Preparation method of Chinese angelica six-yellow decoction reference sample, construction method of fingerprint spectrum and quality control method of preparation

Abstract

The invention discloses a preparation method of an Angelica sinensis-Liuhuang decoction reference sample, a construction method of a fingerprint spectrum and a quality control method of a preparation. The preparation method comprises the following steps: treating a formula of the Chinese angelica and six-yellow decoction, namely crushing raw materials, mixing, adding water for soaking, decocting with strong fire, decocting with slow fire, sieving and the like to obtain a wet material, further drying the wet material by adopting at least three stages of temperature gradient, drying in the first stage at the temperature of less than or equal to-50 DEG C, drying in the second stage at the temperature of-40 DEG C to-10 DEG C, and drying in the third stage at the temperature of-50 DEG C to-10 DEG C to obtain the Chinese angelica and six-yellow decoction. The third-stage drying temperature is 0-30 DEG C, and the radix angelicae sinensis six-yellow decoction reference sample is obtained by controlling the drying procedure. The reference sample prepared by the invention can greatly improve the detection of various active components in the angelica sinensis-Liuhuang decoction, meanwhile, the fingerprint spectrum constructed by adopting the angelica sinensis-Liuhuang decoction reference sample has excellent stability, repeatability and durability, and the comprehensive quality control and evaluation of the angelica sinensis-Liuhuang decoction are realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of quality control of traditional Chinese medicine preparation, and particularly relates to a preparation method of a reference sample of Angelica Six Huang Decoction, a construction method of a fingerprint spectrum, and a quality control method of a preparation. BACKGROUND

[0002] The classical famous prescription Angelica Six Huang Decoction is a "sacred medicine for treating night sweat", which is used for treating night sweat caused by yin deficiency and excessive fire. The prescription is composed of seven medicines, i.e., Angelica sinensis, Radix Rehmanniae, Radix Rehmanniae Preparata, Coptis chinensis, Scutellaria baicalensis, Phellodendri, and Astragalus membranaceus. At present, the Angelica Six Huang Decoction has few studies on the quality control, no perfect standard is established, and the quality evaluation method is difficult to unify the standard, so that the quality of the medicine is difficult to guarantee. Although CN112229934A analyzes a plurality of components in the Angelica Six Huang Decoction, the quality control of the preparation is not simply the detection of the effective components in the preparation, but needs to investigate and verify a plurality of quantitative indexes such as the source of traditional Chinese medicinal materials and different batches, so as to obtain a stable, repeatable and durable quality control means and method. Obviously, the technical scheme does not achieve the purpose. In addition, in the existing related research, there are few studies on active components in the process of quality control and evaluation method of the Angelica Six Huang Decoction, and it is difficult to comprehensively control and evaluate the quality of the Angelica Six Huang Decoction. SUMMARY

[0003] Therefore, it is necessary to provide a quality control and evaluation method of the Angelica Six Huang Decoction which has a plurality of active component detection and can realize stability, repeatability, durability and accuracy.

[0004] In a first aspect, the present application provides a preparation method of a reference sample of Angelica Six Huang Decoction, comprising the following steps:

[0005] The Angelica sinensis, Radix Rehmanniae, Radix Rehmanniae Preparata, Coptis chinensis, Scutellaria baicalensis, Phellodendri, and Astragalus membranaceus are crushed and mixed to prepare a first material;

[0006] Water is added to the first material for soaking, and the material is boiled with a strong fire and a weak fire, and then sieved to prepare a second material;

[0007] The second material is dried to prepare the reference sample of the Angelica Six Huang Decoction;

[0008] The drying includes at least three drying stages, the temperature of the first drying stage is ≤-50℃, the temperature of the second drying stage is-40℃ to-10℃, and the temperature of the third drying stage is 0℃ to 30℃.

[0009] In some embodiments, the mass ratio of the Angelica sinensis, Rehmannia glutinosa, Radix Rehmanniae Recens, Coptis chinensis, Scutellaria baicalensis, Phellodendri chinensis Cortex, and Astragalus membranaceus is 1:1:1:1:1:1:2; and / or, after mixing, the mixture is further sieved through a 4-mesh sieve; and / or, the mass-to-volume ratio of the first material to the water is 1g:(28-30)mL; and / or, the soaking time is 20-40 minutes; and / or, the temperature of the high heat is 110-120℃; and / or, the time of high heat decoction is 13-16 minutes; and / or, the temperature of the low heat is 90-100℃; and / or, the time of high heat decoction is 40-60 minutes; and / or, the volume of the decocted material is 1 / 3 to 2 / 3 of the volume before decoction; and / or, the mesh size of the sieve is 150-250 mesh; and / or, the time of the first drying is 2-4 hours; and / or, the time of the second drying is 70-87 hours; and / or, the time of the third drying is 10-15 hours.

[0010] In a second aspect, the present application further provides a Dangui Liuhuang Decoction reference sample, which is prepared by the preparation method of the first aspect.

[0011] In a third aspect, the present application further provides a method for detecting Dangui Liuhuang Decoction, comprising the following steps:

[0012] A mixture of control sample solution is provided by mixing the control samples of berberine hydrochloride, magnoflorine, 3-O-feruloyl quinic acid, 4-O-feruloyl quinic acid, 5-O-feruloyl quinic acid, palmatine hydrochloride, jatrorrhizine, epiberberine hydrochloride, berberine hydrochloride, palmatine, baicalin, wogonoside A-7-O-glucuronide, and wogonoside with a first solvent, filtering, and taking the filtrate to form the mixture of control sample solution;

[0013] A test sample solution is provided by mixing the Dangui Liuhuang Decoction sample with a second solvent, filtering, and taking the filtrate to form the test sample solution;

[0014] The detection method is selected from liquid chromatography, and the conditions of the liquid chromatography include:

[0015] The elution system includes mobile phase A comprising acetonitrile and mobile phase B comprising water containing 0.2-0.4% phosphoric acid by mass fraction, gradient elution, and the program of the gradient elution includes:

[0016] 0-15 min, the volume percentage of mobile phase A changes from 5% to 12.5%;

[0017] 15-25 min, the volume percentage of mobile phase A changes from 12.5% to 15%;

[0018] 25 min-40 min, the volume percentage of mobile phase A is changed from 15% to 25%;

[0019] 40 min-50 min, the volume percentage of mobile phase A is 25%;

[0020] 50 min-60 min, the volume percentage of mobile phase A is changed from 25% to 35%;

[0021] 60 min-62 min, the volume percentage of mobile phase A is changed from 35% to 90%.

[0022] In some embodiments, the mobile phase B comprises an aqueous solution containing 0.28%-0.32% by mass of phosphoric acid; and / or, the column temperature is 30°C-35°C; and / or, the flow rate is 0.5 mL / min-1.2 mL / min; and / or, the detection wavelength is 275 nm-305 nm; and / or, the theoretical plate number calculated based on the peak of berberine hydrochloride is ≥10000.

[0023] In some embodiments, the Angelica Six Yellow Decoction sample comprises the Angelica Six Yellow Decoction reference sample provided in the second aspect; and / or, the Angelica Six Yellow Decoction sample is prepared by mixing, decocting, and drying Angelica, Rehmannia glutinosa, Radix Rehmanniae Recens, Coptis chinensis, Scutellaria baicalensis, Phellodendri chinensis, and Astragalus membranaceus according to the prescription amount; and / or, the Angelica Six Yellow Decoction sample is prepared by decocting and drying Angelica Six Yellow Decoction pieces, and the dosage form of the Angelica Six Yellow Decoction sample comprises one or more of granules, tablets, decoctions, pills, powders, pastes, and capsules.

[0024] In some embodiments, the first solvent and the second solvent each independently comprise one or more of methanol, ethanol, acetonitrile, dimethyl sulfoxide, and water; and / or, the process of mixing the Angelica Six Yellow Decoction sample with the second solvent further comprises ultrasonic treatment, the power of the ultrasonic treatment being 250 W-350 W, and the frequency of the ultrasonic treatment being 35 kHz-45 kHz.

[0025] In a fourth aspect, the present application further provides a method for constructing a fingerprint of Angelica Six Yellow Decoction, comprising the following steps:

[0026] A control solution and an Angelica Six Yellow Decoction sample solution are respectively provided;

[0027] The control solution is detected by the detection method of the Angelica Six Yellow Decoction provided in the third aspect to obtain a control chromatogram;

[0028] The Angelica Six Yellow Decoction sample solution is detected by the detection method of the Angelica Six Yellow Decoction provided in the third aspect to obtain a sample chromatogram;

[0029] The test product spectrum is subjected to fingerprint similarity analysis to generate a standard fingerprint spectrum, the chromatographic peaks of the standard fingerprint spectrum are identified with reference to the control product spectrum, and a fingerprint spectrum of the Danggui Liuhuang Decoction is constructed.

[0030] In some embodiments, the Danggui Liuhuang Decoction sample comprises the Danggui Liuhuang Decoction reference sample provided in the second aspect.

[0031] In some embodiments, the sample spectrum and / or the standard fingerprint spectrum exhibits 14 characteristic peaks, and the retention time of the characteristic peaks is within the range of -10% to 10% of the retention time value of the corresponding control product chromatographic peak in the standard fingerprint spectrum.

[0032] Compared with the prior art, the technical scheme of the present application has the following beneficial effects:

[0033] The present application provides a preparation method of a Danggui Liuhuang Decoction reference sample, which comprises the following steps: processing the prescription of the Danggui Liuhuang Decoction, including crushing and mixing raw materials, soaking with water, adopting high fire to decoct and low fire to decoct, sieving, and obtaining wet materials, and then drying the wet materials by controlling the drying procedure, wherein at least three drying stages are adopted, the temperature of the first drying stage is ≤-50℃, the temperature of the second drying stage is-40℃ to-10℃, and the temperature of the third drying stage is 0℃ to 30℃. By adopting a specific drying procedure, the present application obtains a Danggui Liuhuang Decoction reference sample, which has high applicability in the process of quality control and evaluation of the preparation, and the Danggui Liuhuang Decoction reference sample prepared by the preparation method can greatly improve the detection and accuracy of active ingredients. Meanwhile, the fingerprint spectrum constructed by using the Danggui Liuhuang Decoction reference sample has excellent stability, repeatability and durability, and realizes the comprehensive quality control and evaluation of the Danggui Liuhuang Decoction. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 The chromatogram of the control solution of the present application.

