阅读说明：本技术 一种利用高效液相色谱波长切换技术同时检测杜仲中多种活性成分的方法 (Method for simultaneously detecting multiple active ingredients in eucommia ulmoides by utilizing high performance liquid chromatography wavelength switching technology ) 是由 王志宏 佘志刚 于 2020-10-27 设计创作，主要内容包括：本发明涉及一种利用高效液相色谱波长切换技术同时检测杜仲中多种活性成分的方法。该方法包括如下步骤：S1：将待测杜仲样品粉末处理制成样品溶液；S2：运用高效液相色谱技术,以对照品溶液为对照,对样品溶液中的活性成分进行洗脱,并利用波长切换技术进行分析。本发明提供的方法通过选用合适的流动相,并利用高效液相色谱波长切换技术可对杜仲中的活性成分的色谱峰型进行改善,可更准确、更全面的对杜仲样品中多种特征成分进行分析检测,具有较高的检测准确度与灵敏度,且检测效率高；能够对杜仲资源的品质更好的监管与控制,对中医药现代化与国际化具有重要的意义。(The invention relates to a method for simultaneously detecting multiple active ingredients in eucommia ulmoides by utilizing a high performance liquid chromatography wavelength switching technology. The method comprises the following steps: s1: processing eucommia ulmoides sample powder to be detected to prepare a sample solution; s2: and (3) eluting the active ingredients in the sample solution by using a high performance liquid chromatography technology and taking a reference substance solution as a reference, and analyzing by using a wavelength switching technology. The method provided by the invention can improve the chromatographic peak pattern of the active ingredients in the eucommia ulmoides by selecting a proper mobile phase and utilizing a high performance liquid chromatography wavelength switching technology, can more accurately and comprehensively analyze and detect various characteristic ingredients in the eucommia ulmoides sample, and has higher detection accuracy and sensitivity and high detection efficiency; can better monitor and control the quality of eucommia ulmoides resources, and has important significance for the modernization and internationalization of Chinese medicines.)

1. A method for simultaneously detecting multiple active ingredients in eucommia ulmoides by utilizing a high performance liquid chromatography wavelength switching technology is characterized by comprising the following steps:

s1: processing eucommia ulmoides sample powder to be detected to prepare a sample solution;

s2: eluting active ingredients in the sample solution by using a high performance liquid chromatography technology and a reference substance solution as a reference, and analyzing by using a wavelength switching technology;

wherein, the mobile phase A is methanol solution, the mobile phase B is 0.1 percent phosphoric acid water solution, and gradient elution is carried out; the conditions for the wavelength switching technique analysis are as follows: 0.01-15 min, 207 nm; 15-23.5 min, 238 nm; 23.5-28.5 min, 326 nm; 28.5-32.00 min, 240 nm; 32.00-40.00 min, 226 nm; 40.00-43.00 min, 254 nm; 43-90 min, 370 nm.

2. The method of claim 1, wherein the gradient elution conditions are: 0.00 → 6.00min, 6.0% mobile phase a, 94% mobile phase B; 6.00 → 13.00min, 6.0 → 13% mobile phase a, 94 → 87% mobile phase B; 13.00 → 14.50min, 13 → 18% mobile phase a, 87 → 82% mobile phase B; 14.50 → 22.00min, 18 → 20% mobile phase a, 82 → 80% mobile phase B; 22.00 → 30.00min, 20 → 40% mobile phase a, 80 → 60% mobile phase B; 30.00 → 32.00min, 40 → 45% mobile phase a, 60 → 55% mobile phase B; 32.00 → 45.00min, 45% mobile phase a, 55% mobile phase B; 45.00 → 46.00min, 45 → 63% mobile phase a, 55 → 37% mobile phase B; 46.00 → 66.00min, 63% mobile phase a, 37% mobile phase B; 66.00 → 68.00min, 63 → 30% mobile phase a, 37 → 70% mobile phase B; 68.00 → 70.00min, 30 → 6.0% mobile phase A, 70 → 94% mobile phase B; 70.99 → 90.00min, 6.0% mobile phase A, 94% mobile phase B.

