A quality testing method for yarrow

Abstract

This invention discloses a quality detection method for yarrow. Through extensive experimentation, the optimal mobile phase composition, elution procedure, flow rate, and chromatographic column conditions were selected. This invention established HPLC fingerprint characteristic spectra of 21 batches of yarrow, identified and designated 13 common characteristic peaks, and established an HPLC method for the simultaneous determination of the content of these six characteristic components with similar polarities in yarrow. This HPLC method exhibits good repeatability, stability, and specificity, and is of great significance for controlling the quality of yarrow and ensuring clinical efficacy.

Description

Technical Field

[0001] This invention relates to a quality control method for traditional Chinese medicine, specifically a quality testing method for yarrow. Background Technology

[0002] Yarrow is the dried aerial part of *Achillea alpina* L., a plant in the Asteraceae family. It is distributed in Henan, Gansu, Inner Mongolia, and Ningxia provinces of my country, with abundant wild resources and significant development value. In the 2020 edition of the *Chinese Pharmacopoeia*, it is listed as having detoxifying, diuretic, blood-activating, and analgesic effects. Early clinical trials have demonstrated that yarrow has good therapeutic effects on snake and insect bites, appendicitis, gastroenteritis, and pyelonephritis.

[0003] In the 2020 edition of the Chinese Pharmacopoeia, chlorogenic acid is listed as a quality control component for yarrow, and its content is determined. However, a single component is insufficient to comprehensively control the overall quality of traditional Chinese medicine. To improve and perfect the quality standard system for traditional Chinese medicine and to refine the methods for quality evaluation and control, quality markers (Q-Markers) for traditional Chinese medicine possess advantages of being holistic, systematic, and quantifiable. They can comprehensively evaluate the quality of traditional Chinese medicine, pointing to a new direction for research on the quality evaluation and control of traditional Chinese medicine.

[0004] The chemical composition of yarrow plants is complex, containing a variety of components such as flavonoids, alkaloids, organic acids, phenylpropanoids, terpenoids, steroids, and volatile oils. Traditional Chinese medicine fingerprinting can accurately describe the authenticity, stability, and uniformity of the quality of traditional Chinese medicine and its preparations, even when the chemical composition of the herbs is unclear.

[0005] Currently, there are no patents for quality testing methods for yarrow. Therefore, in order to fully control the clinical safety of yarrow, its extracts, and preparations, and to protect the interests of patients, it is necessary to research and design a detection method that can accurately and comprehensively detect the effective components of yarrow and its preparations based on existing technologies. Summary of the Invention

[0006] Purpose of the Invention: The purpose of this invention is to overcome the shortcomings of existing technologies. Through extensive experimental screening, this invention provides a method for determining the content of effective components in yarrow and a fingerprint spectroscopy detection method. This method has high detection sensitivity and good stability, and can objectively, comprehensively, and accurately evaluate the quality of yarrow medicinal materials, their extracts, and preparations. It is of great significance for quality control and ensuring efficacy.

[0007] Technical solution: To achieve the above objectives, the technical solution adopted by this invention is as follows:

[0008] A method for quality testing of yarrow, comprising the following steps:

[0009] Step (1): Preparation of mixed reference solution

[0010] Accurately weigh chlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, cypermethrin II and 3,4,5-tricaffeoylquinic acid and dissolve them in methanol to prepare a single reference stock solution; take appropriate amounts of the above six reference stock solutions, filter them through a microporous membrane, and mix them to prepare a mixed reference solution;

[0011] Step (2) Preparation of the test solution

[0012] Take yarrow powder, crush and sieve it, extract it with methanol using ultrasound, filter the filtrate through a microporous membrane to obtain the test solution;

[0013] Step (3) Establishing the linear regression equation

[0014] The mixed reference standard from step (1) was gradually diluted to obtain a series of reference standard solutions of different concentrations. These solutions were then injected into the HPLC system. The mass concentration of the series of reference standards was used as the abscissa X and the corresponding peak area was used as the ordinate Y. Linear regression analysis was performed on each chemical component and the linear regression equation was calculated.

[0015] Step (4) Content determination

[0016] Take the yarrow test solution from step (2) and inject it into HPLC for analysis. Substitute the peak area into the linear regression equation from step (3) to calculate the content of chlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, yarrow extract II and 3,4,5-tricaffeoylquinic acid in the test solution.

