Specific chromatogram construction method and quality control method of radix astragali and cassia twig five-material decoction

Abstract

The invention belongs to the technical field of quality control of traditional Chinese medicines, and particularly relates to a specific chromatogram construction method and a quality control method of radix astragali and cassia twig five-material decoction, and the quality control method comprises a specific chromatogram identification method and a thin-layer chromatography identification method. The UPLC is used for construction and identification of the characteristic spectrum, and the method is short in detection time, high in specificity, rapid, efficient and low in cost. According to the specific chromatogram, 17 common characteristic peaks are stipulated, 12 characteristic peaks are identified, the identification rate is high, and analysis and identification of the material basis of the radix astragali and cassia twig five-material decoction are facilitated. According to the thin-layer chromatography identification method disclosed by the invention, a novel thin-layer identification method for identifying two medicines (astragalus membranaceus and radix paeoniae alba) in a compound by quantitative analysis of multi-components by single marker is successfully created, the detection conditions are simple, rapid and efficient, the specificity is high, the cost is relatively low, and the practicability is relatively high.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine quality control, and particularly relates to a characteristic spectrum construction method and a quality control method of Huangqi Guizhi Wuwu Decoction. Background Art

[0002] Huangqi Guizhi Wuwu Decoction was created by Zhang Zhongjing in the Han Dynasty. It can be found in the "Chapter 6 on Blood Bi, Deficiency and Fatigue, Pulse and Symptoms and Treatment" in Volume 1 of "Golden Chamber Synopsis". The original text is "Blood Bi, Yin and Yang are both weak, Cunkou and Guanshang are weak, Chizhong and Xiao are tight, and the external symptoms are numbness of the body, like wind Bi, Huangqi Guizhi Wuwu Decoction is the main one". In 2018, Huangqi Guizhi Wuwu Decoction was included in the Catalog of Ancient Classic Prescriptions (First Batch), ranking 16th. In 2022, the country announced the prescription information of Huangqi Guizhi Wuwu Decoction for the first time: it is composed of five medicinal flavors: astragalus, white peony, cinnamon twig, ginger, and jujube, and one liang is determined to be 13.8g. Huangqi Guizhi Wuwu Decoction has the effects of invigorating qi and warming the meridians, harmonizing blood and unblocking arthralgia. It is mainly used to treat blood bi, wind bi, arthralgia due to deficiency, stroke (especially in the meridians) and other diseases.

[0003] In recent years, with the increasing interest in the study of classic prescriptions, the research on the quality standard of Huangqi Guizhi Wuwu Decoction has also been deepening. In terms of the quality standard and evaluation of Huangqi Guizhi Wuwu Decoction, the research mainly uses the content indicators of single medicinal materials under the pharmacopoeia for qualitative or quantitative determination, but there are few breakthrough improvements and expansions in the selection of indicator components, especially for the analysis and identification rate of volatile components in the compound, which fails to reflect the integrity of the prescription and the interaction of multiple components.

[0004] Thin layer identification and characteristic spectrum are relatively mature qualitative quality characterization methods used in quality standards. In the prior art, there are certain research foundations and related reports on thin layer identification and characteristic spectrum of Huangqi Guizhi Wuwu Decoction, but there are still some deficiencies and defects.

[0005] 1. Thin layer identification:

[0006] Common drawbacks: Traditional Chinese medicine compound has many medicinal flavors and complex chemical components. There is serious interference between the various medicinal flavors and components. It is difficult to find the exclusive spots of a certain medicinal flavor from multiple medicinal flavors. The current thin-layer research method of compound preparations generally corresponds to one identification method for one medicinal flavor, and several medicinal flavors correspond to several identification methods. Therefore, a large amount of samples, manpower and time are required. In addition, in order to eliminate interference, the pretreatment procedure of compound preparations often adopts multiple purification and enrichment steps. There are many pretreatment steps, many types of organic reagents involved, and large amounts. It takes a long time, pollutes the environment, and harms health. Therefore, finding a simple and fast detection method suitable for water extraction preparations, improving detection efficiency, and reducing detection costs are the key to improving the thin-layer identification method of traditional Chinese medicine compounds.

[0007] Fan Yue et al. [1]A quality standard for Huangqi Guizhi Wuwu Tang granules was established, which used two different thin layer chromatography methods to perform thin layer identification on Astragalus and White Peony Root, respectively. During the pretreatment of Astragalus samples, the test sample was extracted with two organic reagents (ether and n-butanol), washed multiple times, and then concentrated and evaporated, which was cumbersome and complicated. In the identification of White Peony Root, the preparation of the test sample and the control medicinal material also adopted the method of multiple extraction, washing and concentration (n-butanol), which was repetitive and cumbersome. In the development process of White Peony Root, four solvents were used to prepare the developing agent, which had a large number of solvents and was troublesome to prepare. In summary, two different pretreatment methods were used for the two medicinal flavors, and both required multiple extraction, washing and concentration treatments, with many steps, complicated and time-consuming (two medicinal flavors, it is expected to take 2 days).

[0008] 2. Feature map:

[0009] CN110887914A discloses an HPLC detection method for the fingerprint of Huangqi Guizhi Wuwu Decoction freeze-dried powder, and its elution procedure is 77 minutes (the retention time of the last chromatographic peak still needs about 55 minutes), the detection time is long, and the chromatographic peaks in the fingerprint are not clearly identified. CN110887927A discloses an HPLC-Q-TOF-MS / MS method (ultra-fast high-performance liquid chromatography triple quadrupole time-of-flight tandem mass spectrometry method) to analyze and detect Huangqi Guizhi Wuwu Decoction samples, but the detection cost of this LC-MS technology is high, and it cannot be well applied to production detection, and its universality is poor. In addition, the sample preparation process requires steps such as concentration and freeze drying, resulting in a large loss or reduction of the volatile oil components of Guizhi in the compound.

[0010] Yu Aiming et al. [2] The fingerprint of Huangqi Guizhi Wuwu Decoction was established by HPLC, and 17 common peaks were matched. The overall qualitative analysis was of reference significance, but only three components were identified, and there were many unknown peaks, which made it impossible to analyze and identify the material basis. In addition, the elution procedure was 70 minutes, and the detection time was long. Wei Guoli et al. [3] The HPLC fingerprint of Huangqi Guizhi Wuwu Decoction was also established, which used paeoniflorin as the reference peak and marked the 13 common peaks of Huangqi Guizhi Wuwu Decoction. However, except for paeoniflorin, the other 12 common peaks were not identified.