3. The method according to claim 1, wherein the mass concentration of the eucommia ulmoides sample powder in the sample solution in S1 is 1-50 mg/mL.

4. The method according to claim 3, wherein the mass concentration of the eucommia ulmoides sample powder in the sample solution in S1 is 25 mg/mL.

5. The method of claim 1, wherein the sample solution in S1 is prepared by the following process: dissolving Eucommiae cortex sample powder in methanol water solution or ethanol water solution, ultrasonic processing, filtering, and metering volume.

6. The method according to claim 5, wherein the mass fraction of the methanol aqueous solution is 40-80%; the mass fraction of the ethanol water solution is 40-80%.

7. The method of claim 1, wherein the control solution at S2 comprises one or more of aucubin, geniposide, chlorogenic acid, geniposide, pinoresinol diglucoside, genipin, rutin, quercetin or kaempferol.

8. The method of claim 1, wherein the solvent used in the control solution of S2 is methanol or ethanol.

9. The method of claim 1, wherein the chromatography column is an ODS column, octadecylsilane bonded silica packing; the flow rate of the mobile phase was 0.8mL/min, the column temperature was 35 ℃ and the amount of sample was 10. mu.L.

10. The method of claim 1, wherein the active ingredient is one or more of aucubin, geniposide, chlorogenic acid, geniposide, pinoresinol diglucoside, genipin, rutin, quercetin, or kaempferol.

Technical Field

The invention relates to the field of plant active ingredient detection, in particular to a method for simultaneously detecting multiple active ingredients in eucommia ulmoides by utilizing a high performance liquid chromatography wavelength switching technology.

Background

Eucommia ulmoides (Eucommia ulmoides Oliv.) is a perennial deciduous tree of Eucommia of Eucommiaceae, belongs to a unique resource in China, is not only a traditional nourishing medicinal material in China, but also a famous and precious economic crop, has a cultivation area of about 300 ten thousand mu, accounts for more than 95% of the total amount of the world, is very rich in resources, and is mainly distributed in Hunan, Henan, Shaanxi, Guizhou, Hubei and the like. It is listed as the top grade in Shen nong Ben Cao Jing, and has effects of nourishing liver and invigorating kidney, strengthening tendons and bones, strengthening body constitution and resisting aging after long-term administration. Modern researches prove that cortex eucommiae is basically similar to chemical components of leaves in composition, mainly comprises iridoid, lignanoid, phenylpropanoid, polyphenol, flavonoid and the like, and has the effects of reducing blood pressure, blood fat, blood sugar and blood fat, resisting hypertension, resisting hyperlipidemia and resisting hypertension: lowering blood pressure, reducing blood lipid, lowering blood sugar, resisting aging, relieving fatigue, resisting tumor, relieving inflammation, resisting bacteria and virus, and enhancing immunity. For detection and analysis of the above active ingredients, Thin Layer Chromatography (TLC), ultraviolet-visible spectrophotometry (UV), High Performance Liquid Chromatography (HPLC), etc. are generally used.

2015 edition of Chinese pharmacopoeia has definite regulations on the main characteristic components of pinoresinol diglucoside and chlorogenic acid in cortex and folium Eucommiae. The content detection method of pinoresinol diglucoside in cortex Eucommiae is high performance liquid chromatography, using octadecylsilane chemically bonded silica as stationary phase, methanol-water (25:75) as mobile phase, and detecting wavelength of 277 nm; the detection method of chlorogenic acid content in folium Eucommiae is also high performance liquid chromatography, and comprises using octadecyl silane bonded silica gel as stationary phase, acetonitrile-0.4% phosphoric acid water (13:87) as mobile phase, and detection wavelength of 327 nm.

