Content determination method of traditional Chinese medicine composition containing angelica sinensis and cinnamon and application of traditional Chinese medicine composition

Abstract

The invention discloses a method for determining the content of a traditional Chinese medicine composition containing angelica sinensis and cinnamon, which comprises the following steps: taking a test solution and a reference solution of the traditional Chinese medicine composition, and detecting under the chromatographic conditions that a chromatographic column with octadecyl bonded silica gel as a filler is adopted; the mobile phase A is selected from one or more of acetonitrile, methanol and tetrahydrofuran, and the mobile phase B is an acid aqueous solution, an alkali aqueous solution and / or a buffer salt aqueous solution; obtaining content information of a reference substance of the traditional Chinese medicine composition according to a detection result; wherein the traditional Chinese medicine composition comprises angelica sinensis, cinnamon, radix paeoniae alba, ginger, Chinese dates and liquorice, and the reference substances are ferulic acid, cinnamaldehyde, 6-gingerol and ligustilide. The detection method is simple, accurate, efficient, good in stability, high in precision, good in reproducibility, convenient and easy to master, energy can be effectively saved, the analysis cost is reduced, and the technical content is not disclosed in the prior art.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to a method for determining the content of a traditional Chinese medicine composition containing angelica and cinnamon bark and an application thereof. Background Art

[0002] The reference sample carries the effectiveness and safety of the Chinese medicine composition. Quality research has become an important content and key link in the development of Chinese medicine compound preparations, and content determination is the main method to control the quality of the reference sample. Screening quality markers related to effectiveness and safety in Chinese medicine compositions and establishing content determination methods can quantitatively characterize the quality of reference samples.

[0003] Currently, the content determination methods mainly focus on single components, and there are few methods for simultaneously determining the content of multiple effective components. The content determination method and application of the Chinese medicine composition containing angelica and cinnamon bark of the present invention have not been found in the prior art. Summary of the invention

[0004] Based on this, the present invention provides a method for determining the content of a traditional Chinese medicine composition containing angelica and cinnamon, the method comprising the following steps:

[0005] Take the test solution and the reference solution of the Chinese medicine composition for testing.

[0006] The chromatographic conditions of the test are as follows: a chromatographic column with octadecyl bonded silica as filler is used, mobile phase A is selected from one or more of acetonitrile, methanol and tetrahydrofuran, mobile phase B is an acid aqueous solution, an alkaline aqueous solution and / or a buffered saline solution, and the gradient elution program is as follows: 0-10 min, 95% B→94% B; 10-12 min, 94% B→88% B; 12-20 min, 88% B; 20-21 min, 88% B→84% B; 21-38 min, 84% B; 38-48 min, 84% B→80% B; 48-52 min, 84% B→80% B. n, 80% B→75% B; 52~58min, 75% B→71% B; 58~62min, 71% B→68% B; 62~80min, 68% B→44% B; 80~83min, 44% B→26% B; 83~88min, 26% B→15% B; 88~89min, 15% B→95% B; 89~100min, 95% B; flow rate is 0.5~1.5ml / min, column temperature is 10~40℃, detection wavelength is 180~360nm, injection volume is 1~20μl;

[0007] According to the test results, obtaining the content information of the reference substance of the Chinese medicine composition;

[0008] The Chinese medicine composition includes angelica sinensis, cinnamon bark, white peony root, ginger, jujube and licorice, and the reference substance is ferulic acid, cinnamaldehyde, 6-gingerol and ligustilide.

[0009] Furthermore, the licorice is stir-fried licorice.

[0010] Furthermore, the information is calculated according to the external standard method based on the corresponding peak areas in the recorded chromatogram of the test solution of the traditional Chinese medicine composition and the chromatogram of the reference solution.

[0011] Furthermore, the linear equation of ferulic acid is y=47.474x+18.878, R 2 =1.0000.

[0012] Furthermore, the linear equation of the cinnamaldehyde is y=90.332x+44.58, R 2 =1.0000.

[0013] Furthermore, the linear equation of 6-gingerol is y=25.992x+8.2874, R 2 =0.9999.

[0014] Furthermore, the linear equation of the ligustilide is y=10.805x+10.113, R 2 =0.9999.

[0015] Furthermore, the mass ratios of the angelica, cinnamon bark, white peony root, ginger, jujube and liquorice are (45-60):(30-50):(70-90):(30-50):(25-45):(20-40).

[0016] Furthermore, the mass ratio of the angelica sinensis, the cinnamon bark, the white peony root, the ginger, the jujube and the licorice is about 55.2: about 41.4: about 82.8: about 41.4: about 36: about 27.6.

