CN112684053B - A method for simultaneous determination of the content of 7 components in an anti-chronic heart failure drug compound - Google Patents

A method for simultaneous determination of the content of 7 components in an anti-chronic heart failure drug compound Download PDF

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CN112684053B
CN112684053B CN202011582634.4A CN202011582634A CN112684053B CN 112684053 B CN112684053 B CN 112684053B CN 202011582634 A CN202011582634 A CN 202011582634A CN 112684053 B CN112684053 B CN 112684053B
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acetonitrile
liquid chromatography
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heart failure
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周华
瞿惠燕
杨涛
戎靖枫
王长虹
李素丽
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Shuguang Hospital Affiliated to Shanghai University of TCM
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Abstract

本发明涉及一种同时测定抗慢性心衰药复方中7种成分含量的方法。所述7种成分为没食子酸、羟基红花黄色素A、马钱苷、沉香四醇、补骨脂苷、异补骨脂苷和特女贞苷,所述方法为高效液相色谱法,色谱条件为:采用C18色谱柱,流动相乙腈:水,梯度洗脱;流速0.2~1.2mL·min‑1,柱温20~40℃,检测波长为220~360nm。该方法能实现一种抗慢性心衰药复方中7种成分含量的同时测定,具备良好的线性关系,精密度高,稳定性强,重复性好,加样回收率高,简单易行,为抗慢性心衰药复方质量标准的制定提供了依据。

Figure 202011582634

The invention relates to a method for simultaneously measuring the contents of seven components in an anti-chronic heart failure medicine compound. The 7 components are gallic acid, hydroxysafflor yellow A, loganin, agarwood tetraol, psoralen, isopsoraside, and secretinoside, and the method is high performance liquid chromatography, The chromatographic conditions are as follows: a C 18 chromatographic column, mobile phase acetonitrile: water, gradient elution; flow rate 0.2-1.2 mL·min 1 , column temperature 20-40° C., detection wavelength 220-360 nm. The method can realize the simultaneous determination of the contents of 7 components in an anti-chronic heart failure drug compound. The formulation of the quality standard of anti-chronic heart failure drug compound provided the basis.

Figure 202011582634

Description

一种同时测定抗慢性心衰药复方中7种成分含量的方法A method for simultaneous determination of the content of 7 components in an anti-chronic heart failure drug compound

技术领域technical field

本发明涉及中药复方质量控制领域,具体地说,涉及一种同时测定抗慢性心衰药复方中7种成分含量的方法。The invention relates to the field of quality control of traditional Chinese medicine compound prescriptions, in particular to a method for simultaneously measuring the contents of seven components in an anti-chronic heart failure medicine compound prescription.

背景技术Background technique

抗慢性心衰药复方,即补肾活血方,是多年临床治疗慢性心力衰竭的验方,长期应用于心衰患者,临床可靠有效[1,11],已获专利授权。本方在处理标本关系上立足补虚求本,兼顾淤血、水饮等标实;在处理阴阳比重上平补阴阳,以补阳为主,且阴中求阳;在处理全心肾关系上强调心肾相交、心本乎肾,以温肾求强心。该方由鹿角、红花、淫羊藿、补骨脂、女贞子、山茱萸和沉香7味药材组成,方中鹿角咸温入肾,补肾阳、益精血;红花辛温入心,活血通经,两药共为君药;淫羊藿、补骨脂、女贞子、山茱萸共助鹿角平补肾之阴阳,为臣药;沉香纳气归肾以助诸补肾药,行气止痛以助红花活血,并使本方补而不滞,是为佐使药;诸药合用,共奏补肾活血之功效[1]。抗慢性心衰药复方可通过激活gp130/JAK2/STAT3通路以旁分泌形式作用于心肌成纤维细胞,抑制心肌纤维化[2],还可影响血浆RASS产物[3,4],其被认为是导致心肌纤维化的危险因素。The anti-chronic heart failure drug compound, namely Bushen Huoxue Recipe , has been used in clinical treatment of chronic heart failure for many years. In dealing with the relationship between specimens, this prescription is based on tonic and seeks the essence, taking into account congestion, water and other standards; in dealing with the proportion of yin and yang, it balances yin and yang, mainly tonifies yang, and seeks yang in yin; in dealing with the relationship between the whole heart and kidney Emphasizes the intersection of the heart and the kidney, the heart originates from the kidney, and seeks to strengthen the heart by warming the kidney. The prescription is composed of 7 herbs: antler, safflower, epimedium, psoralea, privet japonica, dogwood and agarwood. Activating blood and clearing the meridians, the two medicines are the king medicine; Epimedium, Psoralea, Ligustrum lucidum, and Cornus help the antlers to calm the yin and yang of the kidney, and they are the minister medicine; Agarwood absorbs the qi and returns to the kidney to help the kidney medicine, and promotes qi and relieves pain. Helping safflower to promote blood circulation, and to make this formula tonic without stagnation, it is an adjuvant medicine; the combination of various medicines has the effect of tonifying the kidney and activating blood [1] . The anti-chronic heart failure drug compound can act on myocardial fibroblasts in a paracrine manner by activating the gp130/JAK2/STAT3 pathway to inhibit myocardial fibrosis [2] , and can also affect plasma RASS products [3,4] , which is considered to be a Risk factors for myocardial fibrosis.