[0011] Therefore, the above characteristic spectra are mostly determined by HPLC, which generally has a long analysis cycle and high time cost; there are 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 the 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 Huangqi Guizhi Wuwu Decoction standard decoction and determination 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] Problem that the invention aims to solve

[0016] In order to solve the above problems, the present invention aims to provide a characteristic spectrum construction method and quality control method of Huangqi Guizhi Wuwu Decoction, wherein the quality control method comprises a characteristic spectrum and thin layer chromatography identification: the characteristic spectrum has a short analysis cycle, low detection cost, and a high recognition rate of common peaks, especially a comprehensive analysis of volatile components in the compound; the thin layer chromatography identification method can achieve one-test multiple evaluations of multiple medicinal flavors, which is convenient, fast, cost-saving, highly specific, and highly practical.

[0017] Solutions for solving problems

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

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

[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, calycosin glucoside, cinnamyl alcohol, cinnamaldehyde, cinnamic acid, 6-gingerol, gallic acid, paeoniflorin, coumarin, galloylpeoniflorin, 1,2,3,4,6-penta-galloyl glucoside and calycosin reference substances respectively, add methanol to prepare reference substance solutions;

[0023] (3) Construction of feature maps:

[0024] The Huangqi Guizhi Wuwu Decoction sample solution described in step (1) and the reference solution described in step (2) are subjected to UPLC analysis to obtain a characteristic spectrum of Huangqi Guizhi Wuwu Decoction; wherein the chromatographic conditions of the UPLC analysis include: a chromatographic column using octadecylsilane bonded silica gel as a filler; mobile phase A is methanol, and mobile phase B is a phosphoric acid aqueous solution; gradient elution; and a detection wavelength of 230 to 280 nm.

[0025] Preferably, in step (1), the volume ratio of the Huangqi Guizhi Wuwu Decoction extract to the methanol is 3:1 to 1:4, preferably 1:1 to 1:4, and more preferably 1:1.

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

[0027]

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

[0029]

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

[0031]

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

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

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

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

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

[0037] Preferably, in step (3), the chromatographic conditions of 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 of the UPLC analysis further include: an injection volume of 1 to 3 μl, preferably 2 μl.

[0039] Preferably, in step (3), the characteristic spectrum of Huangqi Guizhi Wuwu Decoction has 17 characteristic peaks, wherein peak 1, peak 4, peak 5, peak 6, peak 8, peak 9, peak 10, peak 11, peak 12, peak 13, peak 15 and peak 17 correspond to the retention times of the chromatographic peaks of gallic acid, paeoniflorin, paeoniflorin, coumarin, calycosin glucoside, galloylpeoniflorin, 1,2,3,4,6-penta-galloyl glucoside, cinnamyl alcohol, cinnamaldehyde, cinnamic acid, calycosin and 6-gingerol reference substances, respectively.

[0040] More preferably, in step (3), the peak (peak 10) corresponding to the chromatographic peak of 1,2,3,4,6-penta-galloyl glucoside reference substance is taken as the S peak, 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 respect to the S peak are within ±15% of the specified value, and 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] Further preferably, in step (3), the peak (peak 10) corresponding to the chromatographic peak of 1,2,3,4,6-penta-galloyl glucoside reference substance is taken as the S peak, and the relative retention time of peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, peak 9, peak 11, peak 12, peak 13, peak 14, peak 15, peak 16 and peak 17 with respect to the S peak is within ±10% of the specified value, and 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] In a second aspect, the present invention provides a quality control method for Huangqi Guizhi Wuwu Decoction, which comprises the following steps:

[0043] (1) Preparation of test solution:

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

[0045] (2) Quality evaluation of test products:

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

[0047] Preferably, in step (1), the volume ratio of the Huangqi Guizhi Wuwu Decoction extract to the methanol is 3:1 to 1:4, preferably 1:1 to 1:4, and more preferably 1:1.

[0048] In a third aspect, the present invention also provides another quality control method for Huangqi Guizhi Wuwu Decoction, which comprises the following steps:

[0049] (1) Preparation of test solution:

[0050] Take the Huangqi Guizhi Wuwu Decoction extract as the test sample, shake and extract it with a water-saturated alcohol solvent, combine the alcohol solvent, wash it with an alkaline test solution, discard the alkaline test solution, evaporate the alcohol solvent to dryness, add methanol to dissolve the residue, and use it as the test sample solution;

[0051] (2) Preparation of control medicinal material solution:

[0052] Astragalus and white peony root as control medicinal materials are respectively taken, methanol is added, ultrasonication is performed, filtration is performed, the filtrate is evaporated to dryness, water is added to the residue to dissolve, extraction is performed by shaking with a water-saturated alcohol solvent, the alcohol solvent is combined, washing is performed with an alkaline test solution, the alkaline test solution is discarded, the alcohol solvent is evaporated to dryness, and methanol is added to the residue to dissolve, thereby obtaining an Astragalus control medicinal material solution and a white peony root control medicinal material solution;

[0053] (3) Preparation of reference solution:

[0054] Astragaloside IV and paeoniflorin as reference substances were taken respectively, and methanol was added to prepare astragaloside IV reference substance solution and paeoniflorin reference substance solution;

[0055] (4) Thin layer chromatography identification:

[0056] The test sample solution described in step (1), the astragalus reference medicinal material solution and the white peony reference medicinal material solution described in step (2), and the astragaloside reference substance solution and the paeoniflorin reference substance solution described in step (3) are respectively spotted on the same thin layer plate, developed, and the determination results of the test sample, the reference medicinal material and the reference substance are compared; wherein the chromatographic conditions of 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 color developer; and inspecting 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 Huangqi Guizhi Wuwu 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.

[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 alkali metal hydroxide aqueous solution, preferably an ammonia test solution.

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

[0062] Preferably, in step (2), the ultrasonication time is 20 to 40 min, preferably 30 min.