[0017] Furthermore, the preparation method of the Chinese medicine composition test solution includes: taking an appropriate amount of a decoction of the Chinese medicine composition containing angelica and cinnamon, placing it in a container, adding a solvent, ultrasonically treating it for a period of time, fixing the volume, shaking it well, centrifuging it, filtering the supernatant, and taking the filtrate to obtain the Chinese medicine composition test solution.

[0018] Furthermore, the container is a volumetric flask.

[0019] Furthermore, the solvent is an alcohol, such as methanol.

[0020] Furthermore, the mass-to-volume ratio between the decoction of the traditional Chinese medicine composition and the solvent is 0.4-0.8, such as about 0.6, in units of g / ml.

[0021] Furthermore, the mass of the decoction of the traditional Chinese medicine composition is 5 to 30 g, for example, about 15 g.

[0022] Furthermore, the power of the ultrasonic treatment is 400-600W, for example, about 500W.

[0023] Furthermore, the frequency of the ultrasonic treatment is 30-50 kHz, for example, about 40 kHz.

[0024] Furthermore, the ultrasonic treatment time is 20 to 40 minutes, for example, about 30 minutes.

[0025] Furthermore, the centrifugal speed is 4000-6000 rpm / min, for example, about 5000 rpm / min.

[0026] Furthermore, the centrifugation time is 5 to 15 minutes, for example, about 10 minutes.

[0027] Furthermore, the preparation method of the decoction of the Chinese medicine composition containing angelica and cinnamon includes: weighing appropriate amounts of angelica, cinnamon, white peony root, ginger, jujube and licorice, crushing them into coarse particles, placing them in a container, soaking them in water, decocting them until boiling and boiling them for a period of time, then keeping them slightly boiling for a period of time, filtering the filtrate with a filter while it is hot, and obtaining the decoction of the Chinese medicine composition containing angelica and cinnamon.

[0028] Furthermore, the volume / mass ratio of the water to the traditional Chinese medicine composition comprising angelica and cinnamon is 5-10, for example, about 7.

[0029] Furthermore, the container is a ceramic medicine decoction pot or an electric ceramic stove.

[0030] Furthermore, the soaking time is 10 to 100 minutes, for example, about 45 minutes.

[0031] Furthermore, the decoction is covered when it boils.

[0032] Furthermore, the power of the container during boiling is 1000-3000W, for example, about 2000W.

[0033] Furthermore, the boiling time is 5 to 15 minutes, for example, about 10 minutes.

[0034] Furthermore, the power of the container when slightly boiling is 500-1500W, for example, about 900W.

[0035] Furthermore, the slight boiling time is 10 to 100 minutes, for example, about 60 minutes.

[0036] Furthermore, the volume / volume ratio of the decoction of the Chinese medicinal composition comprising angelica and cinnamon to the water is 0.1 to 0.5, such as about 0.3.

[0037] Furthermore, the filter is a 200-mesh nylon filter.

[0038] Furthermore, the preparation method of the reference solution includes: weighing appropriate amounts of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide reference substances, adding methanol to prepare the reference solution with concentrations of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide being 1 to 100 μg / ml, respectively.

[0039] Furthermore, the concentration of ferulic acid in the reference solution is about 18 μg / ml.

[0040] Furthermore, the concentration of cinnamaldehyde in the reference solution is about 13 μg / ml.

[0041] Furthermore, the concentration of 6-gingerol in the reference solution is about 6 μg / ml.

[0042] Furthermore, the concentration of ligustilide in the reference solution is about 18 μg / ml.

[0043] Furthermore, the flow rate of the HPLC detection is 0.8-1.2 ml / min, for example, about 1.0 ml / min.

[0044] Furthermore, the column temperature is 20-30°C, for example, about 25°C.

[0045] Further, between the 0th minute and the 60th minute, the detection wavelength is 280-340 nm, for example, about 316 nm.

[0046] Further, between the 60th minute and the 77th minute, the detection wavelength is 260-320 nm, for example, about 290 nm.

[0047] Further, between the 77th minute and the 100th minute, the detection wavelength is 190-230 nm, for example, about 210 nm.

[0048] Furthermore, the injection volume is 5-15 μl, for example, about 10 μl.

[0049] Furthermore, the theoretical plate number of the chromatographic peak corresponding to the ferulic acid is not less than 5000.

[0050] Furthermore, the chromatographic column is an Agilent 5TC-C18 chromatographic column.

