Method for simultaneously determining several chemical components of michelia alba extract by high performance liquid chromatography

Abstract

The invention discloses a method for simultaneously determining several chemical components of a michelia alba extract by high performance liquid chromatography. The method comprises the following steps: a, extracting a michelia alba extract with a solvent to prepare a test solution; b, preparing a mixed reference substance solution from echinacoside, verbascoside and isoacteoside; and c, respectively carrying out high performance liquid chromatography separation on the test solution and the mixed reference substance solution. According to the present invention, the high performance liquid chromatography is adopted to simultaneously determine the several effective components of the michelia alba extract, the echinacoside, the verbascoside, the isoacteoside and other components in the michelia alba extract are determined for the first time, the data analysis is provided for the comprehensive analysis of the michelia alba extract, and the reference is provided for the michelia alba extract. The method provided by the invention is convenient, accurate and efficient, and provides effective data reference for better subsequent research on the michelia alba extract.

Description

Technical Field

[0001] The invention belongs to the detection field, and particularly relates to a method for simultaneously determining several chemical components of a white jasmine extract by using a high performance liquid chromatography method. Background Art

[0002] White orchid is the dried flower of Michelia alba Candolle, a plant of the Magnoliaceae family. [1] , alias: yellow jasmine, white osmanthus, white orchid, orchid, included in "Guangzhou Flora", "Sichuan Chinese Materia Medica", "Chinese Materia Medica Dictionary", etc. [2-4] , has the effects of removing dampness, promoting qi, and relieving cough. The flowering period of white jasmine is from April to October, and the peak bloom period is May. It can be extracted as fragrance or fumigation tea, and can also be extracted into essential oils and extracts as food additives. White jasmine extracts can also be used to prepare fragrances, perfumes, etc. [5] White orchid is native to Java, Indonesia, and is now widely cultivated in Southeast Asia. It is also cultivated in Fujian, Guangdong, Guangxi, Yunnan and other provinces in my country. It is mostly used as a potted ornamental plant in the Yangtze River Basin and other provinces. In recent years, many scholars have conducted research on the chemical composition of white orchid, including different extraction solvents, collection methods, different extraction techniques and different analysis techniques. Summary of the invention

[0003] The purpose of the present invention is to provide a method for simultaneously determining several chemical components of white jasmine extract by high performance liquid chromatography. The method of the present invention is convenient, accurate and efficient, and provides effective data reference for better subsequent research on white jasmine extract.

[0004] The method for simultaneously determining several chemical components of a white jasmine extract by high performance liquid chromatography of the present invention comprises the following steps:

[0005] a. Extract the white jasmine extract with a solvent to prepare a test solution;

[0006] b. Prepare a mixed reference solution by mixing echinacoside, verbascoside and isovalascoside;

[0007] c. The test solution and the mixed reference solution were separated by high performance liquid chromatography, and the elution conditions were as follows: the chromatographic column was a Shimadzu VP-ODS column 150 mm*4.6 mm, 5 um, the mobile phase was methanol (A)-0.2% phosphoric acid aqueous solution (B), gradient elution 0-10 min, 28%-35% A; 10-20 min, 35%-55% A; 20-25 min, 55%-28% A, the flow rate was 1.0 mL / min, the detection wavelength was 330 nm, and the column temperature was 30°C; the contents of echinacoside, verbascoside and isovalascoside were calculated based on the elution results of the chromatographic column.

[0008] Preferably, the step a is to extract by immersion in methanol for 10 minutes, the solid-liquid ratio is 1:90 g / ml, and the immersion liquid is filtered through a 0.22 μm microporous filter membrane to obtain the test solution.

[0009] Preferably, the mixed reference solution uses methanol as a solvent, and each 1 mL contains 0.1999 mg of echinacoside, 0.2489 mg of verbascoside, and 1.4680 mg of isovorascoside.

[0010] The present invention uses high performance liquid chromatography to simultaneously determine several effective components of the white jasmine extract, and for the first time determines echinacoside, verbascoside, isoverbascoside and other components therein, aiming to provide data analysis for a comprehensive analysis of the white jasmine extract and provide a reference for the white jasmine extract. The method of the present invention is convenient, accurate and efficient, and provides an effective data reference for better subsequent research on the white jasmine extract. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is Shimadzu GIST3 shim-pack GIST C18 (250mm*4.6mm, 5um);

[0012] Figure 2 Agilent 5TC-C18(2)(250mm*4.6mm, 5um);

[0013] Figure 3 SHIMADZU VP-ODS (150mm*4.6mm, 5um);

[0014] Figure 4 It is methanol-0.1% formic acid;

[0015] Figure 5 is acetonitrile-0.1% formic acid;

[0016] Figure 6 is acetonitrile-0.2% phosphoric acid;

[0017] Figure 7 It is methanol-0.2% phosphoric acid;

