A02GB143893), terminalia chebula tannic acid (M29GB143373), terminalia chebula tannic acid (J21HB174660), 1,3,6-tri-O-galloyl-β-D-glucose (J11GB154490), and ellagic acid (S24D11G135548) were all purchased from Shanghai Yuanye Biotechnology Co., Ltd.; terminalia chebula acid, 4-galloylshikimic acid, 5-galloylshikimic acid, terminalia chebula, and urolithin M5 were all homemade in our laboratory (purity greater than 98%).
以下实施例中所采用的试药:诃子、西青果药材产地及来源信息如表1所示(HZ代表诃子,XQG代表西青果)。The test drugs used in the following examples: the origin and source information of the medicinal materials of Terminalia chebula and XQG are shown in Table 1 (HZ represents Terminalia chebula, and XQG represents XQG).
表1诃子、西青果药材产地及来源信息Table 1 Origin and source information of Terminalia chebula and Cibotium barometz
以下实施例中所采用的诃子、西青果粉末均为上述药材经粉碎后过3号筛的粉末。The chebula and citronella powders used in the following examples are all powders obtained by pulverizing the above medicinal materials and passing through a No. 3 sieve.
实施例1Example 1
本发明实施例提供了一种诃子中多成分的含量测定方法,包括如下步骤:The embodiment of the present invention provides a method for determining the content of multiple components in Terminalia chebula, comprising the following steps:
步骤a、供试品溶液的制备Step a, preparation of test solution
取诃子粉末0.2g,精密称定,置于50ml容量瓶中,加入适量甲醇,30℃超声提取20min,放置室温后,用甲醇定容至刻度,摇匀,取适量于13700rpm离心10min,取上清液作为供试品溶液;Take 0.2g of Terminalia chebula powder, weigh accurately, place in a 50ml volumetric flask, add appropriate amount of methanol, ultrasonically extract at 30℃ for 20min, leave at room temperature, dilute to scale with methanol, shake well, take appropriate amount, centrifuge at 13700rpm for 10min, and take the supernatant as the test solution;
步骤b、对照品溶液的制备Step b, preparation of reference solution
取12种对照品适量,精密称定,诃子次酸、没食子酸的对照品用水溶解,4-没食子酰莽草酸、5-没食子酰莽草酸、柯里拉京、1,3,6-三-O-没食子酰基-β-D-葡萄糖的对照品用甲醇溶解,安石榴苷(安石榴苷A&B)、诃子宁、尿石素M5、诃子鞣酸、诃子林鞣酸、鞣花酸对照品用二甲基亚砜溶解,分别配制成浓度为10.014mg/ml、7.028mg/ml、1.022mg/ml、1.662mg/ml、1.092mg/ml、0.854mg/ml、4.048mg/ml、3.846mg/ml、1.190mg/ml、1.530mg/ml、1.864mg/ml、0.5844mg/ml的对照品储备液。Take appropriate amount of 12 kinds of reference substances, weigh accurately, dissolve the reference substances of chebulic acid and gallic acid in water, dissolve the reference substances of 4-galloylshikimic acid, 5-galloylshikimic acid, corilagin, 1,3,6-tri-O-galloyl-β-D-glucose in methanol, dissolve the reference substances of punicalagin (punicalagin A & B), chebulic acid, urolithin M5, chebulic acid, chebulic acid, ellagic acid in dimethyl sulfoxide, and prepare respectively. The reference substance stock solutions are prepared with concentrations of 10.014 mg/ml, 7.028 mg/ml, 1.022 mg/ml, 1.662 mg/ml, 1.092 mg/ml, 0.854 mg/ml, 4.048 mg/ml, 3.846 mg/ml, 1.190 mg/ml, 1.530 mg/ml, 1.864 mg/ml and 0.5844 mg/ml.
精密量取诃子次酸、没食子酸、4-没食子酰莽草酸、5-没食子酰莽草酸、柯里拉京、1,3,6-三-O-没食子酰基-β-D-葡萄糖、安石榴苷(安石榴苷A&B)、诃子宁、尿石素M5、诃子鞣酸、诃子林鞣酸、鞣花酸对照品储备液适量,置10ml容量瓶中,加二甲基亚砜定容至刻度,摇匀,得混合对照品溶液。混合对照品溶液中含诃子次酸500.7μg/ml、没食子酸210.84μg/ml、4-没食子酰莽草酸51.1μg/ml、5-没食子酰莽草酸66.48μg/ml、安石榴苷(安石榴苷A&B)242.88μg/ml、柯里拉京65.52μg/ml、1,3,6-三-O-没食子酰基-β-D-葡萄糖68.32μg/ml、诃子宁230.76μg/ml、尿石素M5 35.7μg/ml、诃子鞣酸183.6μg/ml、诃子林鞣酸186.4μg/ml、鞣花酸175.32μg/ml;逐级稀释得到一系列不同浓度的对照品溶液。Accurately measure an appropriate amount of reference substance stock solutions of chebulic acid, gallic acid, 4-galloylshikimic acid, 5-galloylshikimic acid, corilagin, 1,3,6-tri-O-galloyl-β-D-glucose, punicalagin (punicalagin A & B), chebulic acid, urolithin M5, chebulic acid, chebulic acid tannic acid, and ellagic acid, place in a 10 ml volumetric flask, add dimethyl sulfoxide to make up to the mark, shake well, and obtain a mixed reference substance solution. The mixed reference solution contained 500.7μg/ml of terminalic acid, 210.84μg/ml of gallic acid, 51.1μg/ml of 4-galloylshikimic acid, 66.48μg/ml of 5-galloylshikimic acid, 242.88μg/ml of punicalagin (punicalagin A&B), 65.52μg/ml of corilagin, 68.32μg/ml of 1,3,6-tri-O-galloyl-β-D-glucose, 230.76μg/ml of terminalic acid, 35.7μg/ml of urolithin M5, 183.6μg/ml of terminalic acid, 186.4μg/ml of terminalic acid and 175.32μg/ml of ellagic acid; a series of reference solutions of different concentrations were obtained by stepwise dilution.