[0063] Preferably, 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.

[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 alkali metal hydroxide aqueous solution, preferably an ammonia test solution.

[0066] Preferably, in step (4), the spotting volume of the test solution, astragalus and white peony root control medicinal material solutions is 5-10 μl; the spotting volume of the astragaloside IV and paeoniflorin reference solution is 1-3 μl.

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

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

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

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

[0073] Preferably, step (4) further comprises the following steps: comparing the test sample with the test results of the reference medicinal material and the reference substance, and determining whether the thin layer chromatograms of the test sample, the reference medicinal material of Astragalus and the reference substance of Astragaloside IV are f Whether there are spots of the same color at the same position of the value, and whether the thin layer chromatograms of the test sample, white peony reference medicinal material and paeoniflorin reference substance are in the R f Whether there are spots of the same color at the same position.

[0074] In a fourth aspect, the present invention further 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] Effects of the Invention

[0076] The quality control method of Huangqi Guizhi Wuwu Decoction in the present invention comprises identification of characteristic spectrum and thin layer chromatography. The Huangqi Guizhi Wuwu Decoction is subjected to multi-directional qualitative detection by the above two methods, which provides a reference basis for the development research and comprehensive quality control of Huangqi Guizhi Wuwu Decoction.

[0077] The present invention uses UPLC to construct a characteristic spectrum, which has a short detection time, strong specificity, fast and efficient, low cost, and has practical significance in industrial production. The characteristic spectrum stipulates 17 common characteristic peaks, and completes the identification of 12 characteristic peaks, with a high identification rate, which is conducive to the analysis and identification of the material basis of Huangqi Guizhi Wuwu Decoction. In particular, the cinnamyl alcohol component in cinnamon twigs is newly added to the characteristic spectrum, which improves the analysis and research of volatile components in Huangqi Guizhi Wuwu Decoction. In addition, cinnamyl alcohol is characterized and identified in the fingerprint spectrum / characteristic spectrum, and the component has corresponding chromatographic peaks in the characteristic spectrum of standard decoction, extract, and compound preparation samples, which fully reflects the quality and efficacy of a bowl of clinical decoction.

[0078] The thin layer chromatography identification method of the present invention can meet the purpose of extraction by using the same extraction solvent (such as n-butanol), and does not require repeated extraction with multiple solvents. The identification method combines common elements and finds identification differences on the premise of fully analyzing the characteristics of the index components. The same test solution is used on a thin layer plate under two different inspection conditions (sunlight and ultraviolet light) to quickly show good identification and differentiation spots. A new thin layer identification method for identifying two medicinal flavors (astragalus and white peony) in a compound prescription with one test and multiple evaluations is successfully created. The detection conditions are simple, fast, and efficient, the specificity is strong, the cost is low, and the practicality is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 It is used for the thin layer identification (ultraviolet light 365nm) of astragalus and white peony root in Huangqi Guizhi Wuwu Decoction.

[0080] Figure 2 This is the thin layer identification of astragalus and white peony root in Huangqi Guizhi Wuwu Decoction (sunlight).

[0081] Figure 3 These are the results of the chromatographic column investigation of Huangqi Guizhi Wuwu Decoction.

[0082] Figure 4 This is the result of the elution gradient investigation of Huangqi Guizhi Wuwu Decoction.

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

[0084] Figure 6 This is a comparison chart of the characteristic spectrum of Huangqi Guizhi Wuwu Decoction.

[0085] Figure 7 This is the identification spectrum of the characteristic peaks in the characteristic spectrum of Huangqi Guizhi Wuwu Decoction. DETAILED DESCRIPTION

[0086] Various exemplary embodiments, features and aspects of the present invention will be described in detail below. The word "exemplary" used here means "used as an example, embodiment or illustrative". Any embodiment described here as "exemplary" is not necessarily interpreted as being superior or better than other embodiments.

[0087] In addition, in order to better illustrate the present invention, numerous specific details are provided in the following specific embodiments. It should be understood by those skilled in the art that the present invention can be implemented without certain specific details. In other examples, methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the subject matter of the present invention.

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

[0089] In this specification, the word "may" means both performing a certain process and not performing a certain process.

[0090] In this specification, the references to "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", etc., mean that the specific elements (e.g., features, structures, properties and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not exist in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0091] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints numerical values ​​A and B.

[0092] In the present invention, the preparation method of "ammonia test solution" refers to the "ammonia test solution" in the 2020 edition of the "Chinese Pharmacopoeia" Part IV General Rules 8002 Test Solution, and the preparation method is: take 400ml of concentrated ammonia solution, add water to make 1000ml, and obtain.

[0093] In the present invention, the "Huangqi Guizhi Wuwu Decoction Extract" is the decoction of Huangqi Guizhi Wuwu Decoction, and its preparation method is: take appropriate amounts of five medicinal ingredients, including astragalus, white peony root, cinnamon twig, ginger and jujube, add 1200ml of water, boil over high heat, and simmer over low heat until 400ml is obtained.

[0094] In the present invention, the quality control method of Huangqi Guizhi Wuwu Decoction includes a characteristic spectrum identification method and a thin layer chromatography identification method. The above two methods are used to perform multi-faceted qualitative detection on Huangqi Guizhi Wuwu Decoction, which provides a reference basis for the development research and comprehensive quality control of Huangqi Guizhi Wuwu Decoction.

[0095] [Construction of feature maps]

[0096] The present invention establishes a characteristic spectrum of Huangqi Guizhi Wuwu Decoction by using UPLC, which has a short analysis time, is fast and efficient, stipulates 17 characteristic common 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 compound (such as astragaloside IV, calycosin isoflavone glycosides, paeoniflorin and cinnamaldehyde) are mostly pharmacopoeia indicators, and the analytical methods are relatively mature, but the retention and analytical research of various volatile components in cinnamon twigs are often ignored or lost. In existing studies, improper sample preparation methods have led to a large loss of volatile oil components in cinnamon twigs, which are difficult to detect and the component characterization is not comprehensive. The main reason is that the sample preparation process in existing studies has certain drawbacks: generally after water extraction, the sample is obtained by vacuum concentration or vacuum drying. However, the volatile oil components will be lost more during the vacuum filtration process, and the types of volatile oils in the obtained samples are relatively small, which cannot achieve the purpose of comprehensively evaluating the quality of Huangqi Guizhi Wuwu Decoction.

