Method for detecting content of rosmarinic acid in anchusa flowers

Abstract

The invention discloses a method for detecting the content of rosmarinic acid in anchusa coupling flower. The method comprises the following steps: detecting rosmarinic acid in a test solution by using high performance liquid chromatography; the test solution is an oxtongue flower extracting solution; in the high performance liquid chromatography, the detection wavelength is 328 nm; a mobile phase A is a formic acid solution with the volume percentage of 0.05%-0.15%, a mobile phase B is acetonitrile, and gradient elution is carried out. According to the method for detecting the rosmarinic acid in the anchusa flowers, the accuracy, reproducibility and reliability of quantitative analysis of the rosmarinic acid are ensured by optimizing chromatographic conditions and performing methodological verification. The detection method disclosed by the invention is efficient, strong in specificity, high in precision and good in linear relationship, and can provide a scientific basis for measuring the content of rosmarinic acid in a sample.

Description

Technical Field

[0001] This invention relates to a method for detecting the rosmarinic acid content in oxtail grass flowers. Background Technology

[0002] In the 1998 edition of the "Pharmaceutical Standards of the Ministry of Health of the People's Republic of China (Uyghur Medicine Volume)," *Anchusaitalica* flower was not listed as a separate medicinal material. The appendix stated that *Anchusaitalica* flower is derived from the dried flower buds of *Anchusaitalica* Retz., a plant in the Boraginaceae family, and is a commonly used medicinal material in Uyghur medicine. The dried flower buds of *Borageofficinalis* L., also belonging to the Boraginaceae family, are also used as substitutes in Uyghur medicine practice. Currently, the medicinal material standard is included in the first volume of the 2010 edition of the "Uyghur Medicinal Material Standards of Xinjiang Uyghur Autonomous Region," which only tests for appearance and microscopic identification. The existing quality standards are insufficient to comprehensively control the quality of the medicinal material.

[0003] Currently, Yangxin Dawaimishik Honey Ointment is the earliest approved Uyghur medicine classic compound preparation, composed of 26 medicinal materials including musk, sandalwood, agarwood, saffron, ox-tongue flower, and rose, and has been used clinically for decades. Ox-tongue flower, as the main ingredient in this preparation, is used not only for heart diseases but also for mental illnesses that pose challenges to modern medicine, such as anxiety and depression, making it a treasure among Uyghur medicinal materials. However, there are currently issues with the material basis of ox-tongue flower and incomplete quality control standards. Therefore, improving its quality standards is crucial for establishing and perfecting the overall quality control system of this preparation. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the defects of the prior art in the study of oxalis flowers, which have unclear material basis and imperfect quality control standards, and to provide a method for detecting the content of rosmarinic acid in oxalis flowers.

[0005] Boletus lucidum refers to the dried whole herb, while boletus lucidum flowers are the dried flower buds of Boletus lucidum or Borage. Currently, existing technologies only address quality control for the whole herb of Boletus lucidum; there are no methods for controlling the flowers. Since Boletus lucidum and its flowers are two separate medicinal parts, research has found that quality control methods for Boletus lucidum are not applicable to its flowers. For example, chromatographic conditions used in fingerprinting methods for Boletus lucidum cannot effectively separate substances in its flowers when applied to them. Similarly, methods for detecting rosmarinic acid content in Boletus lucidum are also unsuitable for its flowers. Therefore, quality control methods differ between different medicinal parts, and the content and distribution of chemical components in different medicinal parts of the same plant can also vary; the two should not be confused.

[0006] Different medicinal parts of the same plant often use different components as indicators for content determination. For example, *Isatis tinctoria* leaf is the dried leaf of *Isatis tinctoria*, a plant in the Brassicaceae family, while *Isatis tinctoria* root is the dried root of the same plant. Although both belong to the plant *Isatis tinctoria*, different parts are used as separate medicinal materials. The *Chinese Pharmacopoeia* records that *Isatis tinctoria* leaf uses indirubin as the indicator component for content determination, accurately reflecting its intrinsic quality. However, *Isatis tinctoria* root uses the glucosinolate (R,S)-galoidin as the indicator component for content determination. This demonstrates that different medicinal parts of the same plant require different components as content determination indicators and cannot be used interchangeably.