步骤c、用Agilent Infinitiy II 1260超高效液相色谱仪串联6550Q-TOF高分辨率质谱仪对上述混合对照品溶液和供试品溶液进行测定,色谱条件为:Step c: The mixed reference solution and the test solution were measured using an Agilent Infinitiy II 1260 ultra-high performance liquid chromatograph connected in series with a 6550Q-TOF high-resolution mass spectrometer. The chromatographic conditions were:
色谱柱:Cosmosil PBr Packed Column(2.1×100mm,2.6μm);Chromatographic column: Cosmosil PBr Packed Column (2.1×100mm, 2.6μm);
柱温:30℃;Column temperature: 30°C;
进样量:2μL;Injection volume: 2 μL;
流速:0.3ml/min;Flow rate: 0.3ml/min;
流动相A为0.10%v/v甲酸水溶液,流动相B为甲醇,进行线性梯度洗脱,洗脱程序为:Mobile phase A was 0.10% v/v formic acid in water, mobile phase B was methanol, and linear gradient elution was performed. The elution program was:
检测波长为270nm;The detection wavelength is 270nm;
质谱条件为:负离子模式下采集数据,采集模式为Auto MS/MS。离子源参数设置如下:气体温度为200℃;干燥气流速为12L/min;雾化压力为40psi;鞘气温度为350℃;喷嘴电压为-1.5kV;毛细管电压为-4.0kV;Fragmentor为390V;TOF分析仪扫描的质荷比(m/z)范围MS1为100–1500,MS2为50–1500。通过碰撞诱导解离自动选择在MS1谱中强度前3位的离子触发MS/MS裂解,碰撞能量为30V。数据采集在Agilent MassHunter Workstation DataAcquisition软件上实现。The mass spectrometry conditions were as follows: data were collected in negative ion mode, and the acquisition mode was Auto MS/MS. The ion source parameters were set as follows: gas temperature was 200°C; drying gas flow rate was 12 L/min; nebulizer pressure was 40 psi; sheath gas temperature was 350°C; nozzle voltage was -1.5 kV; capillary voltage was -4.0 kV; fragmentor was 390 V; the mass-to-charge ratio (m/z) range scanned by the TOF analyzer was 100–1500 for MS 1 and 50–1500 for MS 2. The top three ions in the MS 1 spectrum were automatically selected by collision-induced dissociation to trigger MS/MS fragmentation, and the collision energy was 30 V. Data acquisition was implemented on the Agilent MassHunter Workstation DataAcquisition software.
检验例1Test Example 1
本发明检验例提供了实施例1中多成分含量测定方法的方法学考察,其中:The test example of the present invention provides a methodological investigation of the multi-component content determination method in Example 1, wherein:
(1)线性关系和检测限、定量限研究(1) Study on linear relationship and detection limit and quantification limit
按照实施例1中步骤b的方法制备系列混合对照品溶液,采用超高效液相色谱法测定峰面积,色谱条件同实施例1中的步骤c。平行进样2次,以各化合物对照品浓度为横坐标x(μg/ml),相应的对照品峰面积为纵坐标y,绘制回归方程;以信噪比S/N=3为检测限(LOD)、S/N=10为定量限(LOQ),测定诃子次酸、没食子酸、4-没食子酰莽草酸、5-没食子酰莽草酸、安石榴苷(安石榴苷A&B)、柯里拉京、1,3,6-三-O-没食子酰基-β-D-葡萄糖、诃子宁、尿石素M5、诃子鞣酸、诃子林鞣酸、鞣花酸的检测限和定量限。结果如表2所示。A series of mixed reference solutions were prepared according to the method of step b in Example 1, and the peak area was determined by ultra-high performance liquid chromatography. The chromatographic conditions were the same as step c in Example 1. The samples were injected twice in parallel, and the concentration of each compound reference substance was taken as the abscissa x (μg/ml), and the corresponding reference substance peak area was taken as the ordinate y to draw a regression equation; the detection limit and quantification limit of chebulic acid, gallic acid, 4-galloylshikimic acid, 5-galloylshikimic acid, punicalagin (punicalagin A & B), corilagin, 1,3,6-tri-O-galloyl-β-D-glucose, chebulic acid, urolithin M5, chebulic acid, chebulic acid, ellagic acid were determined with a signal-to-noise ratio S/N=3 as the detection limit (LOD) and S/N=10 as the quantification limit (LOQ). The results are shown in Table 2.
由表2可知,各成分在各自的浓度范围内线性关系良好(r2≥0.999),检测限的浓度范围为0.0998–0.9779μg/ml,定量限的浓度范围为0.2559–1.956μg/ml,表明本发明提供的诃子中多成分的含量测定方法的灵敏度较高。As shown in Table 2, each component has a good linear relationship within its respective concentration range (r 2 ≥ 0.999), the concentration range of the detection limit is 0.0998–0.9779 μg/ml, and the concentration range of the quantification limit is 0.2559–1.956 μg/ml, indicating that the method for determining the contents of multiple components in Terminalia chebula provided by the present invention has high sensitivity.
