Method for determining content of flavonoid components in rose willow flower extract

Abstract

The invention provides a method for determining the content of flavonoid components in a rose willow flower extract. The flavonoid components are quercetin and isorhamnetin. The method specifically comprises the following steps: preparation of a reference substance solution, preparation of a test solution, establishment of a linear regression equation, detection of the test solution and content calculation. The chromatographic conditions of the high performance liquid chromatography technology are as follows: a Wondasil Cis Herb colomn chromatographic column is adopted, the size is 250 mm * 4.6 mm, and the size is 5 [mu] m; the volume flow rate is 1.0 mL / min; the mobile phase A is methanol, and the mobile phase B is a 0.1% potassium phosphate solution; the column temperature is 30 DEG C; the sample size is 20 [mu] L; and the elution gradient is 0-10 min: 65% A, 10-20 min: 65% A to 48% A, 20-30 min: 48% A, and 30-40 min: 48% A to 65% A. The invention also discloses a method for preparing the traditional Chinese medicine composition. The method has the advantages of high precision, stability, repeatability and accuracy, and is simple and stable.

Description

Technical Field

[0001] The present invention belongs to the technical field of determination and analysis of traditional Chinese medicine content, and particularly relates to a method for determining the content of flavonoid components in the extract of red willow flower. Background Art

[0002] Red willow, scientific name Tamarix chinensis Lour., also known as Tamarix ramosissima Ledeb. or Chinese tamarisk, is an excellent plant for wind prevention and sand fixation and a tree species for afforestation in saline-alkali land. It is also one of the main hosts of the precious traditional Chinese medicine Cistanche tubulosa. It is widely distributed and rich in resources in Ningxia and even the northwest region. Red willow flower refers to the small flowers on the red willow tree, and its color is usually pink or light purple. Red willow flower contains a variety of bioactive substances, mainly including flavonoid compounds, polyphenolic substances, terpene compounds, organic acids, vitamins and minerals, etc. Among them, flavonoid compounds, such as quercetin, isorhamnetin, kaempferol, etc., are the main antioxidant components. Research shows that flavonoid compounds also have certain anti-inflammatory and anti-rheumatic effects. Therefore, it is very important to detect the flavonoid compounds in red willow flower.

[0003] At present, the detection of flavonoids in traditional Chinese medicinal materials generally uses spectrophotometry, high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry, etc. Among them, HPLC is a very commonly used method, which can separate and quantitatively determine flavonoids in complex mixtures. Chinese Patent CN109884199B discloses a method for extracting and analyzing flavonoid components in honey. The flavonoid components are one or more mixtures of quercetin, kaempferol, and isorhamnetin. During the extraction and analysis process, the honey extract is first adsorbed by an adsorbent, and then the adsorbent is eluted to collect the filtrate. After centrifugation, it is detected by a high performance liquid chromatograph. The chromatographic conditions are as follows: chromatographic column: ZORBAX SB-C18 chromatographic column, injection volume: 10 μL, column temperature: 35 °C, flow rate 1 mL / min, mobile phase: a mixture of methanol with a volume ratio of 55:45 and phosphoric acid water with a volume fraction of 0.4%, isocratic elution for 12 min. Chinese Patent CN113358786B discloses a method for determining the content of flavonol glycosides in Ginkgo biloba dropping pills. The above method includes the preparation of a test solution, the establishment of chromatographic conditions and determination, and can simultaneously determine the content of 6 flavonol glycoside components. The chromatographic conditions are as follows: chromatographic column: Agilent InfinityLab Poroshell 120EC-C18, mobile phase: 0.05% formic acid aqueous solution as mobile phase A, acetonitrile-methanol as mobile phase B, the ratio of acetonitrile-methanol is 7:3, gradient elution: 0-6 min, 16% B; 6-20 min, 16-19% B; 20-21 min, 19-20% B; 21-34 min, 20-25% B; 34-35 min, 25-27% B; 35-43 min, 27-35% B; 43-47 min, 35-38% B; 47-50 min, 38-95% B; 50-53 min, 95% B; flow rate: 0.3 mL / min; injection volume: 2 μL; column temperature: 40 °C; detection wavelength: 360 nm. Chinese Patent CN113552256B discloses a method for simultaneously determining the content of 20 flavonoid components in Flos Lonicerae, and also uses high performance liquid chromatography to detect the extract obtained after ultrasonic extraction. The chromatographic conditions are Hola C18 chromatographic column, 2.1 mm × 100 mm, 2.7 μm; injection volume 2 μL; mobile phase A is formic acid aqueous solution, in which the volume fraction of formic acid is 0.08%, B is formic acid acetonitrile solution, in which the volume fraction of formic acid is 0.08%; elution gradient is 0-5 min 10% → 23% B, 5-25 min 23% → 31% B, 25-35 min 31% → 80% B, 35-40 min 80% B; flow rate 0.3 mL / min; column temperature 30 °C.

