A method for constructing a characteristic spectrum of Huangqi Guizhi Wuwu Decoction and a quality control method

Abstract

This invention belongs to the field of traditional Chinese medicine quality control technology, specifically relating to a method for constructing a characteristic chromatogram and a quality control method for Huangqi Guizhi Wuwu Decoction. The quality control method includes a characteristic chromatogram identification method and a thin-layer chromatography (TLC) identification method. This invention uses UPLC for the construction and identification of the characteristic chromatogram, which is fast, efficient, and cost-effective. The characteristic chromatogram specifies 17 common characteristic peaks and identifies 12 of them, demonstrating a high identification rate, which is beneficial for the analysis and identification of the material basis of Huangqi Guizhi Wuwu Decoction. The TLC identification method of this invention successfully creates a novel method for identifying two herbs (Astragalus membranaceus and Paeonia lactiflora) in a compound formula using a single test with multiple evaluations. The detection conditions are simple, fast, efficient, highly specific, cost-effective, and highly practical.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine quality control technology, specifically relating to a method for constructing a characteristic spectrum of Huangqi Guizhi Wuwu Decoction and a quality control method. Background Technology

[0002] Huangqi Guizhi Wuwu Decoction was created by Zhang Zhongjing of the Han Dynasty and is found in the sixth chapter of "Blood Stasis and Deficiency Diseases" in Volume 1 of the *Synopsis of Prescriptions of the Golden Chamber*. The original text states, "Blood stasis, with both Yin and Yang deficient, weak pulse at the Cun and Guan positions, and slightly tight pulse at the Chi position, with external symptoms of numbness in the body, resembling wind-dampness, is treated with Huangqi Guizhi Wuwu Decoction." In 2018, Huangqi Guizhi Wuwu Decoction was included in the first batch of the Catalogue of Ancient Classic Prescriptions, ranking 16th. In 2022, the state published the prescription information for Huangqi Guizhi Wuwu Decoction for the first time: it consists of five herbs: Astragalus membranaceus, Paeonia lactiflora, Cinnamomum cassia, Zingiber officinale, and Ziziphus jujuba, with one liang (approximately 30 grams) equal to 13.8g. Huangqi Guizhi Wuwu Decoction has the effects of tonifying Qi, warming the meridians, harmonizing blood, and relieving stagnation. It is mainly used to treat blood stasis, wind-dampness, stagnation due to deficiency, and stroke (especially affecting the meridians).

[0003] In recent years, with the increasing interest in researching classic prescriptions, research on the quality standards of Huangqi Guizhi Wuwu Tang (Astragalus and Cinnamon Twig Decoction) has also been deepening. Regarding the quality standards and evaluation of Huangqi Guizhi Wuwu Tang, the main focus has been on qualitative or quantitative determination using the content indicators of single medicinal materials from the pharmacopoeia. However, there has been little breakthrough or expansion in the selection of indicator components, particularly regarding the low rate of analysis and identification of volatile components in the compound formula, failing to reflect the integrity of the prescription and the interaction of multiple components.

[0004] Thin-layer chromatography (TLC) identification and characteristic chromatograms are relatively mature qualitative quality characterization methods used in quality standards. While there is some research foundation and related reports on TLC identification and characteristic chromatograms of Huangqi Guizhi Wuwu Decoction in the existing technology, some shortcomings and deficiencies still exist.

[0005] 1. Thin-layer chromatography identification:

[0006] Common drawbacks: Traditional Chinese medicine (TCM) compound preparations often contain numerous herbs and have complex chemical compositions. Significant interference exists between different herbs and components, making it difficult to identify a specific spot associated with a particular herb from a large pool of ingredients. Current thin-layer chromatography (TLC) methods for compound preparations typically employ one identification method per herb, resulting in multiple methods for several herbs. This necessitates significant investment of samples, manpower, and time. Furthermore, to eliminate interference, the pretreatment process for compound preparations often involves multiple purification and enrichment steps. These numerous steps involve a wide variety and large quantities of organic reagents, are time-consuming, and can pollute the environment and pose health risks. Therefore, finding a simple and rapid detection method suitable for water-extracted preparations, improving detection efficiency, and reducing detection costs are crucial for improving TLC identification methods for TCM compound preparations.

[0007] Fan Yue and others [1]A quality standard for Huangqi Guizhi Wuwu Decoction granules was established, employing two different thin-layer chromatography (TLC) methods for the identification of Astragalus membranaceus (Huangqi) and Paeonia lactiflora (Bai Shao). In the pretreatment of Astragalus membranaceus samples, the test sample was extracted with two organic reagents (ether and n-butanol), washed multiple times, and then concentrated and evaporated to dryness—a cumbersome and complex process. Similarly, the identification of Paeonia lactiflora involved multiple extractions, washings, and concentrations with n-butanol, a repetitive and tedious operation. The development process for Paeonia lactiflora required four solvents to prepare the developing solvent, resulting in a large number of solvents and complicated preparation. In summary, the two herbs were treated using two different pretreatment methods, both requiring multiple extractions, washings, and concentrations—a complex and time-consuming process (estimated to take two days for both herbs).

[0008] 2. Feature map:

[0009] CN110887914A discloses an HPLC detection method for the fingerprint chromatogram of lyophilized Astragalus and Cinnamon Twig Five-Ingredient Decoction, with an elution program of 77 minutes (the retention time of the last chromatographic peak still requires about 55 minutes), resulting in a long detection time. Furthermore, neither method explicitly identifies the chromatographic peaks in the fingerprint chromatogram. CN110887927A discloses an HPLC-Q-TOF-MS / MS method (ultra-fast high-performance liquid chromatography-triple quadrupole time-of-flight tandem mass spectrometry) for the analysis and detection of Astragalus and Cinnamon Twig Five-Ingredient Decoction samples. However, this HPLC-MS / MS technique has high detection costs and cannot be well applied to production testing, exhibiting poor universality. Moreover, the sample preparation process requires concentration and freeze-drying steps, leading to a significant loss or reduction in the content of volatile oil components of cinnamon twig in the compound.

[0010] Yu Aiming and others [2] A fingerprint chromatogram of the standard Huangqi Guizhi Wuwu Decoction was established using HPLC, matching 17 common peaks. While the overall qualitative analysis is of reference value, only three components were identified, and numerous unknown peaks prevented analysis and identification of the material basis. Furthermore, the elution procedure was 70 minutes, resulting in a long detection time. (Wei Guoli et al.) [3] Similarly, an HPLC fingerprint of Huangqi Guizhi Wuwu Decoction was established, which used paeoniflorin as a reference peak to identify 13 common peaks of Huangqi Guizhi Wuwu Decoction. However, apart from paeoniflorin, the other 12 common peaks were not identified.

[0011] Therefore, most of the above characteristic spectral studies use HPLC for determination, which generally has a long analysis cycle and high time cost; there are also many unknown characteristic peaks, which is not conducive to the analysis and identification of the material basis.

[0012] [1] Fan Yue, Zhang Wenwei, Tian Ming, et al. Study on quality standard of Huangqi Guizhi Wuwu Decoction granules [J]. Information on Traditional Chinese Medicine, 2019, 36(4):23-26.

[0013] [2] Yu Aiming, Yan Xiangli, Wang Shengxin, et al. Establishment of HPLC fingerprint of standard Huangqi Guizhi Wuwu Decoction and determination of the content of three components [J]. Chinese Journal of Traditional Chinese Medicine, 2019, 37(9):2196-2200.