[0035] Figure 2 The chromatogram of the test solution of the Danggui Liuhuang Decoction reference sample of the present application, wherein: peak 1: phellodendrine hydrochloride; peak 2: magnoflorine; peak 3: 3-O-feruloyl quinic acid; peak 4: 4-O-feruloyl quinic acid; peak 5: 5-O-feruloyl quinic acid; peak 6: berberine hydrochloride; peak 7: epiberberine hydrochloride; peak 8: jatrorrhizine hydrochloride; peak 9: berberine hydrochloride; peak 10: palmatine; peak 11: baicalin; peak 13: fomesone A-7-O-glucuronide; and peak 14: wogonoside.

[0036] Figure 3 The liquid chromatogram for precision investigation of the characteristic spectrum of the Danggui Liuhuang Decoction reference sample in Test Example 1.

[0037] Figure 4 The liquid chromatogram for the reproducibility of the characteristic spectrum of the reference sample of the Danggui Liuhuang Decoction in Test Example 1 is shown.

[0038] Figure 5 The liquid chromatogram for the stability of the characteristic spectrum of the reference sample of the Danggui Liuhuang Decoction in Test Example 1 is shown.

[0039] Figure 6 The liquid chromatogram of the blank control sample (A), the control solution (B) and the test sample solution (C) of the reference sample of the Danggui Liuhuang Decoction in Test Example 2 of the present application is shown.

[0040] Figure 7 The liquid chromatogram of the blank control sample (A), the control solution (B) and the test sample solution (C) of the reference sample of the Danggui Liuhuang Decoction in Test Example 3 of the present application is shown, wherein peak 1 is coptisine hydrochloride and peak 2 is berberine hydrochloride.

[0041] Figure 8 The liquid chromatogram of the blank control sample (A), the control solution (B) and the test sample solution (C) of the reference sample of the Danggui Liuhuang Decoction in Test Example 4 of the present application is shown.

[0042] Figure 9 The liquid chromatogram of the blank control sample (A), the control solution (B) and the test sample solution (C) of the reference sample of the Danggui Liuhuang Decoction in Test Example 5 of the present application is shown.

[0043] Figure 10 The liquid chromatogram of the blank control sample (A), the control solution (B) and the test sample solution (C) of the reference sample of the Danggui Liuhuang Decoction in Test Example 6 of the present application is shown.

[0044] Figure 11 The fingerprint spectrum of the test sample of 17 different reference samples of the Danggui Liuhuang Decoction of the present application is shown. DETAILED DESCRIPTION

[0045] In order to facilitate the understanding of the present application, a more comprehensive description of the present application will be given below with reference to the relevant drawings. The preferred embodiments of the present application are shown in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terms used in the specification of the present application herein are only for the purpose of describing specific embodiments and are not intended to limit the present application.

[0047] As used herein, "optionally", "optional", "optional" means selected from either of the two parallel schemes "have" or "no". If there are multiple "optional" in a technical solution, if there is no special description, and there is no contradictory relationship or mutual restriction, each "optional" is independent. As used herein, the term "and / or" includes any and all combinations of one or more related listed items. As used herein, "multiple", "various" and the like, unless otherwise specified, means greater than 2 or equal to 2 in quantity, for example, "one or more" means one, two or more than two. The open technical features or technical solutions described herein with the words "contain", "include", "comprise" and the like, if there is no other description, can be regarded as providing both the closed features or solutions composed of the listed members, and the open features or solutions including additional members outside the listed members.

[0048] In the present application, the terms "first", "second", "third", "fourth" and the like in "first aspect", "second aspect", "third aspect", "fourth aspect" and the like are only used for description purposes, and cannot be understood as indicating or implying relative importance or quantity, nor can it be understood as implying the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth" and the like only serve the purpose of non-exhaustive enumeration description, and it should be understood that they do not constitute a closed limitation on the quantity.

[0049] If there is no special description, all embodiments and optional embodiments of the present application can be combined with each other to form new technical solutions.

[0050] The quality control and evaluation of the classical famous prescription Danggui Liuhuang Decoction is not perfect at present. The existing control and evaluation method is limited to realizing quality control through detection of active ingredients, and cannot achieve the effect of overall quality control of Danggui Liuhuang Decoction.

[0051] The present application aims to provide a kind of realization Danggui Liuhuang Decoction multi-active ingredient detection, while realizing stable, repeatable and durable fingerprint construction, obtain the overall quality control and evaluation of Danggui Liuhuang Decoction.

[0052] In a first aspect, the present application provides a preparation method of Danggui Liuhuang Decoction reference sample, comprising the following steps:

[0053] S10, Danggui, Shengdihuang, Shudihuang, Huanglian, Huangqi and Huangbai are crushed, mixed, and a first material is prepared.

[0054] S20, water is added to the first material for soaking, and the material is cooked with a strong fire and a weak fire, sieved, and a second material is prepared.

[0055] S30, drying the second material, the drying including at least three stages, the first stage drying at a temperature of ≤-50℃, the second stage drying at a temperature of-40℃ to-10℃, and the third stage drying at a temperature of 0℃ to 30℃, to prepare the Angelica Six Yellow Decoction reference sample.

[0056] The present application can realize the detection of multiple active ingredients by treating the prescription of the Angelica Six Yellow Decoction, and the fingerprint spectrum constructed by using the sample has stability, repeatability and durability, so as to achieve comprehensive quality control and evaluation. In the present application, the drying process during the preparation of the Angelica Six Yellow Decoction sample is controlled, and a three-stage drying procedure is used to maximize the retention of active ingredients and volatile components, reduce the degradation or conversion of components, and avoid the destruction of the microstructure of the sample during the drying process, thereby ensuring the accuracy, stability and repeatability of the fingerprint spectrum. Therefore, the three-stage drying procedure is one of the key means for the quality control of the Angelica Six Yellow Decoction, and the stable Angelica Six Yellow Decoction reference sample prepared thereby can be used as the basis for the construction of the fingerprint spectrum of the Angelica Six Yellow Decoction, and the constructed fingerprint spectrum can truly reflect the types and contents of the chemical components in the Angelica Six Yellow Decoction, so as to realize the stable quality control and evaluation results of the Angelica Six Yellow Decoction sample.

[0057] The application realizes the process of "low-temperature storage → slow sublimation → mild dehydration" by controlling at least three drying procedures, and solves the problem of component loss. The temperature of the first drying procedure is ≤-50°C, which has the effect of low-temperature quick freezing storage and prevents the initial degradation of components. The water in the decoction piece is quickly frozen at ultra-low temperature to avoid the degradation or transformation of components in the early stage of drying. The Danggui Liuhuang Decoction is composed of seven Chinese medicines, including Angelica sinensis, Rehmannia glutinosa, Radix Rehmanniae Preparata, Scutellaria baicalensis, Coptis chinensis, Phellodendri chinense and Astragalus membranaceus, which contain volatile components, phenolic acids, alkaloids and other heat-sensitive components. Drying at an ultra-low temperature of ≤-50°C can freeze the internal water in the decoction piece into fine ice crystals, avoid the formation of large ice crystals by slow freezing of water, which can damage the cell structure, and inhibit the activity of enzymes to prevent them from catalyzing the degradation of heat-sensitive components, and "lock" the initial chemical composition to provide a "baseline state" of components for subsequent fingerprinting. The temperature of the second drying procedure is-40°C to-10°C, which can achieve low-temperature sublimation dehydration, so that the frozen water is removed in the form of sublimation (directly from solid to gas) rather than in the form of liquid after melting, thereby reducing the migration and loss of components; at the same time, sublimation dehydration can avoid the uneven distribution of water-soluble components (such as berberine) due to the migration of water, ensure the uniformity of the components inside the decoction piece, and ensure the stability of the peak area, peak shape and peak time of the "characteristic peak" in the subsequent fingerprint, thereby improving the repeatability, stability and accuracy of the fingerprint.

[0058] In some embodiments, the mass ratio of the Angelica sinensis, Rehmannia glutinosa, Radix Rehmanniae Preparata, Coptis chinensis, Scutellaria baicalensis, Phellodendri chinense and Astragalus membranaceus is 1:1:1:1:1:1:2. As a non-limiting example, 2.58g of Angelica sinensis, 2.58g of Rehmannia glutinosa, 2.58g of Radix Rehmanniae Preparata, 2.58g of Coptis chinensis, 2.58g of Scutellaria baicalensis, 2.58g of Phellodendri chinense and 5.16g of Astragalus membranaceus are crushed and mixed to prepare the first material.

[0059] In some embodiments, after mixing, the mixture is also sieved through a 4-mesh sieve.

[0060] In some embodiments, the mass-to-volume ratio of the first material to water is 1g:(28-30)mL, including but not limited to 1:28, 1:29, 1:30 or a range formed by any two of the foregoing and values within the range.

[0061] In some embodiments, the time of soaking is 20 min - 40 min, including but not limited to 20 min, 25 min, 30 min, 35 min, 40 min, or a range formed by any two of the foregoing and values within the range.

[0062] In some embodiments, the temperature of high heat is 110 °C - 120 °C, including but not limited to 110 °C, 112 °C, 114 °C, 116 °C, 118 °C, 120 °C, or a range formed by any two of the foregoing and values within the range.

[0063] In some embodiments, the time of high heat decocting is 13 min - 16 min, including but not limited to 13 min, 14 min, 15 min, 16 min, or a range formed by any two of the foregoing and values within the range.

[0064] In some embodiments, the temperature of low heat is 90 °C - 100 °C, including but not limited to 90 °C, 92 °C, 94 °C, 96 °C, 98 °C, 100 °C, or a range formed by any two of the foregoing and values within the range.

[0065] In some embodiments, the time of high heat decocting is 40 min - 60 min, including but not limited to 40 min, 45 min, 50 min, 55 min, 60 min, or a range formed by any two of the foregoing and values within the range.

[0066] In some embodiments, the volume of the material after decocting is 1 / 3 to 2 / 3 of the volume before decocting, including but not limited to 1 / 3, 1 / 2, 2 / 3, or a range formed by any two of the foregoing and values within the range.

[0067] In some embodiments, the mesh size of sieving is 150 mesh - 250 mesh, including but not limited to 150 mesh, 200 mesh, 250 mesh, or a range formed by any two of the foregoing and values within the range.

[0068] In some embodiments, the time of the first segment of drying is 2 h - 4 h, including but not limited to 2 h, 2.5 h, 3 h, 4 h, or a range formed by any two of the foregoing and values within the range.

[0069] In some embodiments, the time of the second segment of drying is 70 h - 87 h, including but not limited to 70 h, 72 h, 74 h, 76 h, 78 h, 80 h, 82 h, 84 h, 86 h, 87 h, or a range formed by any two of the foregoing and values within the range.

[0070] In some embodiments, the time of the third segment of drying is 10 h - 15 h, including but not limited to 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, or a range formed by any two of the foregoing and values within the range.