[0004] Although high performance liquid chromatography (HPLC) is a very effective analytical tool for detecting flavonoids, it still faces some challenges and difficulties, as follows: (1) Plant extracts usually contain multiple components, including other phenolic compounds, pigments, proteins, sugars, etc. These components may co-elute with flavonoids or interfere with the detection.

[0005] (2) Flavonoids have a wide range of structural diversities, including aglycones, glycosides, methoxylated, hydroxylated, acylated and other modified forms, which makes their retention behaviors on the chromatographic column different and increases the separation difficulty.

[0006] (3) Due to the large differences in polarity and molecular weight of flavonoids, it is not easy to find a selective stationary phase that can provide good separation for all target compounds.

[0007] For the detection of flavonoids in different traditional Chinese medicine components, different chromatographic conditions need to be established. Currently, there is no publicly disclosed method that can specifically determine the flavonoids in Tamarix ramosissima flowers. Therefore, there is an urgent need for a method for determining flavonoids in Tamarix ramosissima flowers by high performance liquid chromatography. Summary of the Invention

[0008] The purpose of the present invention is to provide a method for determining the content of flavonoid components in an extract of Tamarix ramosissima flowers, so as to solve the problems that the determination of flavonoids by high performance liquid chromatography is relatively complex, and there is no perfect method for detecting flavonoids in Tamarix ramosissima flowers in the prior art.

[0009] To achieve the above purpose, the present invention provides the following technical solutions: A method for determining the content of flavonoid components in an extract of Tamarix ramosissima flowers, wherein the flavonoid components are quercetin and isorhamnetin; Specifically, it includes the following steps: S1: Preparation of reference substance solution: Mix a certain amount of quercetin reference substance and isorhamnetin reference substance and dissolve them in methanol, and dilute to obtain reference substance solutions with different concentrations; S2: Preparation of test sample solution: S21: Crush Tamarix ramosissima flowers to obtain Tamarix ramosissima flower powder; S22: Add an organic solvent to the Tamarix ramosissima flower powder, heat and ultrasonicate, reflux and extract, and obtain an extract of Tamarix ramosissima flowers after rotary evaporation; Add the extract of Tamarix ramosissima flowers to methanol to obtain a test sample solution; S3: Establishment of linear regression equation: S31: Detect and analyze the reference substance solution by high performance liquid chromatography technology; S32: Using the peak areas of the quercetin and isorhamnetin reference standards as the ordinate and the contents of quercetin and isorhamnetin as the abscissa, establish a linear regression equation; S4: Detection of the test solution: Use high-performance liquid chromatography technology to quantitatively analyze quercetin and isorhamnetin in the test solution; S5: Content calculation: Based on the linear regression equation obtained in step S3 and the results of the quantitative analysis of the test solution in step S4, calculate the contents of quercetin and isorhamnetin in the test solution.

[0010] Preferably, in step S1, weigh 5.0 mg of quercetin reference standard and 5.0 mg of isorhamnetin reference standard respectively, place them in a 25 mL volumetric flask, dissolve them with methanol and make up to the mark to obtain a mixed reference standard solution with concentrations of 0.2 mg / mL for quercetin and 0.2 mg / mL for isorhamnetin, which is used as the mixed standard stock solution; Dilute the mixed standard stock solution with methanol to obtain standard series mixed solutions with different concentrations, and then filter through a 0.22 μm microporous membrane to obtain reference standard solutions with different concentrations.