[0017] Step (5) Establishment of fingerprint map

[0018] Export the fingerprint spectrum of the test solution in step 4 and import it into the 2012 version of the Chinese herbal chromatographic fingerprint spectrum similarity evaluation system; using the S1 herbal chromatogram as a reference, a total of 13 common peaks were identified by the median method to obtain the fingerprint spectrum of yarrow; and the chemical components of the peaks in the reference fingerprint spectrum were labeled according to the retention time of the chromatogram of the mixed reference solution.

[0019] As a preferred option, in the above-described method for quality testing of yarrow, step (1) involves the preparation of a mixed reference solution.

[0020] Accurately weigh chlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, cypermethrin II, and 3,4,5-tricaffeoylquinic acid, and dissolve them in methanol to prepare single reference stock solutions. Take appropriate amounts of the above six single reference stock solutions, filter them through a microporous membrane, and mix them to prepare mixed reference solutions with concentrations of 0.5 mg / mL, 0.8 mg / mL, 0.8 mg / mL, 0.5 mg / mL, 0.5 mg / mL, and 0.5 mg / mL, respectively.

[0021] As a preferred embodiment, the method for preparing the test solution in step (2) of the above-described method for detecting the quality of yarrow is as follows:

[0022] Take 21 batches of yarrow powder, crush it through a No. 2 sieve, extract it with 20 times the volume concentration of 70% methanol, and ultrasonically extract it at 40℃ for 45 min. Then filter the filtrate through a microporous membrane to obtain the test solution.

[0023] As a preferred embodiment, the HPLC chromatographic conditions in steps (3) and (4) of the above-described method for quality detection of yarrow are as follows:

[0024] use HSS T3C18 column, 4.6 mm × 150 mm, 5 μm; mobile phase: methanol (phase A) and 0.1% formic acid (phase B), gradient elution; flow rate: 1.0 mL / min. -1 Detection wavelength: 260 nm; column temperature: 35 °C; injection volume: 10 μL.

[0025] Screening experiment:

[0026] 1. Selection of detection wavelength

[0027] The same test solution was used to scan the yarrow sample under UV light at wavelengths of 200–400 nm. Numerous chromatographic peaks and good responses were observed at wavelengths of 260 nm and 290 nm. However, compared to the high response value at 290 nm, the reduced response of peak 13 at 260 nm resulted in better elution of all peaks at this wavelength. Therefore, 260 nm was selected as the detection wavelength in this study.

[0028] 2. Optimization of chromatographic conditions

[0029] This invention investigated the effect of different ratios of acetonitrile-formic acid water and methanol-formic acid water as mobile phases on the separation of all components in yarrow. The results showed that when acetonitrile was used as the organic phase, isochlorogenic acid A and isochlorogenic acid B could not be effectively separated, while methanol-formic acid water as the elution solvent enabled complete separation. After determining the mobile phase, this invention further screened gradient elution methods, finding that different elution methods yielded significantly different degrees of separation. Since yarrow contains multiple quinic acid compounds with similar polarity, and separation on a single column is extremely challenging, this invention screened mobile phase compositions at different time points through extensive experiments to determine the optimal gradient elution method.

[0030] 3. Selection of column temperature and flow rate

[0031] Chromatograms were recorded at different column temperatures (25, 30, 35℃). It was found that the peak shape and resolution of each peak in the HPLC chromatogram of the yarrow sample were better at a temperature of 35℃.

[0032] Beneficial effects: The quality testing method for yarrow provided by this invention has the following advantages compared with the prior art:

[0033] This invention, based on the different active components in yarrow, screened the optimal mobile phase composition, elution procedure, flow rate, chromatographic column, and other analytical conditions through extensive experiments. This invention established HPLC fingerprint characteristic spectra of 21 batches of yarrow, identified and designated 13 common characteristic peaks, and established an HPLC method for the simultaneous determination of the content of these six characteristic components with similar polarities in yarrow. This HPLC method exhibits good repeatability, stability, and specificity, and is of great significance for controlling the quality of yarrow and ensuring clinical efficacy. Attached Figure Description

[0034] Figure 1 This is a high-performance liquid chromatogram of the mixed reference solution.

[0035] Figure 2 Fingerprints of 21 batches of yarrow medicinal materials. Detailed Implementation

[0036] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art will fall within the scope defined by the appended claims.