[0071] The preparation method of the angelica six yellow decoction reference sample provided by the present application can prepare the angelica six yellow decoction reference sample with a dry extract rate of 36% or more. Further, the preparation method of the angelica six yellow decoction reference sample provided by the present application can prepare the angelica six yellow decoction reference sample with a dry extract rate of 36%-45%, and the fingerprint constructed within the dry extract rate range has excellent stability, repeatability and durability.

[0072] In a second aspect, the present application provides an angelica six yellow decoction reference sample prepared by the preparation method of the angelica six yellow decoction reference sample provided in the first aspect.

[0073] In the composition of the angelica six yellow decoction reference sample provided by the present application, the content of phellodendrine hydrochloride ranges from 0.68 mg / g to 1.26 mg / g, the content of baicalin ranges from 19.1 mg / g to 35.0 mg / g, the content of coptisine hydrochloride ranges from 2.6 mg / g to 4.1 mg / g, the content of berberine hydrochloride ranges from 12.6 mg / g to 20.8 mg / g, the content of astragaloside A ranges from 0.47 mg / g to 0.88 mg / g, and the content of senkyunolide I ranges from 0.07 mg / g to 0.13 mg / g.

[0074] In a third aspect, the present application further provides a detection method of the angelica six yellow decoction, which comprises the following steps:

[0075] The control solution is prepared by mixing the control samples including phellodendrine hydrochloride, magnoflorine, 3-O-feruloylquinic acid, 4-O-feruloylquinic acid, 5-O-feruloylquinic acid, coptisine hydrochloride, jatrorrhizine, epiberberine hydrochloride, berberine hydrochloride, palmatine, baicalin, plumericin A-7-O-glucuronide and wogonoside with the first solvent, filtering, and taking the filtrate to form the mixed control solution;

[0076] The test sample solution is prepared by mixing the angelica six yellow decoction sample with the second solvent, filtering, and taking the filtrate to form the test sample solution;

[0077] The detection method is selected from liquid chromatography, and the conditions of the liquid chromatography include:

[0078] The elution system includes: the mobile phase A includes acetonitrile, and the mobile phase B includes an aqueous solution containing 0.2%-0.4% of phosphoric acid by mass fraction, gradient elution, and the gradient elution program includes:

[0079] 0 min-15 min, the volume percentage of the mobile phase A changes from 5% to 12.5%;

[0080] 15 min-25 min, the volume percentage of the mobile phase A changes from 12.5% to 15%;

[0081] 25 min-40 min, the volume percentage of mobile phase A is changed from 15% to 25%;

[0082] 40 min-50 min, the volume percentage of mobile phase A is 25%;

[0083] 50 min-60 min, the volume percentage of mobile phase A is changed from 25% to 35%;

[0084] 60 min-62 min, the volume percentage of mobile phase A is changed from 35% to 90%.

[0085] In some embodiments, the mobile phase B comprises an aqueous solution containing 0.28%-0.32% by mass of phosphoric acid, including but not limited to 0.28%, 0.29%, 0.3%, 0.31%, 0.32% or a range formed by any two of the foregoing and numerical values within the range.

[0086] In some embodiments, the column temperature is 30°C-35°C, including but not limited to 30°C, 31°C, 32°C, 33°C, 34°C, 35°C or a range formed by any two of the foregoing and numerical values within the range.

[0087] In some embodiments, the flow rate is 0.5 mL / min-1.2 mL / min, including but not limited to 0.5 mL / min, 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, 0.9 mL / min, 1 mL / min, 1.1 mL / min, 1.2 mL / min or a range formed by any two of the foregoing and numerical values within the range.

[0088] In some embodiments, the detection wavelength is 275 nm-305 nm, including but not limited to 275 nm, 280 nm, 285 nm, 290 nm, 295 nm, 300 nm, 305 nm or a range formed by any two of the foregoing and numerical values within the range.

[0089] In some embodiments, the theoretical plate number calculated based on the epigoitrin peak is ≥10000.

[0090] In some embodiments, the Angelica Six Huang Decoction sample is prepared by mixing Angelica, Rehmannia glutinosa, Radix Rehmanniae Recens, Coptis, Scutellaria, Phellodendri and Astragalus in the prescription amount, decocting, and drying.

[0091] In some embodiments, the Angelica Six Huang Decoction sample is prepared by decocting and drying Angelica Six Huang Decoction pieces; the dosage form of the Angelica Six Huang Decoction sample includes one or more of granules, tablets, decoctions, pills, powders, pastes and capsules.

[0092] In some embodiments, the Dangui Liuhuang Decoction sample comprises a Dangui Liuhuang Decoction reference sample prepared by the preparation method of the Dangui Liuhuang Decoction reference sample provided in the first aspect or the Dangui Liuhuang Decoction reference sample provided in the second aspect.

[0093] In some embodiments, the first solvent and the second solvent each independently comprises one or more of methanol, ethanol, acetonitrile, dimethyl sulfoxide and water.

[0094] In some embodiments, the mixing of the Dangui Liuhuang Decoction sample with the second solvent further comprises ultrasonic treatment, the power of the ultrasonic treatment is 250W-350W, and the frequency of the ultrasonic treatment is 35kHz-45kHz.

[0095] In the fourth aspect, the present application further provides a method for constructing a fingerprint of Dangui Liuhuang Decoction, comprising the following steps:

[0096] providing a control solution and a Dangui Liuhuang Decoction test solution, respectively;

[0097] detecting the control solution by the detection method of Dangui Liuhuang Decoction provided in the third aspect to obtain a control spectrum;

[0098] detecting the Dangui Liuhuang Decoction test solution by the detection method of Dangui Liuhuang Decoction provided in the third aspect to obtain a control spectrum and a test spectrum;

[0099] performing fingerprint similarity analysis on the test spectrum to generate a standard fingerprint, identifying the chromatographic peaks of the standard fingerprint with reference to the control spectrum, and constructing a fingerprint of Dangui Liuhuang Decoction.

[0100] In some embodiments, the Dangui Liuhuang Decoction test solution is prepared by the Dangui Liuhuang Decoction reference sample provided in the second aspect.

[0101] In some embodiments, the sample spectrum and / or the standard fingerprint exhibits 14 characteristic peaks, and the retention time of the characteristic peaks is within the range of -10% to 10% of the retention time value of the corresponding control chromatographic peak in the standard fingerprint.

[0102] In some embodiments, the similarity between the sample spectrum and the control spectrum in the fingerprint constructed by the present application is ≥0.98.

[0103] It should be noted that the experimental methods in the following embodiments of the present application without specific conditions are usually carried out under conventional conditions or under the conditions recommended by the manufacturers. The various common chemical reagents used in the embodiments are commercially available products or can be prepared by known means by those skilled in the art.

[0104] Baicalin (content 97.2%, 110715-202223, China National Institute for Food and Drug Control), Berberine hydrochloride (content 91.0%, 101035-202305, China National Institute for Food and Drug Control), Phellodendrine hydrochloride (content 94.9%, 111895-201805, China National Institute for Food and Drug Control), Coptisine hydrochloride (content 94.0%, 112026-201802, China National Institute for Food and Drug Control), Magnoflorine (112090-202201, China National Institute for Food and Drug Control), 3-O-feruloylquinic acid (content 98.68%, PS012856, Chengdu Pusai Biological Technology Co., Ltd.), 4-O-feruloylquinic acid (content 99.41%, PS012853, Chengdu Pusai Biological Technology Co., Ltd.), 5-O-feruloylquinic acid (content 99.22%, PS012853, Chengdu Pusai Biological Technology Co., Ltd.), Epiberberine hydrochloride (content 98.5%, 112118-202502, China National Institute for Food and Drug Control), Jatrorrhizine hydrochloride (content 90.3%, 110733-202110, China National Institute for Food and Drug Control), Palmatine hydrochloride (content 85.7%, 110732-201913, China National Institute for Food and Drug Control), Miliradin A-7-O-glucuronide (content 99.24%, PS011092, Chengdu Pusai Biological Technology Co., Ltd.), Wogonoside (content 98.5%, 112002-202303, China National Institute for Food and Drug Control).

[0105] Acetonitrile, phosphoric acid, methanol, potassium dihydrogen phosphate, triethylamine, sodium dodecyl sulfate and other reagents are chromatographic grade, and the experimental water is ultrapure water.

[0106] Example 1 Preparation of Angelica Six Huang Decoction Reference Sample

[0107] The present embodiment provides a preparation method of Angelica Six Huang Decoction reference sample, and the specific steps include:

[0108] Take Angelica 2.58g, Rehmannia glutinosa 2.58g, Radix et Rhizoma Glycyrrhizae 2.58g, Coptis chinensis 2.58g, Scutellaria baicalensis 2.58g, Phellodendron amurense 2.58g, Astragalus membranaceus 5.16g, cut into coarse particles, all pass through a 4-mesh sieve, and reserve. Put 2L electric ceramic decoction pot, add water 600mL, soak for 30 minutes, cover with lid, boil for 14min with strong fire (500W), simmer (95℃) for about 50min, decoct to about 300mL, pass through 200-mesh nylon screen while hot. Pack into a Westlin bottle, freeze-dry, pre-freeze at-50℃ for 3 hours, sublimate at-40℃ for 70h, analyze and dry at 0℃ for 12 hours, and seal the cover. The Angelica Six Huang Decoction reference sample is obtained.

[0109] Example 2 Preparation of Angelica Six Huang Decoction Reference Sample

[0110] The embodiment provides a preparation method of a reference sample of Danggui Liuhuang Decoction, and the specific steps comprise the following steps:

[0111] 2.58 g of angelica, 2.58 g of radix rehmanniae, 2.58 g of radix rehmanniae praeparata, 2.58 g of coptis, 2.58 g of scutellaria, 2.58 g of phellodendri, and 5.16 g of astragalus are cut into coarse particles, and all are passed through a 4-mesh sieve and reserved. A 2L electric ceramic medicine decocting pot is added with 600 mL of water, soaked for 30 minutes, boiled for 13 minutes under a cover with a strong fire (500W), and slowly boiled (90 DEG C) for 50 minutes, and the decoction is about 300 mL, and the hot decoction is passed through a 200-mesh nylon sieve. The sample is divided into several small bottles, and freeze-dried, pre-frozen for 3 hours at-50 DEG C, sublimated for 70 hours at-40 DEG C, and desiccated for 12 hours at 0 DEG C, and the sample is obtained.

