Method for determining chemical component content of engelhardia roxburghiana leaf general flavone extract by HPLC (High Performance Liquid Chromatography) method

Abstract

The invention discloses a method for determining the content of chemical components in engelhardia roxburghiana leaf general flavone extract by HPLC (High Performance Liquid Chromatography), which adopts ultra-high performance liquid chromatography-mass spectrometry to simultaneously determine the content of six chemical components in engelhardia roxburghiana leaf general flavone extract. The six chemical components comprise neo-astilbin, astilbin, neo-iso-astilbin, iso-astilbin, quercitrin and engeletin. By adopting the method disclosed by the invention, the contents of the six chemical components in the engelhardia roxburghiana leaf total flavone extract can be simultaneously determined by reasonably selecting chromatographic conditions and mass spectrum conditions, and the method has the advantages of simplicity, convenience, high sensitivity, high analysis speed, strong specificity and the like, so that the method can be used for controlling the quality of the engelhardia roxburghiana leaf total flavone extract.

Description

Technical Field

[0001] The present invention relates to a method for simultaneously determining the contents of six chemical components in a total flavonoid extract of huangqi leaves using HPLC, and belongs to the field of traditional Chinese medicine component analysis. Background Art

[0002] Engelhardtia roxburghiana (scientific name: Engelhardtia roxburghiana) is a semi-evergreen tree in the Juglandaceae family. It is the dried leaf of the Engelhardtia roxburghiana plant. This tree can reach up to 10 meters tall. It is glabrous and densely covered with orange-yellow peltate glands, with slender, dark brown branches. Its leaves are even-pinnately compound with 3-5 pairs of leaflets. These leaflets are leathery, oblong-lanceolate, glossy on both sides, and have prominent, brittle main veins. Engelhardtia roxburghiana is found in arid, sparse forests at altitudes of 200-1500 meters in southern and southwestern China, and is tolerant of infertile environments. Its leaves are cooling and slightly bitter, with heat-clearing and analgesic properties. They are primarily used to treat colds, fevers, hernias, and abdominal pain. Traditionally, a decoction is taken orally. Modern research confirms that it contains flavonoids such as astilbin and kaempferol, which have antioxidant, anti-radiation, and immunomodulatory properties. The total flavonoids in Engelhardtia roxburghiana leaves may exert protective effects by inhibiting oxidative stress and DNA damage. Therefore, it is necessary to establish an efficient, accurate and highly sensitive method for determining the content of multiple chemical components in the total flavonoids extract of Astragalus membranaceus leaves, so as to reliably and comprehensively control the quality of the total flavonoids extract of Astragalus membranaceus leaves and provide a basis for the quality evaluation of the total flavonoids extract of Astragalus membranaceus leaves. Summary of the Invention

[0003] The object of the present invention is to provide a method for determining the content of chemical components in the total flavonoid extract of Chinese wolfberry leaves, which is used to simultaneously determine the contents of six chemical components, including neoastilbin, astilbin, neoisoastilbin, isostilbin, quercetin and astilbin, in the total flavonoid extract of Chinese wolfberry leaves, and can be used for quality control of the total flavonoid extract of Chinese wolfberry leaves.

[0004] The present application provides a method for determining the content of chemical components in a total flavonoid extract of the leaves of Astragalus membranaceus, which uses ultra-high performance liquid chromatography to simultaneously determine the content of six chemical components in the total flavonoid extract of the leaves of Astragalus membranaceus; the six chemical components include: neoastilbin, astilbin, neoisoastilbin, isostilbin, quercetin and astragalin, and the method comprises:

[0005] 1. Preparation of Reference Stock Solution

[0006] Take appropriate amounts of new astilbin, astilbin, new isoflavin, isoflavin, quercetin, and huangqi glycoside reference substances, accurately weigh them, and dissolve them in methanol to make a mixed reference substance solution containing 0.03099 mg of new astilbin, 0.75273 mg of astilbin, 0.06010 mg of new isoflavin, 0.10 mg of isoflavin, 0.037 mg of quercetin, and 0.38 mg of huangqi glycoside per 1 ml, respectively. Shake well to obtain the solution.