[0051] Furthermore, the specifications of the chromatographic column are: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm.

[0052] Furthermore, the mobile phase A is acetonitrile.

[0053] Furthermore, the acid aqueous solution, alkali aqueous solution and / or buffered saline solution is selected from one or more of weak acids and their salts, weak bases and their salts of different concentrations.

[0054] Further, the acid aqueous solution, alkali aqueous solution and / or buffered saline solution is selected from formic acid, glacial acetic acid, phosphoric acid, trifluoroacetic acid, formic acid and ammonium formate, acetic acid and sodium acetate, acetic acid and ammonium acetate, disodium hydrogen phosphate and sodium dihydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and citric acid, citric acid and sodium citrate, glycine and hydrochloric acid, or phthalic acid and hydrochloric acid in different concentrations.

[0055] Furthermore, the acid aqueous solution is a 0.01% to 1% acid aqueous solution.

[0056] Furthermore, the acid aqueous solution is a 0.01% to 1% phosphoric acid aqueous solution.

[0057] Furthermore, the acid aqueous solution is about 0.1% phosphoric acid aqueous solution.

[0058] Furthermore, the buffered saline solution is an acetate solution and / or an acetate solution.

[0059] Further, the pH value of the buffered saline solution is not greater than 7.0.

[0060] Furthermore, the detection limit of ferulic acid is about 0.4189 μg / ml.

[0061] Furthermore, the detection limit of cinnamaldehyde is about 0.06601 μg / ml.

[0062] Furthermore, the detection limit of 6-gingerol was about 0.1146 μg / ml.

[0063] Furthermore, the detection limit of ligustilide is about 0.1788 μg / ml.

[0064] Furthermore, the limit of quantification of ferulic acid was about 1.3960 μg / ml.

[0065] Furthermore, the limit of quantification of cinnamaldehyde is about 0.2200 μg / ml.

[0066] Furthermore, the limit of quantification of 6-gingerol was about 0.3818 μg / ml.

[0067] Furthermore, the limit of quantification of ligustilide was approximately 0.5961 μg / ml.

[0068] According to another aspect of the present invention, there is provided a use of the above-mentioned determination method in quality detection and / or quality evaluation and / or quality control of a traditional Chinese medicine composition containing angelica and cinnamon bark.

[0069] Beneficial effects of the present invention:

[0070] The detection method of the present invention is simple, accurate, efficient, stable, precise, reproducible, convenient and easy to master, and can effectively save energy and reduce analysis costs. However, the technical content is not disclosed in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without exceeding the scope of protection required by the present invention.

[0072] Figure 1 The present invention is a chromatogram of the content of a reference sample solution of a traditional Chinese medicine composition comprising angelica and cinnamon bark. DETAILED DESCRIPTION

[0073] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0074] Unless otherwise indicated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by those of ordinary skill in the field of the invention or the field in which the terms are used. Although any methods, conditions, substances or materials similar to or equivalent to those disclosed herein may be used in the practice of the present invention, preferred methods, conditions, substances or materials are described herein.

[0075] The present invention is intended to encompass all alternatives, variations and equivalents that may be included in the present invention as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0076] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0077] In the present invention, the term "comprising" is synonymous with "including". As used herein, the terms "comprising", "including", "having", "containing" or any other variation thereof are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus comprising the listed elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0078] As described in the background technology section, the current content determination methods are mainly based on single components, and there are few methods for simultaneously determining the content of multiple effective components. The content determination method of a Chinese medicine composition containing angelica and cinnamon and its application of the present invention have not been found in the prior art. In order to solve the above problems, the present invention provides a content determination method of a Chinese medicine composition containing angelica and cinnamon, and the determination method comprises the following steps:

[0079] Take the test solution and the reference solution of the Chinese medicine composition for testing.

[0080] The chromatographic conditions of the test are as follows: a chromatographic column with octadecyl bonded silica as filler is used, mobile phase A is selected from one or more of acetonitrile, methanol and tetrahydrofuran, mobile phase B is an acid aqueous solution, an alkaline aqueous solution and / or a buffered saline solution, and the gradient elution program is as follows: 0-10 min, 95% B→94% B; 10-12 min, 94% B→88% B; 12-20 min, 88% B; 20-21 min, 88% B→84% B; 21-38 min, 84% B; 38-48 min, 84% B→80% B; 48-52 min, 84% B→80% B. n, 80% B→75% B; 52~58min, 75% B→71% B; 58~62min, 71% B→68% B; 62~80min, 68% B→44% B; 80~83min, 44% B→26% B; 83~88min, 26% B→15% B; 88~89min, 15% B→95% B; 89~100min, 95% B; flow rate is 0.5~1.5ml / min, column temperature is 10~40℃, detection wavelength is 180~360nm, injection volume is 1~20μl;

[0081] According to the test results, obtaining the content information of the reference substance of the Chinese medicine composition;

[0082] The Chinese medicine composition includes angelica, cinnamon bark, white peony root, ginger, jujube and liquorice, and the reference substance includes ferulic acid, cinnamaldehyde, 6-gingerol and ligustilide.