[0014] [3] Wei Guoli, Cao Peng, Huo Jiege, et al. Study on HPLC fingerprint of Huangqi Guizhi Wuwu Decoction [J]. Chinese Journal of Traditional Chinese Medicine, 2013, 31(12):2796-2798. Summary of the Invention

[0015] The problem the invention aims to solve

[0016] To address the aforementioned issues, this invention aims to provide a method for constructing a characteristic chromatogram and a quality control method for Huangqi Guizhi Wuwu Decoction. The quality control method includes characteristic chromatogram and thin-layer chromatography identification: the characteristic chromatogram has a short analysis cycle, low detection cost, and high identification rate of common peaks, especially enabling comprehensive analysis of volatile components in the compound; the thin-layer chromatography identification method can achieve multiple evaluations of multiple medicinal ingredients with a single test, which is convenient, fast, cost-effective, highly specific, and practical.

[0017] Solution for solving the problem

[0018] In a first aspect, the present invention provides a method for constructing a characteristic map of Huangqi Guizhi Wuwu Decoction, which includes the following steps:

[0019] (1) Preparation of sample solution of Huangqi Guizhi Wuwu Decoction:

[0020] Take the extract of Huangqi Guizhi Wuwu Decoction, dilute it with methanol, filter it, and take the filtrate to obtain the final product;

[0021] (2) Preparation of reference solution:

[0022] Take paeoniflorin, verbascoside, cinnamyl alcohol, cinnamaldehyde, cinnamic acid, 6-gingerol, gallic acid, paeoniflorin lactone, coumarin, galloylpaeoniflorin, 1,2,3,4,6-penta-galloyl glucoside and verbascoside reference standards respectively, and add methanol to prepare reference standard solutions.

[0023] (3) Construction of feature maps:

[0024] The sample solution of Huangqi Guizhi Wuwu Decoction in step (1) and the reference solution in step (2) were subjected to UPLC analysis to obtain the characteristic chromatogram of Huangqi Guizhi Wuwu Decoction. The chromatographic conditions of the UPLC analysis included: the column was packed with octadecylsilane-bonded silica gel; mobile phase A was methanol and mobile phase B was aqueous phosphoric acid solution; gradient elution; and the detection wavelength was 230-280 nm.

[0025] Preferably, in step (1), the volume ratio of the Astragalus and Cinnamon Twig Decoction Extract to the Methanol is 3:1 to 1:4, more preferably 1:1 to 1:4, and even more preferably 1:1.

[0026] Preferably, in step (3), the gradient elution procedure is as follows:

[0027]

[0028] More preferably, in step (3), the gradient elution procedure is as follows:

[0029]

[0030] Alternatively, more preferably, in step (3), the gradient elution procedure is as follows:

[0031]

[0032] Preferably, in step (3), the detection wavelength is 250-270 nm, more preferably 260 nm.

[0033] Preferably, in step (3), the mobile phase B is a 0.01% to 0.1% aqueous phosphoric acid solution, more preferably a 0.05% aqueous phosphoric acid solution.

[0034] Preferably, in step (3), the chromatographic column is an HSS T3 column or an Acclaim column. TM RSLC 120C18 column, preferably HSS T3 column.

[0035] Preferably, in step (3), the chromatographic column has the following specifications: column length 100 mm, inner diameter 2.1 mm, and particle size 1.7 to 2.2 μm, preferably 1.7 to 1.8 μm.

[0036] Preferably, in step (3), the chromatographic conditions for the UPLC analysis further include: a column temperature of 25-35°C, preferably 30°C.

[0037] Preferably, in step (3), the chromatographic conditions for the UPLC analysis further include a flow rate of 0.2 to 0.30 ml per minute, preferably 0.25 ml.

[0038] Preferably, in step (3), the chromatographic conditions for the UPLC analysis further include: an injection volume of 1 to 3 μl, preferably 2 μl.

[0039] Preferably, in step (3), the characteristic chromatogram of the Astragalus and Cinnamon Twig Decoction has 17 characteristic peaks, wherein peaks 1, 4, 5, 6, 8, 9, 10, 11, 12, 13, 15 and 17 correspond to the retention times of the chromatographic peaks of gallic acid, paeoniflorin, paeoniflorin, coumarin, versicolor glucoside, galloylpaeoniflorin, 1,2,3,4,6-penta-galloyl glucoside, cinnamyl alcohol, cinnamaldehyde, cinnamic acid, versicolor, and 6-gingerol reference standards.

[0040] More preferably, in step (3), the peak corresponding to the chromatographic peak of 1,2,3,4,6-penta-galloylglucoside reference standard (peak 10) is taken as peak S, and the relative retention times of peaks 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, and 17 with peak S are within ±15% of a specified value, wherein the specified value is as follows: 0.10 (peak 1), 0.11 (peak 2), 0.17 (peak 3), 0.38 (peak 4), 0.51 (peak 5), 0.71 (peak 6), 0.74 (peak 7), 0.81 (peak 8), 0.88 (peak 9), 1.08 (peak 11), 1.15 (peak 12), 1.45 (peak 13), 1.71 (peak 14), 1.78 (peak 15), 1.92 (peak 16) and 1.96 (peak 17).

[0041] More preferably, in step (3), the peak corresponding to the chromatographic peak of 1,2,3,4,6-penta-galloylglucoside reference standard (peak 10) is taken as peak S, and the relative retention times of peaks 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, and 17 with peak S are within ±10% of a specified value, wherein the specified value is as follows: 0.1 0 (peak 1), 0.11 (peak 2), 0.17 (peak 3), 0.38 (peak 4), 0.51 (peak 5), 0.71 (peak 6), 0.74 (peak 7), 0.81 (peak 8), 0.88 (peak 9), 1.08 (peak 11), 1.15 (peak 12), 1.45 (peak 13), 1.71 (peak 14), 1.78 (peak 15), 1.92 (peak 16) and 1.96 (peak 17).

[0042] Secondly, this invention provides a quality control method for Huangqi Guizhi Wuwu Decoction, which includes the following steps:

[0043] (1) Preparation of the test solution:

[0044] Take the extract of Huangqi Guizhi Wuwu Decoction used as the test sample, dilute it with methanol, filter it, and take the filtrate to obtain the final product.

[0045] (2) Quality evaluation of the test sample:

[0046] The test solution described in step (1) is analyzed under the UPLC chromatographic conditions in the characteristic chromatogram construction method described in the first aspect to obtain the test chromatogram, and then compared with the characteristic chromatogram of Huangqi Guizhi Wuwu Decoction in the characteristic chromatogram construction method described in the first aspect.

[0047] Preferably, in step (1), the volume ratio of the Astragalus and Cinnamon Twig Decoction Extract to the Methanol is 3:1 to 1:4, more preferably 1:1 to 1:4, and even more preferably 1:1.

[0048] Thirdly, the present invention also provides another method for quality control of Huangqi Guizhi Wuwu Decoction, which includes the following steps:

[0049] (1) Preparation of the test solution:

[0050] Take the extract of Astragalus and Cinnamon Twig Decoction as the test sample, shake and extract with water-saturated alcohol solvent, combine the alcohol solvents, wash with alkaline test solution, discard the alkaline test solution, evaporate the alcohol solvent to dryness, dissolve the residue in methanol to obtain the test sample solution.

[0051] (2) Preparation of control herbal solution:

[0052] Take Astragalus membranaceus and Paeonia lactiflora as reference herbs, add methanol, sonicate, filter, evaporate the filtrate to dryness, dissolve the residue in water, extract by shaking with water-saturated alcohol solvent, combine the alcohol solvents, wash with alkaline test solution, discard the alkaline test solution, evaporate the alcohol solvent to dryness, dissolve the residue in methanol to obtain Astragalus membranaceus reference herb solution and Paeonia lactiflora reference herb solution.