(2)精密度试验(2) Precision test
(a)日内精密度试验(a) Intra-day precision test
精密吸取同一份上述供试品溶液,重复进样6次,采用超高效液相色谱法测定峰面积,色谱条件同实施例1中的步骤c。分别以峰面积计算RSD值,结果如表3所示。The same sample solution was accurately pipetted and injected 6 times, and the peak area was determined by ultra-high performance liquid chromatography. The chromatographic conditions were the same as those in step c of Example 1. The RSD values were calculated based on the peak areas, and the results are shown in Table 3.
表3日内精密度试验结果(n=6)Table 3 Intra-day precision test results (n=6)
由表3可见,实施例1提供的诃子中多成分的含量测定方法的日内精密度RSD<1.0%。As can be seen from Table 3, the intra-day precision RSD of the method for determining the contents of multiple components in Terminalia chebula provided in Example 1 is less than 1.0%.
(b)日间精密度试验(b) Inter-day precision test
连续3天,每天精密吸取同一份上述供试品溶液,重复进样6次,采用超高效液相色谱法测定峰面积,色谱条件同实施例1中的步骤c。结果如表4所示。For 3 consecutive days, the same sample solution was precisely pipetted every day, and the injection was repeated 6 times. The peak area was determined by ultra-high performance liquid chromatography, and the chromatographic conditions were the same as those in step c of Example 1. The results are shown in Table 4.
表4日间精密度试验结果(n=3)Table 4 Results of inter-day precision test (n=3)
由表4可见,实施例1提供的诃子中多成分的含量测定方法的日间精密度RSD<2.7%,测定方法精密度良好。As can be seen from Table 4, the inter-day precision RSD of the method for determining the contents of multiple components in Terminalia chebula provided in Example 1 is less than 2.7%, and the determination method has good precision.
(3)重复性试验(3) Repeatability test
按照实施例1中步骤a的方法平行制备6份诃子粉末的供试品溶液,采用超高效液相色谱法测定上述供试品溶液,计算各化合物含量,色谱条件同实施例1中的步骤c。以标准曲线计算诃子次酸、没食子酸、4-没食子酰莽草酸、5-没食子酰莽草酸、安石榴苷(安石榴苷A&B)、柯里拉京、1,3,6-三-O-没食子酰基-β-D-葡萄糖、诃子宁、尿石素M5、诃子鞣酸、诃子林鞣酸、鞣花酸的含量并计算RSD值。结果如表5所示。According to the method of step a in Example 1, 6 test solutions of terminalia chebula powder were prepared in parallel, and the test solutions were determined by ultra-high performance liquid chromatography to calculate the content of each compound. The chromatographic conditions were the same as step c in Example 1. The contents of terminalia chebula acid, gallic acid, 4-galloylshikimic acid, 5-galloylshikimic acid, punicalagin (punicalagin A & B), corilagin, 1,3,6-tri-O-galloyl-β-D-glucose, terminalia chebula, urolithin M5, terminalia chebula tannic acid, terminalia chebula tannic acid, and ellagic acid were calculated using a standard curve and the RSD value was calculated. The results are shown in Table 5.
由表5可知,在该试验中诃子次酸41.48mg/g、没食子酸12.65mg/g、4-没食子酰莽草酸2.530mg/g、5-没食子酰莽草酸3.760mg/g、安石榴苷(安石榴苷A&B)9.692mg/g、柯里拉京3.809mg/g、1,3,6-三-O-没食子酰基-β-D-葡萄糖3.467mg/g、诃子宁17.56mg/g、尿石素M5 2.707mg/g、诃子鞣酸9.985mg/g、诃子林鞣酸7.878mg/g、鞣花酸28.76mg/g,各化合物的RSD均小于3.0%,表明该检测方法重复性良好。As shown in Table 5, in this test, the RSDs of the compounds were all less than 3.0%, including 41.48 mg/g of terminalia acid, 12.65 mg/g of gallic acid, 2.530 mg/g of 4-galloylshikimic acid, 3.760 mg/g of 5-galloylshikimic acid, 9.692 mg/g of punicalagin (punicalagin A & B), 3.809 mg/g of corilagin, 3.467 mg/g of 1,3,6-tri-O-galloyl-β-D-glucose, 17.56 mg/g of terminalia chebula, 2.707 mg/g of urolithin M5, 9.985 mg/g of terminalia chebula tannic acid, 7.878 mg/g of terminalia chebula tannic acid and 28.76 mg/g of ellagic acid. This indicated that the detection method had good repeatability.
(4)稳定性试验(4) Stability test
将供试品溶液在10℃下保存,分别于0、2、4、6、8、10、12h精密吸取同一份上述供试品溶液,通过超高效液相色谱法对上述供试品溶液进行测定,色谱条件同实施例1中的步骤c。以诃子次酸、没食子酸、4-没食子酰莽草酸、5-没食子酰莽草酸、安石榴苷(安石榴苷A&B)、柯里拉京、1,3,6-三-O-没食子酰基-β-D-葡萄糖、诃子宁、尿石素M5、诃子鞣酸、诃子林鞣酸、鞣花酸的峰面积计算RSD值。结果如表6所示。The test solution was stored at 10°C, and the same portion of the test solution was accurately drawn at 0, 2, 4, 6, 8, 10, and 12 hours, respectively, and the test solution was measured by ultra-high performance liquid chromatography, and the chromatographic conditions were the same as those in step c of Example 1. The RSD value was calculated based on the peak areas of chebulic acid, gallic acid, 4-galloylshikimic acid, 5-galloylshikimic acid, punicalagin (punicalagin A & B), corilagin, 1,3,6-tri-O-galloyl-β-D-glucose, chebulic acid, urolithin M5, chebulic acid, chebulic acid, ellagic acid. The results are shown in Table 6.