现代药理研究表明,鹿角活性成分鹿茸多肽可介导心肌干细胞分化,修复受损心肌[5];羟基红花黄色素A为单查尔酮苷类结构的化合物,是红花药理功效的最有效水溶性部位,对血管组织细胞有明显的保护作用,具有降血压、扩张血管、改善器官供血、抗凝血、抗炎等功效[6,7];补骨脂有多种生物活性,香豆素类为其主要活性成分,其中补骨脂素对心血管系统具有保护作用[10];淫羊藿主要成分为黄酮类,对抗心力衰竭和保护心脏功能有着很大的作用,其代表性成分淫羊藿苷可增加心脏收缩功能,减轻心脏负荷,使心排量增加,从而保护心脏血管[8,9];山茱萸是治疗糖尿病、冠心病和高血压的主要药材,环烯醚萜类是山茱萸中含量最高的分类群,Kang等[11]研究表明,没食子酸通过上调内皮型一氧化氮合酶(eNOS)磷酸化水平,增加NO含量,同时没食子酸能够抑制血管紧张素Ⅰ转化酶(ACE),显著降低自发性高血压大鼠的血压,且与卡托普利的药理作用强度相当。莫诺苷和马钱苷对糖尿病小鼠心肌有一定的保护作用[12];女贞子所含有效成分既能有效地双向调节人体内分泌系统,又可以使冠脉血流量增加,并对心肌收缩力产生抑制作用,在心功能不全与心律失常等心脏疾病治疗中已有良好成效[10,13],特女贞苷[14]能明显抑制肾小球系膜细胞凋亡,能增加Bcl-2/Bax以及下调cleaved-caspase3蛋白的高表达,从而抑制肾小球系膜细胞过早过多凋亡,起到保护细胞的作用,达到防治肾衰和减少蛋白尿的目的。Modern pharmacological studies have shown that antler polypeptide, the active ingredient of antlers, can mediate the differentiation of myocardial stem cells and repair damaged myocardium [5] ; hydroxysafflor yellow A is a compound with a monochalcone glycoside structure, which is the most effective pharmacological effect of safflower. The water-soluble part has obvious protective effect on vascular tissue cells, and has the functions of lowering blood pressure, dilating blood vessels, improving blood supply to organs, anti-coagulation, and anti-inflammatory [6,7] ; Psoralea has a variety of biological activities, and coumarin Epimedium is its main active ingredient, among which psoralen has a protective effect on the cardiovascular system [10] ; Epimedium is mainly composed of flavonoids, which have a great effect on fighting heart failure and protecting heart function. Its representative ingredients Icariin can increase the systolic function of the heart, reduce the load on the heart, and increase the cardiac output, thereby protecting the heart and blood vessels [8,9] ; Cornus is the main medicinal material for the treatment of diabetes, coronary heart disease and hypertension, and iridoids are The most abundant taxa in Cornus, Kang et al [11] showed that gallic acid increased NO content by up-regulating the phosphorylation level of endothelial nitric oxide synthase (eNOS), while gallic acid could inhibit angiotensin I converting enzyme ( ACE), significantly reduced blood pressure in spontaneously hypertensive rats, and was comparable to the pharmacological effect of captopril. Morroniside and loganin have a certain protective effect on the myocardium of diabetic mice [12] ; the active ingredients contained in Ligustrum lucidum can not only effectively regulate the human endocrine system in both directions, but also increase coronary blood flow, and have a negative effect on myocardial blood flow. It has an inhibitory effect on contractility, and has achieved good results in the treatment of cardiac insufficiency and arrhythmia [10,13] . Teluceoside [14] can significantly inhibit the apoptosis of glomerular mesangial cells and increase Bcl- 2/Bax and down-regulates the high expression of cleaved-caspase3 protein, thereby inhibiting premature and excessive apoptosis of glomerular mesangial cells, protecting cells, preventing renal failure and reducing proteinuria.

中药复方质量控制的研究是中医药现代化和国际化的关键。专利文献CN111175392A,公开日2020.05.19,公开了一种止咳枇杷糖浆的质量控制方法,建立了HPLC法对苦杏仁苷、桑白皮素B和新对叶百部碱进行含量测定。专利文献CN110780018A,公开日2020.02.11,公开了一种精肝颗粒的质量控制方法,建立了HPLC法对大黄素、大黄酚的含量检测方法。专利文献CN112098563A,公开日2020.12.18,公开了一种补肾活血方化学成分的检测方法,补肾活血方中药组分为杜仲、补骨脂、怀牛膝、丹参、威灵仙和木瓜,主治下腰痛,所述检测方法包括以下步骤:将补肾活血方的中药依次进行水浸泡、提取、浓缩、乙醇萃取、静置、离心分离、除去上清液中的乙醇、乙酸乙酯萃取、将所得水相进行正丁醇萃取、浓缩所得水相,得到待测液,然后采用特定的检测条件进行UPLC-MS检测,将所得检测结果对照标准色谱图和天然产物数据库,得到待测液中的化学成分,该发明的优点在于能够同时定性检测出补肾活血方中的93种有效化学成分。但目前未见如本申请所述的抗慢性心衰药复方的质量控制研究,也未见同时测定抗慢性心衰药复方中七种成分没食子酸、羟基红花黄色素A、马钱苷、沉香四醇、补骨脂苷、异补骨脂苷、特女贞苷含量的方法。The research on the quality control of traditional Chinese medicine compounds is the key to the modernization and internationalization of traditional Chinese medicine. The patent document CN111175392A, published on 2020.05.19, discloses a quality control method for cough-relieving loquat syrup, and establishes an HPLC method for the content determination of amygdalin, mulberry B and rabathine. Patent document CN110780018A, published on 2020.02.11, discloses a quality control method for Jinggan Granules, and establishes a method for detecting the content of emodin and chrysophanol by HPLC. Patent document CN112098563A, published on 2020.12.18, discloses a method for detecting the chemical components of Bushen Huoxue Recipe. The traditional Chinese medicine components of Bushen Huoxue Recipe are Eucommia ulmoides, Bakuchichi, Achyranthes huai, Salvia miltiorrhiza, Wei Lingxian and papaya. Low back pain. Phase carrying out n-butanol extraction, concentrating the obtained water phase to obtain the liquid to be tested, and then using specific detection conditions to carry out UPLC-MS detection, the obtained test results are compared with the standard chromatogram and natural product database to obtain the chemical composition in the liquid to be tested , the advantage of the invention is that 93 kinds of effective chemical components in Bushen Huoxue Recipe can be qualitatively detected at the same time. But at present, there is no quality control study on the anti-chronic heart failure drug compound as described in this application, nor has the simultaneous determination of the seven components in the anti-chronic heart failure drug compound gallic acid, hydroxysafflor yellow A, loganin, Method for the content of agarwood tetraol, psoralen, isopsoraside, and tegretinoside.