[0083] In the present invention, when concentration, time, flow rate, column temperature, wavelength, injection volume, ratio, power, frequency, mass, rotation speed, or other values ​​or parameters are expressed as ranges, preferred ranges, or ranges defined by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any upper range limit or preferred value with any lower range limit or preferred value, regardless of whether the range is disclosed separately. For example, when the range "0.5-1.5" is disclosed, the described range should be interpreted as including the ranges "0.5-1.5", "0.5-1.3", "0.5-1.1", "0.5-0.9", "0.5-0.7", "0.7-1.5", "0.7-1.3", "0.7-1.1", "0.7-0.9", "0.9-1.5", "0.9-1.3", "0.9-1.1", "1.1-1.5", "1.1-1.3", "1.3-1.5", etc. When a numerical range is described herein, unless otherwise indicated, that range is intended to include the endpoints and all integers and fractions within that range.

[0084] In a preferred embodiment, the licorice is stir-fried licorice.

[0085] In a preferred embodiment, the information is the content of the reference substance in the traditional Chinese medicine composition calculated according to the external standard method based on the corresponding peak areas in the recorded chromatogram of the test solution of the traditional Chinese medicine composition and the chromatogram of the reference substance solution.

[0086] In a preferred embodiment, the linear equation of ferulic acid is y=47.474x+18.878, R 2 =1.0000.

[0087] In a preferred embodiment, the linear equation of cinnamaldehyde is y=90.332x+44.58, R 2 =1.0000.

[0088] In a preferred embodiment, the linear equation of 6-gingerol is y=25.992x+8.2874, R 2 =0.9999.

[0089] In a preferred embodiment, the linear equation of ligustilide is y=10.805x+10.113, R 2 =0.9999.

[0090] In a preferred embodiment, the mass ratio of the angelica, cinnamon bark, white peony root, ginger, jujube and liquorice is (45-60):(30-50):(70-90):(30-50):(25-45):(20-40).

[0091] In a preferred embodiment, the mass ratio of the angelica sinensis, the cinnamon bark, the white peony root, the ginger, the jujube and the liquorice is about 55.2: about 41.4: about 82.8: about 41.4: about 36: about 27.6.

[0092] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 55.2" includes ±5% of 55.2, or from 52.44 to 57.96; "about 41.4" includes ±5% of 41.4, or from 39.33 to 43.47; "about 82.8" includes ±5% of 82.8, or from 78.66 to 86.94; "about 36" includes ±5% of 36, or from 34.2 to 37.8; "about 27.6" includes ±5% of 27.6, or from 26.22 to 28.98.

[0093] In a preferred embodiment, the preparation method of the Chinese medicine composition test solution comprises: taking an appropriate amount of a decoction of the Chinese medicine composition containing angelica and cinnamon, placing it in a container, adding a solvent, ultrasonically treating it for a period of time, fixing the volume, shaking it well, centrifuging it, filtering the supernatant, and taking the filtrate to obtain the Chinese medicine composition test solution.

[0094] In a preferred embodiment, the container is a volumetric flask.

[0095] In a preferred embodiment, the solvent is an alcohol, such as methanol.

[0096] In a preferred embodiment, the mass-to-volume ratio between the decoction of the traditional Chinese medicine composition and the solvent is 0.4-0.8, such as about 0.6, in units of g / ml.

[0097] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.6" includes ±5% of 0.6, or from 0.57 to 0.63.

[0098] In a preferred embodiment, the mass of the decoction of the traditional Chinese medicine composition is 5 to 30 g, for example, about 15 g.

[0099] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 15" includes ±5% of 15, or from 14.25 to 15.75.

[0100] In a preferred embodiment, the power of the ultrasonic treatment is 400-600W, such as about 500W.

[0101] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 500" includes ±5% of 500, or from 475 to 525.

[0102] In a preferred embodiment, the frequency of the ultrasonic treatment is 30 to 50 kHz, such as about 40 kHz.