[0053] (3) Preparation of reference solution:

[0054] Take astragaloside A and paeoniflorin as reference standards respectively, and add methanol to prepare astragaloside A reference standard solution and paeoniflorin reference standard solution;

[0055] (4) Thin-layer chromatography identification:

[0056] The test solution described in step (1), the Astragalus reference material solution and Paeonia lactiflora reference material solution described in step (2), and the Astragaloside A reference solution and Paeoniflorin reference solution described in step (3) are spotted onto the same thin-layer plate, developed, and the determination results of the test sample, reference materials, and reference standards are compared; wherein, the chromatographic conditions for the thin-layer chromatography identification include: using silica gel as the stationary phase; using chloroform-methanol-water as the developing solvent; using sulfuric acid ethanol solution as the colorimetric reagent; and examining under sunlight and ultraviolet light respectively.

[0057] Preferably, in step (1), the alcohol solvent is n-butanol, n-propanol, isopropanol or isobutanol, preferably n-butanol.

[0058] Preferably, in step (1), the volume ratio of the Astragalus and Cinnamon Twig Decoction extract to the water-saturated alcohol solvent is 3:1 to 3:4, more preferably 3:2 to 3:4, and even more preferably 3:2.

[0059] Preferably, in step (1), the shaking extraction is performed 2 to 4 times, preferably 2 times.

[0060] Preferably, in step (1), the alkaline test solution is an ammonia test solution or an aqueous solution of an alkali metal hydroxide, with ammonia test solution being preferred.

[0061] Preferably, in step (2), the ratio of Astragalus reference material to methanol is 1g:30ml to 1g:90ml, more preferably 1g:60ml to 1g:90ml, and even more preferably 1g:60ml.

[0062] Preferably, in step (2), the ultrasound duration is 20 to 40 minutes, more preferably 30 minutes.

[0063] Preferably, in step (2), the volume ratio of the water to the water-saturated alcohol solvent is 2:1 to 1:4, more preferably 1:1 to 1:2, and even more preferably 1:1.

[0064] Preferably, in step (2), the shaking extraction is performed 2 to 4 times, preferably 2 times.

[0065] Preferably, in step (2), the alkaline test solution is an ammonia test solution or an aqueous solution of an alkali metal hydroxide, with ammonia test solution being preferred.

[0066] Preferably, in step (4), the amount of the test solution, the astragalus and white peony reference solutions is 5-10 μl; and the amount of the astragaloside A and paeoniflorin reference solutions is 1-3 μl.

[0067] Preferably, in step (4), the volume ratio of chloroform-methanol-water in the developing agent is (10-15):(5-10):(1-3), more preferably 13:7:2.

[0068] Preferably, in step (4), the developing agent is a lower layer solution of chloroform-methanol-water placed below 10°C.

[0069] Preferably, in step (4), the volume percentage of the sulfuric acid ethanol solution is 5% to 15%, more preferably 10%.

[0070] Preferably, in step (4), the heating temperature is 90-120°C, more preferably 105°C.

[0071] Preferably, in step (4), the sunlight includes ultraviolet light, visible light and infrared light, and is preferably visible light.

[0072] Preferably, in step (4), the wavelength of the ultraviolet light is 355-375nm, more preferably 365nm.

[0073] Preferably, step (4) further includes the following steps: comparing the determination results of the test sample with those of the reference medicinal material and the reference standard, and determining the thin-layer chromatograms of the test sample, the Astragalus reference medicinal material, and the Astragaloside A reference standard in R f To determine whether spots of the same color exist at the same location, and to judge the thin-layer chromatograms of the test sample, white peony reference material, and paeoniflorin reference standard in R... f Does the same color of spot exist at the same location?

[0074] Fourthly, the present invention also provides a quality control method for Huangqi Guizhi Wuwu Decoction, which includes the quality control method described in the second aspect and the quality control method described in the third aspect.

[0075] The effects of the invention

[0076] The quality control method of Huangqi Guizhi Wuwu Decoction in this invention includes the identification of characteristic chromatograms and thin-layer chromatography. By using the above two methods, Huangqi Guizhi Wuwu Decoction is qualitatively detected from multiple perspectives, providing a reference for the development and research and comprehensive quality control of Huangqi Guizhi Wuwu Decoction.

[0077] This invention utilizes UPLC to construct characteristic spectra, offering advantages such as short detection time, high specificity, speed, efficiency, and low cost, making it practically significant in industrial production. The characteristic spectra define 17 common characteristic peaks, and the identification of 12 of these peaks has been completed, demonstrating a high identification rate and facilitating the analysis and identification of the material basis of Huangqi Guizhi Wuwu Decoction. In particular, the characteristic spectra newly include cinnamyl alcohol from cinnamon twig, enriching the analysis of volatile components in Huangqi Guizhi Wuwu Decoction. Furthermore, cinnamyl alcohol has been characterized and identified in the fingerprint / characteristic spectra; this component has corresponding chromatographic peaks in the characteristic spectra of standard decoctions, extracts, and compound preparations, fully reflecting and restoring the quality and efficacy of a clinically effective decoction.

[0078] The thin-layer chromatography (TLC) identification method of this invention uses only one extraction solvent (such as n-butanol) to achieve the extraction purpose, eliminating the need for repeated extraction with multiple solvents. This identification method, based on a thorough analysis of the characteristics of the indicator components, combines common elements to identify differences. Using the same test solution on a single TLC plate under two different viewing conditions (sunlight and ultraviolet light), it rapidly reveals well-distinguishing spots. This successfully establishes a novel TLC identification method for identifying two herbs (Astragalus membranaceus and Paeonia lactiflora) in a compound prescription, offering multiple evaluations with a single test. The method is simple, rapid, efficient, highly specific, low-cost, and highly practical. Attached Figure Description

[0079] Figure 1 Thin-layer chromatography (UV 365nm) for the identification of Astragalus membranaceus and Paeonia lactiflora in Huangqi Guizhi Wuwu Decoction.

[0080] Figure 2 Thin-layer chromatography (sunlight) for the identification of Astragalus membranaceus and Paeonia lactiflora in Huangqi Guizhi Wuwu Decoction.

[0081] Figure 3 The results of column chromatography for Huangqi Guizhi Wuwu Decoction are presented.

[0082] Figure 4 Results of the washout gradient study for Huangqi Guizhi Wuwu Decoction.

[0083] Figure 5 This is an overlay diagram of multiple batches (S1~S15) of Huangqi Guizhi Wuwu Decoction.

[0084] Figure 6 This is a comparative diagram of the characteristics of Huangqi Guizhi Wuwu Decoction.

[0085] Figure 7 This is an identification map of characteristic peaks in the characteristic map of Huangqi Guizhi Wuwu Decoction. Detailed Implementation

[0086] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0087] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the present invention.

[0088] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0089] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0090] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0091] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0092] In this invention, the preparation method of "ammonia test solution" is as described in Section 8002 of the General Chapter of Part IV of the 2020 edition of the Chinese Pharmacopoeia, which is "ammonia test solution". The preparation method is as follows: take 400 ml of concentrated ammonia solution and add water to make 1000 ml.

[0093] In this invention, the "Astragalus and Cinnamon Twig Decoction Extract" is the decoction of Astragalus and Cinnamon Twig Decoction. The preparation method is as follows: take appropriate amounts of five herbs: Astragalus, White Peony Root, Cinnamon Twig, Ginger, and Jujube, add 1200ml of water, bring to a boil over high heat, and simmer over low heat until reduced to 400ml.

[0094] In this invention, the quality control method of Huangqi Guizhi Wuwu Decoction includes characteristic chromatographic identification and thin-layer chromatography identification. The above two methods are used to perform multi-dimensional qualitative detection of Huangqi Guizhi Wuwu Decoction, providing a reference for the development and research and comprehensive quality control of Huangqi Guizhi Wuwu Decoction.