For the analysis of the ingredients of eucommia ulmoides, the high performance liquid chromatography is a relatively common and accurate analysis method in the reported technologies. However, the types of compounds related to simultaneous detection are limited in the current literature, and the maximum absorption wavelengths of different compounds are different, so when multiple compounds are detected simultaneously, unreasonable detection wavelengths can directly cause corresponding values of compound chromatographic peaks; in addition, because the polarity of some characteristic compounds in eucommia bark is relatively similar, the chromatographic peak separation effect is poor by using a conventional isocratic elution method.

Therefore, the development of a method capable of simultaneously detecting multiple active ingredients in eucommia ulmoides to comprehensively evaluate the authenticity, the excellence and the stability of the quality of raw materials and related samples has important significance in realizing high-value utilization of eucommia ulmoides resources.

Disclosure of Invention

The invention aims to overcome the defect or deficiency of the prior art that the variety of compounds in eucommia ulmoides is limited, and provides a method for simultaneously detecting multiple (9) active ingredients in eucommia ulmoides by using a high performance liquid chromatography wavelength switching technology. According to the method, the appropriate mobile phase is selected, and the high performance liquid chromatography wavelength switching technology is utilized to improve the chromatographic peak pattern of the active ingredients in the eucommia ulmoides, so that various (9) characteristic ingredients in the eucommia ulmoides sample can be analyzed and detected more accurately and comprehensively, and the detection accuracy and sensitivity are higher; can better monitor and control the quality of eucommia ulmoides resources, and has important significance for the modernization and internationalization of Chinese medicines.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for simultaneously detecting multiple active ingredients in eucommia ulmoides by utilizing a high performance liquid chromatography wavelength switching technology comprises the following steps:

s1: processing eucommia ulmoides sample powder to be detected to prepare a sample solution;

s2: eluting active ingredients in the sample solution by using a high performance liquid chromatography technology and a reference substance solution as a reference, and analyzing by using a wavelength switching technology;

wherein, the mobile phase A is methanol solution, the mobile phase B is 0.1 percent phosphoric acid water solution, and gradient elution is carried out; the conditions for the wavelength switching technique analysis are as follows: 0.01-15 min, 207 nm; 15-23.5 min, 238 nm; 23.5-28.5 min, 326 nm; 28.5-32.00 min, 240 nm; 32.00-40.00 min, 226 nm; 40.00-43.00 min, 254 nm; 43-90 min, 370 nm.

The method selects a proper mobile phase and utilizes the high performance liquid chromatography wavelength switching technology to improve the chromatographic peak pattern of the active ingredients in the eucommia ulmoides, more accurately and more comprehensively analyzes and detects various (9) characteristic ingredients in the eucommia ulmoides sample, and has higher detection accuracy and sensitivity and high detection efficiency.

The method provided by the invention can detect the main characteristic components in the eucommia relatively comprehensively, and can ensure that each component is detected at the corresponding maximum absorption wavelength; can better monitor and control the quality of eucommia ulmoides resources, and has important significance for the modernization and internationalization of Chinese medicines.

Preferably, the gradient elution conditions are: 0.00 → 6.00min, 6.0% mobile phase a, 94% mobile phase B; 6.00 → 13.00min, 6.0 → 13% mobile phase a, 94 → 87% mobile phase B; 13.00 → 14.50min, 13 → 18% mobile phase a, 87 → 82% mobile phase B; 14.50 → 22.00min, 18 → 20% mobile phase a, 82 → 80% mobile phase B; 22.00 → 30.00min, 20 → 40% mobile phase a, 80 → 60% mobile phase B; 30.00 → 32.00min, 40 → 45% mobile phase a, 60 → 55% mobile phase B; 32.00 → 45.00min, 45% mobile phase a, 55% mobile phase B; 45.00 → 46.00min, 45 → 63% mobile phase a, 55 → 37% mobile phase B; 46.00 → 66.00min, 63% mobile phase a, 37% mobile phase B; 66.00 → 68.00min, 63 → 30% mobile phase a, 37 → 70% mobile phase B; 68.00 → 70.00min, 30 → 6.0% mobile phase A, 70 → 94% mobile phase B; 70.99 → 90.00min, 6.0% mobile phase A, 94% mobile phase B.