[0007] 2. Preparation of test solution

[0008] Take an appropriate amount of the extract of this product, take about 10 mg, accurately weigh, place in a 25 ml volumetric flask, accurately add methanol to dissolve, dilute to the scale line, shake well, and filter through a 0.45 μm microporous membrane.

[0009] 3. Determination: Use HPLC to precisely pipette the mixed reference solution and test solution, inject into the detector, and determine the result. The chromatographic conditions are as follows.

[0010] Chromatographic column: Pheromones SuperLu C18 (250 mm × 4.5 mm, 5 μm), mobile phase: acetonitrile-0.1% formic acid solution (19:81), flow rate: 1.0 ml / min, column temperature: 30 °C, detection wavelength: 255 nm (quercetin), 290 nm (neoastilbin, astilbin, neoisoastilbin, isoastilbin, quercetin), injection volume 20 μL.

[0011] The above determination method includes the determination of linear relationship; accurately pipette the mixed reference solution, dilute it step by step,

[0012] A series of concentration solutions with dilution multiples of 1, 2, 4, 8, 16, and 32 were obtained.

[0013] The above determination method includes the determination of precision; that is, accurately aspirate a mixed reference solution: each 1 ml of the mixed reference solution contains 0.03099 mg of neoastilbin, 0.75273 mg of astilbin, 0.06010 mg of neoisastilbin, 0.10 mg of isastilbin, 0.037 mg of quercetin and 0.38 mg of citronellol, and inject the sample continuously 6 times under chromatographic conditions to record the peak area.

[0014] The above determination method includes repeatability determination; that is, taking the same batch of extract samples, 6 extract test samples are prepared in parallel, and the samples are measured 6 times under chromatographic conditions, and the chromatographic peak area of ​​each reference substance is calculated.

[0015] The above determination method includes the determination of stability; that is, a test solution of the total flavonoids extract of the leaves of Astragalus membranaceus is taken, placed at room temperature, and sampled at 0, 2, 4, 6, 8, 10, and 12 hours, and the sample is analyzed under chromatographic conditions to record the peak area.

[0016] The above determination method includes a recovery experiment; that is, 5 mg of the total flavonoid extract of Astragalus membranaceus leaves (the contents of neoastilbin, astilbin, neoisoastilbin, isostilbin, quercetin, and astragalus membranaceus are 34.24 mg / g, 191.33 mg / g, 60.99 mg / g, 37.60 mg / g, 16.15 mg / g, and 150.53 mg / g, respectively) are accurately weighed, a total of 6 portions, placed in stoppered conical flasks, and a certain amount of reference substance is added respectively. The test sample solution is prepared according to the test sample preparation method, 10 μl is injected into the liquid chromatograph, the peak area is determined, and the content of each component and the recovery rate are calculated.

[0017] The advantages of the present invention are: 1. Experimental results show that the six chemical components measured in the total flavonoids extract of Astragalus membranaceus leaves can be effectively separated under the isocratic elution conditions of an acetonitrile-0.1% formic acid solution system, and the determination method has a short cycle and high separation efficiency; 3. The present invention simultaneously quantitatively analyzes the six chemical components in the total flavonoids extract of Astragalus membranaceus leaves, has high specificity, simple operation, good repeatability, and accurate and reliable results, and can provide technical support and theoretical reference for the clinical application and quality control of the total flavonoids extract of Astragalus membranaceus leaves. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other embodiments can also be obtained based on these drawings.

[0019] In the attached figure:

[0020] Figure 1 Figure 1 HPLC chromatograms of the mixed reference solution and the test solution of the total flavonoids extract from the leaves of Astragalus membranaceus (1. neoastilbin; 2. astilbin; 3. neoisoastilbin; 4. isoastilbin; 5. quercetin; 6. astragalin). DETAILED DESCRIPTION

[0021] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Example 1: Screening of the preparation process of the total flavonoids extract from the leaves of Astragalus membranaceus

[0023] 1. Instruments and test drugs

[0024] Instruments: Waters high-performance liquid chromatograph (Arc quaternary pump and automatic sampling system, Empower chromatography workstation, 2998 PDA detector, Waters Corporation, USA); Millipore Milli-Q water purifier (Millipore Corporation, USA); METTLER 1 / 100,000 and 1 / 100,000 electronic balances (Mettler, Switzerland); KQ-250E medical ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.).