[0112] Example 3 Preparation of a reference sample of Danggui Liuhuang Decoction

[0113] The embodiment provides a preparation method of a reference sample of Danggui Liuhuang Decoction, and the specific steps comprise the following steps:

[0114] 2.58 g of angelica, 2.58 g of radix rehmanniae, 2.58 g of radix rehmanniae praeparata, 2.58 g of coptis, 2.58 g of scutellaria, 2.58 g of phellodendri, and 5.16 g of astragalus are cut into coarse particles, and all are passed through a 4-mesh sieve and reserved. A 2L electric ceramic medicine decocting pot is added with 600 mL of water, soaked for 30 minutes, boiled for 13 minutes under a cover with a strong fire (500W), and slowly boiled (90 DEG C) for 50 minutes, and the decoction is about 300 mL, and the hot decoction is passed through a 200-mesh nylon sieve. The sample is divided into several small bottles, and freeze-dried, pre-frozen for 3 hours at-50 DEG C, sublimated for 70 hours at-40 DEG C, and desiccated for 12 hours at 0 DEG C, and the sample is obtained.

[0115] Example 4 Preparation of a reference sample of Danggui Liuhuang Decoction

[0116] The embodiment provides a preparation method of a reference sample of Danggui Liuhuang Decoction, and the specific steps comprise the following steps:

[0117] 2.58 g of angelica, 2.58 g of radix rehmanniae, 2.58 g of radix rehmanniae praeparata, 2.58 g of coptis, 2.58 g of scutellaria, 2.58 g of phellodendri, and 5.16 g of astragalus are cut into coarse particles, and all are passed through a 4-mesh sieve and reserved. A 2L electric ceramic medicine decocting pot is added with 600 mL of water, soaked for 30 minutes, boiled for 13 minutes under a cover with a strong fire (500W), and slowly boiled (90 DEG C) for 50 minutes, and the decoction is about 300 mL, and the hot decoction is passed through a 200-mesh nylon sieve. The sample is divided into several small bottles, and freeze-dried, pre-frozen for 3 hours at-50 DEG C, sublimated for 70 hours at-40 DEG C, and desiccated for 12 hours at 0 DEG C, and the sample is obtained.

[0118] Comparative Example 1

[0119] The difference from Example 1 is that after dispensing into the vials, one-stage drying is used, i.e. freeze-drying at -50℃ for 48h, and the remaining preparation steps are consistent with Example 1.

[0120] Comparative Example 2

[0121] The difference from Example 1 is that after dispensing into the vials, two-stage drying is used, pre-freezing at -50℃ for 3h, sublimation at -5℃ for 50h, and the remaining preparation steps are consistent with Example 1.

[0122] Comparative Example 3

[0123] The difference from Example 1 is that after dispensing into the vials, two-stage drying is used, pre-freezing at -50℃ for 3h, desorption drying at -45℃ for 12h, and the remaining preparation steps are consistent with Example 1.

[0124] In order to investigate the chromatographic conditions and system suitability of the Angelica Six-Yellow Decoction reference samples prepared in Examples 1-4 and Comparative Examples 1-3, the present application provides the following multiple sets of method validation test examples.

[0125] Test Example 1 Method Validation I

[0126] Preparation of the reference solution: Take appropriate amounts of the reference substances of phellodendrine, magnoflorine, 3-O-feruloyl quinic acid, 4-O-feruloyl quinic acid, 5-O-feruloyl quinic acid, berberine hydrochloride, epiberberine hydrochloride, jatrorrhizine, berberine hydrochloride, palmatine, baicalin, fargesin A-7-O-glucuronide, and wogonoside, accurately weigh, add 100wt% methanol solution to prepare a mixed solution containing 25μg of phellodendrine hydrochloride, 16μg of berberine hydrochloride, 80μg of berberine hydrochloride, 0.2mg of baicalin, and 80μg of other reference substances per 1mL, shake well, filter, and take the filtrate as the reference solution.

[0127] Preparation of the test solution: Take 0.5g of the Angelica Six-Yellow Decoction reference sample powder of Example 1, accurately weigh, place in a conical flask with a stopper, accurately add 50% methanol 25mL, ultrasonic treat (power 300W, frequency 40kHz) for 30 minutes, cool, make up the weight loss with 50wt% methanol solution, shake well, filter, and take the filtrate as the test solution.

[0128] Chromatographic condition setting: use octadecylsilane-bonded silica gel as the filler (Waters CSH TMC18(3.0x150mm, 1.7μm), mobile phase A: acetonitrile, mobile phase B: 0.3wt% phosphoric acid, gradient elution according to the elution procedure in Table 1, column temperature: 35°C; flow rate: 0.6mL / min, detection wavelength: 285nm, the theoretical plate number should not be less than 10000 calculated by the peak of berberine hydrochloride.

[0129] Table 1: Gradient elution procedure

[0130] Time (min) Mobile phase A (%) Mobile phase B (%) 0～15 5→12.5 95→87.5 15～25 12.5→15 87.5→85 25～40 15→25 85→75 40～50 25 75 50～60 25→35 75→65 60～62 35→90 65→10 62～65 90 10 65～67 90→5 10→95 67～75 5 95

[0131] Take 1μL of the control solution and the test solution respectively, inject into the above set liquid chromatograph for determination, record the chromatogram and analyze, the specific results are as follows:

[0132] (1) Chromatographic peak identification

[0133] Take the control solution and the test solution for analysis according to the above chromatographic conditions, wherein the control characteristic peaks are shown in the attached Figure 1 , and the 13 characteristic peaks are known components of phellodendrine hydrochloride, magnolia base, 3-O-feruloyl quinic acid, 4-O-feruloyl quinic acid, 5-O-feruloyl quinic acid, berberine hydrochloride, epiberberine hydrochloride, jatrorrhizine hydrochloride, berberine hydrochloride, palmatine hydrochloride, baicalin, bretschneiderin A-7-O-glucuronide and wogonoside.

[0134] The 13 characteristic peaks in the test sample spectrum (attached Figure 2 ) are identified by the control characteristic peaks, wherein in the test sample spectrum: peak 1: phellodendrine hydrochloride; peak 2: magnolia base; peak 3: 3-O-feruloyl quinic acid; peak 4: 4-O-feruloyl quinic acid; peak 5: 5-O-feruloyl quinic acid; peak 6: berberine hydrochloride; peak 7: jatrorrhizine; peak 8: columbamine; peak 9: berberine hydrochloride; peak 10: palmatine; peak 11: baicalin; peak 13: bretschneiderin A-7-O-glucuronide; peak 14: wogonoside.

[0135] (2) Precision

[0136] Take the same test sample solution, inject 6 times continuously, determine the retention time and peak area, take peak 6 as the reference peak to calculate the relative retention time and relative peak area RSD value. The results show that the RSD value of the relative retention time of each characteristic peak is less than 2%, and the RSD value of the relative peak area is less than 5%, and the precision of the instrument is good. See the attached Figure 3 for specific results.

[0137] (3) Reproducibility

[0138] Take the same reference sample, prepare 6 test sample solutions according to the test sample solution preparation method, and measure by law. The relative retention time and relative peak area RSD values are calculated with peak 6 as the reference peak. The results show that the RSD values of the relative retention time of each characteristic peak are less than 2%, and the RSD values of the relative peak area are less than 5%, with good repeatability. For specific results, see the following table: Figure 4 .

[0139] (4) Stability

[0140] Take the reference sample, prepare the test sample solution according to the test sample preparation method, and after preparation, sample at 0, 4, 8, 12, 24, 36, 48, 60, 72 hours. The relative retention time and relative peak area RSD values are calculated with peak 6 as the reference peak. The results show that the RSD values of the relative retention time of each characteristic peak are less than 2%, and the RSD values of the relative peak area are less than 5%. The test sample solution has good stability within 72 hours after preparation. For specific results, see the following table: Figure 5 .

[0141] Test Example 2 Methodology Verification II

[0142] Blank control solution: 50wt% methanol solution, filtered, and the filtrate was taken as the blank control solution.

[0143] Preparation of the reference solution: Take a certain amount of phellodendrine hydrochloride reference substance, accurately weigh, add 50wt% methanol solution to prepare a solution containing 25μg of phellodendrine hydrochloride per 1mL, shake well, filter, and take the filtrate as the reference solution.

[0144] Preparation of the test sample solution: Take 0.5g of Angelica Six Huang Decoction reference sample powder from Example 1, accurately weigh, place in a conical flask with a stopper, accurately add 25mL of 50wt% methanol, ultrasonic treat (power 300W, frequency 40kHz) for 30 minutes, cool, make up the weight lost with 50wt% methanol, shake well, filter, and take the filtrate as the test sample solution.

[0145] Chromatographic condition setting: octadecylsilane bonded silica gel as the filler (Waters ACQUITY CSH TM C18(3.0×150mm,1.7μm)), isocratic elution with acetonitrile-triethylamine phosphoric acid aqueous solution (3mL of triethylamine and 3mL of phosphoric acid per 1000mL of water) (8:92) as the mobile phase, column temperature 25℃; flow rate 0.3mL per minute, detection wavelength 285nm, theoretical plate number calculated by phellodendrine hydrochloride peak should not be less than 6000.

[0146] Accurately pipette 1μL of the reference solution and the test sample solution respectively, inject into the above set liquid chromatograph for determination, record the chromatogram and analyze.

[0147] (1) Specificity

[0148] The blank control solution, the control solution and the test solution were analyzed under the above chromatographic conditions, and the results are shown in the following table. The blank control solution had no response at the retention time of phellodendrine hydrochloride peak, and the method had good specificity. Figure 6

[0149] (2) Linearity

[0150] 2.046 mg of phellodendrine hydrochloride reference substance was precisely weighed, and 6 concentrations of phellodendrine hydrochloride reference solution were prepared with 50 wt% methanol solution. 1 μL of each of the above 6 solutions was precisely taken and injected into the above set liquid chromatograph for determination. The standard curve was plotted with the injection concentration (μg / mL) as the abscissa and the peak area as the ordinate. The regression equation was Y = 3.8227X - 2.8350, and the residual sum of squares was 344.91, as shown in Table 2:

[0151] Table 2: Linear relationship results of phellodendrine hydrochloride reference substance

[0152]

[0153] (3) Reproducibility

[0154] The reference sample solution of Angelica Six Huang Decoction prepared in Example 1 was repeatedly prepared for 9 times, and each was injected and determined. The average content of phellodendrine hydrochloride in the 9 test sample solutions was 0.1033%, and the RSD value was less than 3%, indicating that the method had good reproducibility, and the results are shown in Table 3.

[0155] Table 3: Reproducibility relationship results of phellodendrine hydrochloride reference substance

[0156]

[0157] (4) Accuracy

[0158] Nine reference samples of Angelica Six Huang Decoction prepared in Example 1 with known content were taken, and divided into 3 groups, each about 0.2 g, 0.25 g and 0.25 g, and precisely weighed and labeled as 1-9. 4.215 mg of phellodendrine hydrochloride reference substance (purity 94.9%) was precisely weighed, and low, medium and high concentration reference solution was prepared with 50 wt% methanol solution.