[0018] Figure 8 It is 215nm;

[0019] Fig. 9 It is 280nm;

[0020] Fig.10 It is 330nm;

[0021] Fig.11 is 0.8mL / min;

[0022] Fig.12 is 1.0mL / min;

[0023] Fig.13 is 1.2mL / min;

[0024] Fig.14 It is 25℃;

[0025] Fig.15 It is 30℃;

[0026] Fig.16 It's 35℃. DETAILED DESCRIPTION

[0027] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0028] Embodiment 1:

[0029] 1. Instruments and reagents

[0030] 1.1 Instruments Agilent 1260 Agilent Technologies Co., Ltd.; KQ-400E ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); LS220ASCS 1 / 10,000 electronic analytical balance (Pulisis / Shanghai Tianmei Balance Instrument Co., Ltd.); HA225SM-DR 1 / 100,000 electronic analytical balance (Pulisis / Shanghai Tianmei Balance Instrument Co., Ltd.); Center-EDI 45FV water purifier (Shanghai Hetai Instrument Co., Ltd.)

[0031] 1.2 Test drugs: echinacoside (Lemeitian Pharmaceutical | Desit Biotechnology, batch number: DSTDS003801, ≥98%); verbascoside (Shanghai Tauto Biotech Co., Ltd., batch number: 23062021, 99.4%); isoverbascoside (Sichuan Jingcui Tiancheng Pharmaceutical Technology Co., Ltd., batch number: TC0803-240329, ≥98%). Methanol, acetonitrile (chromatographic grade, Fisher Scientific World, USA), water is ultrapure water, and other reagents are analytical grade.

[0032] The white jasmine extract used in the experiment was produced by Guangxi Hualan Biotechnology Co., Ltd. in September 2024.

[0033] 2 Methods and Results

[0034] 2.1 Chromatographic conditions

[0035] The chromatographic column was a Shimadzu SHIMADZU VP-ODS column (150 mm*4.6 mm, 5 um), the mobile phase was methanol (A)-0.2% phosphoric acid aqueous solution (B), gradient elution (0-10 min, 28%-35% A; 10-20 min, 35%-55% A; 20-25 min, 55%-28% A), the flow rate was 1.0 mL / min, the detection wavelength was 330 nm, the column temperature was 30°C, and the injection volume was 3 μL.

[0036] 2.2 Preparation of reference solution

[0037] 2.2.1 Preparation of reference solution

[0038] Accurately weigh appropriate amounts of echinacoside, verbascoside, and isoverbascoside reference substances, place them in 10 mL volumetric flasks, add methanol to dissolve, and prepare stock solutions containing 0.9996 mg, 0.9957 mg, and 5.2430 mg of reference substances per 1 mL, respectively.

[0039] 2.2.2 Preparation of mixed control solution

[0040] Accurately pipette appropriate amount of reference stock solution under "2.2.1" into a 10mL volumetric flask, add methanol to make up to the mark, and prepare mixed reference substance solutions containing 0.1999mg, 0.2489mg, and 1.4680mg per 1mL.

[0041] 2.3 Preparation of test solution

[0042] Take about 1g of each white jasmine extract, weigh accurately, and place in a 150mL stoppered conical flask, with a solid-liquid ratio of 1:90. Prepare the test samples according to the following table, a total of 27 test samples, and filter through a 0.22μm microporous filter membrane to obtain the test sample solution.

[0043]

[0044]

[0045] 2.4 Determination of the contents of three components

[0046] Take the mixed reference solution under "2.2.2" and the test solution under "2.3" according to the determination under "2.1" to calculate the contents of the three components: echinacoside, verbascoside and isovalascoside. The results are shown in the table below.

[0047] Table 3 contents of three components in white orchid extract (mg / g)

[0048]

[0049] Verbascoside 25.9227 26.8964 26.0558 27.8348 27.7130 26.9781 24.010323.7912 23.6882Isovalascoside 148.6845 156.5571 150.6723 167.2261 166.6378162.1037 152.0075 150.6345 150.1652Echinacoside 16.7458 16.9300 16.7516 17.047917.8726 18.0503 5.0221 4.0272 3.6419Verbascoside reflux 25.6353 26.7239 26.816227.1626 28.3324 29.0902 25.6361 24.6862 25.3339Isoverbascoside138.7446 140.1061136.0844 163.5176 170.3390 172.3453 159.0724 157.9932 159.8773

[0050] As can be seen from the table, the contents of the three compounds extracted by reflux methanol for 60 minutes are relatively high, but the content measured by immersion in methanol for 10 minutes is not much different. Considering the cumbersome experimental device, the immersion extraction method is determined. In summary, the best extraction process is extraction solvent: methanol, extraction method: immersion, extraction time: 10 minutes. The content of echinacoside in the white jasmine extract in this experiment is 17.3966 mg / g, the content of verbascoside is 27.8348 mg / g, and the content of isoverbascoside is 167.2261 mg / g.