[0095] [Construction of Feature Maps]

[0096] This invention utilizes UPLC to establish a characteristic spectrum of Huangqi Guizhi Wuwu Decoction, which is quick, efficient, and efficient. It identifies 17 common characteristic peaks and completes the identification of 12 characteristic peaks with a high identification rate.

[0097] The main active ingredients in the Huangqi Guizhi Wuwu Decoction (such as astragaloside A, verbascoside, paeoniflorin, and cinnamaldehyde) are mostly pharmacopoeia indicators, and the analytical methods are relatively mature. However, the preservation and analysis of various volatile components in cinnamon twigs are often neglected or lost. Existing studies have used improper sample preparation methods, resulting in significant losses of volatile oil components in cinnamon twigs, making them difficult to detect and resulting in incomplete component characterization. The main reason is that the sample preparation processes in existing studies have certain drawbacks: generally, after water extraction, vacuum concentration or vacuum drying is used to obtain the sample. However, volatile oil components are largely lost during vacuum filtration, resulting in a limited variety of volatile oils in the obtained sample, which fails to achieve a comprehensive evaluation of the quality of Huangqi Guizhi Wuwu Decoction.

[0098] In the Huangqi Guizhi Wuwu Decoction, cinnamon twig is considered an assistant herb, possessing the functions of inducing sweating and relieving muscle tension, warming and unblocking the meridians, assisting yang and transforming qi, and calming and descending qi. Cinnamon twig volatile oil is the main active substance of cinnamon twig, exhibiting good anti-inflammatory, antibacterial, antiviral, and antitumor effects. This invention adds cinnamyl alcohol from cinnamon twig to the characteristic spectrum, thereby achieving a comprehensive evaluation of the quality of Huangqi Guizhi Wuwu Decoction. Pharmacological studies have shown that cinnamyl alcohol can downregulate the synthesis and secretion of PGE2 in rat brain microvascular endothelial cells induced by IL-1 stimulation, and its effect is related to the inhibition of COX-2 activity. Cinnamyl alcohol can activate K+... + The mediated NO-cGMP-PKG pathway and inhibition of Rho kinase lead to vasodilation.

[0099] Specifically, the construction of the feature map of the present invention includes the following steps:

[0100] (1) Preparation of sample solution of Huangqi Guizhi Wuwu Decoction:

[0101] Take the extract of Huangqi Guizhi Wuwu Decoction, dilute it with methanol, filter it, and take the filtrate to obtain the final product;

[0102] (2) Preparation of reference solution:

[0103] Take paeoniflorin, verbascoside, cinnamyl alcohol, cinnamaldehyde, cinnamic acid, 6-gingerol, gallic acid, paeoniflorin lactone, coumarin, galloylpaeoniflorin, 1,2,3,4,6-penta-galloyl glucoside and verbascoside reference standards respectively, and add methanol to prepare reference standard solutions.

[0104] (3) Construction of feature maps:

[0105] The sample solution of Huangqi Guizhi Wuwu Decoction in step (1) and the reference solution in step (2) were subjected to UPLC analysis to obtain the characteristic chromatogram of Huangqi Guizhi Wuwu Decoction. The chromatographic conditions of the UPLC analysis included: the column was packed with octadecylsilane-bonded silica gel; mobile phase A was methanol and mobile phase B was aqueous phosphoric acid solution; gradient elution; and the detection wavelength was 230-280 nm.

[0106] In some implementations, in step (1), the volume ratio of the Astragalus and Cinnamon Twig Decoction extract to the methanol is 3:1 to 1:4, preferably 1:1 to 1:4, and more preferably 1:1.

[0107] In some implementations, in step (3), the gradient elution procedure is as follows:

[0108]

[0109] In the elution program of this invention, the numerical range of "time (minutes)" refers to any time within that time range. Specifically, "19-20" minutes can be 19 minutes, 19.5 minutes, or 20 minutes, etc.; "33-38" minutes can be 33 minutes, 35 minutes, or 38 minutes, etc. Similarly, the numerical ranges of "mobile phase A (%)" and "mobile phase B (%)" in the elution program also refer to any volume percentage within that volume percentage range. Verification by this invention shows that the above-mentioned gradient elution program can effectively separate 17 characteristic peaks.

[0110] In some preferred embodiments, in step (3), the gradient elution procedure is as follows:

[0111]

[0112] In some other preferred embodiments, in step (3), the gradient elution procedure is as follows:

[0113]

[0114] In some implementations, in step (3), the detection wavelength is 250-270 nm, preferably 260 nm.

[0115] In some embodiments, in step (3), the mobile phase B is a 0.01% to 0.1% aqueous phosphoric acid solution, preferably a 0.05% aqueous phosphoric acid solution.

[0116] In some implementations, in step (3), the chromatographic column is an HSS T3 column or an Acclaim column. TM RSLC120C18 column, preferably HSS T3 column.

[0117] In some implementations, in step (3), the chromatographic column has the following specifications: a column length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.7 to 2.2 μm, preferably 1.7 to 1.8 μm.

[0118] In some implementations, in step (3), the chromatographic conditions for the UPLC analysis further include a column temperature of 25–35°C, preferably 30°C.

[0119] In some implementations, the chromatographic conditions for the UPLC analysis in step (3) further include a flow rate of 0.2 to 0.30 ml per minute, preferably 0.25 ml.

[0120] In some implementations, the chromatographic conditions for the UPLC analysis in step (3) further include an injection volume of 1 to 3 μl, preferably 2 μl.

[0121] In some implementations, in step (3), the characteristic chromatogram of the Astragalus and Cinnamon Twig Decoction has 17 characteristic peaks, wherein peaks 1, 4, 5, 6, 8, 9, 10, 11, 12, 13, 15 and 17 correspond to the retention times of the chromatographic peaks of gallic acid, paeoniflorin, paeoniflorin, coumarin, vermifuran glucoside, galloylpaeoniflorin, 1,2,3,4,6-penta-galloyl glucoside, cinnamyl alcohol, cinnamaldehyde, cinnamic acid, vermifuran and 6-gingerol reference standards.

[0122] In some preferred embodiments, in step (3), the peak (peak 10) corresponding to the chromatographic peak of the 1,2,3,4,6-penta-galloylglucoside reference standard is used as peak S, and the relative retention times of peaks 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, and 17 with peak S are within ±15% of a specified value, which is as follows: 0 0.10 (peak 1), 0.11 (peak 2), 0.17 (peak 3), 0.38 (peak 4), 0.51 (peak 5), 0.71 (peak 6), 0.74 (peak 7), 0.81 (peak 8), 0.88 (peak 9), 1.08 (peak 11), 1.15 (peak 12), 1.45 (peak 13), 1.71 (peak 14), 1.78 (peak 15), 1.92 (peak 16) and 1.96 (peak 17).

[0123] In some preferred embodiments, in step (3), the peak (peak 10) corresponding to the chromatographic peak of 1,2,3,4,6-penta-galloylglucoside reference standard is used as peak S, and the relative retention times of peaks 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, and 17 with peak S are within ±10% of a specified value, which is as follows: 0.10 (peak 1), 0.11 (peak 2), 0.17 (peak 3), 0.38 (peak 4), 0.51 (peak 5), 0.71 (peak 6), 0.74 (peak 7), 0.81 (peak 8), 0.88 (peak 9), 1.08 (peak 11), 1.15 (peak 12), 1.45 (peak 13), 1.71 (peak 14), 1.78 (peak 15), 1.92 (peak 16) and 1.96 (peak 17).