发明内容SUMMARY OF THE INVENTION

本发明的目的是针对现有技术中的不足,提供一种同时测定抗慢性心衰药复方中7种成分含量的方法。The purpose of the present invention is to aim at the deficiencies in the prior art, and provide a method for simultaneously measuring the content of 7 components in the compound prescription of anti-chronic heart failure medicine.

本发明的另一目的是,提供一种抗慢性心衰药复方质量控制方法。Another object of the present invention is to provide a quality control method for an anti-chronic heart failure drug compound.

为实现上述第一个目的,本发明采取的技术方案为:For realizing above-mentioned first purpose, the technical scheme that the present invention takes is:

一种同时测定抗慢性心衰药复方中7种成分含量的高效液相色谱方法,所述7种成分为没食子酸、羟基红花黄色素A、马钱苷、沉香四醇、补骨脂苷、异补骨脂苷和特女贞苷,所述高效液相色谱方法的色谱条件为:采用C18色谱柱,流动相乙腈-0.1%磷酸水,洗脱梯度为0~10min,5%乙腈;10~15min,5%~10%乙腈;15~35min,10%~18%乙腈;35~40min,18%~25%乙腈;40~60min,25%~35%乙腈;流速0.8~1.2mL·min-1,柱温28~32℃,检测波长为220~360nm。A high-performance liquid chromatography method for simultaneously determining the content of 7 components in an anti-chronic heart failure drug compound, wherein the 7 components are gallic acid, hydroxysafflor yellow A, loganin, agarwood tetraol, and psoralen , Isopsoraside and Ateprivaside, the chromatographic conditions of the high performance liquid chromatography method are: adopt C 18 chromatographic column, mobile phase acetonitrile-0.1% phosphoric acid water, elution gradient is 0 ~ 10min, 5% acetonitrile ; 10~15min, 5%~10% acetonitrile; 15~35min, 10%~18% acetonitrile; 35~40min, 18%~25% acetonitrile; 40~60min, 25%~35% acetonitrile; flow rate 0.8~1.2mL ·min -1 , column temperature 28~32℃, detection wavelength 220~360nm.

作为本发明的一种优选实施方案,所述抗慢性心衰药复方中药组成为:鹿角10-20重量份、红花6-12重量份、补骨脂15-25重量份、淫羊藿25-35重量份、山萸肉10-20重量份、女贞子25-35重量份、沉香4-8重量份。As a preferred embodiment of the present invention, the compound traditional Chinese medicine composition of the anti-chronic heart failure drug is: 10-20 parts by weight of antlers, 6-12 parts by weight of safflower, 15-25 parts by weight of psoralen, 25 parts by weight of Epimedium -35 parts by weight, 10-20 parts by weight of dogwood meat, 25-35 parts by weight of Ligustrum lucidum, and 4-8 parts by weight of agarwood.

作为本发明的另一种优选实施方案,所述高效液相色谱方法其供试品溶液的制备方法为:精密称取各药材,加8-15倍水煎煮60-90min,滤过,滤液减压浓缩,转移至容量瓶,定容,摇匀,用微孔滤膜过滤,取续滤液,即得。As another preferred embodiment of the present invention, the method for preparing the test solution of the high performance liquid chromatography method is as follows: accurately weigh each medicinal material, add 8-15 times of water to decoct for 60-90 min, filter, and filter the filtrate. Concentrate under reduced pressure, transfer to a volumetric flask, make up to volume, shake well, filter through a microporous membrane, and take the subsequent filtrate.

作为本发明的另一种优选实施方案,所述高效液相色谱方法其混合对照品溶液的制备方法为:精密称取没食子酸、羟基红花黄色素A、马钱苷、沉香四醇、补骨脂苷、异补骨脂苷、特女贞苷对照品,加甲醇制成浓度分别为2.71、8.61、7.61、643、17.42、11.88、45.00μg/mL混合对照品溶液。As another preferred embodiment of the present invention, the preparation method of the mixed reference solution of the high performance liquid chromatography method is as follows: accurately weighing gallic acid, hydroxysafflor yellow A, loganin, agarwood tetraol, tonic For the reference substances of bakuchioside, isopsoraside, and tegretinoside, methanol was added to prepare mixed reference substance solutions with concentrations of 2.71, 8.61, 7.61, 643, 17.42, 11.88, and 45.00 μg/mL, respectively.

作为本发明的另一种优选实施方案,进样量为1~12μL。As another preferred embodiment of the present invention, the injection volume is 1-12 μL.