由表6可知,实施例1提供的诃子中多成分的含量测定方法的液相色谱条件的稳定性试验RSD<1.2%,说明该供试品溶液在常温条件下12h内稳定性良好。As shown in Table 6, the RSD of the stability test of the liquid chromatography conditions of the method for determining the contents of multiple components in Terminalia chebula provided in Example 1 is less than 1.2%, indicating that the test solution has good stability within 12 hours at room temperature.
(5)加样回收率试验(5) Sample recovery test
取诃子粉末约0.1g,精密称定,加入与样品中各成分质量相当的混合对照品溶液16.6ml(诃子次酸300.4μg/ml、没食子酸84.34μg/ml、4-没食子酰莽草酸18.40μg/ml、5-没食子酰莽草酸29.92μg/ml、安石榴苷(安石榴苷A&B)72.86μg/ml、柯里拉京32.76μg/ml、1,3,6-三-O-没食子酰基-β-D-葡萄糖20.50μg/ml、诃子宁115.4μg/ml、尿石素M5 17.85μg/ml、诃子鞣酸64.26μg/ml、诃子林鞣酸55.92μg/ml、鞣花酸175.3μg/ml)。Take about 0.1g of terminalia chebula powder, weigh it accurately, and add 16.6ml of mixed reference solution with the same mass as the components in the sample (terminal acid 300.4μg/ml, gallic acid 84.34μg/ml, 4-galloylshikimic acid 18.40μg/ml, 5-galloylshikimic acid 29.92μg/ml, punicalagin (punicalagin A&B) 72.86μg/ml, corilagin 32.76μg/ml, 1,3,6-tri-O-galloyl-β-D-glucose 20.50μg/ml, terminalia chebula nin 115.4μg/ml, urolithin M5 17.85μg/ml, terminalia chebula tannic acid 64.26μg/ml, terminalia chebula tannic acid 55.92μg/ml, ellagic acid 175.3μg/ml).
制备6份诃子供试品溶液,每份样品平行测定2次,采用超高效液相色谱法对上述供试品溶液进行测定,色谱条件同实施例1中的步骤c。根据加样回收率公式计算的加样回收率及RSD值,考察该方法的准确性。Six samples of terminalia chebula test solution were prepared, and each sample was measured twice in parallel. The test solution was measured by ultra-high performance liquid chromatography, and the chromatographic conditions were the same as those in step c of Example 1. The accuracy of the method was investigated based on the sample recovery rate and RSD value calculated according to the sample recovery rate formula.
加样回收率公式如下:The recovery rate formula is as follows:
结果如表7所示。The results are shown in Table 7.
由表7可知,实施例1提供的诃子中多成分的含量测定方法的液相色谱条件的平均回收率97.41-113.4%,RSD值均小于6.5%,说明各化合物加样回收率基本符合要求,该方法准确可行。As shown in Table 7, the average recovery rate of the liquid chromatography conditions of the method for determining the content of multiple components in Terminalia chebula provided in Example 1 is 97.41-113.4%, and the RSD values are all less than 6.5%, indicating that the recovery rate of each compound basically meets the requirements and the method is accurate and feasible.
由表2~7可知通过实施例1提供的诃子中多成分的含量测定方法基本满足方法学研究要求,可以用于诃子的定量分析。It can be seen from Tables 2 to 7 that the method for determining the contents of multiple components in Terminalia chebula provided by Example 1 basically meets the requirements of methodological research and can be used for quantitative analysis of Terminalia chebula.
实施例2Example 2
本实施例以实施例1中的含量测定方法检测不同批次诃子中鞣质类成分的含量。步骤同实施例1,结果如表8所示。In this example, the content of tannin components in different batches of Terminalia chebula was detected by the content determination method in Example 1. The steps were the same as in Example 1, and the results are shown in Table 8.
实施例3Example 3
本实施例以实施例1中的含量测定方法检测西青果中的鞣质类成分。In this example, the tannin components in the citrus fruit were detected using the content determination method in Example 1.
步骤a、供试品溶液的制备:同实施例1,区别在于将诃子粉末更换为西青果粉末;Step a, preparation of test solution: same as Example 1, except that the terminalia chebula powder is replaced with citronella powder;
步骤b、对照品溶液的制备:同实施例1;Step b, preparation of reference solution: same as in Example 1;
步骤c、同实施例1,结果如表9所示。Step c: Same as Example 1. The results are shown in Table 9.
各药材供试品溶液的液相色谱图如图1所示,TIC质谱图如图2所示,定性检测结果如表10所示。The liquid chromatogram of each medicinal material test solution is shown in Figure 1, the TIC mass spectrum is shown in Figure 2, and the qualitative detection results are shown in Table 10.
实施例4Example 4
本发明实施例提供了一种诃子特征指纹图谱的构建方法,包括如下步骤:The embodiment of the present invention provides a method for constructing a characteristic fingerprint of Terminalia chebula, comprising the following steps:
步骤1、按实施例1中供试品溶液和对照品溶液的制备方法分别配制诃子粉末的供试品溶液以及混合对照品溶液;Step 1, prepare a test solution of terminalia chebula powder and a mixed reference solution according to the preparation method of the test solution and the reference solution in Example 1;
步骤2、同实施例1中的步骤c,记录特征指纹图谱。Step 2: Same as step c in Example 1, record the characteristic fingerprint.