[0051] discuss

[0052] Determination of chromatographic conditions

[0053] 1. Selection of chromatographic column

[0054] According to the conditions in 2.3, the extraction solvent of white jasmine extract: methanol, extraction method: immersion, extraction time: 10 minutes was used as the test solution and eluted according to the conditions in 2.1, except that the chromatographic column was different.

[0055] This experiment compared Shimadzu Shim-pack GIST C18 (250mm*4.6mm, 5um), Agilent 5TC-C18 (2) (250mm*4.6mm, 5um), and SHIMADZU VP-ODS (150mm*4.6mm, 5um). The SHIMADZU VP-ODS column (150mm*4.6mm, 5um) was finally selected. The peaks of the separation were sharp, the separation degree of each component reached 1.5, and the number of theoretical plates was greater than 7000; see Figures 1 to 3 .

[0056] 2. Mobile phase:

[0057] According to the conditions in 2.3, the extraction solvent of white jasmine extract is methanol, the extraction method is immersion, and the extraction time is 10 minutes. As the test solution, the elution is carried out according to the conditions in 2.1, except that the mobile phase is different, specifically:

[0058] The mobile phases of methanol-0.1% formic acid, acetonitrile-0.1% formic acid, acetonitrile-0.2% phosphoric acid, and methanol-0.2% phosphoric acid gradient elution were compared. It can be seen from the figure that the methanol-0.2% phosphoric acid gradient elution can achieve a separation degree of more than 1.5 for each component. Figures 4 to 7 .

[0059] 3. According to the conditions in 2.3, the extraction solvent of white jasmine extract is methanol, the extraction method is immersion, and the extraction time is 10 minutes. As the test solution, it is eluted according to the conditions in 2.1, except that the detection wavelength is different.

[0061] According to the literature, echinacoside absorbs at 215nm, 280nm, and 330nm, verbascoside absorbs at 280nm and 330nm, and isoverbascoside absorbs at 300nm. By comparing the absorption at these wavelengths, it is determined that the absorption of several components at 330nm is better, the baseline is more stable, and there are fewer stray peaks. Figures 8 to 10 .

[0062] 4. According to the conditions in 2.3, the extraction solvent of white jasmine extract is methanol, the extraction method is immersion, and the extraction time is 10 minutes. As the test solution, it is eluted according to the conditions in 2.1, except that the flow rate and column temperature are different.

[0064] When comparing flow rates of 0.8, 1.0, and 1.2 mL / min, see Figures 11 to 13 , column temperature 25, 30, 35°C, see Figures 14 to 16 Finally, the peak retention time of 1.0 mL / min and 30 °C was reasonable; the final chromatographic conditions were determined.

[0065] In summary, the chromatographic conditions were determined as follows: a SHIMADZU VP-ODS column (150 mm*4.6 mm, 5 um), a mobile phase of methanol (A)-0.2% phosphoric acid aqueous solution (B), a gradient elution (0-10 min, 28%-35% A; 10-20 min, 35%-55% A; 20-25 min, 55%-28% A), a flow rate of 1.0 mL / min, a detection wavelength of 330 nm, a column temperature of 30°C, and an injection volume of 3 μL.

[0066] The method established in this experiment is convenient, accurate and efficient, and can provide effective data reference for better follow-up research on white jasmine extract.

Claims

1. A method for simultaneously determining several chemical components of a white jasmine extract by high performance liquid chromatography, characterized in that: The following steps are involved: a. Extract the white jasmine extract with a solvent to prepare a test solution; b. Prepare a mixed reference solution by mixing echinacoside, verbascoside and isovalascoside; c. The test solution and the mixed reference solution were separated by high performance liquid chromatography, and the elution conditions were as follows: the chromatographic column was a Shimadzu VP-ODS column 150 mm*4.6 mm, 5 um, the mobile phase was methanol (A)-0.2% phosphoric acid aqueous solution (B), gradient elution 0-10 min, 28%-35% A; 10-20 min, 35%-55% A; 20-25 min, 55%-28% A, the flow rate was 1.0 mL / min, the detection wavelength was 330 nm, and the column temperature was 30°C; the contents of echinacoside, verbascoside and isovalascoside were calculated based on the elution results of the chromatographic column.

2. The method according to claim 1, characterized in that The step a is to extract by immersion in methanol for 10 minutes, the solid-liquid ratio is 1:90g / ml, and the immersion liquid is filtered through a 0.22μm microporous filter membrane to obtain the test solution.

3. The method according to claim 1, characterized in that The mixed reference solution uses methanol as a solvent, and each 1 mL contains 0.1999 mg of echinacoside, 0.2489 mg of verbascoside, and 1.4680 mg of isovorascoside.

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