[0011] Preferably, in step S21, the tamarisk flower is crushed and passed through a 40-mesh nylon sieve to obtain tamarisk flower powder.

[0012] Preferably, in step S22, the organic solvent is 95% ethanol, and the mass-to-volume ratio of the tamarisk flower powder to the organic solvent is (3 - 8) g: 100 mL.

[0013] Preferably, in step S22, the heating temperature is 75 - 85 °C and the reflux extraction time is 1 - 10 h.

[0014] Preferably, in step S22, accurately weigh 0.677 g of the tamarisk extract powder, place it in a 25 mL volumetric flask, dissolve it with methanol and make up to the mark to obtain the test solution.

[0015] Preferably, the chromatographic conditions for the high-performance liquid chromatography technology are: Wondasil Cis Herb column, 250 mm × 4.6 mm, 5 μm; mobile phase A is methanol, mobile phase B is 0.1% potassium phosphate solution; column temperature is 30 °C; injection volume is 20 μL; volume flow rate is 1.0 mL / min; elution gradient is 0 - 10 min: 65% A, 10 - 20 min: 65% A → 48% A, 20 - 30 min: 48% A, 30 - 40 min: 48% A → 65% A.

[0016] The present invention sets a specific elution gradient. In the initial stage, a relatively high proportion of methanol (65%) is maintained to quickly elute those compounds with weak retention, reducing background noise. As the analysis time progresses, the proportion of methanol is gradually decreased, while the proportion of potassium phosphate solution is increased, which helps to better separate the flavonoids with larger polarity. Between 20 minutes and 30 minutes, a relatively low proportion of methanol (48%) is maintained to ensure the full separation of the flavonoids with larger polarity. Finally, the proportion of methanol is restored to 65% to clean the chromatographic column and prepare for the next injection.

[0017] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: 1. The present invention uses a method combining heating, ultrasound and reflux extraction, which can effectively extract flavonoids from tamarix chinensis. This method not only improves the extraction efficiency, shortens the extraction time, but also maintains the stability of the target compounds.

[0018] 2. During the high performance liquid chromatography detection of the present invention, a Wondasil Cis Herb chromatographic column suitable for separating flavonoids, as well as an appropriate mobile phase composition and elution gradient, are selected, which can achieve good peak shapes and separation effects, especially for quercetin and isorhamnetin with similar structures. Description of the Drawings

[0019] Figure 1 is the high performance liquid chromatography diagram under the mobile phase of formaldehyde - pure water; Figure 2 is the high performance liquid chromatography diagram under the mobile phase of formaldehyde - 0.1% phosphoric acid water; Figure 3 is the high performance liquid chromatography diagram under the mobile phase of acetonitrile - 0.1% phosphoric acid water; Figure 4 is the high performance liquid chromatography diagram under the mobile phase of methanol - 0.1% potassium phosphate; Figure 5 is the high performance liquid chromatography diagram under the elution gradient a; Figure 6 is the high performance liquid chromatography diagram under the elution gradient b; Figure 7 is the high performance liquid chromatography diagram of the mixed reference substances; Figure 8 is the high performance liquid chromatography diagram of the test sample; Figure 9 is the high performance liquid chromatography diagram of the blank solvent. Detailed Embodiments

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0021] Example 1 This example provides a method for determining the content of flavonoid components in the extract of Salix cheilophila flowers. The flavonoid components are quercetin and isorhamnetin. Specifically, it includes the following steps: S1: Preparation of reference substance solution: Accurately weigh 4.7 mg of quercetin reference substance and 4.8 mg of isorhamnetin reference substance, place them in a 25 mL volumetric flask, dissolve and make up to the mark with methanol to obtain a mixed reference substance solution with concentrations of quercetin 0.188 mg / mL and isorhamnetin 0.192 mg / mL, which is used as the mixed standard stock solution.

[0022] Dilute the mixed reference substance solution with an appropriate amount of methanol to obtain standard series mixed solutions with concentrations of 0.2, 0.1, 0.05, 0.0025, 0.00125, 0.000625, and 0.003125 mg / mL respectively. Pass through a 0.22 μm microporous filter membrane to obtain the reference substance solution.