[0159] Low concentration reference solution was precisely added to 1-3, medium concentration reference solution was precisely added to 4-6, and high concentration reference solution was precisely added to 7-9, and the weight was determined. The determination was carried out according to the above chromatographic method, and the recovery rate was calculated, and the results are shown in Table 4.

[0160] ​Table 4: Accuracy of Phellodendrine Hydrochloride Content Determination

[0161]

[0162] The results show that the recovery rate of phellodendrine hydrochloride meets the requirements of General Rules 9101 of Chinese Pharmacopoeia 2020 (90%~108%), and the RSD values are all less than 3%, indicating good accuracy.

[0163] (5) Stability

[0164] Take the test solution, prepare it at 0, 2, 4, 8, 12, 18, 24, 36, and 48.5 hours, and sample it. Calculate the content and RSD value of phellodendrine hydrochloride at each time point, and the results are shown in Table 5.

[0165] Table 5: Stability Test of Phellodendrine Hydrochloride Content Determination

[0166]

[0167] The results show that the RSD value of the test component in the test solution is less than 3% within 48.5 hours, indicating that the test solution is stable within 48.5 hours after preparation.

[0168] Test Example 3 Methodology Validation Three

[0169] Blank control solution: 70wt% methanol solution, filtered, and the filtrate was taken as the blank control solution.

[0170] Preparation of the reference solution: Take an appropriate amount of baicalin reference substance, accurately weigh it, add 70wt% methanol solution to prepare a solution containing 0.2mg of phellodendrine hydrochloride per 1mL, shake well, filter, and take the filtrate as the reference solution.

[0171] Preparation of the test solution: Take 0.2g of the Angelica Six Huang Decoction reference sample powder of Example 1, accurately weigh it, place it in a conical flask with a stopper, accurately add 25mL of 70wt% methanol, ultrasonically treat it (power 300W, frequency 40kHz) for 15 minutes, cool it, make up the weight loss with 70wt% methanol solution, shake well, filter, and take the filtrate as the test solution.

[0172] Chromatographic condition setting: octadecylsilane-bonded silica gel as the filler (Waters T3, 2.1mm x 150mm, 1.6μm), isocratic elution with 0.2wt% phosphoric acid in methanol-water (volume ratio of methanol to water is 40:60) as the mobile phase, column temperature is 35℃; flow rate is 0.2mL per minute, detection wavelength is 280nm, theoretical plate number calculated by baicalin peak should not be less than 5000.

[0173] Accurately pipette 1 μL of the control solution and the sample solution into the liquid chromatograph set up above, and determine. Record the chromatogram and analyze.

[0174] (1) Specificity

[0175] Analyze the blank control solution, the control solution and the sample solution under the above chromatographic conditions. The results are shown in the following table 4. Figure 7 As shown in the table 4, the blank control solution has no response at the retention time of baicalin peak, and the method has good specificity.

[0176] (2) Linearity

[0177] Accurately weigh 4.147 mg of the baicalin control solution (purity 97.2%), and prepare 6 concentrations of the baicalin control solution with 70 wt% methanol solution. Accurately pipette 1 μL of the above solution into the liquid chromatograph set up above, and determine. Take the sample concentration (μg / mL) as the abscissa, and the peak area as the ordinate, to draw a standard curve. The linear regression equation of baicalin is Y=18059.1206X+22197.2731, and the determination coefficient is 0.9996, which meets the requirements. It shows that baicalin has good linear relationship in the concentration range of 10.08 μg / mL-403.09 μg / mL. The specific results are shown in table 6.

[0178] Table 6: Linear relationship results of the baicalin control solution

[0179]

[0180] (3) Reproducibility

[0181] Prepare 9 portions of the Angelica Six Huang Decoction reference sample prepared in Example 1, and determine by injection. The average content of baicalin in the 9 sample solutions is 2.184%, and the RSD value is less than 3%. The method has good reproducibility, and the results are shown in the following table 7.

[0182] Table 7: Reproducibility relationship results of the baicalin control solution

[0183]

[0184]

[0185] (4) Accuracy

[0186] Take 9 portions of the Angelica Six Huang Decoction reference sample prepared in Example 1 with known content, and divide them into 3 groups, each about 0.1 g. Accurately weigh 26.938 mg of the phellodendrine hydrochloride control solution (purity 97.2%), and prepare low, medium and high concentration control solution with 70 wt% methanol solution.

[0187] Accurately add 25 mL of the low concentration control solution to 1-3, 25 mL of the medium concentration control solution to 4-6, and 25 mL of the high concentration control solution to 7-9, respectively, weigh, determine according to the above chromatographic method, and calculate the recovery rate. The results are shown in Table 8.

[0188] Table 8: Accuracy of determination of baicalin content

[0189]

[0190] The results show that the recovery rate of baicalin meets the requirements of the fourth part of Chinese Pharmacopoeia 2020 (9101) (92% to 105%), the RSD value is less than 2%, and the accuracy is good.

[0191] (5) Stability

[0192] Take the test sample solution, prepare, and sample at 0, 2, 4, 8, 12, 18, 24, 36, and 48 hours, calculate the content and RSD value of baicalin at each time point, and the results are shown in Table 9.

[0193] Table 9: Stability test of determination of baicalin content

[0194]

[0195]

[0196] The results show that the RSD value of the content of the test component in the test sample within 48 hours is less than 2%, indicating that the test sample solution is stable within 48 hours after preparation.

[0197] Test Example 4 Methodology Verification Four

[0198] Blank control solution: a mixed solution of methanol-hydrochloric acid (volume ratio of 100:1), filtered, and the filtrate was taken as the blank control solution.

[0199] Preparation of control solution: take appropriate hydrochloride coptisine and berberine hydrochloride control samples, accurately weigh, add methanol-hydrochloric acid (volume ratio of 100:1) to prepare a mixed solution containing 16 μg of hydrochloride coptisine and 80 μg of berberine hydrochloride per 1 mL, shake well, filter, and take the filtrate as the control solution.

[0200] Preparation of test solution: about 0.2 g of Angelica six Huangqi Decoction reference sample powder of Example 1 was precisely weighed into a conical flask with a stopper, 50 mL of methanol-hydrochloric acid (100:1 by volume) mixed solution was precisely added, the weight was determined, the flask was tightly closed, and ultrasonic treatment (power 300 W, frequency 40 kHz) was performed for 30 minutes. The solution was allowed to cool, the lost weight was made up with methanol-hydrochloric acid (100:1 by volume), and the solution was shaken and filtered. The filtrate was collected to obtain the test solution.

[0201] Chromatographic conditions: isocratic elution was performed with octadecylsilane-bonded silica gel (Agilent Poroshell 120 AQ-C18, 4.6 x 150 mm, 2.7 μm) as the filler and acetonitrile-0.05 mol / L potassium dihydrogen phosphate solution (45:55 by volume) (0.4 g of sodium dodecyl sulfate was added to 100 mL, and the pH value was adjusted to 4.0 with phosphoric acid) as the mobile phase, the column temperature was 25°C, the flow rate was 0.6 mL / min, the detection wavelength was 345 nm, and the theoretical plate number calculated based on the peak of berberine hydrochloride should not be less than 5000.

[0202] About 1 μL of the blank control solution, the reference solution and the test solution was precisely taken and injected into the above set liquid chromatograph for determination. The chromatogram was recorded and analyzed.

[0203] (1) Specificity

[0204] The blank control solution, the reference solution and the test solution were analyzed according to the above chromatographic conditions. The results are shown in the attached Figure 8 Figure, and the blank control solution had no response at the retention time of berberine hydrochloride and berberine hydrochloride, indicating that the method had good specificity.

[0205] (2) Linearity

[0206] Precisely take 1.990 mg of epigoitrin reference substance (content is 94.0%) and 6.385 mg of berberine hydrochloride (content is 91.0%) with methanol to prepare five mixed reference solutions. Precisely take 10 μL of the above solutions and inject into the liquid chromatograph. Take the injection concentration (μg / mL) as the abscissa and the peak area as the ordinate to draw the standard curve. The linear regression equation of epigoitrin is Y = 61704.9355X - 70214.1699, the correlation coefficient is 0.9999, which meets the requirements, showing that epigoitrin has a good linear relationship in the concentration range of 4.677 μg / mL to 93.530 μg / mL. The linear regression equation of berberine hydrochloride is Y = 67413.7110X - 217040.8358, the correlation coefficient is 0.9999, which meets the requirements, showing that berberine hydrochloride has a good linear relationship in the concentration range of 14.526 μg / mL to 290.518 μg / mL. The specific results are shown in Tables 10 and 11.

[0207] Table 10: Linear relationship results of epigoitrin reference substance

[0208]

[0209] Table 11: Linear relationship results of berberine hydrochloride reference substance

[0210]

[0211] (3) Repeatability

[0212] Nine samples of the Angelicae Decoction of Six Yellow Herbs reference sample prepared in Example 1 were prepared repeatedly and injected for determination. The average content of epigoitrin in the nine sample solutions was 0.296%, and the RSD value was less than 3%. The average content of berberine hydrochloride in the nine sample solutions was 1.569%, and the RSD value was less than 3%. The method has good repeatability, and the results are shown in Tables 12 and 13.

[0213] Table 12: Repeatability relationship results of epigoitrin reference substance

[0214]

[0215] Table 13: Repeatability relationship results of berberine hydrochloride reference substance

[0216]

[0217] (4) Accuracy

[0218] Take the known content of the prepared Angelica six Huang decoction reference sample 9 groups, divided into 3 groups, each about 0.05g, 0.1g, 0.1g, precision weighing, marked as 1-9. Precision weighing berberine hydrochloride reference substance 6.448mg, berberine hydrochloride 34.410mg, with methanol: hydrochloric acid (volume ratio of 100:1) preparation of low concentration, medium concentration, high concentration control solution.

[0219] Respectively, 1-3 in the precision of low concentration control solution 50mL, 4-6 in the precision of medium concentration control solution 50mL, 7-9 in the precision of high concentration control solution 50mL, weighing, according to the above chromatographic method for determination, and calculate the recovery rate, the results are shown in table 14 and table 15.

[0220] Table 14: berberine hydrochloride content determination accuracy investigation

[0221]

[0222] Table 15: berberine hydrochloride content determination accuracy investigation

[0223]

[0224] The results show that the recovery rate of berberine hydrochloride, berberine hydrochloride is in line with the provisions of the Chinese Pharmacopoeia four general 9101 (90% ~ 108%, 92% ~ 105%), RSD value is less than 3% (appropriate relaxation), good accuracy.