[0037] Example 1

[0038] Reagents and materials used in this embodiment

[0039] 1. Instruments and reagents

[0040] 1.1 Instruments

[0041] name Production Company model High Performance Liquid Chromatography Waters Company, USA 2695 Photodiode matrix detector Shanghai Waters Technology Co., Ltd. 2998 ultrasonic cleaner Kunshan Ultrasonic Instruments Co., Ltd. KQ-500DE Ultrapure water system Merck Millipore, Germany IQ7000 Electronic analytical balance Mettler Toledo, Switzerland MS105DU Electronic balance Mettler Toledo, Switzerland ME204

[0042] 1.2 Reagents

[0043] name Production Company batch number chlorogenic acid Shanghai Yuanye Biotechnology Co., Ltd. Y20A11K111541 Isochlorogenic acid A Chengdu Lockema Biotechnology Co., Ltd. CHB180921 Isochlorogenic acid B Chengdu Delite Biotechnology Co., Ltd. DST191008-037 Isochlorogenic acid C Chengdu Delite Biotechnology Co., Ltd. DSTDY003801 Bai Rui Cao Su II / / 3,4,5-tricaffeoylquinicacid / /

[0044] 1.3 Yarrow herb

[0045] All the yarrow used in this study were purchased from Tongling Hetian Traditional Chinese Medicine Pieces Co., Ltd., and identified by Professor Yan Yonggang of Shaanxi University of Traditional Chinese Medicine as the dried whole herb of *Achillea millefolium* AL, a plant in the Asteraceae family. Information on 21 batches of yarrow is shown in Table 1.

[0046] Table 1. Batches and Origin of 21 Batches of Yarrow Medicinal Herbs

[0047] Serial Number batch Origin source category S1 20190601 Inner Mongolia Tongling Hetian Traditional Chinese Medicine Pieces Co., Ltd. cultivation S2 20190312 Inner Mongolia Tongling Hetian Traditional Chinese Medicine Pieces Co., Ltd. cultivation S3 20190812 Inner Mongolia Tongling Hetian Traditional Chinese Medicine Pieces Co., Ltd. cultivation S4 20190510 Inner Mongolia Tongling Hetian Traditional Chinese Medicine Pieces Co., Ltd. cultivation S5 20190618 Jilin Tongling Hetian Traditional Chinese Medicine Pieces Co., Ltd. cultivation S6 20190612 Jilin Tongling Hetian Traditional Chinese Medicine Pieces Co., Ltd. cultivation S7 20190606 Jilin Tongling Hetian Traditional Chinese Medicine Pieces Co., Ltd. cultivation S8 20190316 Jilin Tongling Hetian Traditional Chinese Medicine Pieces Co., Ltd. cultivation S9 20190310 Henan Tongling Hetian Traditional Chinese Medicine Pieces Co., Ltd. cultivation S10 20180907 Henan Tongling Hetian Traditional Chinese Medicine Pieces Co., Ltd. cultivation S11 20190810 Henan Tongling Hetian Traditional Chinese Medicine Pieces Co., Ltd. cultivation S12 20190812 Henan Tongling Hetian Traditional Chinese Medicine Pieces Co., Ltd. cultivation S13 20190316 Henan Tongling Hetian Traditional Chinese Medicine Pieces Co., Ltd. cultivation S14 20190606 Henan Tongling Hetian Traditional Chinese Medicine Pieces Co., Ltd. cultivation S15 20190612 Gansu Tongling Hetian Traditional Chinese Medicine Pieces Co., Ltd. cultivation S16 20190810 Gansu Tongling Hetian Traditional Chinese Medicine Pieces Co., Ltd. cultivation S17 20190606 Gansu Tongling Hetian Traditional Chinese Medicine Pieces Co., Ltd. cultivation S18 20180907 Gansu Tongling Hetian Traditional Chinese Medicine Pieces Co., Ltd. cultivation S19 20190618 Gansu Tongling Hetian Traditional Chinese Medicine Pieces Co., Ltd. cultivation S20 20190618 Gansu Tongling Hetian Traditional Chinese Medicine Pieces Co., Ltd. cultivation S21 20190618 Gansu Tongling Hetian Traditional Chinese Medicine Pieces Co., Ltd. cultivation .

[0048] A method for quality testing of yarrow, comprising the following steps:

[0049] Step (1): Preparation of mixed reference solution

[0050] Accurately weigh chlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, cypermethrin II, and 3,4,5-tricaffeoylquinic acid, and dissolve them in methanol to prepare single reference stock solutions. Take appropriate amounts of the above six single reference stock solutions, filter them through a microporous membrane, and mix them to prepare mixed reference solutions with concentrations of 0.5 mg / mL, 0.8 mg / mL, 0.8 mg / mL, 0.5 mg / mL, 0.5 mg / mL, and 0.5 mg / mL, respectively.

