A method for simultaneously controlling the overall quality of a radix astragali and ramulus cinnamomi five-ingredient decoction reference sample in terms of quality and quantity

Abstract

The application discloses a method for simultaneously controlling the qualitative and quantitative overall quality of a Huangqi Guizhi Wuwu Decoction (HGWD) reference sample, and the method is characterized in that: a water decoction process optimized through orthogonal tests is used to prepare HGWD decoctions and freeze-dried powder reference samples of multiple batches of medicinal material combinations from different producing areas; an HPLC characteristic chromatogram is established, 15 common peaks are calibrated by taking cinnamyl aldehyde as a reference peak; based on the same reference sample, the contents of eight index components, i.e., gallic acid, peony lactone glycoside, paeoniflorin, calycosin-7-glucoside, cinnamic acid, cinnamyl aldehyde, formononetin and 6-gingerol, are simultaneously determined by using a multi-wavelength HPLC, and the content of astragaloside A is determined by using an HPLC-ELSD method; the qualitative and quantitative overall quality of the reference sample is simultaneously controlled through characteristic chromatogram similarity evaluation and multi-index component content consistency evaluation. The application realizes the whole-process value transmission evaluation from medicinal material raw materials to final products, and provides a scientific basis for formulating the quality standard of the HGWD reference sample and developing granules.

Description

Technical Field

[0001] This invention belongs to the field of quality control technology for traditional Chinese medicine compound preparations, specifically involving a method for simultaneous qualitative and quantitative overall quality control of Huangqi Guizhi Wuwu Decoction (HGWD) reference samples. Background Technology

[0002] Huangqi Guizhi Wuwu Decoction is the 16th formula in the "List of Famous Ancient Prescriptions (First Batch)" published by the State Administration of Traditional Chinese Medicine, and the 5th formula in the "Key Information Table of Famous Ancient Prescriptions (25 Formulas)" published in 2022. Modern research shows that this formula has activities such as regulating immunity, anti-inflammation, and analgesia. Clinically, it is often used to treat diseases such as diabetic peripheral neuropathy, and has important clinical application value and development prospects.

[0003] With the implementation of the "Technical Guidelines for Pharmaceutical Research of Traditional Chinese Medicine Compound Preparations Managed According to the Catalogue of Ancient Classic Prescriptions (Trial)," systematic requirements have been put forward for the quality control of reference samples of classic prescriptions. It is necessary to establish scientific, standardized, and reproducible quality evaluation methods to support the formulation of reference sample quality standards and the development of granules.

[0004] However, existing research on the quality control of HGWD has several shortcomings. First, the extraction process parameters for the decoction have not been systematically optimized. Differences exist in the form of the reference sample (decoction, concentrate, or lyophilized powder) and the preparation conditions of the lyophilized powder used by different researchers, leading to inconsistencies in the intrinsic quality of the reference samples and making it difficult to guarantee the stability and repeatability of quality standards. Second, some studies have not included characteristic peaks of jujube or astragalus in the established characteristic chromatograms, failing to comprehensively characterize the overall microscopic chemical composition of the compound. Third, some studies separate the characteristic chromatogram analysis from the content determination method, resulting in inefficient and environmentally unfriendly analytical processes. Furthermore, the selection of content determination index components is not comprehensive, failing to fully cover the legally defined content determination components of astragalus, cinnamon twig, and ginger in the Chinese Pharmacopoeia. Finally, most existing studies have not systematically examined the value transfer laws starting from the raw materials, lacking a tracking and evaluation of quality changes throughout the entire preparation process.

[0005] The aforementioned problems make it difficult for existing methods to meet the overall, systematic, and consistent requirements of HGWD reference sample quality control. There is an urgent need to develop a method that can simultaneously achieve qualitative fingerprinting and quantitative determination of multiple components, process optimization, batch consistency evaluation, and whole-process value transfer research for overall quality control. Summary of the Invention

[0006] The purpose of this invention is to overcome the defects of the prior art and provide a method for simultaneous qualitative and quantitative overall quality control of Huangqi Guizhi Wuwu Decoction reference samples.

[0007] The technical solution of the present invention is as follows:

[0008] A method for simultaneous qualitative and quantitative overall quality control of Huangqi Guizhi Wuwu Decoction reference samples includes the following steps:

[0009] (1) Multiple batches of HGWD decoction and lyophilized powder reference samples were prepared using the water decoction process optimized by orthogonal experiment;

[0010] (2) Establish HPLC characteristic chromatograms for multiple batches of HGWD decoction and lyophilized powder reference samples obtained in step (1), and identify 15 common peaks with cinnamaldehyde as the reference peak.

[0011] (3) Based on the reference sample obtained in step (1), prepare the test solution and use multi-wavelength HPLC to simultaneously determine the contents of eight index components: gallic acid, paeoniflorin, paeoniflorin, versicolor glucoside, cinnamic acid, cinnamaldehyde, gentianin and 6-gingerol. The detection wavelengths are 270 nm for gallic acid, 232 nm for paeoniflorin and paeoniflorin, 248 nm for versicolor glucoside and gentianin, 278 nm for cinnamic acid, 290 nm for cinnamaldehyde and 220 nm for 6-gingerol.

[0012] (4) Prepare a test solution from the reference sample obtained in step (1) and determine the content of astragaloside A by HPLC-ELSD method;

[0013] (5) Integrate the feature spectrum data and multi-index component content data obtained from steps (2) to (4), and achieve simultaneous qualitative and quantitative overall quality control of the reference sample through feature spectrum similarity evaluation and multi-index component content consistency evaluation.

[0014] In a preferred embodiment of the present invention, the multiple batches of freeze-dried powder reference samples prepared in step (1) are randomly combined from medicinal materials from different origins.