[0007] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0008] This invention provides a method for detecting rosmarinic acid content in oxtail grass flowers, which includes the following steps: detecting rosmarinic acid in the test sample solution using high performance liquid chromatography;

[0009] The test solution is an extract of *Hedyotis diffusa* flower;

[0010] In the high-performance liquid chromatography, the detection wavelength is 328 nm; the mobile phase A is a 0.05%~0.15% (v / v) formic acid solution, and the mobile phase B is acetonitrile; the gradient elution conditions for the mobile phase A and the mobile phase B are as follows:

[0011]

[0012] The percentages in the table represent the volume percentage of each component in the total volume of mobile phase A and mobile phase B, respectively.

[0013] In this invention, the solvent used in the test solution is preferably an alcohol or an aqueous alcohol solution.

[0014] The alcohol in the alcohol or the aqueous alcohol solution may be methanol or ethanol. The volume percentage of the aqueous alcohol solution may be as is common in the art.

[0015] In this invention, the method for preparing the test solution preferably includes the following steps: ultrasonic extraction of a mixture of ox-tongue flower and solvent, filtration, concentration, drying, and dilution to obtain the test solution.

[0016] The preferred mass-to-volume ratio of the ox-tongue flower to the solvent is (0.8~1.2) g:100 mL, for example, 1 g:100 mL.

[0017] The power of the ultrasonic extraction can be 250 W.

[0018] The frequency of the ultrasonic extraction can be 40 kHz.

[0019] The ultrasonic extraction time can be 0.25~1.0h, for example 0.5h.

[0020] Before filtration, the ultrasonic extract is typically cooled to room temperature naturally.

[0021] The solvent used for redissolution is generally the same as the solvent used for ultrasonic extraction.

[0022] After redissolution, the solution is typically filtered through a 0.22 μm microporous membrane, and the filtrate is collected.

[0023] In a preferred embodiment, the test solution is prepared by the following steps: 1 g of *Houttuynia cordata* flower powder (passed through a No. 4 sieve) and a mixture of methanol are ultrasonically extracted (250 W, 40 kHz) for 30 minutes, cooled to room temperature, filtered, concentrated, dried, dissolved in methanol and diluted to 100 mL, filtered through a 0.22 μm microporous membrane, and the resulting filtrate is the test solution.

[0024] In this invention, the chromatographic column used in the high-performance liquid chromatography (HPLC) detection is preferably a Waters Xbridge Shield RPC18. The column specifications are preferably 1.7 μm, 100 mm × 2.1 mm.

[0025] In this invention, the volume percentage of the mobile phase A in the high-performance liquid chromatography detection is preferably 0.08~0.12%, for example 0.1%.

[0026] In this invention, the injection volume in the high-performance liquid chromatography detection can be conventional in the art, preferably 1~4μL, for example 2μL.

[0027] In this invention, the flow rate of the test solution in the high-performance liquid chromatography detection is preferably 0.1~0.5 mL / min, more preferably 0.25 mL / min.

[0028] In this invention, the column temperature in the high-performance liquid chromatography detection is preferably 32~38℃, for example 35℃.

[0029] In this invention, preferably, during the high-performance liquid chromatography detection, chromatograms are recorded for at least 7.5 minutes.

[0030] In this invention, the preferred linear regression equation and linear range for the *Hylocereus undatus* flower are as follows: where X is the concentration in µg / mL, and Y is the peak area × 10. -4 :

[0031] Y = 1.2338X - 0.0165, linear range 28 μg / mL - 168 μg / mL R 2 = 0.9997.