[0023] S2: Preparation of test sample solution: S21: Crush the Salix cheilophila flowers and pass through a 40-mesh nylon sieve to obtain Salix cheilophila flower powder. S22: Weigh 5 g of the Salix cheilophila flower powder, place it in a three-necked flask, add 100 mL of 95% ethanol, carry out ultrasonic-assisted reflux extraction at 79 °C for 2.19 h, and then obtain the Salix cheilophila flower extract powder after rotary evaporation.

[0024] Accurately weigh 0.677 g of the Salix cheilophila flower extract powder, place it in a 25 mL volumetric flask, dissolve and make up to the mark with methanol to obtain the test sample solution.

[0025] S3: Establishment of linear regression equation: S31: Detect and analyze the reference substance solution by high performance liquid chromatography technology. The chromatographic conditions were as follows: Wondasil Cis Herb column, 250 mm × 4.6 mm, 5 μm; mobile phase A was methanol, and mobile phase B was 0.1% potassium phosphate solution; the column temperature was 30 °C; the injection volume was 20 μL; the volume flow rate was 1.0 mL / min; the elution gradient was 0 - 10 min: 65% A, 10 - 20 min: 65% A → 48% A, 20 - 30 min: 48% A, 30 - 40 min: 48% A → 65% A.

[0026] S32: Taking the peak areas (y) of the reference substances of quercetin and isorhamnetin as the ordinate and the contents (x, mg / mL) of quercetin and isorhamnetin as the abscissa, establish a linear regression equation; The linear regression equations of quercetin and isorhamnetin in the reference substance solution are shown in Table 1 and Table 2.

[0027] Table 1 Concentration and peak area of quercetin

[0028] Table 2 Concentration and peak area of isorhamnetin

[0029] As can be seen from Table 1 and Table 2, the regression equation of quercetin is y = 80448566.90x - 198592.71, and the regression equation of isorhamnetin is y = 82119203.50x - 211750.27, showing a good linear relationship in the range of 0.003125 - 0.20 mg / mL (R 2 = 0.999).

[0030] S4: Detection of the test solution: The test solution was filtered through a 0.22 μm microporous membrane, and high performance liquid chromatography was used to quantitatively analyze quercetin and isorhamnetin in the test solution; S5: Content calculation: Under the chromatographic conditions of Step S31 above, the test solution was continuously measured 3 times, and the measured values were respectively substituted into the regression equations of quercetin and isorhamnetin to calculate their contents.

[0031] The contents of quercetin and isorhamnetin in the test solution are shown in Table 3 and Table 4.

[0032] Table 3 Results of the content determination of quercetin in red willow flowers

[0033] Table 4 Results of the content determination of isorhamnetin in red willow flowers

[0034] Example 2 Specifically, to verify the rationality of this method, the experimental data was also examined as follows.

[0035] (1) Investigation of mobile phase types Using a Wondasil Cis Herb column (250×4.6 mm, 5 μm), the mobile phases methanol - 0.1% potassium phosphate, methanol - 0.1% phosphoric acid water, methanol - pure water, and acetonitrile - 0.1% phosphoric acid water were respectively investigated. The flow rate was 1.0 mL / min, the column temperature was 30 °C, and the wavelength was 360 nm. The resolution and peak shape of the target peak were observed to select the optimal mobile phase type.

[0036] Figures 1 to 4 are the high - performance liquid chromatography (HPLC) diagrams under different types of mobile phases. It can be seen from Figures 1 to 4 that when the mobile phase is methanol - 0.1% potassium phosphate, the resolution of the target peak is greater than 1.5, the peak shape is obvious, meeting the requirements of HPLC quantitative detection.

[0037] (2) Investigation of mobile phase ratios Based on the above mobile phases, the HPLC diagrams were investigated when the mobile phase ratios were elution gradient a (0 - 10 min: 65% A, 10 - 20 min: 65% A → 52% A, 20 - 30 min: 52% A, 30 - 40 min: 52% A → 65% A) or elution gradient b (0 - 10 min: 65% A, 10 - 20 min: 65% A → 48% A, 20 - 30 min: 48% A, 30 - 40 min: 48% A → 65% A). The flow rate was 1.0 mL / min, the detection wavelength was 365 nm, and the column temperature was 30 °C. The influence of the chromatographic conditions on the resolution and peak shape of the target peak was investigated to determine the optimal mobile phase ratio.