[0015] In a preferred embodiment of the present invention, the HPLC characteristic chromatogram in step (2) is established using an Agilent Zorbax SB-C18 column and gradient elution is performed using acetonitrile-0.1% phosphoric acid aqueous solution.

[0016] In a preferred embodiment of the present invention, the 15 common peaks in step (2) are used to classify and identify the medicinal materials through experiments with reference solution, single herbal decoction piece test solution and negative test solution.

[0017] In a preferred embodiment of the present invention, the test solution is prepared by directly diluting the HGWD decoction and the lyophilized powder reference sample solution after water reconstitution with methanol in step (3), and the test solution is prepared by water-saturated n-butanol extraction and ammonia washing purification in step (4).

[0018] In a preferred embodiment of the present invention, in step (5), the similarity of feature maps of multiple batches of reference samples is evaluated, and the similarity between batches is not less than 0.9.

[0019] In a preferred embodiment of the present invention, the method further includes determining the content of the components with the content limit index of the Chinese Pharmacopoeia in the medicinal materials and decoction pieces, and comparing the content with that in the decoction and freeze-dried powder, so as to realize the whole process of value transfer evaluation from the raw materials of medicinal materials to the final reference sample.

[0020] In a preferred embodiment of the present invention, the content consistency evaluation in step (5) is based on the fact that the content of more than 5 of the 9 index components, namely gallic acid, paeoniflorin, paeoniflorin, verbascoflavonoid glucoside, cinnamic acid, cinnamaldehyde, gentianin, 6-gingerol and astragaloside A, is within ±30% of their respective mean values.

[0021] In a preferred embodiment of the present invention, the methodological validation of simultaneous multi-wavelength HPLC determination and HPLC-ELSD determination is further included, and the validation items include specificity, linearity, precision, repeatability, stability and recovery rate.

[0022] In a preferred embodiment of the present invention, the 15 common peaks identified by the HPLC characteristic chromatogram cover the characteristic components of five medicinal herbs: Astragalus membranaceus, Paeonia lactiflora, Cinnamomum cassia, Zingiber officinale, and Ziziphus jujuba.

[0023] The beneficial effects of this invention are:

[0024] 1. This invention employs orthogonal experiments to optimize the traditional decoction process and prepares multiple batches of reference samples with different combinations of medicinal materials from various origins. This improves the representativeness, stability, and batch-to-batch quality consistency of the preparation process of HGWD decoction and freeze-dried powder, providing a reliable process basis for establishing the quality standards of reference samples.

[0025] 2. The HPLC characteristic chromatogram established by this invention contains 15 common peaks, and the medicinal material attribution of each peak is clarified through experiments with reference solution, single herbal pieces and negative samples. It covers the characteristic components of all five medicinal materials: Astragalus membranaceus, Paeonia lactiflora, Cinnamomum cassia, Zingiber officinale and Ziziphus jujuba. Seven common peaks are identified, realizing the overall qualitative characterization of the chemical composition of the compound and effectively overcoming the shortcomings of the incomplete representativeness of the characteristic peaks in the existing chromatograms.

[0026] 3. This invention employs multi-wavelength simultaneous determination technology, which can simultaneously determine the characteristic spectrum of HGWD and the content of 8 indicator components in a single injection. Combined with the dedicated determination method for astragaloside A, it simplifies the analysis process, reduces solvent consumption and analysis time, improves detection efficiency, and meets the requirements of green analysis.

[0027] 4. The index components selected in this invention include gallic acid, paeoniflorin, paeoniflorin, verbascoflavonoid glucoside, cinnamic acid, cinnamaldehyde, gentianin, 6-gingerol, and astragaloside A, covering the components specified in the pharmacopoeia and quality markers based on pharmacological activity, thus achieving a more comprehensive quantitative quality characterization of the five medicinal materials.

[0028] 5. This invention systematically conducts a study on the transfer of quantities throughout the entire process from medicinal materials and decoction pieces to decoctions and freeze-dried powders. Combined with the evaluation of content data from multiple batches, it achieves effective monitoring of quality changes throughout the entire preparation process, thereby improving the traceability and integrity of quality control.

[0029] 6. The methodology of this invention is fully validated and meets the requirements of the 2025 edition of the Chinese Pharmacopoeia. Multiple batches of experimental data support the batch-to-batch consistency evaluation, providing a scientific basis for the formulation of HGWD benchmark sample quality standards and granule development, and has good practicality and application prospects. Attached Figure Description

[0030] Figure 1 The figures show the HPLC chromatograms and their comparative characteristic chromatograms (TR and DR) of 15 batches of HGWD decoction (T1-T15) and lyophilized powder (D1-D15) in Example 3 of this invention, wherein 3-gallic acid; 8-paeonolide; 9-paeonoside; 11-verrucoumarin glucoside; 13-cinnamic acid; 14-cinnamaldehyde; and 15-6-gingerol.

[0031] Figure 2 The image shows the HPLC chromatogram of the mixed reference solution in Example 3 of this invention at 210 nm, containing 3-gallic acid; 8-paeonolide; 9-paeonoside; 11-verrucoumarin glucoside; 13-cinnamic acid; 14-cinnamaldehyde; and 15-6-gingerol.

[0032] Figure 3 The HPLC chromatograms of HGWD single-herb medicinal slices, lyophilized powder, and negative test solution in Example 3 of the present invention are shown, wherein 3-gallic acid; 8-paeonolide; 9-paeonoside; 11-verrucoumarin glucoside; 13-cinnamic acid; 14-cinnamaldehyde; and 15-6-gingerol.

[0033] Figure 4 This is an HPLC chromatogram of the gallic acid content in the HGWD lyophilized powder in Example 4 of the present invention, measured at 270 nm.