将数据导入2012版中药色谱指纹图谱相似度评价系统进行处理,以诃子HZ35的图谱为参照图谱,选择吸收信号较强、分离度与稳定性较好的12个主要特征峰标定为共有峰,见图3。计算诃子指纹图谱与对照指纹图谱间的相似度,结果发现35批诃子样品与对照图谱的相似度大于0.66,说明诃子批次间较大。根据35批诃子药材指纹图谱中各化合物相对保留时间的测定结果,选择峰面积较大、峰形和分离度较好的12个共有色谱峰为特征指纹峰。生成标准对照指纹图谱,以H6峰为参照峰,12个共有色谱峰的相对保留时间、相对峰面积如表11、12所示。通过与对照品色谱图比对,指认各特征指纹峰分别为诃子次酸(H1)、没食子酸(H2)、5-没食子酰基莽草酸(H3)、安石榴苷A(H4)、安石榴苷B(H5)、柯里拉京(H6)、1,3,6-三-O-没食子酰基-β-D-葡萄糖(H7)、诃子宁(H8)、诃子鞣酸(H9)、诃子林鞣酸(H10)、鞣花酸(H11)、4-O-(3”,4”-Di-O-galloyl-α-L-rhamnopyranosyl)ellagic acid(H12)。The data was imported into the 2012 version of the Chinese medicine chromatographic fingerprint similarity evaluation system for processing. The spectrum of Terminalia chebula HZ35 was used as the reference spectrum, and 12 main characteristic peaks with strong absorption signals, good separation and stability were selected as common peaks, as shown in Figure 3. The similarity between the Terminalia chebula fingerprint spectrum and the reference fingerprint spectrum was calculated. The results showed that the similarity between 35 batches of Terminalia chebula samples and the reference spectrum was greater than 0.66, indicating that the difference between Terminalia chebula batches was large. According to the determination results of the relative retention time of each compound in the fingerprint spectrum of 35 batches of Terminalia chebula medicinal materials, 12 common chromatographic peaks with large peak area, good peak shape and separation were selected as characteristic fingerprint peaks. The standard reference fingerprint spectrum was generated, with the H6 peak as the reference peak. The relative retention time and relative peak area of the 12 common chromatographic peaks are shown in Tables 11 and 12. By comparing with the chromatograms of reference substances, the characteristic fingerprint peaks were identified as chebulic acid (H1), gallic acid (H2), 5-galloylshikimic acid (H3), punicalagin A (H4), punicalagin B (H5), corilagin (H6), 1,3,6-tri-O-galloyl-β-D-glucose (H7), chebulin (H8), chebulic tannic acid (H9), chebulin tannic acid (H10), ellagic acid (H11), and 4-O-(3”,4”-Di-O-galloyl-α-L-rhamnopyranosyl)ellagic acid (H12).
实施例5Example 5
本发明实施例提供了一种西青果特征指纹图谱的构建方法,包括如下步骤:The embodiment of the present invention provides a method for constructing a characteristic fingerprint of Xiqing fruit, comprising the following steps:
步骤1、按实施例1中供试品溶液和对照品溶液的制备方法分别配制西青果粉末的供试品溶液以及混合对照品溶液。Step 1: prepare a test solution of Xiqing fruit powder and a mixed reference solution according to the preparation method of the test solution and the reference solution in Example 1.
步骤2、同实施例1中的步骤c,记录特征指纹图谱。Step 2: Same as step c in Example 1, record the characteristic fingerprint.
将数据导入2012版中药色谱指纹图谱相似度评价系统进行处理,以西青果XQG24的图谱为参照图谱,选择吸收信号较强、分离度与稳定性较好的11个主要特征峰标定为共有峰,见图4。计算各样品UPLC图谱与对照指纹图谱间的相似度,结果发现28批西青果样品与对照图谱的相似度大于0.86,相似度良好。根据28批西青果药材指纹图谱中各化合物相对保留时间的测定结果,选择峰面积较大、峰形和分离度较好的11个共有色谱峰为特征指纹峰。生成标准对照指纹图谱,以X5峰(柯里拉京)为参照峰,11个共有色谱峰的相对保留时间、相对峰面积如表13、14所示。通过与对照品色谱图比对,指认各特征指纹峰分别为诃子次酸(X1)、没食子酸(X2)、安石榴苷A(X3)、安石榴苷B(X4)、柯里拉京(X5)、1,3,6-三-O-没食子酰基-β-D-葡萄糖(X6)、诃子宁(X7)、尿石素M5(X8)、诃子鞣酸(X9)、诃子林鞣酸(X10)、鞣花酸(X11)。The data was imported into the 2012 version of the Chinese medicine chromatographic fingerprint similarity evaluation system for processing. The spectrum of Xiqingguo XQG24 was used as the reference spectrum, and 11 main characteristic peaks with strong absorption signals, good separation and stability were selected as common peaks, as shown in Figure 4. The similarity between the UPLC spectrum of each sample and the reference fingerprint spectrum was calculated. The results showed that the similarity between the 28 batches of Xiqingguo samples and the reference spectrum was greater than 0.86, and the similarity was good. According to the determination results of the relative retention time of each compound in the fingerprint spectrum of 28 batches of Xiqingguo medicinal materials, 11 common chromatographic peaks with large peak area, good peak shape and separation were selected as characteristic fingerprint peaks. The standard reference fingerprint spectrum was generated, with the X5 peak (corilagin) as the reference peak. The relative retention time and relative peak area of the 11 common chromatographic peaks are shown in Tables 13 and 14. By comparing with the chromatogram of the reference substance, the characteristic fingerprint peaks were identified as chebulic acid (X1), gallic acid (X2), punicalagin A (X3), punicalagin B (X4), corilagin (X5), 1,3,6-tri-O-galloyl-β-D-glucose (X6), chebulic acid (X7), urolithin M5 (X8), chebulic acid (X9), chebulic acid (X10), and ellagic acid (X11).