[0225] (5) stability

[0226] Take the test solution, after preparation in the 0, 2, 4, 8, 12, 18, 24, 36, 48 hours sample, statistics of each time point berberine hydrochloride, berberine hydrochloride peak area, content and RSD value calculation, the results are shown in table 16.

[0227] Table 16: berberine hydrochloride, berberine hydrochloride content determination stability test

[0228]

[0229]

[0230] The results show that the content of berberine hydrochloride, berberine hydrochloride in the test sample within 48 hours, the content of no obvious change, indicating that the test solution is stable within 48 hours after preparation.

[0231] Test example 5 methodology validation five

[0232] Blank control solution: 100wt% methanol solution, filter, take the filtrate, as a blank control solution.

[0233] Preparation of reference solution: Take an appropriate amount of astragaloside A reference standard, accurately weigh it, and add methanol to prepare a solution containing 0.1 mg per ml, which is the reference solution.

[0234] Preparation of the test solution: Take 1.0 g of the Dang Gui Liu Huang Tang reference sample powder from Example 1, accurately weigh it, add 50 ml of water, place it in a round-bottom flask, add 20 ml of ammonia water and 50 ml of water-saturated n-butanol, mix well, stopper tightly, weigh, reflux for 1 h, cool, weigh again, add water-saturated n-butanol to make up the weight loss, shake well, wait for the layers to separate, accurately measure 25 ml of the upper layer solution, evaporate to dryness, dissolve the residue in methanol, transfer to a 2 ml volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate to obtain the test solution.

[0235] Chromatographic conditions: Octadecylsilane-bonded silica gel as the stationary phase (reference column: Agilent ZORBAX Extend-C18, 4.6 × 250 mm, 5 μm); acetonitrile-water (32:68) as the mobile phase; flow rate: 1.0 mL / min; column temperature: 30 °C; evaporative light scattering detector (reference parameters: drift tube temperature 80 °C; nebulizer temperature 70 °C; gas flow rate: 1.6 SLM). The theoretical plate number, calculated based on the astragaloside A peak, should be no less than 5000.

[0236] Accurately pipette 2 μl (or 5 μl) and 10 μl (or 20 μl) of the reference solution and 5–20 μl of the test solution into the liquid chromatograph, respectively, and determine the result by calculating the logarithmic equation using the external standard two-point method.

[0237] (1) Exclusivity

[0238] The blank control solution, reference solution, and test solution were analyzed under the chromatographic conditions described above. The results are shown in the appendix. Figure 9 As shown, the blank control solution showed no response at the retention time of the astragaloside A peak, indicating that this method has good specificity.

[0239] (2) Linear relationship

[0240] Accurately weigh 4.147 mg of astragaloside A reference standard (purity 97.2%) and prepare six concentrations of astragaloside A reference standard solution with 70 wt% methanol solution. Accurately pipette 1 μL of each solution and inject it into the pre-set liquid chromatograph for determination. Plot a standard curve with the injection concentration (μg / mL) on the x-axis and the peak area on the y-axis. The linear regression equation for baicalin is Y = 18059.1206X + 22197.2731, with a coefficient of determination of 0.9996, which meets the requirements. This shows that baicalin has a good linear relationship in the concentration range of 10.08 μg / mL-403.09 μg / mL. The specific results are shown in Table 17.

[0241] Table 17: Linearity Results of Astragaloside A Reference Standard

[0242]

[0243] (3) Repeatability

[0244] Nine replicates of the Danggui Liuhuang Decoction reference sample solution prepared in Example 1 were prepared and injected for determination. The average content of astragaloside A in the nine sample solutions was 2.184%, and the RSD value was less than 3%. The method has good repeatability. The results are shown in Table 18 below.

[0245] Table 18: Repeatability results of astragaloside A

[0246]

[0247] (4) Accuracy

[0248] Nine reference samples of Dang Gui Liu Huang Tang prepared in Example 1 with known content were taken and divided into three groups (labeled as 1-9), each weighing approximately 0.5g, 0.5g, and 1.0g. The samples were accurately weighed and placed in stoppered conical flasks (numbered 1-9).

[0249] Medium-concentration reference solution: Accurately weigh 2.449 mg of astragaloside A reference standard (content calculated as 96.2%), place it in a 100 ml volumetric flask, add an appropriate amount of water-saturated n-butanol, sonicate to dissolve, dilute to the mark with water-saturated n-butanol, and shake well to obtain the astragaloside A standard solution (labeled as solution A); accurately pipette 50 ml of solution A, place it in a 200 ml volumetric flask, add an appropriate amount of water-saturated n-butanol, dilute to the mark with water-saturated n-butanol, and shake well to obtain the medium-concentration reference solution (labeled as solution B).

[0250] High-concentration reference solution: Accurately weigh 2.453 mg of astragaloside A reference standard (content calculated as 96.2%), place it in a 200 ml volumetric flask, add an appropriate amount of water-saturated n-butanol, sonicate to dissolve, dilute to the mark with water-saturated n-butanol, and shake well to obtain the high-concentration reference solution (labeled as solution C).

[0251] Low concentration reference standard spiking solution: Accurately pipette 50 ml of solution C into a 200 ml volumetric flask, add an appropriate amount of water-saturated n-butanol.

[0252] Accurately add 50 ml of water to each of the conical flasks numbered 1-9, place them in a round-bottom flask, and then accurately add 20 ml of ammonia water sequentially. Add 50 ml of solution D, solution B, and solution C to conical flasks 1-3, 4-6, and 7-9 respectively, mix well, stopper tightly, weigh, reflux for 1 hour, cool, weigh again, make up the weight loss with water-saturated n-butanol, shake well, wait for separation, accurately measure 25 ml of the upper layer solution, evaporate to dryness, dissolve the residue in methanol, transfer to a 2 ml volumetric flask, add methanol to the mark, shake well, filter, and collect the filtrate. Simultaneously, prepare two aliquots of the test solution as accompanying samples according to the test sample preparation method. Calculate the recovery rate using the content of the accompanying samples; the recovery rate results are shown in Table 19 below.

[0253] Table 19: Accuracy Study of Astragaloside A Content Determination

[0254]

[0255] The results showed that the recovery rate of astragaloside A met the requirements of General Chapter 9101 of Part IV of the 2020 edition of the Chinese Pharmacopoeia (92%–105%), with an RSD value of less than 2%, indicating good accuracy.

[0256] (5) Stability

[0257] The test solution was prepared and injected at 0, 2, 4, 8, 12, 18, 24, 36 and 48 hours. The peak area of ​​astragaloside A at each time point was counted, and the content and RSD value were calculated. The results are shown in Table 20.

[0258] Table 20: Stability Test for Astragaloside A Content Determination

[0259]

[0260] The results showed that the RSD value of the test component in the sample was less than 3% within 48 hours, indicating that the sample solution had good stability within 48 hours after preparation.

[0261] Experimental Example 6: Methodological Validation

[0262] Blank control solution: 100 wt% methanol solution, filtered, and the filtrate is used as the blank control solution.

[0263] Preparation of reference solution: Take an appropriate amount of ligustrazine lactone I reference standard, accurately weigh it, and add methanol to prepare a solution containing 2 μg per 1 ml, which is the reference solution.

[0264] Preparation of the test solution: Take 0.5g of the Danggui Liuhuang Decoction reference sample powder from Example 1, accurately weigh it, place it in a stoppered conical flask, accurately add 20ml of methanol, seal tightly, weigh it, and extract it by ultrasonication (power 300W, frequency 40kHz) for 30 minutes. After cooling, weigh it again, replenish the lost weight with methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0265] Chromatographic conditions: Octadecylsilane-bonded silica gel as the stationary phase (reference column: Agilent ZORBAX Extend-C18, 4.6 × 250 mm, 5 μm); acetonitrile-water (32:68) as the mobile phase; flow rate: 1.0 mL / min; column temperature: 30 °C; evaporative light scattering detector (reference parameters: drift tube temperature 80 °C; nebulizer temperature 70 °C; gas flow rate: 1.6 SLM). The theoretical plate number, calculated based on the astragaloside A peak, should be no less than 5000.

[0266] Accurately pipette 2 μL each of the reference solution and the test solution, inject them into the liquid chromatograph, and determine the result.

[0267] (1) Exclusivity

[0268] The blank control solution, reference solution, and test solution were analyzed under the chromatographic conditions described above. The results are shown in the appendix. Figure 10 As shown, the blank control solution showed no response at the retention time of the Ligusticum lactone I peak, indicating that the method has good specificity.

[0269] (2) Linear relationship

[0270] Accurately weigh 4.147 mg of ligustrazine lactone I reference standard (purity 97.2%), and prepare six concentrations of ligustrazine lactone I reference standard solutions with methanol solution. Accurately pipette 1 μL of each of the above solutions and inject them into the pre-set liquid chromatograph for determination. Plot a standard curve with the injection concentration (μg / mL) as the x-axis and the peak area as the y-axis. The linear regression equation for ligustrazine lactone I is Y = 18059.1206X + 22197.2731, with a coefficient of determination of 0.9996, which meets the requirements. This shows that ligustrazine lactone I has a good linear relationship in the concentration range of 10.08 μg / mL-403.09 μg / mL. The specific results are shown in Table 21.

[0271] Table 21: Linearity results of Ligusticum lactone I reference standard

[0272]

[0273] (3) Repeatability

[0274] Nine replicates of the Danggui Liuhuang Decoction reference sample solution prepared in Example 1 were prepared and injected for determination. The average content of ligustrazine I in the nine sample solutions was 2.184%, and the RSD value was less than 3%. The method has good repeatability. The results are shown in Table 22 below.

[0275] Table 22: Repeatability results of ligustilide I

[0276]

[0277] The results showed that the RSD values ​​of the analyte content in the nine test sample solutions were less than 4%, indicating good repeatability.

[0278] (4) Accuracy

[0279] Nine samples prepared in Example 1 with known content were taken and divided into three groups (labeled as 1-9), each weighing approximately 0.25g. They were accurately weighed and placed in stoppered conical flasks (numbered 1-9). Accurately weigh 4.108 mg (99.5% purity) of ligustrazine lactone I reference standard, place it in a 10 ml volumetric flask, add an appropriate amount of methanol to dissolve and dilute to the mark, shake well to obtain the high-concentration reference standard stock solution (labeled as solution A); accurately pipette 1 ml of solution A, place it in a 100 ml volumetric flask, dilute to the mark with methanol, shake well to obtain the high-concentration reference standard solution (labeled as solution B); accurately weigh 2.279 mg (99.5% purity) of ligustrazine lactone I reference standard, place it in a 10 ml volumetric flask, add an appropriate amount of methanol, sonicate to dissolve, dilute to the mark with methanol, shake well to obtain the medium-concentration reference standard stock solution (labeled as solution C); accurately pipette 1 ml of solution C, place it in a 100 ml volumetric flask, dilute to the mark with methanol, shake well to obtain the medium-concentration reference standard solution (labeled as solution D); accurately pipette solution C... 20 ml of the solution was placed in a 100 ml volumetric flask, diluted to the mark with methanol, and shaken well to obtain a low-concentration reference solution (labeled as solution E).