[0124] [Characteristic Mapping Identification Method]

[0125] This invention provides a quality control method for Huangqi Guizhi Wuwu Decoction. Specifically, the quality control method is a characteristic chromatographic identification method, which includes the following steps:

[0126] (1) Preparation of the test solution:

[0127] Take the extract of Huangqi Guizhi Wuwu Decoction used as the test sample, dilute it with methanol, filter it, and take the filtrate to obtain the final product.

[0128] (2) Quality evaluation of the test sample:

[0129] The test solution described in step (1) is analyzed under the UPLC chromatographic conditions in the characteristic chromatogram construction method described in the first aspect to obtain the test chromatogram, and then compared with the characteristic chromatogram of Huangqi Guizhi Wuwu Decoction in the characteristic chromatogram construction method described in the first aspect.

[0130] In some implementations, in step (1), the volume ratio of the Astragalus and Cinnamon Twig Decoction extract to the methanol is 3:1 to 1:4, preferably 1:1 to 1:4, and more preferably 1:1.

[0131] [Thin-layer chromatography identification method]

[0132] Considering that the active ingredients in Astragalus membranaceus and Atractylodes macrocephala are both water-soluble, and that astragaloside A and paeoniflorin are both saponins with similar physicochemical properties, the thin-layer chromatography method of this invention can achieve the extraction objective using only one extraction solvent (n-butanol), eliminating the need for repeated extraction with multiple solvents. Furthermore, this invention successfully established a novel thin-layer chromatography identification method for identifying two medicinal ingredients in a compound formula using the same test solution on a single thin-layer plate under two different inspection conditions, enabling multiple evaluations with a single test.

[0133] Therefore, the present invention also provides another quality control method for Huangqi Guizhi Wuwu Decoction. Specifically, the quality control method is a thin-layer chromatography identification method, which includes the following steps:

[0134] (1) Preparation of the test solution:

[0135] Take the extract of Astragalus and Cinnamon Twig Decoction as the test sample, shake and extract with water-saturated alcohol solvent, combine the alcohol solvents, wash with alkaline test solution, discard the alkaline test solution, evaporate the alcohol solvent to dryness, dissolve the residue in methanol to obtain the test sample solution.

[0136] (2) Preparation of control herbal solution:

[0137] Take Astragalus membranaceus and Paeonia lactiflora as reference herbs, add methanol, sonicate, filter, evaporate the filtrate to dryness, dissolve the residue in water, extract by shaking with water-saturated alcohol solvent, combine the alcohol solvents, wash with alkaline test solution, discard the alkaline test solution, evaporate the alcohol solvent to dryness, dissolve the residue in methanol to obtain Astragalus membranaceus reference herb solution and Paeonia lactiflora reference herb solution.

[0138] (3) Preparation of reference solution:

[0139] Take astragaloside A and paeoniflorin as reference standards respectively, and add methanol to prepare astragaloside A reference standard solution and paeoniflorin reference standard solution;

[0140] (4) Thin-layer chromatography identification:

[0141] The test solution described in step (1), the Astragalus reference material solution and Paeonia lactiflora reference material solution described in step (2), and the Astragaloside A reference solution and Paeoniflorin reference solution described in step (3) are spotted onto the same thin-layer plate, developed, and the determination results of the test sample, reference materials, and reference standards are compared; wherein, the chromatographic conditions for the thin-layer chromatography identification include: using silica gel as the stationary phase; using chloroform-methanol-water as the developing solvent; using sulfuric acid ethanol solution as the colorimetric reagent; and examining under sunlight and ultraviolet light respectively.

[0142] In some embodiments, in step (1), the alcohol solvent is n-butanol, n-propanol, isopropanol or isobutanol, preferably n-butanol.

[0143] In some implementations, in step (1), the volume ratio of the Astragalus and Cinnamon Twig Decoction extract to the water-saturated alcohol solvent is 3:1 to 3:4, preferably 3:2 to 3:4, and more preferably 3:2.

[0144] In some implementations, the shaking extraction is performed 2 to 4 times in step (1), preferably 2 times.

[0145] In some embodiments, in step (1), the alkaline test solution is an ammonia test solution or an aqueous solution of an alkali metal hydroxide, preferably an ammonia test solution.

[0146] In some implementations, in step (2), the ratio of the Astragalus reference material to methanol is 1g:30ml to 1g:90ml, preferably 1g:60ml to 1g:90ml, and more preferably 1g:60ml.

[0147] In some implementations, in step (2), the ultrasound duration is 20 to 40 minutes, preferably 30 minutes.

[0148] In some embodiments, in step (2), the volume ratio of the water to the water-saturated alcohol solvent is 2:1 to 1:4, preferably 1:1 to 1:2, and more preferably 1:1.

[0149] In some implementations, in step (2), the shaking extraction is performed 2 to 4 times, preferably 2 times.

[0150] In some embodiments, in step (2), the alkaline test solution is an ammonia test solution or an aqueous solution of an alkali metal hydroxide, preferably an ammonia test solution.

[0151] In steps (1) and (2), the purpose of washing with alkaline test solution is to make the solution weakly alkaline, so that alkaloids and other non-saponin components can be released into the alkaline test solution under alkaline conditions, thereby achieving the purpose of separation.

[0152] In some implementations, in step (4), the amount of the test solution, the astragalus and white peony reference solutions is 5-10 μl; and the amount of the astragaloside A and paeoniflorin reference solutions is 1-3 μl.

[0153] In some embodiments, in step (4), the volume ratio of chloroform-methanol-water in the developing agent is (10-15):(5-10):(1-3), preferably 13:7:2.

[0154] In some embodiments, in step (4), the developing agent is a lower layer solution of chloroform-methanol-water placed below 10°C.

[0155] In some embodiments, in step (4), the volume percentage of the sulfuric acid ethanol solution is 5% to 15%, preferably 10%.

[0156] In some implementations, in step (4), the heating temperature is 90 to 120°C, preferably 105°C.

[0157] In some implementations, in step (4), the wavelength of the ultraviolet light is 355-375 nm, preferably 365 nm.

[0158] In some implementations, step (4) further includes the following steps: comparing the determination results of the test sample with those of the reference medicinal material and the reference standard, and determining the thin-layer chromatograms of the test sample, the Astragalus reference medicinal material, and the astragaloside A reference standard in R f To determine whether spots of the same color exist at the same location, and to judge the thin-layer chromatograms of the test sample, white peony reference material, and paeoniflorin reference standard in R... f Does the same color of spot exist at the same location?

[0159] [A quality control method combining characteristic chromatographic identification and thin-layer chromatography identification methods]

[0160] In some implementation schemes, characteristic chromatographic identification methods and thin-layer chromatography identification methods can be combined to perform multi-faceted qualitative detection of Huangqi Guizhi Wuwu Decoction, thereby achieving the purpose of comprehensively evaluating its quality.

[0161] Therefore, the present invention also provides a quality control method for Huangqi Guizhi Wuwu Decoction, which includes the above-mentioned characteristic chromatographic identification method and the above-mentioned thin-layer chromatography identification method.

[0162] The names, CAS numbers, and structural formulas of the components in the Huangqi Guizhi Wuwu Decoction involved in this invention are shown in the table below:

[0163]

[0164]

[0165] Example

[0166] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0167] Example 1: Thin-layer chromatography identification of Astragalus and White Peony in Huangqi Guizhi Wuwu Decoction

[0168] 1.1 Instruments and Reagents

[0169] Thin-layer automated imaging system (CAMAG TLC VISUALIZER), ultrasonic cleaner (KQ-250E, Kunshan Ultrasonic Instrument Co., Ltd.), 0.1% balance (AR223CN, Ohaus), AE-2401 electronic analytical balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.), GKC temperature-controlled water bath (Nantong Huatai Experimental Instrument Co., Ltd.).