以柯里拉京的峰为参照峰(X5),各共有峰与参照峰的相对保留时间在规定值的±5%的范围之内;规定值为:X1峰(诃子次酸)为0.2232-0.2466,X2峰(没食子酸)为0.4239-0.4686,X3峰(安石榴苷A)为0.7401-0.818,X4峰(安石榴苷B)为0.8442-0.933,X5峰(柯里拉京)为1.000,X6峰(1,3,6-三-O-没食子酰基-β-D-葡萄糖)为1.057-1.168,X7峰(诃子宁)为1.167-1.290,X8峰(尿石素M5)为1.201-1.327,X9峰(诃子鞣酸)为1.307-1.444,H10峰(诃子林鞣酸)为1.503-1.662,X11峰(鞣花酸)为1.533-1.694。The peak of corilagin is used as the reference peak (X5), and the relative retention time of each common peak and the reference peak is within the range of ±5% of the specified value; the specified values are: X1 peak (chebulic acid) is 0.2232-0.2466, X2 peak (gallic acid) is 0.4239-0.4686, X3 peak (punicalagin A) is 0.7401-0.818, X4 peak (punicalagin B) is 0.8442-0.933, X5 peak (corilagin) is 1 .000, X6 peak (1,3,6-tri-O-galloyl-β-D-glucose) was 1.057-1.168, X7 peak (chebula tannin) was 1.167-1.290, X8 peak (urolithin M5) was 1.201-1.327, X9 peak (chebula tannic acid) was 1.307-1.444, H10 peak (chebula tannic acid) was 1.503-1.662, and X11 peak (ellagic acid) was 1.533-1.694.
实施例6Example 6
本发明实施例提供了一种诃子中多成分的含量测定方法,包括如下步骤:The embodiment of the present invention provides a method for determining the contents of multiple components in Terminalia chebula, comprising the following steps:
步骤a、供试品溶液的制备:同实施例1;Step a, preparation of test solution: same as in Example 1;
步骤b、对照品溶液的制备:同实施例1;Step b, preparation of reference solution: same as in Example 1;
步骤c、用Waters ACQUITY UPLC H-Class PLUS超高效液相色谱仪对上述混合对照品溶液和供试品溶液进行测定,色谱条件与实施例1基本相同,区别在于柱温为40℃。Step c: The mixed reference solution and the test solution were measured using a Waters ACQUITY UPLC H-Class PLUS ultra-high performance liquid chromatograph. The chromatographic conditions were substantially the same as those in Example 1, except that the column temperature was 40°C.
实施例7Example 7
本发明实施例提供了一种诃子中多成分的含量测定方法,包括如下步骤:The embodiment of the present invention provides a method for determining the contents of multiple components in Terminalia chebula, comprising the following steps:
步骤a、供试品溶液的制备:同实施例1;Step a, preparation of test solution: same as in Example 1;
步骤b、对照品溶液的制备:同实施例1;Step b, preparation of reference solution: same as in Example 1;
步骤c、用Waters ACQUITY UPLC H-Class PLUS超高效液相色谱仪对上述混合对照品溶液和供试品溶液进行测定,色谱条件与实施例1基本相同,区别在于柱温为50℃。Step c: The mixed reference solution and the test solution were measured using a Waters ACQUITY UPLC H-Class PLUS ultra-high performance liquid chromatograph. The chromatographic conditions were substantially the same as those in Example 1, except that the column temperature was 50°C.
实施例8Example 8
本发明实施例提供了一种诃子中多成分的含量测定方法,包括如下步骤:The embodiment of the present invention provides a method for determining the contents of multiple components in Terminalia chebula, comprising the following steps:
步骤a、供试品溶液的制备:同实施例1;Step a, preparation of test solution: same as in Example 1;
步骤b、对照品溶液的制备:同实施例1;Step b, preparation of reference solution: same as in Example 1;
步骤c、用Waters ACQUITY UPLC H-Class PLUS超高效液相色谱仪对上述混合对照品溶液和供试品溶液进行测定,色谱条件与实施例1基本相同,区别在于柱温为60℃。Step c: The mixed reference solution and the test solution were measured using a Waters ACQUITY UPLC H-Class PLUS ultra-high performance liquid chromatograph. The chromatographic conditions were substantially the same as those in Example 1, except that the column temperature was 60°C.
实施例1、6、7、8中供试品溶液的液相色谱图如图5所示,在30~60℃柱温条件下均可使13种鞣质类化合物的峰分离,其中在30℃柱温条件下13种化合物的峰型分离度更好。The liquid chromatograms of the test solutions in Examples 1, 6, 7, and 8 are shown in FIG5 . The peaks of the 13 tannin compounds can be separated at a column temperature of 30 to 60° C., and the peak separation of the 13 compounds is better at a column temperature of 30° C.