[0034] Figure 5 This is an HPLC chromatogram at 232 nm for the determination of paeoniflorin and paeoniflorin content in HGWD lyophilized powder in Example 4 of the present invention.

[0035] Figure 6 This is an HPLC chromatogram at 248 nm for the determination of the content of verbascoside and gentianin in HGWD lyophilized powder in Example 4 of the present invention.

[0036] Figure 7 This is an HPLC chromatogram of the determination of cinnamic acid content in HGWD lyophilized powder at 278 nm in Example 4 of the present invention.

[0037] Figure 8 This is an HPLC chromatogram of the determination of cinnamaldehyde content in HGWD lyophilized powder at 290 nm in Example 4 of the present invention.

[0038] Figure 9 This is an HPLC chromatogram of the determination of 6-gingerol content in HGWD lyophilized powder at 220 nm in Example 4 of the present invention.

[0039] Figure 10 This is an HPLC chromatogram showing the determination of astragaloside A content in HGWD lyophilized powder in Example 5 of the present invention. Detailed Implementation

[0040] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0041] Example 1: Instruments and Materials

[0042] 1.1 Instruments: Agilent 1260 high-performance liquid chromatograph with DAD detector, Chem Station workstation, Agilent Technologies, USA; 3300 evaporative light scattering detector, Alltech, USA; XS205DU electronic analytical balance, d=0.01 mg, Mettler Toledo, Switzerland; KQ-500VDB three-frequency digitally controlled ultrasonic cleaner, Kunshan Ultrasonic Instrument Co., Ltd.; DZG-DN50D decoction pot, Shantou Mengqi Technology Co., Ltd.; Savant Modulyo D freeze dryer, Thermo Fisher Scientific.

[0043] 1.2 Materials and Reference Standards: Astragaloside A (batch number 110781-202219, mass fraction 96.2%), Verbena isoflavone glucoside (batch number 111920-202308, mass fraction 96.9%), Paeoniflorin (batch number 110736-202246, mass fraction 96.7%), Cinnamaldehyde (batch number 110710-202424, mass fraction 99.2%), 6-Gingerol (batch number 111833-202007, mass fraction 99.3%), China National Institutes for Food and Drug Control; Gallic acid (… Batch No. MUST-23112611, mass fraction 99.96%), paeoniflorin (batch No. MUST-24081811, mass fraction 99.53%), cinnamic acid (batch No. MUST-24071910, mass fraction 99.99%), and gentianin (batch No. MUST-23033005, mass fraction 99.03%), from Manster (Chengdu) Biotechnology Co., Ltd.; methanol and phosphoric acid, analytical grade, from Xilong Scientific Co., Ltd.; methanol and acetonitrile, chromatographic grade, from Tianjin Biaoshiqi Technology Development Co., Ltd.

[0044] The medicinal materials were identified by the Xiamen Institute of Pharmaceutical Research as follows: Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao (Fabaceae family); Paeonia lactiflora (White peony root) (Ranunculaceae family); Cinnamomum cassia Presl (Cinnamomum cassia Presl (Lauraceae family); Zingiber officinale Rosc. (Zingiberaceae family); and Ziziphus jujuba Mill. (Rhamnaceae family). Based on the key information, the medicinal materials were processed into decoction pieces according to the corresponding processing techniques in the 2025 edition of the Chinese Pharmacopoeia. Information on the origins of the qualified medicinal materials is shown in Table 1, all of which are major producing areas.

[0045] Table 1. Batch numbers and origins of 15 batches of HGWD-formulated medicinal materials

[0046]

[0047] Example 2: Preparation of HGWD decoction and lyophilized powder

[0048] 2.1 Determination of the Origin and Batch of Medicinal Materials Astragalus has two origins. Based on its quality and yield, Mongolian Astragalus was selected as the origin of Astragalus. The other four herbs are all single origins. Based on the content test results of multiple batches of medicinal slices, slices with a content within the range of mean ±30% were selected and randomly combined using a random number table method. The batch numbers of the medicinal slices for 15 batches of HGWD decoction (T1~T15) are shown in Table 1 of Example 1.

[0049] 2.2 Determination of the content of index components in medicinal materials and decoction pieces: The content of five index components, namely astragaloside A, verbascoside, paeoniflorin, cinnamaldehyde, and 6-gingerol, in 15 batches of HGWD-formulated medicinal materials and decoction pieces were determined according to the method of the 2025 edition of the Chinese Pharmacopoeia. The results are shown in Table 2.

[0050] Table 2. Content of five indicator components in 15 batches of HGWD-formulated medicinal materials and processed medicinal slices, calculated on a dried basis.

[0051]

[0052] 2.3 Preparation of HGWD decoction and freeze-dried powder: Using the previously orthogonally optimized HGWD decoction process, 41.40 g of Astragalus membranaceus, 41.40 g of Cinnamomum cassia, 41.40 g of Paeonia lactiflora, 82.80 g of Zingiber officinale, and 36.00 g of Ziziphus jujuba were weighed and placed in a decoction pot. 1200 mL of water was added, and the mixture was soaked for 30 min. The decoction was then boiled at 250 W with the lid on (about 55 min), then the power was adjusted to 500 W, and the mixture was allowed to simmer gently until the end of the decoction period (about 60 min). The mixture was filtered through a 200-mesh pharmacopoeia sieve while hot, and water was added to adjust the volume to obtain 400 mL of decoction. Half of the decoction was frozen for later use; the other half was dispensed into freeze-drying trays, pre-frozen at −22 ℃ for 12 h, freeze-dried for 72 h, and then pulverized to obtain freeze-dried powder. Prepare 15 batches of HGWD decoction (T1~T15) and 15 batches of HGWD lyophilized powder (D1~D15) in parallel according to the combinations in Table 1, seal them and set them aside for later use.