[0025] Reagents: Acetonitrile was chromatographically pure, ultrapure water was used, and all other reagents were of analytical grade. Neoastilbin reference substance (purity >98.0%, batch number: PS010667), astilbin reference substance (batch number: PS010703, purity >98.0%), neoisoastilbin reference substance (batch number: PS010708, purity ≥98.0%), isoastilbin reference substance (batch number: PS011862, purity >98.0%), quercetin reference substance (batch number: PS000430, purity >98.0%), and chrysanthemin reference substance (batch number: PS000469, purity >98.0%) were purchased from Chengdu Pusi Biotechnology Co., Ltd. for content determination.

[0026] 2. Chromatographic conditions

[0027] Chromatographic column: Phenylephrine SuperLu C18 (4.6×250mm, 5μm); mobile phase: acetonitrile-0.1% formic acid solution (19:81); flow rate: 1.0ml / min; column temperature: 30℃; detection wavelength: referring to the high performance liquid chromatography content determination method of flavonoids in Astragalus membranaceus leaves in the references, the detection wavelengths were determined to be 255nm (quercetin), 290nm (neoastilbin, astilbin, neoisoastilbin, isoastilbin, astilbin).

[0028] Under these chromatographic conditions, neoastilbin, astilbin, neoisoastilbin, isoastilbin, quercetin, and chrysantheminin were baseline-separated from adjacent peaks with a resolution greater than 1.5. The HPLC chromatograms of the reference and test samples are shown in Figure 1.

[0029] 3. Preparation of reference solution and test solution

[0030] The preparation methods of reference substance and test substance solutions are as follows: Preparation of reference substance stock solution: Take appropriate amount of neoastilbin, astilbin, neoisoastilbin, isoastilbin, quercetin, and chrysotoxin reference substances, weigh accurately, add methanol to dissolve to make a mixed reference substance solution containing 0.03099 mg of neoastilbin, 0.75273 mg of astilbin, 0.06010 mg of neoisoastilbin, 0.10 mg of isoastilbin, 0.037 mg of quercetin, and 0.38 mg of chrysotoxin per 1 ml, respectively; shake well to obtain the solution.

[0031] Preparation of test solution: Take an appropriate amount of the extract of this product, take about 10 mg, accurately weigh, place in a 25 ml volumetric flask, accurately add methanol to dissolve, dilute to the scale line, shake well, and filter through a 0.45 μm microporous membrane.

[0032] 4. Linear range investigation

[0033] Accurately pipette 1 ml of the reference substance stock solution into a 2 ml volumetric flask. Dilute to the mark with methanol and shake well. Repeat this method for serial dilutions. The concentrations of the prepared reference substances are shown in Table 39. Pipet 10 μl of each of the above reference substance solutions into a liquid chromatograph and measure the peak area. Perform linear regression using the injection volume (μg) as the abscissa (x) and the peak area as the ordinate (y) to determine the regression equation. The correlation coefficients (r) for the regression equations of the six components ranged from 0.9999 to 1.0000, indicating good linearity for neoastilbin within the range of 0.0097 to 0.3099 μg; for astilbin within the range of 0.2352 to 7.5273 μg; for neoisoastilbin within the range of 0.0188 to 0.6010 μg; for isoastilbin within the range of 0.0315 to 1.0094 μg; for quercetin within the range of 0.0117 to 0.3754 μg; and for chrysanthemin within the range of 0.1184 to 3.7874 μg. The results are shown in Table 1.

[0034] Table 1 Linearity investigation results

[0035]

[0036] 5. Precision test

[0037] Accurately pipette 10 μl of the reference solution into the liquid chromatograph for six consecutive injections. The peak area RSDs were calculated. The results showed that the peak area RSDs for the six components (neoastilbin, astilbin, neoisoastilbin, isoastilbin, quercetin, and chrysanthemin) were all less than 2.0%, indicating good precision for this method. (See Table 2.)