[0051] Step (2) Preparation of the test solution

[0052] Take 2g of yarrow powder from 21 batches listed in Table 1 above, pulverize them and pass them through a No. 2 sieve. Extract them with 40mL of 70% methanol at 40℃ for 45min using ultrasound. Then, filter the filtrate through a microporous membrane to obtain the test solution.

[0053] Step (3) Establishing the linear regression equation

[0054] The mixed reference standard from step (1) was serially diluted to obtain a series of reference standard solutions. These solutions were then injected into the HPLC system. The mass concentration of the series of reference standards was used as the abscissa X and the corresponding peak area was used as the ordinate Y. Linear regression analysis was performed on each chemical component and the linear regression equation was calculated as shown in Table 2 below.

[0055] Table 2

[0056] Element Linear equations r <![CDATA[Linear range / μg·mL -1 > chlorogenic acid <![CDATA[y=2×10 7 x+2.4750×10 4 ]]> 0.9998 4～500 Isochlorogenic acid B <![CDATA[y=2×10 7 x-7.2940×10 4 ]]> 0.9997 6～800 Isochlorogenic acid A <![CDATA[y=2×10 7 x-2.0215×10 4 ]]> 0.9999 6～800 Isochlorogenic acid C <![CDATA[y=3×10 7 x-7.6133×10 4 ]]> 0.9999 4～500 Bai Rui Cao Su II <![CDATA[y=2×10 7 x-1.33869×10 5 ]]> 0.9992 4～500 3,4,5-tricaffeoylquinicacid <![CDATA[y=1×10 7 x-5.1433×10 4 ]]> 0.9995 4～500

[0057] Step (4) Content determination

[0058] Take 21 batches of yarrow test solution from step (2) and inject them into HPLC for analysis. Substitute the peak area into the linear regression equation of step (3) to calculate the content of chlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, cypermethrin II and 3,4,5-tricaffeoylquinic acid in the test solution, as shown in Table 3 below.

[0059] Table 3. Content of components in 21 batches of yarrow herbal materials (mg / g) -1

[0060]

[0061]

[0062] Step (5) Establishment of fingerprint map

[0063] The fingerprint chromatogram of the test solution in step 4 was exported and imported into the 2012 version of the Chinese herbal chromatographic fingerprint chromatogram similarity evaluation system. Using the S1 herbal chromatogram as a reference, 13 common peaks were identified by the median method to obtain the fingerprint chromatogram of yarrow. The similarity of 21 batches of yarrow fingerprint chromatograms was evaluated using the above software, and the similarity of yarrow fingerprint chromatograms was found to be between 0.910 and 0.990, as shown in Table 4.

[0064] The chemical composition of the peaks in the reference fingerprint chromatogram was labeled according to the retention time of the chromatogram of the mixed reference solution. Peak 2 was chlorogenic acid, peak 5 was isochlorogenic acid B, peak 6 was isochlorogenic acid A, peak 9 was isochlorogenic acid C, peak 10 was cypermethrin II, and peak 13 was 3,4,5-tricaffeoylquinic acid.

[0065] Table 4

[0066] serial number Similarity serial number Similarity S1 0.979 S12 0.985 S2 0.978 S13 0.987 S3 0.983 S14 0.986 S4 0.982 S15 0.934 S5 0.985 S16 0.987 S6 0.986 S17 0.910 S7 0.988 S18 0.990 S8 0.985 S19 0.987 S9 0.983 S20 0.990 S10 0.983 S21 0.986 S11 0.986 .

[0067] The HPLC chromatographic conditions in steps (3) and (4) above are as follows:

[0068] use HSS T3C18 column, 4.6 mm × 150 mm, 5 μm; mobile phase: methanol (phase A) and 0.1% formic acid (phase B), gradient elution; flow rate: 1.0 mL / min. -1 Detection wavelength: 260 nm; column temperature: 35℃; injection volume: 10 μL. Gradient elution method:

[0069]

[0070]

[0071] Example 2 Methodological Investigation

[0072] (1) Precision test

[0073] Take S5 batch of yarrow powder and inject it 6 times consecutively according to the extraction method and chromatographic conditions of Example 1. Using peak No. 2 as a control, calculate the relative retention time and relative peak area of ​​each common peak. The relative standard deviation (RSD) is less than 1%, indicating that the instrument has good precision.