[0053] Example 3: Establishment of Characteristic Chromatography for HGWD Decoction and Lyophilized Powder

[0054] 3.1 Chromatographic Conditions: The chromatographic column was an Agilent Zorbax SB-C18 column (250 mm × 4.6 mm, 5 μm); the mobile phase was acetonitrile-0.1% phosphoric acid aqueous solution, with gradient elution: 0–7.0 min, 2.8% acetonitrile; 7.0–31.5 min, 2.8%–15.0% acetonitrile; 31.5–46.0 min, 15.0%–20.8% acetonitrile; 46.0–61.5 min, 20.8%–38.0% acetonitrile; 61.5–62.0 min, 38.0%–49.0% acetonitrile; 62.0–67.0 min, 49.0% acetonitrile; 67.0–70.0 min, 49.0%–50.0% acetonitrile; 70.0–71.0 min, 50.0%–100.0% acetonitrile; 71.0–74.0 min… 74.0–74.1 min, 100.0%–2.8% acetonitrile; 74.1–80.0 min, 2.8%–2.8% acetonitrile; flow rate 1.0 mL / min; column temperature 30 ℃; detection wavelength 210 nm; injection volume 5 μL.

[0055] 3.2 Preparation of the test solution

[0056] (1) HGWD lyophilized powder test solution: Accurately weigh 0.075 g of HGWD lyophilized powder, place it in a 10 mL volumetric flask, add 1.645 mL of ultrapure water, shake well to reconstitute, let stand for 30 min, add methanol to the mark, let stand for 30 min, filter through a 0.45 μm nylon membrane, and take the filtrate to obtain the test solution.

[0057] (2) HGWD decoction test solution: Accurately measure 1.645 mL of HGWD decoction into a 10 mL volumetric flask, add methanol to the mark, and then follow the same procedure as in item “3.2 (1)” of this example.

[0058] 3.3 Preparation of test solutions of single-herb medicinal slices: Weigh each single-herb medicinal slice according to the prescription amount, and prepare the decoction and freeze-dried powder of each single-herb medicinal slice according to the method under "2.3" of Example 2. Prepare the solution according to the method under "3.2" of this Example.

[0059] 3.4 Preparation of negative test solution: Weigh the other 4 medicinal slices that are missing the corresponding medicinal slices according to the prescription amount, and prepare each negative decoction and lyophilized powder according to the method under "2.3" in Example 2. Prepare according to the method under "3.2" in this Example.

[0060] 3.5 Preparation of Reference Solutions Accurately weigh appropriate amounts of gallic acid, paeoniflorin, paeoniflorin, verbascoflavonoid glucoside, cinnamic acid, cinnamaldehyde, gentianin, and 6-gingerol reference standards. Prepare reference stock solutions with methanol at mass concentrations of 1065.57, 1054.02, 1035.66, 1075.59, 411.16, 598.77, 203.21, and 1150.35 μg / mL, respectively. Then prepare mixed reference solutions with methanol at mass concentrations of 20.00, 50.00, 49.99, 5.00, 5.00, 5.01, 0.80, and 10.01 μg / mL, respectively.

[0061] 3.6 Precision test: The same HGWD lyophilized powder test solution was injected and measured 6 times consecutively under the chromatographic conditions in section “3.1” of this example. Peak 14 (cinnamaldehyde) was used as a reference. The results showed that the RSD of the relative retention time of each common peak was ≤4.32%, and the RSD of the relative peak area was ≤4.50%, which met the requirements of fingerprint chromatographic methodology.

[0062] 3.7 Stability Study: The same HGWD lyophilized powder test solution was used and chromatographically analyzed at 0, 8, 17, 21, 25, and 41 h after preparation, according to the chromatographic conditions in section “3.1” of this embodiment. The results showed that the RSD of the relative retention time of each common peak was ≤3.79%, and the RSD of the relative peak area was ≤4.85%, which met the requirements of the fingerprint chromatographic method.

[0063] 3.8 Repeatability test: Take the same batch of HGWD lyophilized powder and prepare 6 test solutions in parallel according to the method in section “3.2 (1)” of this example. Inject and determine according to the chromatographic conditions in section “3.1” of this example. The results show that the RSD of the relative retention time of each common peak is ≤3.67% and the RSD of the relative peak area is ≤4.55%, which meets the requirements of fingerprint chromatographic methodology.

[0064] 3.9 Establishment and Similarity Evaluation of HGWD Characteristic Chromatography Fifteen batches each of HGWD lyophilized powder (D1-D15) and HGWD decoction (T1-T15) were accurately weighed and prepared and measured according to the methods described in sections "3.2" and "3.1" of this embodiment. The HPLC chromatograms of each sample were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version)" software in AIA format. Using D1 and T1 as references, a time width of 0.1 min was set. The median method was used, combined with peak multi-point correction, to perform full peak matching, obtaining characteristic chromatograms for 15 batches (D1-D15 and T1-T15), and generating control characteristic chromatograms (DR and TR). Based on the HPLC chromatograms, chromatographic peaks with good stability and obvious characteristics were selected as common peaks, and 15 common peaks were identified. Peak 14 (cinnamaldehyde), with a larger peak area and better resolution, was selected as the reference peak (S). Similarity analysis was performed on the characteristic chromatograms of 15 batches of HGWD freeze-dried powder (D1–D15) and decoction (T1–T15). The results showed that the similarity of the characteristic chromatograms of the 15 batches of freeze-dried powder and decoction with DR and TR were ≥0.976 and 0.974, respectively. The similarity between batches of freeze-dried powder and decoction was ≥0.918 and 0.909, respectively. This indicates that the preparation process of HGWD freeze-dried powder and decoction is stable, and the differences in characteristic components among HGWD freeze-dried powder and decoction prepared from different origins and batches of medicinal materials are small, indicating good consistency in quality between and within batches of HGWD freeze-dried powder and decoction. (See results below.) Figure 1 Tables 3 and 4.