[0098] Cinnamon twig is a ministerial drug in the Huangqi Cinnamon Twig Five Ingredients Decoction, and has the effects of sweating and relieving muscles, warming and unblocking the meridians, assisting yang and transforming qi, and balancing and descending qi. Cinnamon twig volatile oil is the main active substance of cinnamon twig, which has good anti-inflammatory, antibacterial, antiviral and anti-tumor effects. The characteristic spectrum of the present invention has newly added cinnamyl alcohol in cinnamon twig, so as to achieve a comprehensive evaluation of the quality of Huangqi Cinnamon Twig Five Ingredients Decoction. Pharmacological studies have shown that cinnamyl alcohol can down-regulate the synthesis and secretion of PGE2 in rat brain microvascular endothelial cells caused by IL-1 stimulation, and its effect is related to the inhibition of COX-2 activity. Cinnamyl alcohol can activate K + It mediates the NO-cGMP-PKG pathway and inhibits Rho kinase, thereby dilating blood vessels.

[0099] Specifically, the construction of the characteristic spectrum of the present invention comprises the following steps:

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

[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, calycosin glucoside, cinnamyl alcohol, cinnamaldehyde, cinnamic acid, 6-gingerol, gallic acid, paeoniflorin, coumarin, galloylpeoniflorin, 1,2,3,4,6-penta-galloyl glucoside and calycosin reference substances respectively, add methanol to prepare reference substance solutions;

[0104] (3) Construction of feature maps:

[0105] The Huangqi Guizhi Wuwu Decoction sample solution described in step (1) and the reference solution described in step (2) are subjected to UPLC analysis to obtain a characteristic spectrum of Huangqi Guizhi Wuwu Decoction; wherein the chromatographic conditions of the UPLC analysis include: a chromatographic column using octadecylsilane bonded silica gel as a filler; mobile phase A is methanol, and mobile phase B is a phosphoric acid aqueous solution; gradient elution; and a detection wavelength of 230 to 280 nm.

[0106] In some embodiments, in step (1), the volume ratio of the Huangqi Guizhi Wuwu 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 embodiments, in step (3), the elution procedure of the gradient elution is as follows:

[0108]

[0109] In the elution program of the present invention, the numerical range in "time (minutes)" refers to any time within the 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 in "mobile phase A (%)" and "mobile phase B (%)" in the elution program also refer to any volume percentage within the volume percentage range. After verification by the present invention, the above-mentioned gradient elution elution program can achieve effective separation of 17 characteristic peaks.

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

[0111]

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

[0113]

[0114] In some embodiments, 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% phosphoric acid aqueous solution, preferably a 0.05% phosphoric acid aqueous solution.

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

[0117] In some embodiments, in step (3), the specifications of the chromatographic column are: column length 100 mm, inner diameter 2.1 mm, particle size 1.7-2.2 μm, preferably 1.7-1.8 μm.

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

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

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

[0121] In some embodiments, in step (3), the characteristic spectrum of Huangqi Guizhi Wuwu Decoction has 17 characteristic peaks, wherein peak 1, peak 4, peak 5, peak 6, peak 8, peak 9, peak 10, peak 11, peak 12, peak 13, peak 15 and peak 17 correspond to the retention times of the chromatographic peaks of gallic acid, paeoniflorin, paeoniflorin, coumarin, calycosin glucoside, galloylpeoniflorin, 1,2,3,4,6-penta-galloyl glucoside, cinnamyl alcohol, cinnamaldehyde, cinnamic acid, calycosin and 6-gingerol reference substances, respectively.

[0122] In some preferred embodiments, in step (3), the peak (peak 10) corresponding to the chromatographic peak of 1,2,3,4,6-penta-galloyl glucoside reference substance is taken as the S peak, 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 respect to the S peak are within ±15% of the specified value, and the specified value is as follows: .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 more preferred embodiments, in step (3), the peak (peak 10) corresponding to the chromatographic peak of the 1,2,3,4,6-penta-galloyl glucoside reference substance is taken as the S peak, 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 respect to the S peak are within ±10% of the specified values, and the specified values ​​are 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] [Feature Spectrum Identification Method]

[0125] The present invention provides a quality control method for Huangqi Guizhi Wuwu Decoction. Specifically, the quality control method is a characteristic spectrum identification method, which comprises the following steps:

[0126] (1) Preparation of test solution:

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

[0128] (2) Quality evaluation of test products:

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

[0130] In some embodiments, in step (1), the volume ratio of the Huangqi Guizhi Wuwu 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 and Atractylodes are all water-soluble ingredients, and astragaloside IV and paeoniflorin are both saponin components with similar physical and chemical properties, the thin layer chromatography method of the present invention uses the same extraction solvent (n-butanol) to meet the extraction purpose, and does not require repeated extraction with multiple solvents. In addition, the present invention uses the same test solution on a thin layer plate under two different inspection conditions to successfully create a new thin layer identification method for identifying two medicinal flavors in a compound with one test and multiple evaluations.

[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 comprises the following steps:

[0134] (1) Preparation of test solution:

[0135] Take the Huangqi Guizhi Wuwu Decoction extract as the test sample, shake and extract it with a water-saturated alcohol solvent, combine the alcohol solvent, wash it with an alkaline test solution, discard the alkaline test solution, evaporate the alcohol solvent to dryness, add methanol to dissolve the residue, and use it as the test sample solution;

[0136] (2) Preparation of control medicinal material solution:

[0137] Astragalus and white peony root as control medicinal materials are respectively taken, methanol is added, ultrasonication is performed, filtration is performed, the filtrate is evaporated to dryness, water is added to the residue to dissolve, extraction is performed by shaking with a water-saturated alcohol solvent, the alcohol solvent is combined, washing is performed with an alkaline test solution, the alkaline test solution is discarded, the alcohol solvent is evaporated to dryness, and methanol is added to the residue to dissolve, thereby obtaining an Astragalus control medicinal material solution and a white peony root control medicinal material solution;

[0138] (3) Preparation of reference solution:

[0139] Astragaloside IV and paeoniflorin as reference substances were taken respectively, and methanol was added to prepare astragaloside IV reference substance solution and paeoniflorin reference substance solution;

[0140] (4) Thin layer chromatography identification:

[0141] The test sample solution described in step (1), the astragalus reference medicinal material solution and the white peony reference medicinal material solution described in step (2), and the astragaloside reference substance solution and the paeoniflorin reference substance solution described in step (3) are respectively spotted on the same thin layer plate, developed, and the determination results of the test sample, the reference medicinal material and the reference substance are compared; wherein the chromatographic conditions of 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 color developer; and inspecting 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 embodiments, in step (1), the volume ratio of the Huangqi Guizhi Wuwu 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 embodiments, in step (1), the shaking extraction is performed 2 to 4 times, preferably 2 times.