[0032] In a preferred embodiment, the detection method may include the following steps:

[0033] Step (1): Prepare a series of reference solutions and test solutions of different concentrations, and analyze the reference solutions and test solutions using the above detection method; measure the peak area of ​​rosmarinic acid in the reference solutions and test solutions;

[0034] Step (2): Based on the peak area and corresponding concentration of rosmarinic acid in the reference solution, perform linear regression to obtain the standard curve equation;

[0035] Step (3): The peak area measured in step (1) × 10 -4 Substituting the Y value into the standard curve equation obtained in step (2), the concentration of rosmarinic acid in the test solution is obtained. The content of rosmarinic acid in the medicinal material can be calculated from the concentration of rosmarinic acid in the test solution using conventional methods.

[0036] In step (1), the preparation of the reference solution or the test solution can be done using a conventional solvent that can dissolve it, such as methanol.

[0037] The concentration of the reference solution can be referenced from the concentration used in preparing the standard curve equation of concentration versus peak area in the art, for example, 20~130 μg / mL, or for example, 28 μg / mL, 56 μg / mL, 84 μg / mL, 140 μg / mL or 168 μg / mL.

[0038] In step (2), the standard curve equation can be obtained by referring to conventional calculation methods in the art. For example, the concentration of rosmarinic acid in the reference solution can be used as the abscissa, and the peak area corresponding to the corresponding rosmarinic acid concentration can be multiplied by 10. -4 Using the vertical axis as the ordinate, perform linear regression to obtain the standard curve equation.

[0039] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0040] The reagents and raw materials used in this invention are all commercially available.

[0041] The positive and progressive effects of this invention are as follows:

[0042] To control the safety of *Hylocereus undatus* flowers and their extracts, the method for detecting rosmarinic acid in *Hylocereus undatus* flowers in this invention optimizes chromatographic conditions and validates the methodology, ensuring the accuracy, reproducibility, and reliability of the quantitative analysis of rosmarinic acid. This invention's detection method is efficient, specific, precise, and exhibits good linearity, providing a scientific basis for determining the content of rosmarinic acid in samples. Attached Figure Description

[0043] Figure 1 The UV spectrum of rosmarinic acid.

[0044] Figure 2 This is the standard curve for rosmarinic acid.

[0045] Figure 3 This is an HPLC chromatogram of rosmarinic acid reference standard and oxtail grass flower. The black curve represents oxtail grass flower, and the blue curve represents rosmarinic acid reference standard. Detailed Implementation

[0046] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0047] Instruments and Materials

[0048] Instruments: Sartorius CPA124s electronic analytical balance (Sartorius, Germany); DZF-6050B vacuum drying oven (Shanghai Qixin Scientific Instruments Co., Ltd., China); SK7210LH ultrasonic cleaner (Shanghai Kedao Ultrasonic Instruments Co., Ltd., China).

[0049] Reagents: Chromatographic grade acetonitrile, methanol, and formic acid (Merck, Germany); ultrapure water (Watsons, China); rosmarinic acid reference standard (batch number 23020112, purchased from Sichuan Weikeqi Biotechnology Co., Ltd.); all other analytical grade reagents (Tianjin Guangfu Fine Chemical Research Institute, China).

[0050] Materials: The medicinal herbs were sourced from Iran and Pakistan, and were obtained through conventional commercial channels. Relevant sample numbers and origin information are as follows:

[0051] surface Source and batch number of ox tongue grass flower medicinal material

[0052]

[0053] Example 1: Preparation of Standard Curve

[0054] Accurately weigh 0.0070 g of rosmarinic acid reference standard into a 25 mL volumetric flask, dissolve in methanol and dilute to the mark, shake well, and prepare a solution containing 280 µg of reference standard per 1 mL.

[0055] Accurately measure 1 mL, 2 mL, 3 mL, 5 mL, and 6 mL of rosmarinic acid reference solution respectively, place them in 10 mL volumetric flasks, and dilute with methanol to prepare reference solutions with concentrations of 28 µg / mL, 56 µg / mL, 84 µg / mL, 140 µg / mL, and 168 µg / mL.