实施例9Example 9
本发明实施例提供了一种诃子中多成分的含量测定方法,包括如下步骤:The embodiment of the present invention provides a method for determining the content of multiple components in Terminalia chebula, comprising the following steps:
步骤a、供试品溶液的制备:取诃子粉末0.2g,精密称定,置于25ml容量瓶中,加入适量甲醇,30℃超声提取20min,放置室温后,用甲醇定容至刻度,摇匀,取适量于13700rpm离心10min,取上清液作为供试品溶液;同法制备以60%v/v、80%v/v和100%v/v为提取溶剂的供试品溶液。Step a, preparation of the test solution: take 0.2g of Terminalia chebula powder, accurately weigh it, place it in a 25ml volumetric flask, add appropriate amount of methanol, ultrasonically extract at 30℃ for 20min, leave it at room temperature, dilute to the scale with methanol, shake well, take appropriate amount, centrifuge at 13700rpm for 10min, and take the supernatant as the test solution; prepare test solutions with 60% v/v, 80% v/v and 100% v/v as extraction solvents in the same way.
步骤b、对照品溶液的制备:同实施例1。Step b, preparation of reference solution: same as in Example 1.
步骤c、用Waters ACQUITY UPLC H-Class PLUS超高效液相色谱仪对上述混合对照品溶液和供试品溶液进行测定,色谱条件同实施例1。Step c: using Waters ACQUITY UPLC H-Class PLUS ultra-high performance liquid chromatograph to measure the mixed reference solution and the test solution, the chromatographic conditions are the same as those in Example 1.
各供试品溶液所得液相色谱图中,各成分的峰面积如表15所示。The peak areas of the components in the liquid chromatograms obtained from the test solutions are shown in Table 15.
表15不同提取溶剂下所得供试品溶液的鞣质类成分峰面积Table 15 Peak areas of tannin components in the test solution obtained under different extraction solvents
由表15可见,甲醇作为提取溶剂时,鞣质类成分峰面积最高,提取效果最优。As can be seen from Table 15, when methanol is used as the extraction solvent, the peak area of tannin components is the highest and the extraction effect is the best.
实施例10Example 10
本发明实施例提供了一种诃子中多成分的含量测定方法,包括如下步骤:The embodiment of the present invention provides a method for determining the contents of multiple components in Terminalia chebula, comprising the following steps:
步骤a、供试品溶液的制备:精密称取诃子粉末1、0.2、0.2、0.1g,分别置于50ml、25ml、50ml、50ml容量瓶中,分别精密加入甲醇适量,30℃超声提取20min,放置室温后,用甲醇定容至刻度,摇匀,得到料液比为1:50、1:125、1:250、1:500的溶液,分别取适量于13700rpm离心10min,取上清液作为供试品溶液。Step a, preparation of test solution: accurately weigh 1, 0.2, 0.2, 0.1g of Terminalia chebula powder, and place them in 50ml, 25ml, 50ml, and 50ml volumetric flasks, respectively, accurately add appropriate amount of methanol, ultrasonically extract at 30℃ for 20min, leave at room temperature, dilute to the scale with methanol, shake well to obtain solutions with solid-liquid ratio of 1:50, 1:125, 1:250, and 1:500, respectively, take appropriate amount, centrifuge at 13700rpm for 10min, and take the supernatant as the test solution.
步骤b、对照品溶液的制备:同实施例1。Step b, preparation of reference solution: same as in Example 1.
步骤c、用Waters ACQUITY UPLC H-Class PLUS超高效液相色谱仪对上述混合对照品溶液和供试品溶液进行测定,色谱条件同实施例1。Step c: using Waters ACQUITY UPLC H-Class PLUS ultra-high performance liquid chromatograph to measure the mixed reference solution and the test solution, the chromatographic conditions are the same as those in Example 1.
各供试品溶液所得液相色谱图中,各成分的峰面积如表16所示。The peak areas of the components in the liquid chromatograms obtained from the test solutions are shown in Table 16.
表16不同料液比下所得供试品溶液的鞣质类成分峰面积Table 16 Peak areas of tannin components in the test solution obtained under different material-liquid ratios
由表16可见,料液比为1:250和1:500时,鞣质类成分峰面积均较高,提取效果较优,优选料液比为1:250。。As can be seen from Table 16, when the solid-liquid ratio is 1:250 and 1:500, the peak area of tannin components is relatively high, the extraction effect is better, and the preferred solid-liquid ratio is 1:250.
实施例11Embodiment 11
本发明实施例提供了一种诃子中多成分的含量测定方法,包括如下步骤:The embodiment of the present invention provides a method for determining the content of multiple components in Terminalia chebula, comprising the following steps:
步骤a、供试品溶液的制备:取诃子粉末0.2g,精密称定,置于50ml容量瓶中,加甲醇适量,分别于30℃超声提取10、20、30min,放置室温后,用甲醇定容至刻度,摇匀,分别取适量于13700rpm离心10min,取上清液作为供试品溶液。Step a, preparation of test solution: take 0.2g of Terminalia chebula powder, accurately weigh it, put it in a 50ml volumetric flask, add an appropriate amount of methanol, ultrasonically extract at 30℃ for 10, 20, and 30min respectively, after leaving it at room temperature, add methanol to the scale, shake well, take an appropriate amount, centrifuge at 13700rpm for 10min, and take the supernatant as the test solution.
步骤b、对照品溶液的制备:同实施例1。Step b, preparation of reference solution: same as in Example 1.