[0074] (2) Repeatability test

[0075] Take S4 batch of yarrow powder and inject it continuously according to the method and chromatographic conditions of Example 1. Using peak 2 as the reference peak, calculate the relative retention time and relative peak area of ​​each common peak. The RSD value of the relative retention time is less than 1% and the RSD value of the relative peak area is less than 5%, indicating that the method has good repeatability.

[0076] (3) Stability test

[0077] Take S5 batch of yarrow powder and inject it at 0, 2, 4, 6, 8, 12, 16 and 24 h after chromatographic conditions according to Example 1. Using peak 2 as the reference peak, calculate the relative retention time and relative peak area of ​​each common peak. The RSD value of the relative retention time is less than 1% and the RSD value of the relative peak area is less than 2%, indicating that the prepared test sample is stable within 1 day.

[0078] The detection method provided by this invention can simultaneously and accurately detect the content of six active ingredients in yarrow, and establishes a fingerprint spectroscopy detection method for yarrow. Methodological testing shows that the method has high precision, high sensitivity and high stability, and can objectively, comprehensively and accurately evaluate the quality of yarrow medicinal materials and their extracts and preparations, which is of great significance for accurately controlling the quality of yarrow medicinal materials and their preparations.

[0079] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for quality testing of yarrow, characterized in that, Includes the following steps: Step (1): Preparation of mixed reference solution Accurately weigh chlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, cypermethrin II and 3,4,5-tricaffeoylquinic acid and dissolve them in methanol to prepare a single reference stock solution; take appropriate amounts of the above six reference stock solutions, filter them through a microporous membrane, and mix them to prepare a mixed reference solution; Step (2) Preparation of the test solution Take 21 batches of yarrow powder, pulverize them and pass them through a No. 2 sieve. Extract them with 10 to 20 times the volume of 70% methanol at 40°C for 45 minutes by ultrasonication. Then filter the filtrate through a microporous membrane to obtain the test solution. Step (3) Establishing the linear regression equation The mixed reference standard from step (1) was gradually diluted to obtain a series of reference standard solutions of different concentrations. These solutions were then injected into the HPLC system. The mass concentration of the series of reference standards was used as the abscissa X and the corresponding peak area was used as the ordinate Y. Linear regression analysis was performed on each chemical component and the linear regression equation was calculated. Step (4) Content determination Take the yarrow test solution from step (2) and inject it into HPLC for analysis. Substitute the peak area into the linear regression equation from step (3) to calculate the content of chlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, yarrow extract II and 3,4,5-tricaffeoylquinic acid in the test solution. Step (5) Establishment of fingerprint map Export the fingerprint spectrum of the test solution in step 4 and import it into the Chinese herbal chromatographic fingerprint spectrum similarity evaluation system 2012; using the S1 herbal chromatogram as a reference, a total of 13 common peaks were identified using the median method to obtain the fingerprint spectrum of yarrow; and label the chemical components of the peaks in the reference fingerprint spectrum according to the retention time of the chromatogram of the mixed reference solution. The HPLC chromatographic conditions in steps (3) and (4) are as follows: use HSS T3 C18 column, 4.6 mm × 150 mm, 5 μm; mobile phase: methanol (phase A) and 0.1% formic acid (phase B), gradient elution; flow rate: 1.0 mL / min. -1 Detection wavelength: 260 nm; column temperature: 35 °C; injection volume: 10 μL.

2. The method for quality testing of yarrow according to claim 1, characterized in that, Step (1) Preparation of mixed reference solution Accurately weigh chlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, cypermethrin II, and 3,4,5-tricaffeoylquinic acid, and dissolve them in methanol to prepare single reference stock solutions. Take appropriate amounts of the above six single reference stock solutions, filter them through a microporous membrane, and mix them to prepare mixed reference solutions with concentrations of 0.5 mg / mL, 0.8 mg / mL, 0.8 mg / mL, 0.5 mg / mL, 0.5 mg / mL, and 0.5 mg / mL, respectively.

3. The method for quality testing of yarrow according to claim 1, characterized in that, In step (5), the retention time of each reference standard was compared with the fingerprint spectrum of each medicinal material by HPLC, and six common peaks were identified: peak 2 was chlorogenic acid, peak 5 was isochlorogenic acid B, peak 6 was isochlorogenic acid A, peak 9 was isochlorogenic acid C, peak 10 was cyclohexane II, and peak 13 was 3,4,5-tricaffeoylquinic acid.

4. The method for quality testing of yarrow according to claim 1, characterized in that, The standard curve established in step (3) is as follows:

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