Preferably, the mass concentration of the eucommia ulmoides sample powder in the sample solution in S1 is 1-50 mg/mL.

More preferably, the mass concentration of the eucommia ulmoides sample powder in the sample solution in the S1 is 25 mg/mL.

Preferably, the sample solution in S1 is prepared by the following process: dissolving Eucommiae cortex sample powder in methanol water solution or ethanol water solution, ultrasonic processing, filtering, and metering volume.

More preferably, the mass fraction of the methanol aqueous solution is 40-80%; the mass fraction of the ethanol water solution is 40-80%.

Preferably, the control solution in S2 contains one or more of aucubin, geniposide, chlorogenic acid, geniposide, pinoresinol diglucoside, genipin, rutin, quercetin or kaempferol.

Preferably, the solvent used for the control solution in S2 is methanol aqueous solution or ethanol aqueous solution.

Preferably, the chromatographic column is an ODS column, and octadecylsilane bonded silica gel packing; the flow rate of the mobile phase was 0.8mL/min, the column temperature was 35 ℃ and the amount of sample was 10. mu.L.

Preferably, the active ingredients are aucubin, geniposide, chlorogenic acid, geniposide, pinoresinol diglucoside, genipin, rutin, quercetin and kaempferol.

Compared with the prior art, the invention has the following beneficial effects:

the method selects a proper mobile phase and utilizes the high performance liquid chromatography wavelength switching technology to improve the chromatographic peak pattern of the active ingredients in the eucommia ulmoides, can more accurately and comprehensively analyze and detect various (9) characteristic ingredients in the eucommia ulmoides sample, and has higher detection accuracy and sensitivity and high detection efficiency.

The method provided by the invention can detect the main characteristic components in the eucommia more comprehensively, and can ensure that each component is detected at the corresponding maximum absorption wavelength; can better monitor and control the quality of eucommia ulmoides resources, and has important significance for the modernization and internationalization of Chinese medicines.

Drawings

FIG. 1 is a high performance liquid chromatogram of a control solution;

FIG. 2 is a high performance liquid chromatogram of a sample solution.

Detailed Description

The invention is further illustrated by the following examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the examples below, generally according to conditions conventional in the art or as suggested by the manufacturer; the raw materials, reagents and the like used are, unless otherwise specified, those commercially available from the conventional markets and the like. Any insubstantial changes and substitutions made by those skilled in the art based on the present invention are intended to be covered by the claims.

Example 1

The embodiment provides a method for simultaneously detecting multiple active ingredients in eucommia ulmoides by utilizing a high performance liquid chromatography wavelength switching technology, which comprises the following steps:

1. preparation of control solutions

Accurately weighing the reference substances including aucubin, geniposide, chlorogenic acid, geniposide, pinoresinol diglucoside, genipin, rutin, quercetin and kaempferol respectively 2.23mg, 4.27mg, 4.10mg, 1.33mg, 1.68mg, 1.44mg, 1.58mg, 1.07mg and 1.00mg, dissolving in 50% methanol water solution, fully dissolving, and fixing the volume to the scale (10 mL). The obtained solution is a reference solution of a reference substance, and is stored at 4 ℃ for later use.

2. Preparation of test sample solutions

Weighing 507mg of eucommia ulmoides sample powder to be detected, respectively placing the eucommia ulmoides sample powder to be detected in conical bottles with stoppers, dissolving the eucommia ulmoides sample powder in 50% methanol, dissolving the eucommia ulmoides sample powder for 30min with ultrasonic assistance, cooling the eucommia ulmoides sample powder at room temperature after full dissolution, filtering the eucommia ulmoides sample powder, and fixing the volume to 25mL to obtain a sample solution to be detected, and storing the sample solution at 4 ℃ for later use.