[0103] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 40" includes ±5% of 40, or from 38 to 42.

[0104] In a preferred embodiment, the ultrasonic treatment time is 20 to 40 minutes, such as about 30 minutes.

[0105] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 30" includes ±5% of 30, or from 28.5 to 31.5.

[0106] In a preferred embodiment, the centrifugal rotation speed is 4000-6000 rpm / min, such as about 5000 rpm / min.

[0107] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 5000" includes ±5% of 5000, or from 4750 to 5250.

[0108] In a preferred embodiment, the centrifugation time is 5 to 15 min, such as about 10 min.

[0109] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0110] In a preferred embodiment, the preparation method of the decoction of the Chinese medicine composition containing angelica and cinnamon comprises: weighing appropriate amounts of angelica, cinnamon, white peony root, ginger, jujube and licorice, crushing them into coarse particles, placing them in a container, soaking them in water, decocting them until boiling and boiling them for a period of time, then keeping them slightly boiling for a period of time, filtering the filtrate with a filter while it is hot, and obtaining the decoction of the Chinese medicine composition containing angelica and cinnamon.

[0111] In a preferred embodiment, the volume / mass ratio of the water to the traditional Chinese medicine composition comprising angelica and cinnamon is 5-10, such as about 7.

[0112] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 7" includes ±5% of 7, or from 6.65 to 7.35.

[0113] In a preferred embodiment, the container is a ceramic medicine decoction pot or an electric ceramic stove.

[0114] In a preferred embodiment, the soaking time is 10 to 100 minutes, such as about 45 minutes.

[0115] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 45" includes ±5% of 45, or from 42.75 to 47.25.

[0116] In a preferred embodiment, the decocting vessel is covered when boiling.

[0117] In a preferred embodiment, the power of the container during boiling is 1000-3000W, for example, about 2000W.

[0118] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 2000" includes ±5% of 2000, or from 1900 to 2100.

[0119] In a preferred embodiment, the boiling time is 5 to 15 minutes, such as about 10 minutes.

[0120] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0121] In a preferred embodiment, the power of the container when slightly boiling is 500-1500W, such as about 900W.

[0122] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 900" includes ±5% of 900, or from 855 to 945.

[0123] In a preferred embodiment, the slight boiling time is 10 to 100 minutes, for example, about 60 minutes.

[0124] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 60" includes ±5% of 60, or from 57 to 63.

[0125] In a preferred embodiment, the volume / volume ratio of the decoction of the traditional Chinese medicine composition comprising angelica and cinnamon to the water is 0.1-0.5, such as about 0.3.

[0126] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.3" includes ±5% of 0.3, or from 0.285 to 0.315.

[0127] In a preferred embodiment, the filter is a 200 mesh nylon filter.

[0128] In a preferred embodiment, the preparation method of the reference solution includes: weighing appropriate amounts of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide reference substances, adding methanol to prepare the reference solution with concentrations of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide being 1 to 100 μg / ml, respectively.

[0129] In a preferred embodiment, the concentration of ferulic acid in the reference solution is about 18 μg / ml.

[0130] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 18" includes ±5% of 18, or from 17.1 to 18.9.

[0131] In a preferred embodiment, the concentration of cinnamaldehyde in the reference solution is about 13 μg / ml.

[0132] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 13" includes ±5% of 13, or from 12.35 to 13.65.

[0133] In a preferred embodiment, the concentration of 6-gingerol in the reference solution is about 6 μg / ml.

[0134] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 6" includes ±5% of 6, or from 5.7 to 6.3.

[0135] In a preferred embodiment, the concentration of ligustilide in the reference solution is about 18 μg / ml.

[0136] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 18" includes ±5% of 18, or from 17.1 to 18.9.

[0137] In a preferred embodiment, the flow rate of the HPLC detection is 0.8-1.2 ml / min, for example, about 1.0 ml / min.

[0138] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1.0" includes ±5% of 1.0, or from 0.95 to 1.05.

[0139] In a preferred embodiment, the column temperature is 20-30°C, such as about 25°C.

[0140] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 25" includes ±5% of 25, or from 23.75 to 26.25.

[0141] In a preferred embodiment, between the 0th minute and the 60th minute, the detection wavelength is 280-340 nm, for example, about 316 nm.

[0142] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 316" includes ±5% of 316, or from 300.2 to 331.8.

[0143] In a preferred embodiment, between the 60th minute and the 77th minute, the detection wavelength is 260-320 nm, such as about 290 nm.