更优选地,进样量为10μL。More preferably, the injection volume is 10 μL.

作为本发明的另一种优选实施方案,流速为1.0mL·min-1As another preferred embodiment of the present invention, the flow rate is 1.0 mL·min -1 .

作为本发明的另一种优选实施方案,柱温为30℃。As another preferred embodiment of the present invention, the column temperature is 30°C.

作为本发明的另一种优选实施方案,检测波长为220~240nm。As another preferred embodiment of the present invention, the detection wavelength is 220-240 nm.

为实现上述第二个目的,本发明采取的技术方案为:For realizing the above-mentioned second purpose, the technical scheme adopted by the present invention is:

一种抗慢性心衰药复方的质量控制方法,包含如上任一所述高效液相色谱方法的步骤。A quality control method for an anti-chronic heart failure drug compound, comprising the steps of any one of the high performance liquid chromatography methods described above.

本发明优点在于:The advantages of the present invention are:

1、本发明建立了抗慢性心衰药复方中七种成分没食子酸、羟基红花黄色素A、马钱苷、沉香四醇、补骨脂苷、异补骨脂苷、特女贞苷含量测定的方法,并通过对七项指标的检测方法进行系统适应性、专属性、线性、精密度、重复性、稳定性的方法学验证,以确定七个成分检测方法的可行性,结果表明该方法具备良好的线性关系,精密度高,稳定性强,重复性好,加样回收率高,简单易行。1. The present invention establishes the contents of seven components gallic acid, hydroxysafflor yellow A, loganin, agarwood tetraol, psoralen, isopsoraside, and privatide in the anti-chronic heart failure medicine compound. The method of determination, and the methodological verification of system adaptability, specificity, linearity, precision, repeatability and stability of the detection method of the seven indicators to determine the feasibility of the detection method of the seven components. The method has a good linear relationship, high precision, strong stability, good repeatability, high sample recovery rate, and is simple and easy to implement.

2、本发明检测方法所涉及的参数是在发明人研究经验的基础上得出的,该检测条件下实现了优异的检测效果。2. The parameters involved in the detection method of the present invention are obtained on the basis of the inventor's research experience, and an excellent detection effect is achieved under this detection condition.

3、本发明为抗慢性心衰药复方质量标准的制定提供了依据。3. The present invention provides a basis for the formulation of the quality standard of the anti-chronic heart failure drug compound.

附图说明Description of drawings

图1:混合对照品HPLC图(A)和供试品HPLC图(B)。1.没食子酸;2.羟基红花黄色素A;3.马钱苷;4.沉香四醇;5.补骨脂苷;6.异补骨脂苷;7.特女贞苷。Figure 1: Mixed reference HPLC chart (A) and test product HPLC chart (B). 1. Gallic acid; 2. Hydroxysafflor yellow A; 3. Loganin; 4. Agarwood tetraol;

图2:同一色谱条件不同流动相下的色谱图。A:0.1%甲酸-(B)甲醇系统;B:0.1%甲酸-乙腈系统。Figure 2: Chromatograms under the same chromatographic conditions with different mobile phases. A: 0.1% formic acid-(B) methanol system; B: 0.1% formic acid-acetonitrile system.

图3:不同洗脱梯度下的色谱图。A:洗脱梯度为0~8min,5%乙腈;8~60min,5%~50%乙腈;记录时间为0~60min;B:洗脱程序为流动相为乙腈-0.1%磷酸水,洗脱梯度为0~5min,5%乙腈;5~15min,5%~15%乙腈;15~60min,15%~45%乙腈;记录时间为0~60min;C:洗脱程序为流动相为乙腈-0.1%磷酸水,洗脱梯度为0~5min,5%乙腈;5~15min,5%~15%乙腈;15~41min,15%~27%乙腈;41~43min,27%~30%乙腈;43~60min,30%~40%乙腈;60~61min,40%~5%乙腈;记录时间为0~60min;D:洗脱程序为流动相为乙腈-0.1%磷酸水,洗脱梯度为0~10min,5%乙腈;10~15min,5%~10%乙腈;15~35min,10%~18%乙腈;35~40min,18%~25%乙腈;40~60min,25%~35%乙腈;记录时间为0~60min。Figure 3: Chromatograms at different elution gradients. A: The elution gradient is 0~8min, 5% acetonitrile; 8~60min, 5%~50% acetonitrile; The recording time is 0~60min; B: The elution procedure is acetonitrile-0.1% phosphoric acid water, elution Gradient: 0~5min, 5% acetonitrile; 5~15min, 5%~15% acetonitrile; 15~60min, 15%~45% acetonitrile; recording time: 0~60min; C: The elution program is acetonitrile- 0.1% phosphoric acid water, elution gradient: 0~5min, 5% acetonitrile; 5~15min, 5%~15% acetonitrile; 15~41min, 15%~27% acetonitrile; 41~43min, 27%~30% acetonitrile; 43~60min, 30%~40% acetonitrile; 60~61min, 40%~5% acetonitrile; recording time is 0~60min; D: The elution program is acetonitrile-0.1% phosphoric acid water, the elution gradient is 0 ~10min, 5% acetonitrile; 10~15min, 5%~10% acetonitrile; 15~35min, 10%~18% acetonitrile; 35~40min, 18%~25% acetonitrile; 40~60min, 25%~35% acetonitrile ; The recording time is 0~60min.