3. High performance liquid chromatography analysis method

Accurately transferring 2.0mL of reference solution or sample solution of the test sample, filtering with 0.45 μm membrane, and analyzing with high performance liquid chromatography wavelength switching technology to obtain corresponding chromatogram. The specific conditions are as follows:

stationary phase: octadecylsilane bonded silica packing (ODS column).

Mobile phase: the phase A is methanol, the phase B is 0.1% phosphoric acid water solution, and gradient elution is carried out.

The gradient elution procedure was: 0.00 → 6.00min, 6.0% mobile phase a, 94% mobile phase B; 6.00 → 13.00min, 6.0% → 13% mobile phase a, 94% → 87% mobile phase B; 13.00 → 14.50min, 13% → 18% mobile phase a, 87% → 82% mobile phase B; 14.50 → 22.00min, 18% → 20% mobile phase a, 82% → 80% mobile phase B; 22.00 → 30.00min, 20% → 40% mobile phase a, 80% → 60% flow

Moving phase B; 30.00 → 32.00min, 40% → 45% mobile phase a, 60% → 55% mobile phase B; 32.00 → 45.00min, 45% mobile phase a, 55% mobile phase B; 45.00 → 46.00min, 45% → 63% mobile phase a, 55% → 37% mobile phase B; 46.00 → 66.00min, 63% mobile phase a, 37% mobile phase B;

66.00 → 68.00min, 63% → 30% mobile phase a, 37% → 70% mobile phase B; 68.00 → 70.00min, 30% → 6.0% mobile phase a, 70% → 94% mobile phase B; 70.99 → 90.00min, 6.0% mobile phase A, 94% mobile phase B.

Detection wavelength: 0.01-15 min, 207 nm; 15-23.5 min, 238 nm; 23.5-28.5 min, 326 nm; 28.5-32.00 min, 240 nm; 32.00-40.00 min, 226 nm; 40.00-43.00 min, 254 nm; 43-90 min, 370 nm.

In the process of high performance liquid chromatography analysis, the mobile phase is 1.0mL/min, the column temperature is 35 ℃, the flow rate is 0.8mL/min, and the sample injection amount is 10 muL.

According to the above method, FIG. 1 is a high performance liquid chromatogram of a reference solution, wherein compound 1 is aucubin, 2 is geniposide, 3 is chlorogenic acid, 4 is geniposide, 5 is pinoresinol diglucoside, 6 is genipin, 7 is rutin, 8 is quercetin, and 9 is kaempferol. The detection results are subjected to integration treatment, and the relevant parameters of standard curves of different active ingredients in eucommia ulmoides are shown in table 1.

TABLE 1 relevant parameters of standard curves for different active ingredients in eucommia ulmoides

And (3) detecting and analyzing the sample solution to obtain a high performance liquid chromatogram of the sample solution, as shown in figure 2. It can be seen that the chromatographic peak separation degree of 9 characteristic components in the eucommia ulmoides sample is high, and the peak shape is good. The method can be used for qualitatively or quantitatively analyzing the active ingredients in the eucommia ulmoides sample, and has the advantages of higher detection accuracy and sensitivity and high detection efficiency.

In conclusion, the invention successfully establishes a method for simultaneously detecting multiple (9) components in eucommia ulmoides by utilizing a high performance liquid chromatography wavelength switching technology, can simply, rapidly, efficiently and accurately simultaneously detect multiple active components in eucommia ulmoides, can more comprehensively monitor natural effective components of different eucommia ulmoides samples, can more accurately and truly reflect the quality of the samples, and provides effective guarantee for quality control by taking eucommia ulmoides as a development raw material.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.