[0038] Table 2 Precision test (n=6)

[0039]

[0040] 6. Repeatability test

[0041] Accurately weigh 10 mg of powdered total flavonoid extract from the leaves of Astragalus membranaceus (6 replicates) and place in a 25 ml volumetric flask. Prepare the test solution according to the test solution preparation method. A 10 μl aliquot was injected into a liquid chromatograph to measure the peak areas of neoastilbin, astilbin, neoisoastilbin, isostilbin, quercetin, and astragalin. The content and relative standard deviation (RSD) of each component in the sample were calculated. The RSD values ​​for the content of each component ranged from 0.62% to 1.97%, and the RSD for total flavonoids was 0.59%, indicating good reproducibility of this method. (See Table 3.)

[0042] Table 3 Repeatability test (n=6, mg / mg)

[0043]

[0044] 7. Stability test

[0045] Approximately 10 mg of the total flavonoid extract from the leaves of Astragalus membranaceus was accurately weighed and placed in a stoppered conical flask. A test solution was prepared according to the test solution preparation method. The peak areas of the six components (neoastilbin, astilbin, neoisoastilbin, isoastilbin, quercetin, and astragalin) were measured at 0, 2, 4, 6, 8, 10, and 12 hours, and their relative standard deviations (RSDs) were calculated. The RSDs for each component ranged from 0.58% to 1.19%, indicating that the test solution was stable within 12 hours. See Table 4.

[0046] Table 4 Stability test (n=6)

[0047]

[0048] 8. Sample recovery test

[0049] Accurately weigh 5 mg of the total flavonoid extract from the leaves of Astragalus membranaceus (contents of neoastilbin, astilbin, neoisoastilbin, isoastilbin, quercetin, and astragalin were 34.24 mg / g, 191.33 mg / g, 60.99 mg / g, 37.60 mg / g, 16.15 mg / g, and 150.53 mg / g, respectively). Six aliquots were placed into stoppered conical flasks. A predetermined amount of reference substance was added to each aliquot. Test solutions were prepared according to the test sample preparation method. 10 μl of the aliquot was injected into a liquid chromatograph, and the peak areas were measured. The content and recovery of each component were calculated. The results showed recoveries ranging from 97.12% to 104.04%, with RSDs ranging from 1.16% to 1.68%, indicating high accuracy of this recipe. See Tables 5-10.

[0050] Table 5 Recovery rate test of neoastilbin (n=6)

[0051]

[0052] Table 6 Recovery rate test of astilbin (n=6)

[0053]

[0054] Table 7 Recovery rate test of neoastilbin (n=6)

[0055]

[0056] Table 8 Recovery rate test of isoflavone glycosides (n=6)

[0057]

[0058] 9. Sample Measurement

[0059] Four batches of Chinese samples of total flavonoids extract from Chinese wolfberry leaves (Z240301, Z240302, Z240303, and Z240401) were taken to prepare test solutions. The total flavonoids contents in the four batches of samples were determined according to the proposed content determination method. The total flavonoids contents were 54.53%, 52.83%, 51.72%, and 53.89%, respectively. The transfer rates of total flavonoids were calculated, and the results are shown in Table 11.

[0060] Table 11 Results of flavonoid content determination in four batches of pilot samples (n=2)

[0061]

[0062] In summary, this application establishes an HPLC analysis method that can simultaneously determine the contents of six chemical components in the extract of the Chinese wolfberry leaves, including neoastilbin, astilbin, neoisoastilbin, isostilbin, quercetin, and astilbin, so as to better apply it to the quality evaluation of the Chinese wolfberry leaf extract; this method is simple, highly sensitive, fast in analysis speed, and highly specific.

[0063] The above description is only a preferred embodiment of the present application and is not intended to limit the scope of protection of the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application are included in the scope of protection of the present application.