[0065] Table 3. Similarity of 15 batches of HGWD lyophilized powder (D1~D15)

[0066]

[0067] Table 4. Similarity of 15 batches of HGWD decoction (T1~T15)

[0068]

[0069] 3.10 Characteristic Peak Assignment and Identification: Take the mixed reference solution, the individual herbal decoction pieces test solution, the HGWD lyophilized powder test solution, and the negative test solution respectively, and inject them according to the chromatographic conditions described in section "3.1" of this embodiment for determination. Comparative analysis shows that... Figure 2 and Figure 3 As shown, the 15 characteristic peaks were assigned and identified. Peaks 1 and 11 (verrucaside) were assigned to Astragalus membranaceus; peaks 3 (gallic acid), 4 and 8 (paeoniflorin), 9 (paeoniflorin), 10 and 12 were assigned to Paeonia lactiflora; peaks 6 and 13 (cinnamic acid) and 14 (cinnamaldehyde) were assigned to Cinnamomum cassia; peak 15 (6-gingerol) was assigned to Zingiber officinale; peak 2 was a peak shared by Astragalus membranaceus and Ziziphus jujuba; and peaks 5 and 7 were peaks shared by Paeonia lactiflora and Cinnamomum cassia.

[0070] Example 4: Simultaneous multi-wavelength determination of the content of eight indicator components in HGWD decoction and lyophilized powder.

[0071] 4.1 The chromatographic conditions and detection wavelengths were 270 nm (gallic acid), 232 nm (paeoniflorin, paeoniflorin), 248 nm (verructin, isoflavone glucoside, gentianin), 278 nm (cinnamic acid), 290 nm (cinnamaldehyde), and 220 nm (6-gingerol), and the rest were the same as in item “3.1” of Example 3.

[0072] 4.2 The preparation of the mixed reference solution is the same as in item “3.5” of Example 3.

[0073] 4.3 The preparation of the test solution is the same as in sections “3.2” and “3.4” of Example 3.

[0074] 4.4 Specificity Assessment: The mixed reference solution, HGWD lyophilized powder test solution, negative test solution, and methanol blank solvent were injected separately and analyzed according to the chromatographic conditions described in section 4.1 of this embodiment. The results are as follows: Figures 4 to 9 As shown, the resolution of all eight indicator components was greater than 1.5, there was no interference from negative results, the baseline was stable, the specificity was strong, and it met the requirements of the 2025 edition of the Chinese Pharmacopoeia.

[0075] 4.5 Linearity Examination The mixed reference solution from section "3.5" of Example 3 was precisely pipetted and injected according to the method described in section "4.1" of this Example. A standard curve was plotted with the mass concentration of the reference solution as the abscissa (X) and the peak area as the ordinate (Y). Linear regression was performed to obtain the regression equation, correlation coefficient (r), and linear range. The results were as follows: Gallic acid Y = 16.04X - 4.60, r = 0.9998, linear range 2.00–119.98 μg / mL; Paeoniflorin Y = 3.581X - 1.96, r = 0.9998, linear range 5.00–299.97 μg / mL; Paeoniflorin Y = 6.76X - 9.22, r = 0.9999, linear range 2.00–299.93 μg / mL; Verbenaside Y = 18.34 X + 2.11, r = 0.9997, linear range 0.50–29.99 μg / mL; Cinnamic acid Y = 42.96 X + 4.81, r = 0.9998, linear range 0.50–30.01 μg / mL; Cinnamaldehyde Y = 52.91 X + 6.20, r = 0.9997, linear range 0.50–30.06 μg / mL; Acanthocyanin Y = 30.58 X + 0.40, r = 0.9998, linear range 0.08–4.80 μg / mL; 6-Gingerol Y = 8.14 X + 4.74, r = 0.9997, linear range 1.00–60.05 μg / mL. All components showed good linearity within their respective linear ranges. The mixed reference solution was serially diluted, and the mass concentration of each reference standard at a signal-to-noise ratio of 3 and 10 was used as the limit of detection (LOD) and limit of quantitation (LOQ). The results showed that the LODs for gallic acid, paeoniflorin, paeoniflorin, verbascoflavonoid glucoside, cinnamic acid, cinnamaldehyde, gentianin, and 6-gingerol were 0.06, 0.42, 0.23, 0.05, 0.02, 0.02, 0.11 μg / mL, respectively, and the LOQs were 0.21, 1.39, 0.75, 0.16, 0.05, 0.05, 0.05, and 0.35 μg / mL, respectively.

[0076] 4.6 Instrument Precision Assessment: A mixed reference solution was injected and measured six times consecutively under the chromatographic conditions described in section 4.1 of this embodiment. The RSDs of the peak areas of gallic acid, paeoniflorin, paeoniflorin, verrucoside glucoside, cinnamic acid, cinnamaldehyde, gentianin, and 6-gingerol were 0.46%, 0.45%, 0.36%, 0.17%, 0.32%, 0.28%, 0.57%, and 0.85%, respectively, indicating that the instrument has good precision.

[0077] 4.7 Precision Test: The 10th batch of HGWD lyophilized powder test solution was injected and measured 6 times consecutively under the chromatographic conditions described in section “4.1” of this example. The RSDs of the peak areas of gallic acid, paeoniflorin, paeoniflorin, verrucoside, cinnamic acid, cinnamaldehyde, gentianin, and 6-gingerol were 0.32%, 0.65%, 0.57%, 2.72%, 0.27%, 0.60%, 0.41%, and 1.81%, respectively, indicating good injection precision.