图4:抗慢性心衰药复方不同波长下的色谱图。A.211nm;B.230nm;C.254nm;D.265nm;E.280nm;F.300nm;G.330nm。Figure 4: Chromatograms of the anti-chronic heart failure drug compound at different wavelengths. A. 211 nm; B. 230 nm; C. 254 nm; D. 265 nm; E. 280 nm; F. 300 nm; G. 330 nm.

具体实施方式Detailed ways

下面结合附图对本发明提供的具体实施方式作详细说明。The specific embodiments provided by the present invention will be described in detail below with reference to the accompanying drawings.

实施例1Example 1

1材料与方法1 Materials and methods

1.1材料与仪器1.1 Materials and Instruments

Agilent 1100series型高效液相色谱仪(Agilent DAD检测器,美国安捷伦科技有限公司);BSA124S-CW电子分析天平(万分之一)、BT25S电子分析天平(十万分之一,赛多利斯科学仪器(北京)有限公司)。羟基红花黄色素A(批号AF9081601,含有量98%)、马钱苷(批号AF7110703,含有量98%)、补骨脂苷(批号AF9102107,含有量98%)、异补骨脂苷(批号AF9102108,含有量98%)均购于上海鸿永生物科技有限公司;沉香四醇(批号DST190801-042,含有量99%)、没食子酸(批号DST190715-008,含有量99%)均购于成都乐美天医药科技有限公司;特女贞苷(批号2018011201,含有量95%)对照品购于成都普利斯生物科技有限公司。鹿角、红花、补骨脂、淫羊藿、山茱萸、女贞子、沉香饮片均购于上海康桥中药饮片有限公司,经上海中药标准化研究中心王长虹研究员鉴定为正品。乙腈(美国Fisher公司);甲酸(批号:G0960200,含有量98%)购于上海安谱科学仪器有限公司;其他试剂均为分析纯;水为超纯水。Agilent 1100series high performance liquid chromatograph (Agilent DAD detector, Agilent Technologies, USA); BSA124S-CW electronic analytical balance (1/10,000), BT25S electronic analytical balance (1/100,000, Sartorius Scientific Instruments) (Beijing) Co., Ltd.). Hydroxysafflor yellow A (batch number AF9081601, content 98%), loganin (batch number AF7110703, content 98%), psoralen (batch number AF9102107, content 98%), isopsorain (batch number AF9102107, content 98%) AF9102108, content 98%) were purchased from Shanghai Hongyong Biotechnology Co., Ltd.; Agarwood tetraol (batch number DST190801-042, content 99%) and gallic acid (batch number DST190715-008, content 99%) were purchased in Chengdu Lemeitian Pharmaceutical Technology Co., Ltd.; special privet glycosides (batch number 2018011201, content 95%) reference substance were purchased from Chengdu Pulis Biotechnology Co., Ltd. Deer antler, safflower, psoralen, epimedium, dogwood, privet lucidum, and agarwood decoction pieces were all purchased from Shanghai Kangqiao Traditional Chinese Medicine Pieces Co., Ltd., and were identified as genuine by researcher Wang Changhong of Shanghai Standardization Research Center of Traditional Chinese Medicine. Acetonitrile (Fisher Company, USA); formic acid (batch number: G0960200, content 98%) were purchased from Shanghai Anpu Scientific Instrument Co., Ltd.; other reagents were analytically pure; water was ultrapure water.

1.2试验方法与结果1.2 Test methods and results

1.2.1色谱条件1.2.1 Chromatographic conditions

色谱柱为ZORBAX SB-Aq(4.6mm×250mm,5μm),流动相为乙腈-0.1%磷酸水,洗脱梯度为0~10min,5%乙腈;10~15min,5%~10%乙腈;15~35min,10%~18%乙腈;35~40min,18%~25%乙腈;40~60min,25%~35%乙腈;流速1.0mL·min-1,柱温30℃,检测波长为230nm,进样10μL,混合对照品溶液及供试品溶液的HPLC色谱图见图1。The chromatographic column is ZORBAX SB-Aq (4.6mm×250mm, 5μm), the mobile phase is acetonitrile-0.1% phosphoric acid water, the elution gradient is 0~10min, 5% acetonitrile; 10~15min, 5%~10% acetonitrile; 15 ~35min, 10%~18% acetonitrile; 35~40min, 18%~25% acetonitrile; 40~60min, 25%~35% acetonitrile; flow rate 1.0mL·min -1 , column temperature 30℃, detection wavelength 230nm, 10 μL was injected, and the HPLC chromatogram of the mixed reference solution and the test solution was shown in Figure 1.

1.2.2混合对照品溶液的制备1.2.2 Preparation of mixed reference solution

精密称取没食子酸、羟基红花黄色素A、马钱苷、沉香四醇、补骨脂苷、异补骨脂苷、特女贞苷对照品适量,加甲醇制成浓度分别为2.71、8.61、7.61、643、17.42、11.88、45.00μg/mL混合对照品溶液。Accurately weigh the appropriate amount of gallic acid, hydroxysafflor yellow A, loganin, agarwood tetraol, psoralen, isopsoraside, and secretinoside, and add methanol to make the concentrations of 2.71 and 8.61, respectively. , 7.61, 643, 17.42, 11.88, 45.00μg/mL mixed reference solution.

1.2.3供试品溶液制备1.2.3 Preparation of the test solution

按处方量(鹿角15g、红花9g、补骨脂20g、淫羊藿30g、山萸肉15g、女贞子30g、沉香6g)精密称取各药材适量,加12倍水煎煮75min,滤过,滤液减压浓缩,转移至500mL量瓶中,定容,摇匀,0.45μm微孔滤膜过滤,取续滤液,即得。Precisely weigh an appropriate amount of each medicinal material according to the prescribed amount (15g antler, 9g safflower, 20g psoralen, 30g epimedium, 15g dogwood meat, 30g Ligustrum lucidum, 6g agarwood), add 12 times of water and cook for 75min, filter After passing, the filtrate was concentrated under reduced pressure, transferred to a 500 mL volumetric flask, adjusted to volume, shaken well, filtered through a 0.45 μm microporous membrane, and the subsequent filtrate was obtained.