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

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

[0147] In some embodiments, in step (2), the ultrasonication time is 20 to 40 min, preferably 30 min.

[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 embodiments, 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 alkali metal hydroxide aqueous solution, preferably an ammonia test solution.

[0151] In step (1) and step (2), the purpose of washing with an alkaline test solution is to make the solution weakly alkaline, and to release alkaloids and other non-saponin components into the alkaline test solution under alkaline conditions, thereby achieving the purpose of separation.

[0152] In some embodiments, in step (4), the spotting amount of the test solution, astragalus and white peony root control medicinal material solutions is 5-10 μl; the spotting amount of the astragaloside IV and paeoniflorin reference solution is 1-3 μl.

[0153] In some embodiments, in step (4), the volume ratio of chloroform-methanol-water in the developing solvent 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 embodiments, in step (4), the heating temperature is 90-120°C, preferably 105°C.

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

[0158] In some embodiments, step (4) further comprises the following steps: comparing the test sample with the test results of the reference medicinal material and the reference substance, and determining whether the thin layer chromatograms of the test sample, the reference medicinal material of Astragalus and the reference substance of Astragaloside IV are in the range of R f Whether there are spots of the same color at the same position of the value, and whether the thin layer chromatograms of the test sample, white peony reference medicinal material and paeoniflorin reference substance are in the R f Whether there are spots of the same color at the same position.

[0159] [Quality control method combining characteristic spectrum identification method and thin layer chromatography identification method]

[0160] In some embodiments, the characteristic spectrum identification method and the thin layer chromatography identification method can be combined to perform multi-faceted qualitative testing on 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 spectrum identification method and the above-mentioned thin layer chromatography identification method.

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

[0163]

[0164]

[0165] Example

[0166] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0167] Example 1 Thin layer chromatography identification of astragalus and white peony root in Huangqi Guizhi Wuwu Decoction

[0168] 1.1 Instruments and reagents

[0169] Thin layer automatic imaging instrument (CAMAG TLC VISUALIZER), ultrasonic cleaner (KQ-250E, Kunshan Ultrasonic Instrument Co., Ltd.), one thousandth balance (AR223CN, Ohaus), AE-2401 electronic analytical balance (Mettler-Toledo Instrument (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, and silica gel G thin layer plates were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0171] Astragalus membranaceus reference material (120974-201612), white peony root reference material (120905-201610), astragaloside reference substance (110781-201717), and paeoniflorin reference substance (110736-201741) were purchased from China Food and Drug Inspection Institutes.

[0172] The batch numbers of 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 batch number of the negative control lacking astragalus is HQGZ-(lack of astragalus), and the batch number of the negative control lacking white peony is HQGZ-(lack of white peony).

[0173] 1.2 Solution preparation

[0174] 1.2.1 Preparation of test solution

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

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

[0177] Take 0.5g of Astragalus membranaceus reference medicinal material, add 30ml of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, add 20ml of water to dissolve the residue, shake and extract twice with water-saturated n-butanol, 20ml each time, combine the n-butanol solution, wash with 20ml of ammonia test solution, discard the ammonia test solution, evaporate the n-butanol solution to dryness, add 1ml of methanol to dissolve the residue, and use it as the reference medicinal material solution. Take 1g of white peony reference medicinal material, and prepare the white peony reference medicinal material solution by the same method. Take astragaloside IV and paeoniflorin reference substances, add methanol to make a solution containing 1mg of astragaloside IV and paeoniflorin per 1ml, as the reference substance solution.

[0178] 1.2.3 Preparation of negative sample solution

[0179] Take 30 ml of Huangqi Guizhi Wuwu Decoction extract lacking Astragalus, and prepare a negative control solution lacking Astragalus in the same way as "1.2.1 Preparation of test solution". Take 30 ml of Huangqi Guizhi Wuwu Decoction extract lacking White Peony, and prepare a negative control solution lacking White Peony in the same way.

[0180] 1.3 Thin layer chromatography conditions

[0181] According to the thin layer chromatography method (General Rules 0502 of the 2015 edition of the Chinese Pharmacopoeia), 5-10 μl of the test sample solution, 8 μl of the astragalus reference medicinal material solution, 10 μl of the white peony reference medicinal material solution, 2 μl each of astragaloside IV and paeoniflorin reference material solutions were respectively spotted on the same silica gel G thin layer plate, and the lower layer solution of chloroform-methanol-water (13:7:2) placed below 10°C was used as the developing agent. The plate was developed, taken out, dried, sprayed with 10% ethanolic sulfuric acid solution, heated at 105°C until the spots were clearly colored, and examined under sunlight and ultraviolet light (365nm), respectively.

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

[0183] Different batches of Huangqi Guizhi Wuwu Decoction samples were subjected to TLC identification of Astragalus and White Peony Root. Figure 1 and Figure 2 .exist Figure 1 and Figure 2 Among them, 1 to 15 are 15 batches of Huangqi Guizhi Wuwu Decoction samples, 16 is a negative control lacking astragalus, S1 is an astragalus control medicinal material, S2 is a white peony control medicinal material, 17 is a negative control lacking white peony, T1 is an astragaloside reference substance, and T2 is a paeoniflorin reference substance.