[0280] Then, precisely add 20 ml of solution B to conical flasks numbered 1-3, precisely add 20 ml of solution D to conical flasks numbered 4-6, and precisely add 20 ml of solution E to conical flasks numbered 7-9. Weigh the solutions, sonicate (300 W, 40 kHz) for 30 minutes, cool, weigh again, replenish the lost weight with methanol, shake well, filter, and collect the filtrate to obtain the accuracy test sample. Simultaneously, prepare three test solutions as accompanying samples according to the preparation method of the test solution. Calculate the recovery rate using the content of the accompanying samples; the recovery rate results are shown in Table 23 below.

[0281] Table 23: Accuracy Study of Ligusticum striatum I Content Determination

[0282]

[0283]

[0284] The results showed that the recovery rate of ligustilide I met the requirements of General Chapter 9101 of Part IV of the 2020 edition of the Chinese Pharmacopoeia (85%–110%), with an RSD value of less than 4%, indicating good accuracy.

[0285] (5) Stability

[0286] Take the test solution, prepare it, and inject it at 0, 2, 4, 8, 12, 18, 24, 36, and 48 hours. Count the peak area of ​​Ligusticum lactone I at each time point, calculate the content and RSD value. The results are shown in Table 24.

[0287] Table 24: Stability Test for Determination of Ligusticum striatum I Content

[0288]

[0289] The results showed that the RSD value of the test component in the sample was less than 4% within 48 hours, indicating that the sample solution had good stability within 48 hours after preparation.

[0290] Experimental Example 7: Methodological Validation VII

[0291] The reference samples of Danggui Liuhuang Decoction prepared in Examples 2-4 and Comparative Examples 1-3 were tested. Compared with Example 1, Examples 2-4 showed no significant difference in the content deviation of the index components (baicalin, berberine hydrochloride, coptisine hydrochloride, berberine hydrochloride, astragaloside A, and ligustrazine lactone I) with deviations of less than 5%; their characteristic chromatograms all showed 14 characteristic peaks, indicating consistent characteristic components. Compared with Example 1, Comparative Examples 1-3 had some samples that were not completely freeze-dried, resulting in lower contents of the index components (baicalin, berberine hydrochloride, coptisine hydrochloride, berberine hydrochloride, astragaloside A, and ligustrazine lactone I). Their characteristic chromatograms all showed 14 characteristic peaks, but the peak area response of the characteristic components was generally lower, and the linear correlation was reduced. The accuracy of component detection in these samples was low, and the accuracy, repeatability, and stability of the detection were poor, indicating insufficient comprehensive quality control and evaluation of Danggui Liuhuang Decoction.

[0292] Experimental Example 8: Methodological Validation

[0293] The difference from Experimental Example 1 is that the composition of the mobile phase is different. The mobile phase used in this experiment is composed of acetonitrile-0.5% formic acid.

[0294] Octadecylsilane-bonded silica gel (Agilent ZORBAX Eclipse Plus C18 (3.0×150mm, 1.8μm)) was used as the packing material, acetonitrile was used as mobile phase A, and 0.5wt% formic acid aqueous solution was used as mobile phase B. The flow rate was 0.8 ml per minute. The elution procedure was the same as Table 1 of Experimental Example 1, and the remaining steps were the same as those in Experimental Example 1.

[0295] The experimental results showed that the separation of characteristic peaks at 14.5 minutes and 37 minutes was poor, failing to meet the requirements of the characteristic spectral analysis method.

[0296] Experimental Example 9: Methodological Validation 9

[0297] The difference from Experimental Example 1 is that the composition of the mobile phase is different. The mobile phase used in this experiment is composed of acetonitrile-0.5% acetic acid.

[0298] Octadecylsilane-bonded silica gel (Agilent ZORBAX Eclipse Plus C18 (3.0×150mm, 1.8μm)) was used as the packing material, acetonitrile was used as mobile phase A, and 0.5wt% aqueous acetic acid solution was used as mobile phase B. The flow rate was 0.8 ml per minute. The elution procedure was the same as in Table 1 of Experimental Example 1, and the remaining steps were the same as in Experimental Example 1.

[0299] The experimental results showed that the characteristic peak separation was poor at 14.5 minutes and the characteristic peak symmetry was poor at 34 minutes, failing to meet the requirements of the characteristic spectrum analysis method.

[0300] Experimental Example 10: Methodological Validation

[0301] The difference from Example 1 is that the composition of the mobile phase is different. The mobile phase used in this example is: acetonitrile-0.5% phosphoric acid.

[0302] Octadecylsilane-bonded silica gel (Agilent ZORBAX Eclipse Plus C18 (3.0×150mm, 1.8μm)) was used as the packing material, acetonitrile was used as mobile phase A, and 0.5wt% phosphoric acid aqueous solution was used as mobile phase B. The flow rate was 0.8 ml per minute. The elution procedure was the same as Table 1 of Experimental Example 1, and the remaining steps were the same as those in Experimental Example 1.

[0303] The experimental results showed that the separation effect was good when 0.5% phosphoric acid was used as the mobile phase. However, the pH value of the mobile phase exceeded the pH tolerance of the chromatographic column. Considering the long-term monitoring of column wear, the additive concentration was adjusted to 0.3% for data collection.

[0304] Based on the methodological investigation of the preparation method and chromatographic conditions of the above-mentioned Dang Gui Liu Huang Tang reference sample, it was verified that the Dang Gui Liu Huang Tang reference sample prepared by the preparation method provided by the present invention in Example 1 has excellent methodological stability under the chromatographic conditions of Example 1. Further, multiple Dang Gui Liu Huang Tang samples were prepared using the aforementioned preparation method and chromatographic conditions, and fingerprint chromatograms were constructed to provide an accurate quality control and evaluation method for Dang Gui Liu Huang Tang.

[0305] Example 5: Construction of fingerprint map

[0306] Multiple batches of Angelica sinensis, Rehmannia glutinosa (raw and processed), Coptis chinensis, Scutellaria baicalensis, Phellodendron chinense, and Astragalus membranaceus were taken and prepared as reference samples of Dang Gui Liu Huang Tang according to the preparation method in Example 1. Among them, batches 1-17 were produced as follows: Angelica sinensis from Gansu and Qinghai provinces, Rehmannia glutinosa from Henan and Shaanxi provinces, Phellodendron chinense from Chongqing and Sichuan provinces, Scutellaria baicalensis from Shaanxi province, Coptis chinensis from Hubei province and Chongqing municipality, and Astragalus membranaceus from Shaanxi province and Inner Mongolia province.

[0307] The characteristic chromatograms and content of various indicators of multiple batches of Dang Gui Liu Huang Tang reference samples were determined according to the chromatographic conditions and detection methods of Example 1. After quality studies of the reference samples, the contents of some active ingredients in 17 batches of Dang Gui Liu Huang Tang reference samples are shown in Table 25.

[0308] Table 25: Research Results of 17 Batches of Dang Gui Liu Huang Tang Reference Samples

[0309]

[0310] As shown in Table 25, the content of berberine hydrochloride in the 17 batches of Dang Gui Liu Huang Tang reference samples fluctuated between 0.74 and 1.28 mg / g, with an average of 0.97 mg / g. Based on ±30% of the average, the limit range for berberine hydrochloride content is 0.68 to 1.26 mg / g. One batch of reference samples (1.28 mg / g) had a slightly higher berberine hydrochloride content than the upper limit, showing dispersion. After excluding outliers, the mean ±30% range for 16 batches of reference samples was 0.67 to 1.24 mg / g, which is basically consistent with the mean ±30% range for the 17 batches (0.68 to 1.26 mg / g). Therefore, the berberine hydrochloride content range for reference samples is specified as ±30% of the mean, i.e., calculated on a dried basis, the content of berberine hydrochloride in the reference samples is calculated based on berberine hydrochloride (C...). 20 H 23 Based on NO4·HCl, the concentration should be 0.68–1.26 mg / g.

[0311] As shown in Table 25, the baicalin content of the 17 batches of Dang Gui Liu Huang Tang reference samples fluctuated between 19.1 and 35.0 mg / g, with an average of 25.8 mg / g. The range calculated using ±30% of the average was 18.1 to 33.6 mg / g. All 17 batches of reference samples met the specified range. Therefore, the specified range for baicalin content in reference samples is calculated using ±30% of the average, i.e., based on the dried product, the content of baicalin in the reference samples is calculated as follows: Baicalin (C...) 21 H 18 O 11 The calculated value should be 18.1–33.6 mg / g.

[0312] As shown in Table 25, the berberine hydrochloride content of the 17 batches of Dang Gui Liu Huang Tang reference samples fluctuated between 2.6 and 4.1 mg / g, with an average of 3.2 mg / g. The range calculated using ±30% of the average was 2.2 to 4.1 mg / g, and all 17 batches of reference samples met the specified range. Therefore, the specified range for berberine hydrochloride content in the reference samples is calculated using ±30% of the average, i.e., based on the dried product, the content of berberine hydrochloride in the reference samples is calculated as follows: (C...) 19 H 13 Based on NO4·HCl, the concentration should be 2.2–4.1 mg / g.

[0313] As shown in Table 25, the berberine hydrochloride content of the 17 batches of Dang Gui Liu Huang Tang reference samples fluctuated between 12.6 and 20.8 mg / g, with an average of 15.8 mg / g. The range calculated using ±30% of the average was 11.1 to 20.5 mg / g. All 17 batches of reference samples met the specified range. Therefore, the specified range for berberine hydrochloride content in the reference samples is calculated using ±30% of the average, i.e., based on the dried product, the content of Coptis chinensis and Phellodendron amurense is calculated as berberine hydrochloride (C... 20 H 17 Based on NO4·HCl, the concentration should be 11.1–20.5 mg / g.

[0314] As shown in Table 25, the astragaloside A content of the 17 batches of Dang Gui Liu Huang Tang reference samples fluctuated between 0.45 and 0.91 mg / g, with an average of 0.67 mg / g. The range calculated using ±30% of the average was 0.47–0.88 mg / g. One batch of reference samples (0.45 mg / g) had an astragaloside A content slightly below the lower limit, indicating dispersion. After excluding outliers, the mean ±30% range of 16 batches of reference samples was 0.48–0.90 mg / g, which is basically consistent with the mean ±30% range of 0.47–0.88 mg / g for the 17 batches. Therefore, it is stipulated that the astragaloside A content range of the reference samples should be calculated based on the mean ±30%, i.e., calculated on a dried basis, the content of astragaloside A in the reference samples should be calculated as astragaloside A (C... 41 H 68 O 14 The calculated value should be 0.47–0.88 mg / g.