[0078] 4.8 Repeatability Test: Six portions of the 10th batch of HGWD lyophilized powder were accurately weighed. Test solutions were prepared and injected for determination according to the methods described in "3.2" of Example 3 and "4.1" of this Example. The RSDs of the mass fractions of gallic acid, paeoniflorin, paeoniflorin, verbascoflavonoid glucoside, cinnamic acid, cinnamaldehyde, gentianin, and 6-gingerol were 1.23%, 1.53%, 1.00%, 0.99%, 1.17%, 1.39%, 3.24%, and 1.85%, respectively. The results indicate that the experiment has good repeatability.

[0079] 4.9 Stability Study: The test solution of the 10th batch of HGWD lyophilized powder repeatability test was taken and analyzed at 0, 8, 17, 21, 25, and 41 h after preparation, according to the chromatographic conditions in section “4.1” of this example. The RSDs of the peak areas of gallic acid, paeoniflorin, paeoniflorin, verbascoflavonoid glucoside, cinnamic acid, cinnamaldehyde, gentianin, and 6-gingerol were 0.49%, 0.60%, 0.88%, 5.29%, 0.68%, 1.09%, 2.00%, and 2.04%, respectively. The results indicate that the test solution has good stability within 41 h.

[0080] 4.10 Intermediate Precision Assessment: On the second day, different personnel accurately weighed the 10th batch of HGWD lyophilized powder and prepared six HGWD lyophilized powder test solutions according to the method in section "3.2". The solutions were then injected six times consecutively under the chromatographic conditions in section "4.1" of this embodiment. The RSDs of the peak areas for gallic acid, paeoniflorin, paeoniflorin, verrucoside, cinnamic acid, cinnamaldehyde, gentianin, and 6-gingerol were 0.75%, 0.80%, 0.69%, 0.85%, 0.74%, 1.08%, 0.99%, and 1.24%, respectively, indicating good intermediate precision.

[0081] 4.11 Recovery Test Accurately weigh 38.0 mg of HGWD lyophilized powder with known content of each indicator component, in 6 portions, and place them in 10 mL volumetric flasks. Add 1.645 mL of ultrapure water, shake well to reconstitute, and let stand for 30 min. Add the 8 reference stock solutions under "3.5" of Example 3 according to 100% of the lyophilized powder, add methanol to the mark, shake well, and let stand for 30 min. Filter through a 0.45 μm nylon membrane, and take the filtrate to obtain the test solution for the recovery test. According to the chromatographic conditions under section "4.1" of this embodiment, the average recovery rate and RSD of each index component were calculated. The results showed that the average recovery rates of gallic acid, paeoniflorin, paeoniflorin, verbascoflavonoid glucoside, cinnamic acid, cinnamaldehyde, gentianin, and 6-gingerol were 101.58%, 96.08%, 104.56%, 93.47%, 109.69%, 104.62%, 83.89%, and 84.74%, respectively, and the RSDs were 0.82%, 1.67%, 0.62%, 1.71%, 0.59%, 0.75%, 2.66%, and 0.38%, respectively. It can be seen that all of them meet the requirements of the 2025 edition of the Chinese Pharmacopoeia, indicating that the experiment has good accuracy.

[0082] 4.12 Content determination: Take 15 batches each of HGWD decoction and HGWD lyophilized powder, prepare test solutions according to the method under “3.2” in Example 3, and inject them according to the chromatographic conditions under “4.1” to determine the content of each index component. The results are shown in Tables 5 and 6.

[0083] Table 5. Content of 9 indicator components in 15 batches of HGWD decoction, calculated according to the corresponding amount of medicinal slices.

[0084]

[0085] Table 6. Content of 9 indicator components in 15 batches of HGWD lyophilized powder, calculated on a dried basis.

[0086]

[0087] As shown in Table 5, among the 15 batches of HGWD decoction, the number of batches with contents exceeding ±30% for astragaloside A, paeoniflorin, verbascoside, cinnamic acid, and cinnamaldehyde were 3, 6, 1, 1, and 2, respectively; the contents of the remaining batches were all within ±30%. As shown in Table 6, among the 15 batches of HGWD freeze-dried powder, the number of batches with contents exceeding ±30% for astragaloside A, paeoniflorin, verbascoside, cinnamaldehyde, gentianin, and 6-gingerol were 3, 7, 2, 5, 1, and 1, respectively; the contents of the remaining batches were all within ±30%.

[0088] Example 5: Determination of Astragaloside A Content in HGWD Decoction and Lyophilized Powder

[0089] 5.1 Chromatographic conditions: The chromatographic column was an Agilent Zorbax SB-C18 column (250 mm × 4.6 mm, 5 μm); the mobile phase was acetonitrile-water (34:66); the flow rate was 1.0 mL / min; the column temperature was 35 ℃; the injection volume was 10 μL for the test solution and 2, 5, and 10 μL for the reference solutions; the evaporative light scattering detector parameters were: drift tube temperature 65 ℃, carrier gas flow rate 1.5 L / min, and gain value 4.

[0090] 5.2 Preparation of reference solution: Accurately weigh an appropriate amount of astragaloside A reference standard and prepare a stock solution of astragaloside A reference standard with methanol at a concentration of 1031.26 μg / mL. Accurately pipette the stock solution and prepare a reference solution of astragaloside A reference standard with methanol at a concentration of 250.08 μg / mL.

[0091] 5.3 Preparation of the test solution

[0092] (1) HGWD lyophilized powder test solution: Accurately weigh 1.1 g of HGWD lyophilized powder, place it in a 100 mL stoppered conical flask, add 25 mL of ultrapure water, shake well to reconstitute, let stand for 30 min, transfer to a 250 mL separatory funnel, extract 4 times with 40 mL of water-saturated n-butanol each time, separate and combine the upper n-butanol layer, wash twice with 40% ammonia water each time, discard the ammonia water, evaporate the n-butanol solution to dryness in a water bath, dissolve it in methanol and transfer it to a 5 mL volumetric flask, add methanol to the mark, shake well, filter through a 0.45 μm nylon membrane, and take the filtrate to obtain the test solution.