[0056] Selection of detection wavelength: The absorption wavelength of rosmarinic acid reference standard was scanned using a UV spectrophotometer. The results showed that, in addition to terminal absorption at 210 nm, rosmarinic acid reference standard exhibited the highest absorption at 328 nm (see...). Figure 1 , Figure 1 (This is the UV spectrum of rosmarinic acid), therefore 328 nm was chosen as the detection wavelength for content determination.

[0057] Chromatographic conditions: Column: Waters Xbridge Shield RPC18 (1.7 μm, 100 mm × 2.1 mm); Mobile phase: 0.1% formic acid solution (A) - acetonitrile (B); Column temperature: 35℃; Flow rate: 0.25 mL / min; Injection volume: 2 μL; Detection wavelength: 328 nm. Gradient elution mode was used, with the following elution gradient:

[0058] Table 2

[0059]

[0060] Perform the determination under the above chromatographic conditions, record the peak area, and plot the reference standard concentration (X) on the x-axis, then multiply the reference standard peak area by 10. -4 Linear regression was performed on the ordinate, and the results are shown in the table below. Figure 2 .

[0061] Table 3. Concentrations and corresponding peak areas of rosmarinic acid reference standards

[0062]

[0063] Table 4. Linear equation and linear range of rosmarinic acid reference standard

[0064] (Y is the peak area × 10) -4 (where X is the concentration in μg / mL)

[0065]

[0066] Figure 2 This is the standard curve for rosmarinic acid.

[0067] Example 2

[0068] (1) Preparation of test solution: Weigh 1 g of ox tongue grass flower powder (passed through No. 4 sieve), accurately weigh it, place it in a 100 mL volumetric flask, add an appropriate amount of methanol, extract by ultrasonication (250 W, 40 kHz) for 30 minutes, cool to room temperature, filter, concentrate, dry, dilute with methanol to the mark, filter with a 0.22 μm microporous membrane, and take the filtrate.

[0069] Accurately weigh 10 batches of *Hedyotis diffusa* flower sample powder from Table 1, and prepare the sample solution according to the above preparation method.

[0070] (2) Preparation of rosmarinic acid reference solution: Accurately weigh an appropriate amount of rosmarinic acid, dissolve it in methanol, and prepare a reference stock solution containing 500 μg of reference standard per 1 mL. Accurately pipette the reference stock solution and dilute it with methanol to different concentrations, and shake well.

[0071] (3) Accurately pipette 10 μL each of the reference solution and the test solution and inject them into the high-performance liquid chromatograph (HPLC) for determination. The specific methods and conditions for HPLC detection are the same as in Example 1. Determine the content according to the external standard method of HPLC (0512) in Part IV of the 2020 edition of the Chinese Pharmacopoeia. Calculate the content based on the peak area by multiplying the peak area of ​​the test solution by 10. -4 Substituting Y into the standard curve of Example 1, we obtained X, which is the concentration of rosmarinic acid in the test solution. The results are shown in the table below.

[0072] Table 5. Determination of rosmarinic acid content in *Hedyotis diffusa* flower medicinal material

[0073]

[0074] Experimental results showed that rosmarinic acid (C) was present in 10 batches of medicinal materials. 18 H 16 The content of rosmarinic acid (C8) is between 0.48% and 0.74%, with an average content of 0.60%. Using 80% as the limit (this limit standard can ensure that the content in more than 80% of batches meets the standard), it is recommended to... 18 H 16 The content of O8 shall not be less than 0.5%.

[0075] Note: The percentage of rosmarinic acid in the table represents the mass percentage of rosmarinic acid in the medicinal material (i.e., the number of grams of rosmarinic acid contained in each 1g of medicinal material).

[0076] Figure 3The figures show the HPLC chromatograms of rosmarinic acid reference standard and *Hedyotis diffusa* flower (sample number S01). The black curve represents *Hedyotis diffusa* flower, and the blue curve represents rosmarinic acid reference standard. This indicates that under these chromatographic conditions, the chromatograms of *Hedyotis diffusa* flower and the reference standard show peaks at the same retention times, with no interference from other components, good peak shapes, and complete separation.