[0315] As shown in Table 25, the content of ligustrolactone I in the 17 batches of Dang Gui Liu Huang Tang reference samples fluctuated between 0.05 and 0.15 mg / g, with an average of 0.10 mg / g. The range calculated using ±30% of the average was 0.07–0.15 mg / g. Three batches of reference samples (0.05, 0.06, and 0.06 mg / g) had ligustrolactone content slightly below the lower limit, exhibiting dispersion. After excluding outlier batches, the mean ±30% range of 14 batches of reference samples was 0.08–0.14 mg / g, which is basically consistent with the mean ±30% range of 0.07–0.15 mg / g for the 17 batches. Therefore, it is stipulated that the ligustrolactone I content range of the reference samples should be calculated using ±30% of the mean, i.e., based on the dried product, the content of ligustrolactone I in Dang Gui Liu Huang Tang should be calculated as follows: (C...) 12 H 16 The concentration of O4 should be 0.07–0.15 mg / g.

[0316] The characteristic chromatograms of 17 batches of reference samples were determined according to the established characteristic chromatogram analysis method. The MARK peak matching of the traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) was used to generate reference sample comparison chromatograms (see appendix). Figure 11 The characteristic spectra of 17 batches of reference samples showed a total of 14 characteristic peaks. See the appendix for the experimental results. Figure 11 Tables 26, 27 and 28.

[0317] Table 26: Similarity Results of Feature Maps of 17 Batches of Reference Samples

[0318]

[0319]

[0320] Table 27: Relative Retention Time Results of Characteristic Spectra of 17 Batches of Reference Samples

[0321]

[0322]

[0323] Table 28: Relative Peak Area Results of Characteristic Spectra of 17 Batches of Reference Samples

[0324]

[0325]

[0326] As shown in the table, taking peak 6 (berberine hydrochloride) as S1 peak, the relative retention time RSD values ​​for peak 2, 3, 4, 5, 8, 10, 12, 13, and 14 are all less than 3.0%, meeting the requirements for establishing a standard for characteristic spectra of reference samples. There are certain differences in the relative peak areas of the characteristic peaks among different batches of reference samples. Specifically, the relative peak area RSD value for peak 2 is 20.0%, with a range of 0.17-0.35; the relative peak area RSD value for peak 3 is 13.6%, with a range of... The relative peak area RSD of peak 4 is 14.9%, with a relative peak area range of 0.46-0.75; the relative peak area RSD of peak 5 is 22.6%, with a relative peak area range of 1.10-2.35; the relative peak area RSD of peak 8 is 18.7%, with a relative peak area range of 0.23-0.52; the relative peak area RSD of peak 10 is 12.8%, with a relative peak area range of 1.00- 1.76; the relative peak area RSD value of peak 12 is 17.8%, and the relative peak area range is 0.052-0.118; the relative peak area RSD value of peak 13 is 28.4%, and the relative peak area range is 0.041-0.086; the relative peak area RSD value of peak 14 is 11.0%, and the relative peak area range is 0.214-0.32; therefore, the relative peak area limit of each characteristic peak is not specified in the standard of characteristic spectrum of reference sample for the time being.

[0327] In summary, the standard for determining the characteristic chromatogram of the reference sample is as follows: the chromatogram of the test sample should show 14 characteristic peaks; the retention times of peaks 1, 6, 7, 9, and 11 should correspond to the retention times of the reference peaks of berberine hydrochloride, coptisine hydrochloride, cypermethrin, berberine hydrochloride, and baicalin, respectively. The peak corresponding to the reference peak of berberine hydrochloride is peak S1. Calculate the relative retention times of peaks 2, 3, 4, 5, 8, and 10 with peak S1. The relative retention times should be within ±10% of the specified values, which are 0.46 (peak 2), 0.55 (peak 3), 0.78 (peak 4), 0.89 (peak 5), 1.14 (peak 8), and 1.41 (peak 10). The peak corresponding to the reference peak of baicalin is peak S2. Calculate the relative retention times of peaks 12, 13, and 14 with peak S2. The relative retention times should be within ±10% of the specified values, which are 1.07 (peak 12), 1.13 (peak 13), and 1.18 (peak 14).

[0328] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0329] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for preparing a reference sample of Dang Gui Liu Huang Tang, characterized in that, Includes the following steps: Angelica sinensis, Rehmannia glutinosa (raw and processed), Coptis chinensis, Scutellaria baicalensis, Phellodendron chinense, and Astragalus membranaceus are pulverized and mixed to prepare the first material; Soak the first material in water, then boil it over high heat and low heat, and sieve it to prepare the second material. The second material was dried to prepare a reference sample of Danggui Liuhuang Decoction; The drying process includes at least three stages: the first stage is dried at a temperature of ≤-50℃, the second stage is dried at a temperature of -40℃ to -10℃, and the third stage is dried at a temperature of 0℃ to 30℃.

2. The method for preparing the reference sample of Dang Gui Liu Huang Tang according to claim 1, characterized in that, The mass ratio of Angelica sinensis, Rehmannia glutinosa (raw), Rehmannia glutinosa (processed), Coptis chinensis, Scutellaria baicalensis, Phellodendron chinense, and Astragalus membranaceus is 1:1:1:1:1:1:2; and / or, after mixing, the mixture is passed through a 4-mesh sieve; and / or, the mass-to-volume ratio of the first material to the water is 1g:(28-30)mL; and / or, the soaking time is 20min-40min; and / or, the temperature of the high heat is 110℃-120℃; and / or, the decocting time of the high heat is 13min. n-16min; and / or, the temperature of the simmering heat is 90℃-100℃; and / or, the simmering time is 40min-60min; and / or, the volume of the material after simmering is 1 / 3 to 2 / 3 of that before simmering; and / or, the mesh size of the sieve is 150-250 mesh; and / or, the drying time of the first stage is 2h-4h; and / or, the drying time of the second stage is 70h-87h; and / or, the drying time of the third stage is 10h-15h.

3. The reference sample of Dang Gui Liu Huang Tang, characterized in that, It was prepared by the method for preparing the reference sample of Danggui Liuhuang Decoction as described in claim 1 or 2.

4. A method for detecting Dang Gui Liu Huang Tang, characterized in that, Includes the following steps: Berberine hydrochloride, magnoflorine, 3-O-feruloylquinic acid, 4-O-feruloylquinic acid, 5-O-feruloylquinic acid, berberine hydrochloride, epiberberine hydrochloride, cypermethrin, berberine hydrochloride, palmatine, baicalin, scutellarin A-7-O-glucuronide, and wogonin were mixed with the first solvent, filtered, and the filtrate was collected to form a mixed reference solution. The Danggui Liuhuang Decoction sample was mixed with the second solvent, filtered, and the filtrate was collected to form the test solution. The detection method is selected from liquid chromatography, and the conditions of the liquid chromatography include: Elution system: Mobile phase A comprises acetonitrile, mobile phase B comprises an aqueous solution containing 0.2%-0.4% phosphoric acid by mass, and gradient elution is performed. The gradient elution procedure includes: From 0 min to 15 min, the volume percentage of mobile phase A changed from 5% to 12.5%; Between 15 and 25 minutes, the volume percentage of mobile phase A changed from 12.5% ​​to 15%. Between 25 and 40 minutes, the volume percentage of mobile phase A changed from 15% to 25%. For 40-50 minutes, the volume percentage of mobile phase A is 25%. Between 50 and 60 minutes, the volume percentage of mobile phase A changed from 25% to 35%. Between 60 and 62 minutes, the volume percentage of mobile phase A changed from 35% to 90%.

5. The detection method for Dang Gui Liu Huang Tang according to claim 4, characterized in that, The mobile phase B comprises an aqueous solution containing 0.28%-0.32% phosphoric acid by mass; and / or, a column temperature of 30℃-35℃; and / or, a flow rate of 0.5mL / min-1.2mL / min; and / or, a detection wavelength of 275nm-305nm; and / or, a theoretical plate number calculated based on the berberine hydrochloride peak ≥10000.

6. The detection method for Dang Gui Liu Huang Tang according to claim 4, characterized in that, The Dang Gui Liu Huang Tang sample includes the Dang Gui Liu Huang Tang reference sample as described in claim 3; and / or, the Dang Gui Liu Huang Tang sample is prepared by mixing, decocting, and drying Dang Gui, Sheng Di Huang, Shu Di Huang, Huang Lian, Huang Qin, Huang Bai, and Huang Qi according to the prescription dosage; and / or, the Dang Gui Liu Huang Tang sample is prepared by decocting and drying Dang Gui Liu Huang Tang slices; and / or, the dosage form of the Dang Gui Liu Huang Tang sample includes one or more of granules, tablets, decoctions, pills, powders, ointments, and capsules.

7. The detection method for Dang Gui Liu Huang Tang according to claim 4, characterized in that, The first solvent and the second solvent each independently include one or more of methanol, ethanol, acetonitrile, dimethyl sulfoxide and water; and / or, the process of mixing the Danggui Liuhuang Decoction sample with the second solvent also includes ultrasonic treatment, with an ultrasonic power of 250W-350W and an ultrasonic frequency of 35kHz-45kHz.

8. A method for constructing the fingerprint spectrum of Dang Gui Liu Huang Tang, characterized in that, Includes the following steps: Provide reference solution and Dang Gui Liu Huang Tang test solution respectively; The reference solution was tested using the detection method of Danggui Liuhuang Decoction according to any one of claims 4 to 7 to obtain the reference chromatogram; The chromatogram of the sample solution of Danggui Liuhuang Decoction was obtained by using the detection method of any one of claims 4 to 7. The fingerprint spectrum of the test sample is subjected to fingerprint spectrum similarity analysis to generate a standard fingerprint spectrum. The chromatographic peaks of the standard fingerprint spectrum are identified by referring to the reference spectrum to construct the fingerprint spectrum of Dang Gui Liu Huang Tang.

9. The method for constructing the fingerprint spectrum of Dang Gui Liu Huang Tang according to claim 8, characterized in that, The Danggui Liuhuang Decoction sample includes the Danggui Liuhuang Decoction reference sample as described in claim 3.

10. The method for constructing the fingerprint spectrum of Dang Gui Liu Huang Tang according to claim 8, characterized in that, The sample chromatogram and / or the standard fingerprint chromatogram exhibit 14 characteristic peaks, and the retention time of the characteristic peaks is in the range of -10% to 10% of the retention time value of the corresponding reference peak in the standard fingerprint chromatogram.

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