Claims

1. A method for simultaneously determining the contents of six chemical components in a total flavonoid extract from the leaves of Astragalus membranaceus using HPLC, characterized in that: The steps include: (1) Preparation of reference substance stock solution Preparation of reference solution: Take appropriate amount of neoastilbin, astilbin, neoisoastilbin, isoastilbin, quercetin, and cyperitoside reference substances, accurately weigh them, and dissolve them in methanol to prepare a mixed reference solution containing 0.03099 mg of neoastilbin, 0.75273 mg of astilbin, 0.06010 mg of neoisoastilbin, 0.10 mg of isoastilbin, 0.037 mg of quercetin, and 0.38 mg of cyperitoside per 1 ml, respectively. Shake well to obtain the solution. (2) Preparation of test solution Preparation of test solution: Take an appropriate amount of the extract of this product, take about 10 mg, accurately weigh, place in a 25 ml volumetric flask, accurately add methanol to dissolve, dilute to the scale line, shake well, and filter through a 0.45 μm microporous membrane; (3) Determination method Chromatographic conditions: The concentrations of the mixed reference and test samples were determined by HPLC. The chromatographic conditions used were as follows: chromatographic column: Phenylephrine SuperLu C18 (250 mm × 4.5 mm, 5 μm), mobile phase: acetonitrile-0.1% formic acid solution (19:81), flow rate: 1 ml / min, column temperature: 30 °C, detection wavelength: 255 nm (quercetin), 290 nm (neoastilbin, astilbin, neoisoastilbin, isoastilbin, and quercetin), injection volume: 10 μL.

2. A method for simultaneously determining the contents of six chemical components in the total flavonoids extract of Aralia dahurica leaves using HPLC as claimed in claim 1, characterized in that: The above determination method includes the determination of linear relationship; accurately pipette the mixed reference solution, dilute it step by step, and obtain a series of concentration solutions with dilution multiples of 1, 2, 4, 8, 16, and 32.

3. A method for simultaneously determining the contents of six chemical components in the total flavonoids extract of Aralia dahurica leaves using HPLC as claimed in claim 1, characterized in that: The above determination method includes the determination of precision; that is, accurately aspirate a mixed reference solution: each 1 ml of the mixed reference solution contains 0.03099 mg of neoastilbin, 0.75273 mg of astilbin, 0.06010 mg of neoisastilbin, 0.10 mg of isastilbin, 0.037 mg of quercetin and 0.38 mg of citrarin, and inject the sample continuously 6 times under chromatographic conditions to record the peak area.

4. A method for simultaneously determining the contents of six chemical components in the total flavonoids extract of Aralia dahurica leaves using HPLC as claimed in claim 1, characterized in that: The above determination method includes repeatability determination; that is, taking the same batch of extract samples, 6 extract test samples are prepared in parallel, and the samples are measured 6 times under chromatographic conditions, and the chromatographic peak area of ​​each reference substance is calculated.

5. A method for simultaneously determining the contents of six chemical components in the total flavonoids extract of Aralia dahurica leaves using HPLC as claimed in claim 1, characterized in that: The above determination method includes the determination of stability; that is, a test solution of the total flavonoids extract of the leaves of Astragalus membranaceus is taken, placed at room temperature, and sampled at 0, 2, 4, 6, 8, 10, and 12 hours, and the sample is analyzed under chromatographic conditions to record the peak area.

6. A method for simultaneously determining the contents of six chemical components in the total flavonoids extract from the leaves of Astragalus membranaceus using HPLC as claimed in claim 1, characterized in that: The above determination method includes a recovery experiment; that is, 6 portions of the total flavonoid extract of Astilbin leaves, including neoastilbin, neoisoastilbin, isostilbin, quercetin and astilbin, with contents of 34.24 mg / g, 191.33 mg / g, 60.99 mg / g, 37.60 mg / g, 16.15 mg / g and 150.53 mg / g, respectively, are accurately weighed and placed in stoppered conical flasks. A certain amount of reference substance is added respectively, and the test sample solution is prepared according to the test sample preparation method. 10 μl is injected into a liquid chromatograph, the peak area is determined, and the content of each component and the recovery rate are calculated.

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