[0184] Conclusion: Figure 1 and Figure 2 It can be seen that the R f The value is 0.4, showing red spots under sunlight and light yellow spots under 365nm; the R fThe value is 0.57, showing dark purple spots under sunlight and dark blue spots at 365nm. Different batches of Huangqi Guizhi Wuwu Decoction show spots of the same color at the corresponding positions of the control medicinal material chromatogram and the reference substance chromatogram. The negative sample does not show spots of the same color at the corresponding position of the sample chromatogram. This shows that the thin layer chromatography method of the present invention has strong specificity and can identify the astragalus and white peony root in Huangqi Guizhi Wuwu Decoction.

[0185] Example 2: Development of the characteristic spectrum method of Huangqi Guizhi Wuwu Decoction

[0186] 1.1 Instruments and reagents

[0187] Waters ACQUITY UPLC H-Class ultra-high performance liquid chromatography system; Waters QuaternarySolvent 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); ultrapure water; methanol (chromatographic grade, Thermo Fisher); phosphoric acid (chromatographic grade, Aladdin).

[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, and HQGZ-F15.

[0189] 1.2 Source of reference substances

[0190] Paeoniflorin (batch number: 110736-201943, purity 95.1%, China National Institute for Food and Drug Control); calycosin glucoside (batch number: 111920-201606, purity 97.6%, China National Institute for Food and Drug Control); 1,2,3,4,6-penta-galloyl glucoside (batch number: ST16430120, purity 98%, Standard); cinnamyl alcohol (batch number: ST06990320, purity 90.0%, Standard); cinnamaldehyde (batch number: 110710-201821, purity 99.6%, China National Institute for Food and Drug Control); cinnamic acid (batch number: 110786-201604, purity 98.8%, China National Institute for Food and Drug Control); 6-gingerol (batch number: 111833-201705, purity 96.8%, China National Institute for Food and Drug Control).

[0191] 1.3 Determined chromatographic conditions

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

[0193]

[0194] 1.4 Preparation of reference solution

[0195] Preparation of reference substance solution: Take appropriate amount of paeoniflorin reference substance, calycosin glucoside reference substance, 1,2,3,4,6-penta-galloyl glucoside, cinnamyl alcohol reference substance, cinnamaldehyde reference substance, cinnamic acid reference substance, and 6-gingerol reference substance, weigh them accurately, add methanol to make reference substance solutions containing 200 μg of paeoniflorin, 3 μg of calycosin glucoside, 150 μg of 1,2,3,4,6-penta-galloyl glucoside, 3 μg of cinnamyl alcohol, 3 μg of cinnamaldehyde, 4 μg of cinnamic acid, and 15 μg of 6-gingerol per 1 ml, shake well to obtain.

[0196] 1.5 Preparation of test solution

[0197] 1.5.1 Investigation of different extraction solvents

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

[0199] Accurately pipette 5ml of Huangqi Guizhi Wuwu Decoction extract, place it in a 10ml volumetric flask, add 50% methanol and methanol to the mark, mix well, and get 25% methanol and 50% methanol test solution; accurately pipette 2ml of Huangqi Guizhi Wuwu Decoction extract, place it in a 10ml volumetric flask, dilute to the mark with methanol, mix well, and get 80% methanol test solution. Take the above test solution and pass it through a 0.22μm microporous filter membrane to get it. Accurately pipette 1μl of each test solution, inject it into the liquid chromatograph, and measure it according to the above chromatographic conditions. The results are shown in Table 1.

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

[0201]

[0202] The results showed that except for the lower extraction efficiency of 25% methanol, the extraction efficiency of 50% methanol and 80% methanol was equivalent, among which the total peak area of ​​80% methanol was higher. Considering comprehensively, 50% methanol was selected as the extraction solvent.

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

[0204] Accurately pipette 5 ml of Huangqi Guizhi Wuwu Decoction extract into a 10 ml volumetric flask, add methanol to dilute to the scale, mix well, shake well, filter, and take the filtrate to obtain the final product.

[0205] 1.6 Methodology

[0206] 1.6.1 Precision investigation

[0207] Take the same batch of samples and prepare the test solution according to the test solution preparation method. Inject 6 times in a row, 1 μl each time, and record the chromatogram. Take the peak corresponding to the 1,2,3,4,6-penta-galloyl glucoside peak (peak 10) as the reference peak, calculate the relative retention time and relative peak area of ​​each common peak, and the results are shown in Table 2 and Table 3.

[0208] Table 2 Precision test results (relative retention time)

[0209]

[0210] Table 3 Precision test results (relative peak area)

[0211]

[0212] The results showed that the RSD of the relative retention time and relative peak area of ​​each characteristic peak was less than 3%, and the precision was good.

[0213] 1.6.2 Stability test

[0214] Take the same batch of samples and prepare the test solution according to the test solution preparation method. Inject 1 μl every few hours for a total of 24 hours and record the chromatogram. Take the peak corresponding to the 1,2,3,4,6-penta-galloyl glucoside peak (peak 10) as the reference peak, calculate the relative retention time and relative peak area of ​​each common peak, and the results are shown in Table 4 and Table 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 RSDs of the relative retention times and relative peak areas of most characteristic peaks were less than 3%, and the test solution had good stability within 24 hours.

[0220] 1.6.3 Repeatability test

[0221] Take the same batch of samples and prepare the test solution according to the test solution preparation method. Make 6 parallels, inject 1μl each time, and record the chromatogram. Take the peak corresponding to the 1,2,3,4,6-penta-galloyl glucoside peak (peak 10) as the reference peak, calculate the relative retention time and relative peak area of ​​each common peak, and the results are shown in Table 6 and Table 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 RSDs of the relative retention time and relative peak area of ​​each characteristic peak were less than 3%, and the test solution had good repeatability.

[0227] 1.6.4 Durability test

[0228] 1.6.4.1 Chromatographic column inspection

[0229] The same batch of samples were taken and the test solution was prepared according to the test solution preparation method. Four chromatographic columns were used, namely 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 effects of the four chromatographic columns with different fillers were compared. The results are shown in Figure 3 .

[0230] The results showed that the characteristic peaks had good separation effects on the HSS T3 column and the Acclaim RSLC 120C18 column, while some chromatographic peaks had poor separation and few peaks on the EP C18 column and the SB Aq column. Therefore, it is recommended to use the HSS T3 column or the Acclaim RSLC 120C18 column for the characteristic spectrum study of Huangqi Guizhi Wuwu Decoction.