1.2.4线性关系考察1.2.4 Investigation of Linear Relationship

取“1.2.2”项下混合对照品溶液,稀释后摇匀,在“1.2.1”项色谱条件下进样10μL测定。以溶液质量浓度为横坐标(X),峰面积积分值为纵坐标(Y)进行回归,绘制标准曲线,结果见表1。Take the mixed reference solution under item "1.2.2", dilute and shake well, and inject 10 μL of sample under the chromatographic conditions of item "1.2.1" for determination. Take the solution mass concentration as the abscissa (X) and the integral value of the peak area as the ordinate (Y) for regression, and draw a standard curve. The results are shown in Table 1.

表1抗慢性心衰药复方中7个成分的线性关系考察结果Table 1 The results of the linear relationship investigation of the 7 components in the anti-chronic heart failure drug compound

Figure BDA0002864748030000051
Figure BDA0002864748030000051

1.2.5精密度试验1.2.5 Precision test

取高(H)、中(M)、低(L)质量浓度对照品溶液,同1d内在“1.2.1”项色谱条件下进样6次、连续测定3天,测得各成分峰面积RSD表明仪器日内精密度良好,结果见表2。Take high (H), medium (M) and low (L) mass concentration reference substance solutions, inject 6 times under the chromatographic conditions of "1.2.1" within the same 1d, and measure continuously for 3 days, and measure the peak area RSD of each component. It shows that the intraday precision of the instrument is good, and the results are shown in Table 2.

表2抗慢性心衰药复方中7个成分的精密度考察结果Table 2 The results of the precision investigation of the 7 components in the anti-chronic heart failure drug compound

Figure BDA0002864748030000061
Figure BDA0002864748030000061

1.2.6重复性试验1.2.6 Repeatability test

按“1.2.3”项下方法制备供试品溶液,平行6份,在1.2.1项色谱条件下进样测定,测得没食子酸、羟基红花黄色素A、马钱苷、沉香四醇、补骨脂苷、异补骨脂苷、特女贞苷含有量的RSD分别为2.04%、2.78%、2.51%、2.78%、0.75%、2.49%、0.96%,表明方法的重复性良好符合试验要求。The test solution was prepared according to the method under "1.2.3", 6 parallels were prepared, and the samples were injected and measured under the chromatographic conditions of 1.2.1, and gallic acid, hydroxysafflor yellow A, loganin, agarwood tetraol were measured. The RSDs of the contents of , psoralen, isopsoraside, and secretinoside are 2.04%, 2.78%, 2.51%, 2.78%, 0.75%, 2.49%, and 0.96%, respectively, indicating that the method has good repeatability. Test requirements.

1.2.7稳定性试验1.2.7 Stability test

按“1.2.3”项下方法制备供试品溶液,于0、1、4、6、12、16、24h在1.2.1项色谱条件下进样测定,测得没食子酸、羟基红花黄色素A、马钱苷、沉香四醇、补骨脂苷、异补骨脂苷、特女贞苷峰面积RSD分别为1.6%、2.7%、2.5%、2.9%、0.9%、1.8%、1.3%,表明溶液在24h内稳定性良好。Prepare the test solution according to the method under item "1.2.3", inject samples at 0, 1, 4, 6, 12, 16, and 24 h under the chromatographic conditions of item 1.2.1, and measure gallic acid, hydroxysafflor yellow The RSDs of the peak areas of pigment A, loganin, agarwood tetraol, psoralen, isopsoraside, and teruquitoside were 1.6%, 2.7%, 2.5%, 2.9%, 0.9%, 1.8%, and 1.3, respectively. %, indicating that the solution has good stability within 24h.

1.2.8加样回收率试验1.2.8 Sample addition recovery test

精密量取“1.2.3”项下含有量已知的供试品溶液5mL,置于10mL容量瓶中,加入50%、100%、150%水平的对照品溶液,在1.2.1项色谱条件下进样测定,计算回收率。结果,没食子酸、羟基红花黄色素A、马钱苷、沉香四醇、补骨脂苷、异补骨脂苷、特女贞苷平均加样回收率分别为102.58%、98.85%、100.17%、98.87%、98.61%、97.32%、98.53%,RSD分别为3.72%、3.08%、3.92%、2.02%、1.21%`、2.02%、1.14%。结果见表3。Precisely measure 5mL of the test solution with known content under "1.2.3", put it in a 10mL volumetric flask, add the reference solution at 50%, 100%, 150% level, under the chromatographic conditions of 1.2.1 The sample was injected and measured, and the recovery rate was calculated. As a result, the average loading recoveries of gallic acid, hydroxysafflor yellow A, loganin, agarwood tetraol, psoralen, isopsoraside, and estrogenin were 102.58%, 98.85%, and 100.17%, respectively. , 98.87%, 98.61%, 97.32%, 98.53%, and the RSDs were 3.72%, 3.08%, 3.92%, 2.02%, 1.21%, 2.02%, 1.14%, respectively. The results are shown in Table 3.