步骤c、用Waters ACQUITY UPLC H-Class PLUS超高效液相色谱仪对上述混合对照品溶液和供试品溶液进行测定,色谱条件同实施例1。Step c: using Waters ACQUITY UPLC H-Class PLUS ultra-high performance liquid chromatograph to measure the mixed reference solution and the test solution, the chromatographic conditions are the same as those in Example 1.
各供试品溶液所得液相色谱图中,各成分的峰面积如表17所示。The peak areas of the components in the liquid chromatograms obtained from the test solutions are shown in Table 17.
表17不同超声时间下所得供试品溶液的鞣质类成分峰面积Table 17 Peak areas of tannin components in the test solution obtained at different ultrasonic times
由表17可见,超声时间20min和30min时,鞣质类成分峰面积均较高,提取效果较优,选择超声时间20min。As can be seen from Table 17, when the ultrasonic time is 20 min and 30 min, the peak area of tannin components is higher and the extraction effect is better, so the ultrasonic time of 20 min is selected.
实施例12Example 12
本发明实施例提供了一种诃子中多成分的含量测定方法,包括如下步骤:The embodiment of the present invention provides a method for determining the contents of multiple components in Terminalia chebula, comprising the following steps:
步骤a、供试品溶液的制备:取诃子粉末0.2g,精密称定,置于50ml容量瓶中,加甲醇适量,分别于30、40、50℃超声提取20min,放置室温后,用甲醇定容至刻度,摇匀,分别取适量于13700rpm离心10min,取上清液作为供试品溶液。Step a, preparation of test solution: take 0.2g of Terminalia chebula powder, accurately weigh it, put it in a 50ml volumetric flask, add appropriate amount of methanol, ultrasonically extract at 30, 40, and 50℃ for 20min respectively, after leaving it at room temperature, dilute to the scale with methanol, shake well, take appropriate amount respectively, centrifuge at 13700rpm for 10min, and take the supernatant as the test solution.
步骤b、对照品溶液的制备:同实施例1。Step b, preparation of reference solution: same as in Example 1.
步骤c、用Waters ACQUITY UPLC H-Class PLUS超高效液相色谱仪对上述混合对照品溶液和供试品溶液进行测定,色谱条件同实施例1。Step c: using Waters ACQUITY UPLC H-Class PLUS ultra-high performance liquid chromatograph to measure the mixed reference solution and the test solution, the chromatographic conditions are the same as those in Example 1.
各供试品溶液所得液相色谱图中,各成分的峰面积如表18所示。The peak areas of the components in the liquid chromatograms obtained from the test solutions are shown in Table 18.
表18不同超声温度下所得供试品溶液的鞣质类成分峰面积Table 18 Peak areas of tannin components in the test solution obtained at different ultrasonic temperatures
由表18可见,超声温度为30℃时,鞣质类成分峰面积最高,提取效果最优。As can be seen from Table 18, when the ultrasonic temperature is 30°C, the peak area of tannin components is the highest and the extraction effect is optimal.
对比例13Comparative Example 13
本发明实施例提供了一种诃子中多成分的含量测定方法,包括如下步骤:The embodiment of the present invention provides a method for determining the content of multiple components in Terminalia chebula, comprising the following steps:
步骤a、待测溶液的制备:同实施例1中对照品溶液的制备;Step a, preparation of the test solution: the same as the preparation of the reference solution in Example 1;
步骤b、用Waters ACQUITY UPLC H-Class PLUS超高效液相色谱仪对上述待测溶液进行测定,色谱条件为:Step b: using Waters ACQUITY UPLC H-Class PLUS ultra-high performance liquid chromatograph to measure the above-mentioned test solution, the chromatographic conditions are:
色谱柱:ZORBAX SB-C18(4.6x 100mm,1.8μm),ACQUITY UPLC HSS T3(2.1x100mm,1.8μm)或Cosmosil PBr Packed Column(2.1x 100mm,2.6μm);Chromatographic column: ZORBAX SB-C18 (4.6x 100mm, 1.8μm), ACQUITY UPLC HSS T3 (2.1x100mm, 1.8μm) or Cosmosil PBr Packed Column (2.1x 100mm, 2.6μm);
柱温:30℃Column temperature: 30°C
进样量:2μL;Injection volume: 2 μL;
流速:0.3ml/min;Flow rate: 0.3ml/min;
流动相A为0.1%v/v甲酸水溶液,流动相B为甲醇,进行线性梯度洗脱,洗脱程序为:Mobile phase A was 0.1% v/v formic acid in water, mobile phase B was methanol, and linear gradient elution was performed. The elution program was:
色谱柱为ZORBAX SB-C18时得到的色谱图如图6中的A图所示;色谱柱为ACQUITYUPLC HSS T3的色谱图如图6中的B图所示;色谱柱为Cosmosil PBr Packed Column的色谱图如图6中的C图所示。The chromatogram obtained when the chromatographic column is ZORBAX SB-C18 is shown in Figure 6A; the chromatogram when the chromatographic column is ACQUITYUPLC HSS T3 is shown in Figure 6B; and the chromatogram when the chromatographic column is Cosmosil PBr Packed Column is shown in Figure 6C.
由图6可见,Cosmosil PBr Packed Column色谱柱更适用于诃子中鞣质类化合物(极性相似)的分离且分离度较好。As shown in Figure 6, the Cosmosil PBr Packed Column is more suitable for the separation of tannin compounds (similar polarity) in Terminalia chebula and has a better separation degree.
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.
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