[0231] 1.6.4.2 Elution gradient investigation

[0232] Take the same batch of samples and prepare the test solution according to the test solution preparation method. Inject according to the following elution procedure. The other chromatographic conditions are the same as "Chromatographic conditions determined in 1.3". Inject 1μl and record the chromatogram. The results are shown in Figure 4 The peak corresponding to the 1,2,3,4,6-penta-galloyl glucoside peak (peak 10) was used as the reference peak to calculate the relative retention time of each common peak.

[0233]

[0234] The results showed that the characteristic peaks could also be effectively separated under the above gradient conditions. Taking the peak corresponding to 1,2,3,4,6-penta-galloyl glucoside peak (peak 10) as the reference peak, the relative retention times of the characteristic peaks 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 samples of different batches of Huangqi Guizhi Wuwu Decoction and determination of characteristic peaks

[0236] The results were analyzed using the “Chinese Herbal Medicine Chromatographic Characteristic Spectrum Similarity Evaluation System (2012 Edition)” recommended by the National Pharmacopoeia Committee. Combined with the peak attribution, the common peaks were selected, and the 1,2,3,4,6-penta-galloyl glucoside 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 common peak superposition spectrum was fitted. Figure 5 ; Generate a feature comparison map of the common pattern, see Figure 6 The results are shown below (Table 8, Table 9):

[0237] Table 8 Multi-batch experimental results (relative retention time)

[0238]

[0239] Table 9 Multi-batch experimental results (relative peak area)

[0240]

[0241] It can be seen from the relative retention time and relative peak area of ​​each characteristic peak that the retention time ratio of the characteristic peaks in 15 batches of samples is relatively stable, while the relative peak areas of the characteristic peaks 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 samples from different origins and combinations vary greatly and are not suitable for inclusion in the standard.

[0242] 1.8 Identification of characteristic peak reference substances

[0243] The preparation of the reference solution is as described in the preparation of the reference solution under the item "1.4 Reference Solution" of this embodiment. Take appropriate amounts of gallic acid, paeoniflorin, coumarin, galloyl paeoniflorin, and calycosin isoflavone reference substances, accurately weigh them, and add methanol to prepare reference solutions containing 50 μg of gallic acid, 150 μg of galloyl paeoniflorin, 50 μg of paeoniflorin, 200 μg of calycosin, and 50 μg of coumarin per 1 ml, shake well, and obtain. Inject 1 μl of the above reference solution respectively, record the chromatogram, and compare it with the characteristic spectrum of Huangqi Guizhi Wuwu Decoction to identify the characteristic peaks.

[0244] Results Figure 7, 17 characteristic peaks: peak 1 corresponds to the peak of gallic acid reference substance, peak 4 corresponds to the peak of paeoniflorin reference substance, peak 5 corresponds to the peak of paeoniflorin reference substance, peak 6 corresponds to the peak of coumarin reference substance, peak 8 corresponds to the peak of calycosin glucoside reference substance, peak 9 corresponds to the peak of galloyl paeoniflorin reference substance, peak 10 corresponds to the peak of 1,2,3,4,6-penta-galloyl glucoside reference substance, peak 11 corresponds to the peak of cinnamyl alcohol reference substance, peak 12 corresponds to the peak of cinnamaldehyde reference substance, peak 13 corresponds to the peak of cinnamic acid reference substance, peak 15 corresponds to the peak of calycosin isoflavone reference substance, and peak 17 corresponds to the peak of 6-gingerol reference substance. It can be seen that the characteristic spectrum in the present invention realizes the identification of 12 characteristic peaks, and the identification rate is high.

[0245] It should be noted that although the technical solutions of the present invention are described with specific examples, those skilled in the art will appreciate that the present invention should not be limited thereto. The various embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Without departing from the scope and spirit of the described embodiments, many modifications and changes are obvious to those of ordinary skill in the art. The choice of terms used herein is intended to best explain the principles of the embodiments, practical applications, or technical improvements in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A method for constructing a characteristic spectrum of Huangqi Guizhi Wuwu Decoction, comprising the following steps: (1) Preparation of Huangqi Guizhi Wuwu Decoction sample solution: Take the Huangqi Guizhi Wuwu Decoction extract, dilute it with methanol, filter it, and take the filtrate to obtain; preferably, the volume ratio of the Huangqi Guizhi Wuwu Decoction extract to the methanol is 3:1 to 1:4, preferably 1:1 to 1:4, and more preferably 1:1; (2) Preparation of reference solution: Take paeoniflorin, calycosin glucoside, cinnamyl alcohol, cinnamaldehyde, cinnamic acid, 6-gingerol, gallic acid, paeoniflorin, coumarin, galloylpeoniflorin, 1,2,3,4,6-penta-galloyl glucoside and calycosin reference substances respectively, add methanol to prepare reference substance solutions; (3) Construction of feature maps: The Huangqi Guizhi Wuwu Decoction sample solution in step (1) and the reference solution in step (2) are subjected to UPLC analysis to obtain a characteristic spectrum of Huangqi Guizhi Wuwu Decoction; wherein the chromatographic conditions of the UPLC analysis include: a chromatographic column using octadecylsilane bonded silica gel as a filler; mobile phase A is methanol, and mobile phase B is a phosphoric acid aqueous solution; gradient elution, and the preferred elution procedure is as follows: The detection wavelength is 230 to 280 nm, preferably 250 to 270 nm, and more preferably 260 nm.

2. The method for constructing a characteristic map according to claim 1, characterized in that: In step (3), the elution procedure is: or 3. The method for constructing a characteristic spectrum according to claim 1 or 2, characterized in that: In step (3), The chromatographic conditions include at least one of the following conditions: The mobile phase B is 0.01% to 0.1% phosphoric acid aqueous solution, preferably 0.05% phosphoric acid aqueous solution; The chromatographic column is HSS T3 chromatographic column or Acclaim TM RSLC 120C18 column, preferably HSS T3 column; The specifications of the chromatographic column are: column length 100 mm, inner diameter 2.1 mm, particle size 1.7-2.2 μm, preferably 1.7-1.8 μm.