[0144] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 290" includes ±5% of 290, or from 275.5 to 304.5.

[0145] In a preferred embodiment, between the 77th minute and the 100th minute, the detection wavelength is 190-230 nm, for example, about 210 nm.

[0146] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 210" includes ±5% of 210, or from 199.5 to 220.5.

[0147] In a preferred embodiment, the injection volume is 5-15 μl, such as about 10 μl.

[0148] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0149] In a preferred embodiment, the theoretical plate number of the chromatographic peak corresponding to the ferulic acid is not less than 5000.

[0150] In a preferred embodiment, the chromatographic column is an Agilent 5TC-C18 chromatographic column.

[0151] In a preferred embodiment, the specifications of the chromatographic column are: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm.

[0152] In a preferred embodiment, mobile phase A is acetonitrile.

[0153] In a preferred embodiment, the acid aqueous solution, base aqueous solution and / or buffered saline solution is selected from one or more of weak acids and their salts, weak bases and their salts of different concentrations.

[0154] In a preferred embodiment, the acid aqueous solution, alkali aqueous solution and / or buffered saline solution is selected from formic acid, glacial acetic acid, phosphoric acid, trifluoroacetic acid, formic acid and ammonium formate, acetic acid and sodium acetate, acetic acid and ammonium acetate, disodium hydrogen phosphate and sodium dihydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and citric acid, citric acid and sodium citrate, glycine and hydrochloric acid, or phthalic acid and hydrochloric acid in different concentrations.

[0155] In a preferred embodiment, the acid aqueous solution is a 0.01% to 1% acid aqueous solution.

[0156] In a preferred embodiment, the acid aqueous solution is a 0.01% to 1% phosphoric acid aqueous solution.

[0157] In a preferred embodiment, the aqueous acid solution is about 0.1% phosphoric acid aqueous solution.

[0158] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.1%" includes ±5% of 0.1%, or from 0.095% to 0.105%.

[0159] In a preferred embodiment, the buffered saline solution is an acetate aqueous solution and / or an acetate aqueous solution.

[0160] In a preferred embodiment, the pH of the buffered saline solution is no greater than 7.0.

[0161] In a preferred embodiment, the detection limit of ferulic acid is about 0.4189 μg / ml.

[0162] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.4189" includes ±5% of 0.4189, or from 0.397955 to 0.439845.

[0163] In a preferred embodiment, the detection limit of cinnamaldehyde is about 0.06601 μg / ml.

[0164] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.06601" includes ±5% of 0.06601, or from 0.0627095 to 0.0693105.

[0165] In a preferred embodiment, the detection limit of 6-gingerol is about 0.1146 μg / ml.

[0166] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.1146" includes ±5% of 0.1146, or from 0.10887 to 0.12033.

[0167] In a preferred embodiment, the detection limit of ligustilide is about 0.1788 μg / ml.

[0168] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.1788" includes ±5% of 0.1788, or from 0.16986 to 0.18774.

[0169] In a preferred embodiment, the limit of quantification of ferulic acid is about 1.3960 μg / ml.

[0170] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1.3960" includes ±5% of 1.3960, or from 1.3262 to 1.4658.

[0171] In a preferred embodiment, the limit of quantification of cinnamaldehyde is about 0.2200 μg / ml.

[0172] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.2200" includes ±5% of 0.2200, or from 0.2090 to 0.2310.

[0173] In a preferred embodiment, the limit of quantification of 6-gingerol is about 0.3818 μg / ml.

[0174] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.3818" includes ±5% of 0.3818, or from 0.36271 to 0.40089.

[0175] In a preferred embodiment, the limit of quantification of ligustilide is about 0.5961 μg / ml.

[0176] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.5961" includes ±5% of 0.5961, or from 0.566295 to 0.625905.

[0177] According to another aspect of the present invention, there is provided a use of the above-mentioned determination method in quality detection and / or quality evaluation and / or quality control of a traditional Chinese medicine composition containing angelica and cinnamon bark.

[0178] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions or conditions recommended by the manufacturer.