[0170] Methanol (Sinopharm Chemical Reagent Co., Ltd.), n-butanol (Sinopharm Chemical Reagent Co., Ltd.), and chloroform (Shanghai Lingfeng Chemical Reagent Co., Ltd.) were all of analytical grade. Silica gel G thin-layer plates were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0171] Astragalus reference material (120974-201612), Paeonia lactiflora reference material (120905-201610), astragaloside A reference standard (110781-201717), and paeoniflorin reference standard (110736-201741) were purchased from the National Institutes for Food and Drug Control.

[0172] The batch numbers for Huangqi Guizhi Wuwu Decoction are HQGZ-F1, HQGZ-F2, HQGZ-F3, HQGZ-F4, HQGZ-F5, HQGZ-F6, HQGZ-F7, HQGZ-F8, HQGZ-F9, HQGZ-F10, HQGZ-F11, HQGZ-F12, HQGZ-F13, HQGZ-F14, and HQGZ-F15. The negative control batch number lacking Huangqi is HQGZ-(lacking Huangqi), and the negative control batch number lacking Baishao is HQGZ-(lacking Baishao).

[0173] 1.2 Solution Preparation

[0174] 1.2.1 Preparation of the test solution

[0175] Take 30 ml of Huangqi Guizhi Wuwu Decoction extract, shake it twice with water-saturated n-butanol, 20 ml each time, combine the n-butanol extracts, wash with 20 ml of ammonia test solution, discard the ammonia test solution, evaporate the n-butanol extract to dryness, add 0.5 ml of methanol to dissolve the residue, and use it as the test solution.

[0176] 1.2.2 Preparation of reference medicinal material solution and reference standard solution

[0177] Take 0.5g of Astragalus membranaceus reference material, add 30ml of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 20ml of water, and extract twice with 20ml of water-saturated n-butanol each time. Combine the n-butanol extracts, wash with 20ml of ammonia solution, discard the ammonia solution, evaporate the n-butanol extract to dryness, and dissolve the residue in 1ml of methanol to obtain the reference material solution. Take 1g of Paeonia lactiflora reference material and prepare the Paeonia lactiflora reference material solution using the same method. Take astragaloside A and paeoniflorin reference standards, add methanol to prepare a solution containing 1mg of each of astragaloside A and paeoniflorin per ml, and use this as the reference solution.

[0178] 1.2.3 Preparation of negative sample solution

[0179] Take 30 ml of the extract of Astragalus and Cinnamon Twig Decoction (lacking Astragalus) and prepare a negative control solution lacking Astragalus in the same manner as described in "1.2.1 Preparation of Test Solution". Take 30 ml of the extract of Astragalus and Cinnamon Twig Decoction (lacking Paeonia lactiflora) and prepare a negative control solution lacking Paeonia lactiflora in the same manner.

[0180] 1.3 Thin-layer chromatography conditions

[0181] Perform the thin-layer chromatography test (General Chapter 0502, Chinese Pharmacopoeia 2015 Edition). Take 5-10 μl of the test solution, 8 μl of Astragalus membranaceus reference material solution, 10 μl of Paeonia lactiflora reference material solution, and 2 μl each of Astragaloside A and Paeoniflorin reference solutions, and spot them separately on the same silica gel G thin-layer plate. Use the lower layer solution of chloroform-methanol-water (13:7:2) placed below 10℃ as the developing solvent. Develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly visible. Examine under sunlight and ultraviolet light (365nm) respectively.

[0182] 1.4 Identification of Astragalus and White Peony in Multiple Batches of Huangqi Guizhi Wuwu Decoction

[0183] Thin-layer chromatography was performed on different batches of Huangqi Guizhi Wuwu Tang samples to identify the components of Astragalus membranaceus and Paeonia lactiflora. (See attached figures.) Figure 1 and Figure 2 .exist Figure 1 and Figure 2 In the table, 1-15 are 15 batches of Huangqi Guizhi Wuwu Decoction samples, 16 is a negative control lacking Huangqi, S1 is Huangqi reference material, S2 is Baishao reference material, 17 is a negative control lacking Baishao, T1 is astragaloside A reference standard, and T2 is paeoniflorin reference standard.

[0184] Conclusion: From Figure 1 and Figure 2 It can be known that the R of astragaloside A f The value is 0.4, showing red spots under sunlight and light yellow spots at 365nm; the R of paeoniflorin fThe value is 0.57, showing deep purple spots under sunlight and deep blue spots at 365 nm. Different batches of Huangqi Guizhi Wuwu Tang show spots of the same color at the corresponding positions as the reference herb chromatogram and the reference standard chromatogram. The negative sample does not show spots of the same color at the corresponding positions as the sample chromatogram. This indicates that the thin-layer chromatography method of the present invention has strong specificity and can identify Astragalus and Paeonia lactiflora in Huangqi Guizhi Wuwu Tang.

[0185] Example 2: Development of a Characteristic Mapping Method for Huangqi Guizhi Wuwu Decoction

[0186] 1.1 Instruments and Reagents

[0187] Waters ACQUITY UPLC H-Class ultra-high performance liquid chromatography system; Waters Quaternary Solvent Manager quaternary pump; Sample Manager-FTN autosampler; Waters UPLC PDA detector; Empower 3 chromatography workstation; KQ-250E ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); temperature-controlled water bath (Nantong Huatai Experimental Instrument Co., Ltd.); pure water system (Sartorius); TGL-16C centrifuge (Shanghai Anting Scientific Instrument Factory); water is ultrapure water; methanol (chromatographic grade, Thermo Fisher Scientific); phosphoric acid (chromatographic grade, Aladdin Scientific).

[0188] The extract of Huangqi Guizhi Wuwu Decoction was prepared and provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd. The batch numbers of compound S1 to S15 are as follows: HQGZ-F1, HQGZ-F2, HQGZ-F3, HQGZ-F4, HQGZ-F5, HQGZ-F6, HQGZ-F7, HQGZ-F8, HQGZ-F9, HQGZ-F10, HQGZ-F11, HQGZ-F12, HQGZ-F13, HQGZ-F14, HQGZ-F15.

[0189] 1.2 Source of reference standard

[0190] Paeoniflorin (batch number: 110736-201943, purity 95.1%, National Institutes for Food and Drug Control); Versicolor isoflavone glucoside (batch number: 111920-201606, purity 97.6%, National Institutes for Food and Drug Control); 1,2,3,4,6-penta-galloyl glucoside (batch number: ST16430120, purity 98%, Shidande); Cinnamyl alcohol (batch number: ST06990320 (purity 90.0%, Shidande); Cinnamaldehyde (batch number: 110710-201821, purity 99.6%, National Institutes for Food and Drug Control); Cinnamic acid (batch number: 110786-201604, purity 98.8%, National Institutes for Food and Drug Control); 6-Gingerol (batch number: 111833-201705, purity 96.8%, National Institutes for Food and Drug Control).

[0191] 1.3 Determined chromatographic conditions

[0192] Octadecylsilane-bonded silica gel was used as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm); Waters HSS T3 column was used as the chromatographic column, methanol was used as mobile phase A, and 0.05% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the specifications in the table below; the flow rate was 0.25 ml per minute; the column temperature was 30 °C.

[0193]

[0194] 1.4 Preparation of reference solution

[0195] Preparation of reference solutions: Accurately weigh appropriate amounts of paeoniflorin reference standard, verbascoside glucoside reference standard, 1,2,3,4,6-penta-galloyl glucoside, cinnamyl alcohol reference standard, cinnamaldehyde reference standard, cinnamic acid reference standard, and 6-gingerol reference standard. Add methanol to prepare reference solutions containing 200 μg paeoniflorin, 3 μg verbascoside glucoside, 150 μg 1,2,3,4,6-penta-galloyl glucoside, 3 μg cinnamyl alcohol, 3 μg cinnamaldehyde, 4 μg cinnamic acid, and 15 μg 6-gingerol per ml. Shake well to obtain the reference solution.