4. The method for constructing a characteristic spectrum according to any one of claims 1 to 3, characterized in that: In step (3), The chromatographic conditions also include at least one of the following conditions: The column temperature is 25-35°C, preferably 30°C; The flow rate is 0.2-0.30 ml per minute, preferably 0.25 ml; The injection volume is 1-3 μl, preferably 2 μl.

5. The method for constructing a characteristic map according to any one of claims 1 to 4, characterized in that: In step (3), The characteristic spectrum of Huangqi Guizhi Wuwu Decoction has 17 characteristic peaks, wherein peak 1, peak 4, peak 5, peak 6, peak 8, peak 9, peak 10, peak 11, peak 12, peak 13, peak 15 and peak 17 correspond to the retention times of the chromatographic peaks of gallic acid, paeoniflorin, paeoniflorin, coumarin, calycosin glucoside, galloylpeoniflorin, 1,2,3,4,6-penta-galloyl glucoside, cinnamyl alcohol, cinnamaldehyde, cinnamic acid, calycosin and 6-gingerol reference substances, respectively; Further, the peak (peak 10) corresponding to the chromatographic peak of 1,2,3,4,6-penta-galloyl glucoside reference substance is taken as the S peak, 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 respect to the S peak are within ±15% of the prescribed value, and the prescribed 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).

6. A quality control method for Huangqi Guizhi Wuwu Decoction, comprising the following steps: (1) Preparation of test solution: Taking the Huangqi Guizhi Wuwu Decoction extract as a test sample, diluting it with methanol, filtering it, and taking the filtrate to obtain; preferably, the volume ratio of the Huangqi Guizhi Wuwu Decoction extract to the methanol is 3:1 to 1:4, preferably 1:1 to 1:4, and more preferably 1:1; (2) Quality evaluation of test products: The test solution described in step (1) is analyzed according to the UPLC chromatographic conditions in the characteristic spectrum construction method described in any one of claims 1 to 5 to obtain a test sample spectrum, and the spectrum is compared with the characteristic spectrum of Huangqi Guizhi Wuwu Decoction in the characteristic spectrum construction method described in any one of claims 1 to 5.

7. A quality control method for Huangqi Guizhi Wuwu Decoction, comprising the following steps: (1) Preparation of test solution: Take the Huangqi Guizhi Wuwu Decoction extract as the test sample, shake and extract it with a water-saturated alcohol solvent, combine the alcohol solvent, wash it with an alkaline test solution, discard the alkaline test solution, evaporate the alcohol solvent to dryness, add methanol to dissolve the residue, and use it as the test sample solution; (2) Preparation of control medicinal material solution: Astragalus and white peony root as control medicinal materials are respectively taken, methanol is added, ultrasonication is performed, filtration is performed, the filtrate is evaporated to dryness, water is added to the residue to dissolve, extraction is performed by shaking with a water-saturated alcohol solvent, the alcohol solvent is combined, washing is performed with an alkaline test solution, the alkaline test solution is discarded, the alcohol solvent is evaporated to dryness, and methanol is added to the residue to dissolve, thereby obtaining an Astragalus control medicinal material solution and a white peony root control medicinal material solution; (3) Preparation of reference solution: Astragaloside IV and paeoniflorin as reference substances were taken respectively, and methanol was added to prepare astragaloside IV reference substance solution and paeoniflorin reference substance solution; (4) Thin layer chromatography identification: The test sample solution described in step (1), the astragalus reference medicinal material solution and the white peony reference medicinal material solution described in step (2), and the astragaloside reference substance solution and the paeoniflorin reference substance solution described in step (3) are respectively spotted on the same thin layer plate, developed, and the determination results of the test sample, the reference medicinal material and the reference substance are compared; wherein the chromatographic conditions of 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 color developer; and inspecting under sunlight and ultraviolet light respectively.

8. The quality control method according to claim 7, characterized in that: Step (1) includes at least one of the following conditions: The alcohol solvent is n-butanol, n-propanol, isopropanol or isobutanol, preferably n-butanol; The volume ratio of the Huangqi Guizhi Wuwu 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; The shaking extraction is performed 2 to 4 times, preferably 2 times; The alkaline test solution is an ammonia test solution or an alkali metal hydroxide aqueous solution, preferably an ammonia test solution.

9. The quality control method according to claim 7 or 8, characterized in that: Step (2) includes at least one of the following conditions: The ratio of the astragalus reference medicinal material to methanol is 1g:30ml to 1g:90ml, preferably 1g:60ml to 1g:90ml, more preferably 1g:60ml; The ultrasonic time is 20 to 40 minutes, preferably 30 minutes; 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; The shaking extraction is performed 2 to 4 times, preferably 2 times; The alkaline test solution is an ammonia test solution or an alkali metal hydroxide aqueous solution, preferably an ammonia test solution.

10. The quality control method according to any one of claims 7 to 9, characterized in that: Step (4) includes at least one of the following conditions: The sample volume of the test solution, astragalus and white peony root control medicinal material solution is 5-10 μl; the sample volume of astragaloside IV and paeoniflorin reference solution is 1-3 μl; The volume ratio of chloroform-methanol-water in the developing agent is (10-15):(5-10):(1-3), preferably 13:7:2; further, the developing agent is a lower layer solution of chloroform-methanol-water placed below 10°C; The volume percentage of the sulfuric acid ethanol solution is 5% to 15%, preferably 10%; The heating temperature is 90-120°C, preferably 105°C; The wavelength of the ultraviolet light is 355-375 nm, preferably 365 nm.

11. The quality control method according to any one of claims 7 to 10, characterized in that: Step (4) also includes the following steps: Compare the test results of the test sample with those of the reference medicinal material and the reference substance to determine whether the thin layer chromatograms of the test sample, the reference medicinal material of Astragalus and the reference substance of Astragaloside IV are f Whether there are spots of the same color at the same position of the value, and whether the thin layer chromatograms of the test sample, white peony reference medicinal material and paeoniflorin reference substance are in the R f Whether there are spots of the same color at the same position.

12. A quality control method for Huangqi Guizhi Wuwu Decoction, comprising the quality control method according to claim 6 and the quality control method according to any one of claims 7-11.

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