[0077] Effect Example

[0078] (1) Precision test

[0079] Accurately weigh the powdered *Houttuynia cordata* flower sample (S01), extract it using the method described in "Example 2: Preparation of Test Solution", and continuously inject it 6 times under the chromatographic conditions of "Example 1" to determine the peak area. Calculate the concentration and content of rosmarinic acid, and then calculate its relative standard deviation. The results are shown in the table below. The RSD of the rosmarinic acid peak area is 0.75%, indicating good instrument precision.

[0080] Table 6 Precision test results

[0081]

[0082] (2) Repeatability test

[0083] Accurately weigh six portions of the same batch of *Hedyotis diffusa* flower sample powder (S01), and extract them using the method described in "Example 2: Preparation of Test Solution". Measure the peak area according to the chromatographic conditions described in "Example 1", calculate the concentration and content of rosmarinic acid, and then calculate the relative standard deviation. The results are shown in the table below. The RSD of rosmarinic acid is 2.10%, indicating good method repeatability.

[0084] Table 7 Repeatability Experiments

[0085]

[0086] (3) Stability test

[0087] Accurately weigh the *Hylocereus undatus* flower sample powder (S01) and extract it according to the method described in "Example 2 Test Solution Preparation Method". Peak areas were measured at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h, and 48 h according to the chromatographic conditions described in "Example 1". The concentration and content of rosmarinic acid were calculated, and its relative standard deviation was also calculated. The results are shown in the table below. The RSD of the rosmarinic acid peak area was 0.54%, indicating that the test solution was basically stable within 48 h.

[0088] Table 8. Stability test of rosmarinic acid

[0089]

[0090] (4) Spike recovery experiment

[0091] Accurately weigh 9 portions of *Hedyotis diffusa* flower powder (S01), each approximately 0.5g. Separately mix the rosmarinic acid reference solution (C...) with the powder. 迷迭香酸 =1.24 mg / mL) was added to 9 portions of medicinal materials in high (1:1.2), medium (1:1), and low (1:0.8) ratios. Extraction was performed according to the "Preparation Method of Test Solution in Example 2". Peak area was determined according to the "Chromatographic Conditions in Example 1". Recovery rate and relative standard deviation were calculated. The recovery rate was 102.01% and the RSD was 2.90%, as shown in the table below.

[0092] Table 9. Recovery rate of rosmarinic acid in *Hedyotis diffusa* flower (n=3)

[0093]

[0094] Note: In the table, the measured value represents the total mass (mg) of rosmarinic acid in the test solution, calculated from the peak area using a standard curve, and then multiplied by the solution volume. In HPLC spiking recovery experiments, "measured value" refers to the total mass (unit: mg) of rosmarinic acid measured from the test solution, including the rosmarinic acid contained in the herb itself (background value) and the mass of the added reference standard.

Claims

1. A method for detecting the rosmarinic acid content in *Hedyotis diffusa* flowers, characterized in that, It includes the following steps: detecting rosmarinic acid in the test solution using high performance liquid chromatography; The test solution is an extract of *Hedyotis diffusa* flower; In the high-performance liquid chromatography, the detection wavelength is 328 nm; the mobile phase A is a 0.05%~0.15% (v / v) formic acid solution, and the mobile phase B is acetonitrile; the gradient elution conditions for the mobile phase A and the mobile phase B are as follows: ； The percentages in the table represent the volume percentage of each component in the total volume of mobile phase A and mobile phase B, respectively.

2. The method for detecting rosmarinic acid content in *Hedyotis diffusa* flowers as described in claim 1, characterized in that, In the high-performance liquid chromatography detection, the volume percentage of mobile phase A is 0.08~0.12%, preferably 0.1%.

3. The method for detecting rosmarinic acid content in *Hedyotis diffusa* flowers as described in claim 1, characterized in that, The linear regression equation and linear range of the *Hedyotis diffusa* flower are as follows: X is the concentration in µg / mL, Y is the peak area × 10. -4 : Y = 1.2338X - 0.0165, linear range 28 μg / mL - 168 μg / mL R 2 = 0.9997.