[0196] 1.5 Preparation of the test solution

[0197] 1.5.1 Investigation of different extraction solvents

[0198] This experiment investigated the effects of different extraction solvents on the characteristic chromatograms of Huangqi Guizhi Wuwu Decoction. 25% methanol, 50% methanol, and 80% methanol were selected as extraction solvents, and the effects of different extraction solvents on the characteristic chromatograms of Huangqi Guizhi Wuwu Decoction were compared by the total peak area of ​​17 main chromatographic peaks.

[0199] Accurately pipette 5 ml of the extract of Astragalus and Cinnamon Twig Decoction into a 10 ml volumetric flask, add 50% methanol and methanol to the mark respectively, mix well to obtain 25% methanol and 50% methanol test solutions; accurately pipette 2 ml of the extract of Astragalus and Cinnamon Twig Decoction into a 10 ml volumetric flask, add methanol to the mark, mix well to obtain 80% methanol test solution. Take the above test solutions and filter them through a 0.22 μm microporous membrane. Accurately pipette 1 μl of each test solution and inject it into the liquid chromatograph, and determine it according to the above chromatographic conditions. The results are shown in Table 1.

[0200] Table 1 Comparison of total peak areas with different extraction solvents

[0201]

[0202] The results showed that, except for the lower extraction efficiency of 25% methanol, the extraction efficiencies of 50% methanol and 80% methanol were comparable, with 80% methanol having a higher total peak area. Considering all factors, 50% methanol was selected as the extraction solvent.

[0203] Based on the above research results, the preparation method of the test solution for the characteristic spectrum study of Huangqi Guizhi Wuwu Decoction was determined as follows:

[0204] Accurately pipette 5 ml of the extract of Huangqi Guizhi Wuwu Decoction into a 10 ml volumetric flask, dilute with methanol to the mark, mix well, shake well, filter, and collect the filtrate.

[0205] 1.6 Methodology

[0206] 1.6.1 Precision Examination

[0207] Samples from the same batch were used to prepare test solutions according to the test solution preparation method. Six consecutive injections of 1 μl each were performed, and the chromatograms were recorded. The peak corresponding to 1,2,3,4,6-penta-galloylglucoside (peak 10) was used as the reference peak. The relative retention time and relative peak area of ​​each common peak were calculated, and the results are shown in Tables 2 and 3.

[0208] Table 2. Precision Experiment Results (Relative Retention Time)

[0209]

[0210] Table 3. Precision Experiment Results (Relative Peak Area)

[0211]

[0212] The results show that the relative retention time and relative peak area of ​​each characteristic peak are all less than 3%, indicating good precision.

[0213] 1.6.2 Stability Test

[0214] Samples from the same batch were prepared into test solutions according to the test solution preparation method. 1 μl of sample was injected every few hours for a total of 24 hours, and the chromatograms were recorded. The peak corresponding to 1,2,3,4,6-penta-galloylglucoside (peak 10) was used as the reference peak. The relative retention time and relative peak area of ​​each common peak were calculated, and the results are shown in Tables 4 and 5.

[0215] Table 4. Stability test results (relative retention time)

[0216]

[0217] Table 5. Stability test results (relative peak area)

[0218]

[0219] The results showed that the relative retention time and relative peak area of ​​most characteristic peaks had an RSD of less than 3%, indicating that the test solution had good stability within 24 hours.

[0220] 1.6.3 Repeatability Test

[0221] Samples from the same batch were prepared into test solutions according to the test solution preparation method. Six parallel solutions were prepared, with 1 μl injected each time, and the chromatograms were recorded. The peak corresponding to the 1,2,3,4,6-penta-galloylglucoside peak (peak 10) was used as the reference peak. The relative retention time and relative peak area of ​​each common peak were calculated, and the results are shown in Tables 6 and 7.

[0222] Table 6. Stability test results (relative retention time)

[0223]

[0224] Table 7. Stability test results (relative peak area)

[0225]

[0226] The results showed that the RSD of the relative retention time and relative peak area of ​​each characteristic peak was less than 3%, indicating good repeatability of the test solution.

[0227] 1.6.4 Durability Test

[0228] 1.6.4.1 Chromatographic column investigation

[0229] Samples from the same batch were used to prepare test solutions according to the test solution preparation method. Four different chromatographic columns were used: ACQUITY UPLCHSS T3 (2.1×100mm, 1.8μm), Acclaim RSLC 120C18 (2.1×100mm, 2.2μm), Eclipse PlusC18 RRHD (2.1×100mm, 1.8μm), and ZORBAX SB-Aq RRHD (2.1×100mm, 1.8μm). The separation performance of the four different packed columns was compared, and the results are shown in [Figure number missing]. Figure 3 .

[0230] The results showed that the characteristic peaks were well separated on the HSS T3 column and the Acclaim RSLC 120C18 column. However, the EP C18 column and the SB Aq column showed poor separation of some chromatographic peaks and a lack of peaks. Therefore, it is recommended to use the HSS T3 column or the Acclaim RSLC 120C18 column for the characteristic chromatographic study of Huangqi Guizhi Wuwu Decoction.

[0231] 1.6.4.2 Investigation of Elution Gradient

[0232] Take samples from the same batch number and prepare test solutions according to the test solution preparation method. Inject the sample according to the following elution procedure, with other chromatographic conditions the same as those determined in "1.3". Inject 1 μl of sample and record the chromatogram. The results are shown in [Figure / Reference]. Figure 4 Using the peak corresponding to 1,2,3,4,6-penta-galloylglucoside (peak 10) as the reference peak, the relative retention time of each common peak was calculated.

[0233]

[0234] The results showed that the characteristic peaks could be effectively separated under the above gradient conditions. Taking the peak corresponding to 1,2,3,4,6-penta-galloylglucoside (peak 10) as the reference peak, the relative retention times of each characteristic peak were: 0.09 (peak 1), 0.10 (peak 2), 0.16 (peak 3), 0.39 (peak 4), 0.56 (peak 5), 0.76 (peak 6), 0.83 (peak 7), 0.89 (peak 8), 0.94 (peak 9), 1.19 (peak 11), 1.22 (peak 12), 1.37 (peak 13), 1.46 (peak 14), 1.51 (peak 15), 1.72 (peak 16) and 1.80 (peak 17).

[0235] 1.7 Determination of characteristic peaks in different batches of Huangqi Guizhi Wuwu Decoction samples

[0236] The results were analyzed using the "Similarity Evaluation System for Chromatographic Characteristic Magnetisms of Traditional Chinese Medicine (2012 Edition)" recommended by the National Pharmacopoeia Commission. Based on peak attribution, common peaks were selected, with the 1,2,3,4,6-penta-galloylglucoside peak (peak 10) used as the reference peak. The relative retention time and relative peak area of ​​each common peak were calculated, and a superimposed chromatogram of the common peaks was fitted. (See attached image). Figure 5 ; Generate a feature comparison map of shared patterns, see Figure 6 The results are shown below (Tables 8 and 9):

[0237] Table 8. Results of multiple batches of experiments (relative retention time)

[0238]

[0239] Table 9. Results of multiple batches of experiments (relative peak area)

[0240]

[0241] The relative retention times and relative peak areas of each characteristic peak show that the retention time ratios of the characteristic peaks in the 15 batches of samples are relatively stable, while the relative peak areas of each characteristic peak vary greatly, with RSD values ​​ranging from 25.24% to 82.58%. This indicates that the relative peak area ratios of the components contained in the samples from different origins and combinations vary greatly, making them unsuitable for inclusion in the standard.