[0093] (2) HGWD decoction test solution: Accurately pipette 25 mL of HGWD decoction into a 250 mL separatory funnel and prepare it by the same method as in item “5.3 (1)” of this example.

[0094] (3) Negative test solution: The negative decoction and negative lyophilized powder for Astragalus deficiency prepared under “3.4” in Example 3 were used to prepare negative test solutions for Astragalus deficiency according to the methods under “5.3 (1)” and “(2)” in this Example.

[0095] 5.4 Specificity Assessment: Accurately pipette the astragaloside A reference solution, HGWD test solution, astragalus-deficient negative test solution, and blank solvent, and inject them according to the chromatographic conditions described in section "5.1" of this embodiment for determination. Figure 10 As shown, the astragaloside A chromatographic peak was resolved to other chromatographic peaks by more than 1.5, with no interference from negative and blank solvents, a stable baseline, and strong specificity.

[0096] 5.5 Linearity Examination Accurately measure the reference stock solution from section "5.2" of this embodiment, dilute it to prepare reference solutions of different mass concentrations, and inject them for determination according to the chromatographic conditions in section "5.1" of this embodiment. Plot a standard curve with the logarithm of the mass concentration of the astragaloside A reference solution as the abscissa (X) and the logarithm of the peak area as the ordinate (Y). Calculate the regression equation using the least squares method, obtaining the linear regression equation as Y = 1.43X - 3.19, r = 0.9980, with a linear range of 49.92–500.25 μg / mL.

[0097] 5.6 Instrument precision test: The same astragaloside A reference solution was injected and measured 6 times consecutively under the chromatographic conditions in section "5.1" of this example. The RSD of the peak area of ​​astragaloside A was 0.79%, indicating that the instrument has good precision.

[0098] 5.7 Precision test: The 10th batch of HGWD decoction sample solution was injected and measured 6 times consecutively according to the chromatographic conditions in section "5.1" of this example. The RSD of the peak area of ​​astragaloside A was 1.59%, which indicates that the injection precision of this method is good.

[0099] 5.8 Determination of Detection Limit and Quantification Limit The astragaloside A reference stock solution under section “5.2” of this embodiment was serially diluted according to the ratio and injected for determination according to the chromatographic conditions under section “5.1” of this embodiment. The detection limit and quantification limit of astragaloside A were calculated to be 11.86 and 39.55 µg / mL, respectively, with signal-to-noise ratios of 3 and 10.

[0100] 5.9 Repeatability test: Take the 10th batch of HGWD decoction and prepare 6 test solutions according to the method in section "5.3 (2)" of this example. The samples were injected and determined according to the chromatographic conditions in section "5.1" of this example. The RSD of the mass fraction of astragaloside A was 4.35%, indicating that the method has good repeatability.

[0101] 5.10 Stability Study: The 10th batch of HGWD decoction test solution was taken and chromatographically analyzed at 0, 5, 8, 14, 24, 29, and 41 h after preparation, according to the chromatographic conditions in section "5.1" of this embodiment. The RSD of the peak area of ​​astragaloside A was 5.83%, indicating that the test solution was stable within 41 h.

[0102] 5.11 Intermediate precision assessment: On the second day, different personnel accurately measured the 10th batch of HGWD decoction and prepared 6 test solutions according to the method in section "5.3 (2)" of this embodiment. The samples were injected and measured according to the chromatographic conditions in section "5.1" of this embodiment. The RSD of the peak area of ​​astragaloside A was 3.06%, indicating that the intermediate precision of the method was good.

[0103] 5.12 Recovery Rate Study 12.5 mL of HGWD decoction with known astragaloside A content was precisely measured and placed in a 250 mL separatory funnel. 12.5 mL of purified water was added and mixed well. Then, 366 µL of the stock solution from section “5.2” of this embodiment was added and mixed well. The solution was extracted four times with 40 mL of water-saturated n-butanol each time, following the same method as in section “5.3(2)” of this embodiment, to prepare the test solution for the recovery rate determination. The sample was injected and determined according to the chromatographic conditions in section “5.1” of this embodiment. The average recovery rate of astragaloside A was calculated to be 95.15%, with an RSD of 5.09%, which meets the requirements of the 2025 edition of the Chinese Pharmacopoeia. The results indicate that the method has good accuracy.

[0104] 5.13 Content Determination: Accurately take 15 batches each of HGWD decoction and lyophilized powder, and prepare test solutions according to the method in section "5.3" of this embodiment. Accurately inject 10 μL of the test solution and 2, 5, and 10 μL of the reference solution in section "5.2" of this embodiment into the chromatograph. Determine the content of astragaloside A according to the chromatographic conditions in section "5.1". The results are shown in Tables 5 and 6.

[0105] Example 6: Method Optimization, Index Component Selection, and Overall Quality Evaluation

[0106] In establishing the method of this invention, this embodiment investigated the extraction effects of different solvents (water, methanol with different volume fractions), ultrasonic frequencies (0, 45, 80, 100 kHz) on the test samples. The effects of mobile phases such as methanol or acetonitrile-0.1% phosphoric acid or 0.2% phosphoric acid aqueous solution, seven detection wavelengths, five different chromatographic columns, and the number of extractions with water-saturated n-butanol were compared. The results showed that the method presented here is superior. The flow rate, column temperature, and phosphoric acid volume fraction exhibit good robustness, while the column robustness is moderate. An Agilent Zorbax SB-C18 column or a column with equivalent performance should be consistently used to determine the content of eight components in HGWD decoction and lyophilized powder. The volume fraction, column temperature, and chromatographic column exhibit good robustness for HPLC determination of astragaloside A content.