表3抗慢性心衰药复方中指标成分含量测定的加样回收率试验Table 3 The recovery rate test of sample addition for the determination of the content of the index components in the anti-chronic heart failure drug compound

Figure BDA0002864748030000062
Figure BDA0002864748030000062

Figure BDA0002864748030000071
Figure BDA0002864748030000071

Figure BDA0002864748030000081
Figure BDA0002864748030000081

Figure BDA0002864748030000091
Figure BDA0002864748030000091

1.2.9样品测定1.2.9 Sample determination

按1.2.3项下方法制备供试品溶液,在1.2.1项色谱条件下进样测定,结果见表4。Prepare the test solution according to the method in Item 1.2.3, inject the sample under the chromatographic conditions of Item 1.2.1, and see the results in Table 4.

表4抗慢性心衰药复方中7个指标成分的含量测定(n=3)Table 4 Determination of the content of 7 index components in the anti-chronic heart failure drug compound (n=3)

Figure BDA0002864748030000092
Figure BDA0002864748030000092

2讨论2 Discussion

(1).慢性心力衰竭按严重程度可分为A,B,C,D共4个阶段[15],其中B阶段的中药治疗尚属市场空白期,临床应用和动物试验证明抗慢性心衰药复方不仅能改善症状,提高生活质量,还可延缓心肌重构的发展。该方由鹿角、红花、淫羊藿、补骨脂、女贞子、山茱萸和沉香7味药材组成,方中鹿角咸温入肾,补肾阳、益精血;红花辛温入心,活血通经,两药共为君药;淫羊藿、补骨脂、女贞子、山茱萸共助鹿角平补肾之阴阳,为臣药;沉香纳气归肾以助诸补肾药,行气止痛以助红花活血,并使本方补而不滞,是为佐使药。(1). Chronic heart failure can be divided into 4 stages A, B, C, and D according to the severity [15] . Among them, the traditional Chinese medicine treatment in stage B is still in the blank period of the market, and clinical application and animal tests have proved that it is resistant to chronic heart failure. Drug compounds can not only improve symptoms, improve quality of life, but also delay the development of myocardial remodeling. The prescription is composed of 7 herbs: antler, safflower, epimedium, psoralea, privet japonica, dogwood and agarwood. Activating blood and clearing the meridians, the two medicines are the king medicine; Epimedium, Psoralea, Ligustrum lucidum, and Cornus help the antlers to calm the yin and yang of the kidney, and they are the minister medicine; Agarwood absorbs the qi and returns to the kidney to help the kidney medicine, and promotes qi and relieves pain. Helping safflower to activate blood circulation, and make this prescription tonic without stagnation, it is an adjuvant medicine.

(2).依据《中国药典》2015版,羟基红花色素A、补骨脂素、异补骨脂素、淫羊藿苷、马钱苷、莫诺苷、特女贞苷均为相应药材的质控指标;沉香四醇作为沉香里的主要药效成分,2015年版《中国药典》首次将沉香四醇的含量测定作为质控指标[16],沉香对抗心律失常、冠心病有治疗作用,将沉香四醇的含量测定作为指标成分的定量分析是建立沉香药材有效质量评价体系的重要组成部分。根据前期实验结果,补骨脂素、异补骨脂素、淫羊藿苷、莫诺苷在抗慢性心衰药复方水煎液中含量较低,不宜作为其复方的质控指标,补骨脂苷、异补骨脂苷在体内可转化为补骨脂素和异补骨脂素[17],且在水煎液中的含量较高,故选择没食子酸、羟基红花黄色素A、马钱苷、沉香四醇、补骨脂苷、异补骨脂苷、特女贞苷作为抗慢性心衰药复方的质控指标。(2). According to the 2015 edition of the Chinese Pharmacopoeia, hydroxysafflorin A, psoralen, isopsoralen, icariin, loganin, morroniside, and quinoside are all corresponding medicinal materials The quality control index of agarwood tetraol; agarwood tetraol as the main medicinal component in agarwood, the 2015 edition of "Chinese Pharmacopoeia" for the first time determined the content of agarwood tetraol as a quality control index [16] , agarwood has a therapeutic effect against arrhythmia and coronary heart disease, The quantitative analysis of the content of agarwood tetraol as the index component is an important part of establishing the effective quality evaluation system of agarwood. According to the previous experimental results, the contents of psoralen, isopsoralen, icariin and morroniside in the decoction of the anti-chronic heart failure drug compound are relatively low, and they should not be used as the quality control indicators of the compound. Liposide and isopsoraside can be converted into psoralen and isopsoralen in vivo [17] , and the content in decoction is high, so gallic acid, hydroxysafflor yellow A, Loganin, agarwood tetraol, psoralen, isopsoraside, and tequilagenin were used as the quality control indicators of the anti-chronic heart failure drug compound.

(3).流动相的选择:经比较、筛选,发现0.1%甲酸-乙腈系统以线性梯度洗脱,所得液相色谱图基线较平稳、色谱峰峰形对称、分离度较好,因此选择0.1%甲酸-乙腈系统为流动相,比较结果见图2。(3). Selection of mobile phase: After comparison and screening, it was found that the 0.1% formic acid-acetonitrile system was eluted with a linear gradient, and the obtained liquid chromatogram had a relatively stable baseline, symmetrical chromatographic peaks, and good resolution. Therefore, 0.1 was selected. The % formic acid-acetonitrile system was used as the mobile phase, and the comparison results are shown in Figure 2.