[0179] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0180] The above features mentioned in the present invention or the features mentioned in the embodiments can be combined in any way. All the features disclosed in this patent specification can be used in combination with any combination form, and each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

[0181] Example

[0182] 1. Instruments and reagents

[0183] 1.1 Instruments

[0184] High performance liquid chromatograph, model: Agilent1290_DAD HPLC (Agilent Technologies Co., Ltd.); High performance liquid chromatograph, model: Waters 2695_2996 (Waters Technologies Co., Ltd.); Chromatographic column: Agilent 5TC-C18(2)250×4.6mm, SN:731424 / 735516 / 735519; Electronic balance, model: BCE224I-1CCN (Sartorius Group, Germany); Electronic balance, model: JY2002 (Shanghai Puchun Metrology Instrument Co., Ltd.); Desktop low-speed centrifuge, model: TD4N (Changsha Yingtai Instrument Co., Ltd.); CNC ultrasonic cleaner, model: KQ-500DE (Kunshan Ultrasonic Instrument Co., Ltd.); Digital display constant temperature water bath, model: HH-6 (Shanghai Boxun Medical Biological Instrument Co., Ltd.); Diaphragm oil-free vacuum pump model: MT-10 Yancheng Xinmingte Glass Instrument Co., Ltd.

[0185] 1.2 Reagents and test drugs

[0186] Methanol and acetonitrile were of chromatographic grade, methanol and phosphoric acid were of analytical grade, and water was purified water; ferulic acid (batch number: 110773-202316, content: 99.3%, China Food and Drug Inspection Institutes); cinnamaldehyde (batch number: 110710-202223, content: 98.9%, China Food and Drug Inspection Institutes); ligustilide (batch number: 111737-202311, content: 100.0%, China Food and Drug Inspection Institutes); 6-gingerol (batch number: 111833-202007, content: 99.3%, China Food and Drug Inspection Institutes).

[0187] 1.3. Sample information

[0188] The Chinese medicine composition granule reference sample containing angelica and cinnamon of the present invention (batch number: 2024062801-L source: homemade); the Chinese medicine composition granule reference sample containing angelica and cinnamon of the present invention (batch number: 2024072501-L source: homemade); the Chinese medicine composition granule reference sample containing angelica and cinnamon of the present invention (batch number: 2024082904-L source: homemade); the Chinese medicine composition granule containing angelica and cinnamon of the present invention lacking angelica negative reference sample (batch number: 202409 0201-L Source: homemade); the Chinese medicine composition granules containing angelica and cinnamon of the present invention lack cinnamon negative benchmark sample (batch number: 2024090204-L Source: homemade); the Chinese medicine composition granules containing angelica and cinnamon of the present invention lack white peony negative benchmark sample (batch number: 2024090202-L Source: homemade); the Chinese medicine composition granules containing angelica and cinnamon of the present invention lack ginger negative benchmark sample (batch number: 2024090301-L Source: homemade); the Chinese medicine composition granules containing angelica and cinnamon of the present invention lack ginger negative benchmark sample (batch number: 2024090302-L Source: homemade); The Chinese medicine composition granules containing angelica and cinnamon lack the negative reference sample of jujube (batch number: 2024090302-L source: homemade); the Chinese medicine composition granules containing angelica and cinnamon of the present invention lack the negative reference sample of fried licorice (batch number: 2024090203-L source: homemade); angelica slices (batch number: ZA00323102101-01, origin: Min County, Dingxi City, Gansu Province, source: homemade); cinnamon slices (batch number: ZA04223112801, origin: Ping, Guigang City, Guangxi Zhuang Autonomous Region, source : homemade); white peony slices (batch number: ZA00123121502, origin: Qiaocheng District, Bozhou City, Anhui Province, source: homemade); jujube slices (batch number: ZA00223121402, origin: Ningyang County, Tai'an City, Shandong Province, source: homemade); ginger slices (batch number: ZA00524071501, origin: Wanyuan County, Dazhou City, Sichuan Province, source: homemade); stir-fried licorice slices (batch number: ZD00423090401-01, origin: Hangjin Banner, Ordos, Inner Mongolia, source: homemade).

[0189] 1.4. Solution preparation

[0190] (1) Preparation method of reference sample decoction

[0191] Take 55.20g of angelica, 41.40g of cinnamon, 82.80g of white peony root, 41.40g of ginger, 36.00g of jujube, and 27.60g of stir-fried licorice, grind them into coarse particles, put them in a ceramic decoction pot, add 2000ml of water, cover, soak for 45 minutes, boil at 2000W in an electric ceramic stove for about 10 minutes, keep it at a slight boil at 900W and simmer until 600ml (about 60 minutes), filter through a 200-mesh nylon filter while hot.

[0192] (2) Preparation of reference solution:

[0193] Take appropriate amounts of ferulic acid, cinnamaldehyde, 6-gingerol and ligustilide reference substances, weigh them accurately, add methanol to prepare a mixed solution containing 18 μg of ferulic acid, 13 μg of cinnamaldehyde, 6 μg of 6-gingerol and 18 μg of ligustilide per 1 ml as the reference solution.