[0107] The selection criteria for the indicator components are as follows: HGWD is a traditional Chinese medicine formula for treating "blood stasis". Among its components, astragaloside A can protect nerve cells; verrucoside isoflavone glucoside can improve microcirculation and relieve pain; gentianin can regulate vasomotor activity and promote bone health; cinnamaldehyde can dilate blood vessels, promote sweating, and relieve pain; cinnamic acid can reduce inflammation and inhibit platelet aggregation; paeoniflorin and paeoniflorin lactone can reduce inflammation and relieve pain; gallic acid can reduce inflammation and protect nerve cells; and 6-gingerol can dilate blood vessels and inhibit the release of inflammatory factors. National standards and literature on jujube formula granules have been used to determine cyclic adenosine monophosphate (cAMP) in jujubes. However, through a series of investigations including chromatographic column analysis, mobile phase testing, and sample preparation methods, cAMP was not detected in HGWD decoction, nor were kaempferol and quercetin detected. Based on the prediction of HGWD quality markers and the content indicators in the 2025 edition of the Chinese Pharmacopoeia, this experiment identified astragaloside A, gallic acid, paeoniflorin, paeoniflorin, verbascoside, cinnamic acid, cinnamaldehyde, gentiopicrin, and 6-gingerol as nine indicator components to characterize the quality of HGWD decoctions and freeze-dried powders.

[0108] This invention introduces a novel HPLC method to comprehensively determine the similarity of characteristic chromatograms and the content of nine quality marker components in 15 batches of HGWD decoctions and lyophilized powders. This provides data for the overall quality evaluation of HGWD reference samples and granule development. The inter-batch similarity of the characteristic chromatograms of the 15 batches of lyophilized powder and decoctions was ≥0.909, and the intra-batch similarity was ≥0.983, indicating good inter-batch and intra-batch quality consistency of HGWD lyophilized powder and decoctions. The 15 characteristic peaks established in this invention cover all medicinal slices, showcasing one characteristic peak shared with jujube, and this invention detects the most indicator components.

[0109] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A method for simultaneous qualitative and quantitative overall quality control of a Huangqi Guizhi Wuwu Decoction reference sample, characterized in that: Includes the following steps: (1) Multiple batches of HGWD decoction and lyophilized powder reference samples were prepared using the water decoction process optimized by orthogonal experiment; (2) Establish HPLC characteristic chromatograms for multiple batches of HGWD decoction and lyophilized powder reference samples obtained in step (1), and identify 15 common peaks with cinnamaldehyde as the reference peak. (3) Based on the reference sample obtained in step (1), prepare the test solution and use multi-wavelength HPLC to simultaneously determine the contents of eight index components: gallic acid, paeoniflorin, paeoniflorin, verbascoflavonoid glucoside, cinnamic acid, cinnamaldehyde, gentianin and 6-gingerol. The detection wavelengths are 270 nm for gallic acid, 232 nm for paeoniflorin and paeoniflorin, 248 nm for verbascoflavonoid glucoside and gentianin, 278 nm for cinnamic acid, 290 nm for cinnamaldehyde and 220 nm for 6-gingerol. (4) Prepare a test solution from the reference sample obtained in step (1) and determine the content of astragaloside A by HPLC-ELSD method; (5) Integrate the feature spectrum data and multi-index component content data obtained from steps (2) to (4), and achieve simultaneous qualitative and quantitative overall quality control of the reference sample through feature spectrum similarity evaluation and multi-index component content consistency evaluation.

2. The method as described in claim 1, characterized in that: The multiple batches of freeze-dried powder reference samples prepared in step (1) were randomly combined from medicinal materials from different origins.

3. The method as described in claim 1, characterized in that: In step (2), the HPLC characteristic chromatogram was established using an Agilent Zorbax SB-C18 column and gradient elution was performed using acetonitrile-0.1% phosphoric acid aqueous solution.

4. The method as described in claim 1, characterized in that: The 15 common peaks in step (2) were compared and identified as medicinal materials through a comparison experiment of reference solution, single herbal decoction piece test solution and negative test solution.

5. The method as described in claim 1, characterized in that: In step (3), the HGWD decoction and the lyophilized powder reference sample solution after water reconstitution are prepared by directly diluting with methanol to prepare the test solution. In step (4), the test solution is prepared by water-saturated n-butanol extraction and ammonia washing and purification.

6. The method as described in claim 1, characterized in that: In step (5), the similarity of feature maps of multiple batches of reference samples is evaluated, and the similarity between batches is not less than 0.

9.

7. The method as described in claim 1, characterized in that: It also includes determining the content of components with content limits in the Chinese Pharmacopoeia in medicinal materials and decoction pieces, and comparing them with the content in decoctions and freeze-dried powders, so as to realize the whole process of value transfer evaluation from raw medicinal materials to the final reference sample.

8. The method as described in claim 1, characterized in that: The consistency evaluation of the content in step (5) is based on the fact that the content of more than 5 of the 9 index components, namely gallic acid, paeoniflorin, paeoniflorin, verbascoflavonoid glucoside, cinnamic acid, cinnamaldehyde, gentianin, 6-gingerol and astragaloside A, is within ±30% of their respective mean values.

9. The method as described in claim 1, characterized in that: It also includes methodological validation for simultaneous multi-wavelength HPLC determination and HPLC-ELSD determination, with validation items including specificity, linearity, precision, repeatability, stability, and recovery rate.

10. The method as described in claim 1, characterized in that: The 15 common peaks identified by the HPLC characteristic chromatograms cover the characteristic components of five medicinal herbs: Astragalus membranaceus, Paeonia lactiflora, Cinnamomum cassia, Zingiber officinale, and Ziziphus jujuba.

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