(4).流动相洗脱梯度的摸索:经系统摸索、优化,现发现如下线性梯度洗脱程序既能较全面的检出制剂中的化学成分,又可使这些成分获得较为理想的分离效果:洗脱程序确定为流动相为乙腈-0.1%磷酸水,洗脱梯度为0~10min,5%乙腈;10~15min,5%~10%乙腈;15~35min,10%~18%乙腈;35~40min,18%~25%乙腈;40~60min,25%~35%乙腈,记录时间为0~60min,比较结果见图3。(4) Exploration of the mobile phase elution gradient: After systematic exploration and optimization, it is found that the following linear gradient elution procedure can not only detect the chemical components in the preparation more comprehensively, but also obtain a relatively ideal separation effect for these components. : The elution procedure is determined as the mobile phase is acetonitrile-0.1% phosphoric acid water, the elution gradient is 0~10min, 5% acetonitrile; 10~15min, 5%~10% acetonitrile; 15~35min, 10%~18% acetonitrile; 35~40min, 18%~25% acetonitrile; 40~60min, 25%~35% acetonitrile, the recording time is 0~60min, the comparison result is shown in Figure 3.

(5).色谱波长的选择:经液相色谱—紫外光谱三维图谱的比较分析,发现在211nm、330nm下基线不平、分离度差;在254nm、265nm、280nm、300nm的制剂图谱中基线较平,峰形尚可,但出峰数偏少,不能全面反映制剂的成分,在230nm下基线较平,除已知成分没食子酸、羟基红花黄色素A、马钱苷、沉香四醇、补骨脂苷、异补骨脂苷、特女贞苷、槲皮素等均能较好的检出外,其它成分指纹峰分离度也较好,综合考虑色谱峰的数目、基线及信号相应强弱,确定230nm为制剂指纹图谱的最佳检测波长,该波长相对于其他波长下峰数目多,各成分响应、分离度均好,比较结果见图4。(5) Selection of chromatographic wavelength: The comparison and analysis of the three-dimensional spectrum of liquid chromatography-ultraviolet spectrum shows that the baseline is uneven and the separation degree is poor at 211nm and 330nm; the baseline is relatively flat in the preparation atlas of 254nm, 265nm, 280nm and 300nm. , the peak shape is acceptable, but the number of peaks is too small, which cannot fully reflect the composition of the preparation. The baseline is relatively flat at 230 nm, except for the known components of gallic acid, hydroxysafflor yellow A, loganin, agarwood tetraol, tonic In addition to the good detection of skoraloside, isopsoraside, quincetin, quercetin, etc., the fingerprint peak resolution of other components is also good. Considering the number of chromatographic peaks, the baseline and the corresponding strong signal Weak, 230nm was determined to be the best detection wavelength for the fingerprint of the preparation. Compared with other wavelengths, this wavelength has more peaks, and the response and resolution of each component are good. The comparison results are shown in Figure 4.

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以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员,在不脱离本发明方法的前提下,还可以做出若干改进和补充,这些改进和补充也应视为本发明的保护范围。The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the method of the present invention, several improvements and supplements can be made, and these improvements and supplements should also be regarded as It is the protection scope of the present invention.

Claims (8)

1. A high performance liquid chromatography method for simultaneously determining the content of 7 components in an anti-chronic heart failure medicine compound is characterized in that the 7 components are gallic acid, hydroxysafflor yellow A, loganin, agaroterol, psoralen glycoside, isopsoralen glycoside and specnuezhenide, and the chromatographic conditions of the high performance liquid chromatography method are as follows: adopting a C18 chromatographic column, wherein the mobile phase acetonitrile-0.1% phosphoric acid water is adopted, the elution gradient is 0-10 min, and the elution gradient is 5% acetonitrile; 10-15 min,5% -10% acetonitrile; 15-35 min,10% -18% acetonitrile; 35-40 min,18% -25% acetonitrile; 40-60 min, 25-35% acetonitrile; the flow rate is 0.8-1.2 mL/min < -1 >, the column temperature is 28-32 ℃, the detection wavelength is 220-360 nm, and the anti-chronic heart failure compound traditional Chinese medicine comprises the following components in percentage by weight: 10-20 parts of antler, 6-12 parts of safflower, 15-25 parts of fructus psoraleae, 25-35 parts of epimedium herb, 10-20 parts of cornus officinalis, 25-35 parts of glossy privet fruit and 4-8 parts of agilawood, and the preparation method of the test solution by the high performance liquid chromatography method comprises the following steps: precisely weighing the above materials, adding 8-15 times of water, decocting for 60-90min, filtering, concentrating the filtrate under reduced pressure, transferring to volumetric flask, metering volume, shaking, filtering with microporous membrane, and collecting the filtrate.
2. The high performance liquid chromatography method according to claim 1, wherein the mixed reference solution is prepared by: accurately weighing gallic acid, hydroxy safflower yellow A, loganin, linalool, psoralen, isopsoralen, and specnuezhenide, and adding methanol to obtain mixed reference solutions with concentrations of 2.71, 8.61, 7.61, 643, 17.42, 11.88, and 45.00 μ g/mL.
3. The high performance liquid chromatography method according to claim 1, wherein the amount of sample is 1 to 12. mu.L.
4. The high performance liquid chromatography method according to claim 3, wherein the amount of the sample is 10. mu.L.
5. The high performance liquid chromatography method according to claim 1, wherein the flow rate is 1.0mL _ min "1.
6. The high performance liquid chromatography method of claim 1, wherein the column temperature is 30 ℃.
7. The high performance liquid chromatography method according to claim 1, wherein the detection wavelength is 220 to 240 nm.
8. A quality control method of a compound anti-chronic heart failure medicine, which is characterized by comprising the steps of the high performance liquid chromatography method of any one of claims 1 to 7.
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