[0038] Figures 5 to 6 are the HPLC diagrams under different elution gradients. The chromatographic conditions for a are: 0 - 10 min: 65% A, 10 - 20 min: 65% A → 52% A, 20 - 30 min: 52% A, 30 - 40 min: 52% A → 65% A; the chromatographic conditions for b are: 0 - 10 min: 65% A, 10 - 20 min: 65% A → 48% A, 20 - 30 min: 48% A, 30 - 40 min: 48% A → 65% A.

[0039] On the basis of methanol - 0.1% potassium phosphate as the mobile phase, the effects of different mobile phase ratios on the resolution and peak shape of the target peak were investigated, and the optimal chromatographic conditions were determined as follows: 0 - 10 min: 65% A, 10 - 20 min: 65% A → 48% A, 20 - 30 min: 48% A, 30 - 40 min: 48% A → 65% A, flow rate: 1.0 mL / min, detection wavelength: 365 nm, column temperature: 30 °C, injection volume: 20 μL. The results are as Figure 6 shown, and the target peak shape has no tailing and the resolution is good.

[0040] (3)Investigation of specificity Precisely pipette the test solution, mixed reference solution, and an appropriate amount of methanol solution respectively, filter through a 0.22 μm microporous filter membrane, and inject for analysis under the chromatographic conditions of Example 1.

[0041] Figures 7 to 9 are the high - performance liquid chromatograms of different samples, where: 1 is quercetin and 2 is isorhamnetin. As Figures 7 to 9 can be seen, the peak emergence times of the test solution of red willow flower and the mixed reference solution coincide, the peak shapes are good, and the blank solvent does not interfere with the determination of the target peak, indicating that the HPLC detection method for two flavonoid compounds in red willow flower established in this experiment has good specificity.

[0042] (4)Precision test Precisely pipette the test solution, filter through a 0.22 μm microporous filter membrane, and inject continuously 6 times under the chromatographic conditions of Example 1 above, and calculate the RSD values of the concentrations of quercetin and isorhamnetin.

[0043] Table 5 Results of precision investigation of quercetin

[0044] Table 6 Results of precision investigation of isorhamnetin

[0045] It can be seen from Table 5 and Table 6 that the average contents of quercetin and isorhamnetin in red willow flower are 0.0090 mg / mL and 0.0073 mg / mL respectively, and the RSD values of their precisions are 1.3% and 0.8% respectively, indicating that the precision of the instrument is good.

[0046] (5)Repeatability test Extract the flavonoid components in red willow according to the method of Example 1, in parallel 6 times. Filter the prepared test solution through a 0.22 μm microporous filter membrane, inject under the chromatographic conditions of Example 1 above, measure the peak areas of quercetin and isorhamnetin, and calculate the RSD value.

[0047] Table 7 Results of repeatability investigation of quercetin

[0048] Table 8 Results of reproducibility investigation of isorhamnetin

[0049] As can be seen from Table 7 and Table 8, the average contents of quercetin and isorhamnetin in tamarix chinensis are 0.0092 mg / mL and 0.0078 mg / mL respectively, and the RSD values are 2.0% and 1.8% respectively, indicating good reproducibility.

[0050] (6)Spiked recovery test Precisely pipette the test solution prepared in Example 1, and add 3 portions of mixed standard solutions at low, medium, and high (80%, 100%, 120%) levels respectively. Filter through a 0.22 μm microporous filter membrane and determine according to the chromatographic conditions in Example 1, and calculate the spiked recoveries of quercetin and isorhamnetin respectively.

[0051] Table 9 Results of spiked addition investigation of quercetin

[0052] Table 10 Results of spiked addition investigation of isorhamnetin

[0053] As can be seen from Table 9 and Table 10, the average recoveries of quercetin and isorhamnetin are 101.3% and 99.3% respectively, and their RSD values are 3.7% and 2.37% respectively. The recoveries are high and meet the analysis requirements.