[0242] 1.8 Identification of Characteristic Peak Reference Standards

[0243] For the preparation of the reference solution, refer to the preparation of the reference solution under "1.4 Reference Solution" in this embodiment. Separately, accurately weigh appropriate amounts of gallic acid, paeoniflorin, coumarin, galloylpaeoniflorin, and verbascoside reference standards, and add methanol to prepare reference solutions containing 50 μg gallic acid, 150 μg galloylpaeoniflorin, 50 μg paeoniflorin, 200 μg verbascoside, and 50 μg coumarin per ml, respectively. Shake well. Inject 1 μl of each of the above reference solutions, record the chromatograms, and compare them with the characteristic chromatogram of Huangqi Guizhi Wuwu Decoction to identify the characteristic peaks.

[0244] See results Figure 7Of the 17 characteristic peaks: peak 1 corresponds to the gallic acid reference standard; peak 4 corresponds to the paeoniflorin reference standard; peak 5 corresponds to the paeoniflorin reference standard; peak 6 corresponds to the coumarin reference standard; peak 8 corresponds to the verbascoside glucoside reference standard; peak 9 corresponds to the galloylpaeoniflorin reference standard; peak 10 corresponds to the 1,2,3,4,6-penta-galloyl glucoside reference standard; peak 11 corresponds to the cinnamyl alcohol reference standard; peak 12 corresponds to the cinnamaldehyde reference standard; peak 13 corresponds to the cinnamic acid reference standard; peak 15 corresponds to the verbascoside reference standard; and peak 17 corresponds to the 6-gingerol reference standard. Therefore, the characteristic spectrum of this invention achieves the identification of 12 characteristic peaks with a high identification rate.

[0245] It should be noted that although specific examples have been used to illustrate the technical solutions of the present invention, those skilled in the art will understand that the present invention is not limited thereto. Various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, and are not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical applications, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for constructing a characteristic map of Huangqi Guizhi Wuwu Decoction, comprising the following steps: (1) Preparation of sample solution of Huangqi Guizhi Wuwu Decoction: Take the extract of Huangqi Guizhi Wuwu Decoction, dilute it with methanol, filter it, and take the filtrate to obtain the final product; (2) Preparation of reference solution: Take paeoniflorin, verbascoside, cinnamyl alcohol, cinnamaldehyde, cinnamic acid, 6-gingerol, gallic acid, paeoniflorin lactone, coumarin, galloylpaeoniflorin, 1,2,3,4,6-penta-galloyl glucoside and verbascoside reference standards respectively, and add methanol to prepare reference standard solutions. (3) Construction of feature maps: The sample solution of Huangqi Guizhi Wuwu Decoction described in step (1) and the reference solution described in step (2) were subjected to UPLC analysis to obtain the characteristic chromatogram of Huangqi Guizhi Wuwu Decoction. The chromatographic conditions for the UPLC analysis included: an HSS T3 column with a particle size of 1.8 μm, or an Acclaim RSLC 120 C18 column with a particle size of 2.2 μm; a detection wavelength of 230–280 nm; mobile phase A being methanol, and mobile phase B being a 0.01%–0.1% aqueous phosphoric acid solution; gradient elution with the following elution program: or 。 2. The feature map construction method according to claim 1, characterized in that, In step (1), the volume ratio of the Astragalus and Cinnamon Twig Decoction Extract to the Methanol is 3:1 to 1:

4.

3. The feature map construction method according to claim 2, characterized in that, In step (1), the volume ratio of the Astragalus and Cinnamon Twig Decoction Extract to the Methanol is 1:1 to 1:

4.

4. The feature map construction method according to claim 3, characterized in that, In step (1), the volume ratio of the Astragalus and Cinnamon Twig Decoction Extract to the Methanol is 1:

1.

5. The feature map construction method according to claim 1, characterized in that, In step (3), the detection wavelength is 250-270 nm.

6. The feature map construction method according to claim 5, characterized in that, In step (3), the detection wavelength is 260 nm.

7. The feature map construction method according to any one of claims 1-6, characterized in that, In step (3), the specifications of the chromatographic column are: column length 100mm and inner diameter 2.1mm.

8. The feature map construction method according to any one of claims 1-6, characterized in that, In step (3), the mobile phase B is a 0.05% aqueous solution of phosphoric acid.

9. The feature map construction method according to any one of claims 1-6, characterized in that, In step (3), the chromatographic column is an HSS T3 chromatographic column.

10. The feature map construction method according to any one of claims 1-6, characterized in that, In step (3), the chromatographic conditions further include at least one of the following conditions: The column temperature is 25–35℃; The flow rate is 0.2–0.30 ml per minute; The injection volume is 1–3 μl.

11. The feature map construction method according to claim 10, characterized in that, In step (3), the column temperature is 30°C.

12. The feature map construction method according to claim 10, characterized in that, In step (3), the flow rate is 0.25 ml per minute.

13. The feature map construction method according to claim 10, characterized in that, In step (3), the injection volume is 2 μl.

14. The feature map construction method according to any one of claims 1-6, characterized in that, In step (3), the characteristic chromatogram of the Astragalus and Cinnamon Twig Decoction has 17 characteristic peaks, among which peaks 1, 4, 5, 6, 8, 9, 10, 11, 12, 13, 15 and 17 correspond to the retention times of the chromatographic peaks of gallic acid, paeoniflorin, paeoniflorin, coumarin, vermifuran glucoside, galloylpaeoniflorin, 1,2,3,4,6-penta-galloyl glucoside, cinnamyl alcohol, cinnamaldehyde, cinnamic acid, vermifuran and 6-gingerol reference standards.

15. The feature map construction method according to claim 14, characterized in that, Peak 10, corresponding to the chromatographic peak of 1,2,3,4,6-penta-galloylglucoside reference standard, was designated as peak S. The relative retention times of peaks 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, and 17 with peak S were within ±15% of the specified values, which were as follows: peak 1 = 0.10, peak 2 = 0.11, peak 3 = 0.17, peak 4 = 0.38, peak 5 = 0.51, peak 6 = 0.71, peak 7 = 0.74, peak 8 = 0.81, peak 9 = 0.88, peak 11 = 1.08, peak 12 = 1.15, peak 13 = 1.45, peak 14 = 1.71, peak 15 = 1.78, peak 16 = 1.92, and peak 17 = 1.

96.

16. A quality control method for Huangqi Guizhi Wuwu Decoction, comprising the following steps: (1) Preparation of the test solution: Take the extract of Huangqi Guizhi Wuwu Decoction used as the test sample, dilute it with methanol, filter it, and take the filtrate to obtain the final product. (2) Quality evaluation of the test sample: The test solution described in step (1) is analyzed under the UPLC chromatographic conditions of the characteristic spectrum construction method according to any one of claims 1-15 to obtain the test spectrum, and it is compared with the characteristic spectrum of Huangqi Guizhi Wuwu Decoction in the characteristic spectrum construction method according to any one of claims 1-15.

17. The quality control method according to claim 16, characterized in that, In step (1), the volume ratio of the Astragalus and Cinnamon Twig Decoction Extract to the Methanol is 3:1 to 1:

4.

18. The quality control method according to claim 17, characterized in that, In step (1), the volume ratio of the Astragalus and Cinnamon Twig Decoction Extract to the Methanol is 1:1 to 1:

4.

19. The quality control method according to claim 18, characterized in that, In step (1), the volume ratio of the Astragalus and Cinnamon Twig Decoction Extract to the Methanol is 1:1.

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