4. The method for detecting rosmarinic acid content in *Hedyotis diffusa* flowers as described in claim 1, characterized in that, The detection method satisfies one or more of the following conditions: (1) In the high performance liquid chromatography detection, the chromatographic column is a Waters Xbridge Shield RPC18; (2) In the high performance liquid chromatography detection, the injection volume is 1~4 μL; (3) In the high-performance liquid chromatography detection, the flow rate of the test solution is 0.1~0.5 mL / min; and, (4) In the high performance liquid chromatography detection, the column temperature is 32~38℃.

5. The method for detecting rosmarinic acid content in *Hedyotis diffusa* flowers as described in claim 1, characterized in that, The detection method satisfies one or more of the following conditions: (1) In the high performance liquid chromatography detection, the chromatographic column has a size of 1.7 μm and a diameter of 100 mm × 2.1 mm; (2) In the high performance liquid chromatography detection, the injection volume is 2 μL; (3) In the high-performance liquid chromatography detection, the flow rate of the test solution is 0.25 mL / min; and, (4) In the high performance liquid chromatography detection, the column temperature is 35℃.

6. The method for detecting rosmarinic acid content in *Hedyotis diffusa* flowers as described in claim 1, characterized in that, The detection method satisfies (1) and / or (2) of the following conditions: (1) The solvent used in the test solution is an alcohol or an aqueous alcohol solution; the alcohol in the alcohol or the aqueous alcohol solution is preferably methanol or ethanol; (2) The preparation method of the test solution includes the following steps: ultrasonic extraction of a mixture of ox tongue grass flower and solvent, filtration, concentration, drying and dilution to obtain the test solution.

7. The method for detecting rosmarinic acid content in *Hedyotis diffusa* flowers as described in claim 6, characterized in that, The method for preparing the test solution satisfies one or more of the following conditions: (1) The mass-to-volume ratio of the ox-tongue flower to the solvent is (0.8~1.2) g:100 mL, preferably 1 g:100 mL; (2) The power of the ultrasonic extraction is 250 W; (3) The frequency of the ultrasonic extraction is 40 kHz; and, (4) The ultrasonic extraction time is 0.25~1.0h, preferably 0.5h.

8. The method for detecting rosmarinic acid content in *Hedyotis diffusa* flowers as described in claim 1, characterized in that, The preparation method of the test solution includes the following steps: 1 g of ox tongue grass flower powder and methanol mixture, ultrasonically extracted at 250 W and 40 kHz for 30 minutes, cooled to room temperature, filtered, concentrated, dried, dissolved in methanol and diluted to 100 mL, filtered through a 0.22 μm microporous membrane, and the resulting filtrate is the test solution.

9. The method for detecting rosmarinic acid content in *Hedyotis diffusa* flowers as described in claim 1, characterized in that, The detection method may include the following steps: Step (1): Prepare a series of reference solutions and test solutions of different concentrations, and analyze and detect the reference solutions and test solutions using the detection method. The peak areas of rosmarinic acid in the reference solution and the test solution were measured. Step (2): Based on the peak area and corresponding concentration of rosmarinic acid in the reference solution, perform linear regression to obtain the standard curve equation; Step (3): The peak area measured in step (1) × 10 -4 Substitute the Y value into the standard curve equation obtained in step (2) to obtain the concentration of rosmarinic acid in the test solution.

10. The method for detecting rosmarinic acid content in *Hedyotis diffusa* flowers as described in claim 9, characterized in that, In step (1), the concentration of the reference solution is 20~130 μg / mL, preferably 28 μg / mL, 56 μg / mL, 84 μg / mL, 140 μg / mL or 168 μg / mL; And / or, in step (2), the concentration of rosmarinic acid in the reference solution is used as the abscissa, and the peak area corresponding to the corresponding rosmarinic acid concentration is multiplied by 10. -4 Using the vertical axis as the ordinate, perform linear regression to obtain the standard curve equation.

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