[0054] (7)Stability test Prepare a test solution with a concentration of 2.7 mg / mL according to the method in Example 1, and dilute the prepared solution with an appropriate amount of methanol to prepare test solutions at 3 concentrations of 2.7, 1.35, and 0.675 mg / mL at high, medium, and low levels. Filter through a 0.22 μm microporous filter membrane and place at room temperature for 0, 2, 4, and 8 h. Each concentration is determined in parallel 3 times according to the chromatographic conditions in Example 1, and the RSD values of quercetin and isorhamnetin at high, medium, and low concentrations are measured.

[0055] Table 11 Results of stability investigation of quercetin

[0056] Table 12 Results of stability investigation of isorhamnetin

[0057] As can be seen from Table 11 and Table 12, the RSDs of quercetin and isorhamnetin at high, medium, and low concentrations are all less than 5%, indicating good stability of the sample within 8 h.

[0058] The above are the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for determining the content of flavonoids in red willow flower extract, characterized in that: The flavonoid components are quercetin and isorhamnetin; The specific steps include: S1: Preparation of reference solution: a certain amount of quercetin reference substance and isorhamnetin reference substance were mixed and dissolved in methanol, and diluted to obtain reference solution of different concentrations; S2: Preparation of test solution: S21: crushing the red willow flowers to obtain red willow flower powder; S22: adding an organic solvent to the red willow flower powder, performing ultrasonic heating, performing reflux extraction, and obtaining a red willow flower extract after rotary evaporation; Add the red willow flower extract to methanol to obtain a test solution; S3: Linear regression equation establishment: S31: detecting and analyzing the reference solution by high performance liquid chromatography; S32: Establish a linear regression equation with the peak area of ​​the reference substances of quercetin and isorhamnetin as the ordinate and the content of quercetin and isorhamnetin as the abscissa; S4: Test solution detection: Quantitative analysis of quercetin and isorhamnetin in the test solution was performed using high performance liquid chromatography; S5: Assay calculation: The contents of quercetin and isorhamnetin in the test solution are calculated by combining the linear regression equation obtained in step S3 and the results of the quantitative analysis of the test solution in step S4.

2. The method for determining the content of flavonoids in a red willow flower extract according to claim 1, characterized in that: In step S1, 5.0 mg of quercetin reference substance and 5.0 mg of isorhamnetin reference substance were weighed respectively, placed in a 25 mL volumetric flask, and dissolved to the mark with methanol to form a mixed reference substance solution with a concentration of 0.2 mg / mL for quercetin and 0.2 mg / mL for isorhamnetin, as a mixed standard stock solution; The mixed standard stock solution was diluted with methanol to obtain a series of standard mixed solutions of different concentrations, and then filtered through a 0.22 μm microporous filter membrane to obtain reference substance solutions of different concentrations.

3. The method for determining the content of flavonoids in a red willow flower extract according to claim 1, characterized in that: In step S21, the red willow flowers are crushed and passed through a 40-mesh nylon sieve to obtain red willow flower powder.

4. The method for determining the content of flavonoids in a red willow flower extract according to claim 1, characterized in that: In step S22, the organic solvent is 95% ethanol, and the mass volume ratio of red willow flower powder to the organic solvent is (3-8) g:100 mL.

5. The method for determining the content of flavonoids in a red willow flower extract according to claim 1, characterized in that: In step S22, the heating temperature is 75-85°C, and the reflux extraction time is 1-10 h.

6. The method for determining the content of flavonoids in a red willow flower extract according to claim 1, characterized in that: In step S22, 0.677 g of red willow extract powder was accurately weighed and placed in a 25 mL volumetric flask, and dissolved in methanol to the mark to obtain the test solution.

7. The method for determining the content of flavonoids in a red willow flower extract according to claim 1, characterized in that: The chromatographic conditions of the HPLC technique were as follows: Wondasil Cis Herb column, 250 mm×4.6 mm, 5 μm; mobile phase A was methanol, and mobile phase B was 0.1% potassium phosphate solution; column temperature was 30 °C; injection volume was 20 μL; volume flow rate was 1.0 mL / min; elution gradient was 0~10 min: 65% A, 10~20 min: 65% A→48% A, 20~30 min: 48% A, 30~40 min: 48% A→65% A.

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