[0194] (3) Preparation of test solution

[0195] Take about 15g of the reference sample decoction, place it in a 25ml volumetric flask, add an appropriate amount of methanol, ultrasonically (500W, 40kHz) for 30 minutes, cool, make up to volume, shake well, centrifuge at 5000rpm / min for 10min, filter the supernatant, and take the filtrate to obtain the product.

[0196] (4) Preparation of negative control solution

[0197] Preparation of negative sample solution lacking Angelica sinensis: weigh other medicinal ingredients lacking Angelica sinensis according to the prescription, prepare negative sample lacking Angelica sinensis according to the reference sample preparation method, and prepare negative sample solution lacking Angelica sinensis according to item "(3)".

[0198] Negative control solutions lacking angelica, cinnamon bark, white peony root, ginger, jujube and fried licorice were prepared in the same way.

[0199] (5) Blank solvent: methanol.

[0200] 2. Establishment of chromatographic conditions for content determination

[0201] 2.1. Study on chromatographic conditions for content determination

[0202] The proposed initial chromatographic conditions are:

[0203] Determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Rules 0512).

[0204] Chromatographic conditions and system suitability test: Octadecyl bonded silica gel was used as filler (Agilent 5TC-C18 4.6×250 mm 5 μm); acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in Table 1; the flow rate was 1 ml per minute; the column temperature was 25° C.; and full wavelength scanning was performed.

[0205] Table 1 Chromatographic conditions Gradient 1

[0206]

[0207]

[0208] The summary table of the optimal absorption wavelengths of the reference solution is shown in Table 2.

[0209] Table 2 Summary of the best absorption wavelengths of reference solutions

[0210] Reference solution Peak time Optimum absorption wavelength (nm) Ferulic acid 34.661 218、236、322 Cinnamaldehyde 61.018 206、222、290 6-Gingerol 77.183 282 Ligustilide 84.164 208、282、326

[0211] Considering that the detection wavelength of ferulic acid content in Angelica sinensis in the Chinese Pharmacopoeia is 316nm, and the optimal wavelength of ferulic acid reference solution is around 316nm; the optimal wavelength of cinnamaldehyde reference solution is 290nm; the optimal wavelength of ligustilide reference is 210nm, considering that the elution time of 6-gingerol is close to that of ligustilide, and it has good absorption at 210nm; therefore, the detection wavelengths for content determination are determined to be 316nm for ferulic acid, 290nm for cinnamaldehyde, and 210nm for 6-gingerol and ligustilide. In view of the poor separation of the characteristic peaks at 30 to 40 minutes, it was further optimized to the gradient 2 in Table 3.

[0212] Table 3 Gradient 2

[0213]

[0214]

[0215] The results showed that under gradient 2, the separation and response values ​​of ferulic acid, cinnamaldehyde, 6-gingerol and ligustilide had reached the requirements of the liquid phase content determination spectrum; considering that the peak time of ligustilide was around 85 minutes and had returned to the starting gradient, it was further optimized to gradient 3 in Table 4.

[0216] After the above screening, the following chromatographic conditions for content determination were determined:

[0217] Determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Rules 0512).

[0218] Chromatographic conditions and system suitability test: Octadecyl bonded silica gel was used as filler (Agilent 5TC-C18 4.6×250mm 5μm); acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in Table 4; the flow rate was 1 ml per minute; the column temperature was 25°C; the detection wavelength was 316 nm from 0 to 60 min, 290 nm from 60 to 77 min, and 210 nm from 77 to 100 min. The theoretical plate number calculated based on the ferulic acid peak should be no less than 5000.

[0219] Table 4 Final elution gradient

[0220] Time (min) Acetonitrile (%) 0.1% Phosphoric acid (%) 0→10 5→6 95→94 10→12 6→12 94→88 12→20 12 88 20→21 12→16 88→84 21→38 16 84 38→48 16→20 84→80 48→52 20→25 80→75 52→58 25→29 75→71 58→62 29→32 71→68 62→80 32→56 68→44 80→83 56→74 44→26 83→88 74→85 26→15 88→89 85→5 15→95 89→100 5 95

[0221] The results show that under this gradient, the symmetry and separation of ferulic acid, cinnamaldehyde, 6-gingerol and ligustilide in the Chinese medicine composition granules containing angelica and cinnamon of the present invention are good, and have met the requirements for liquid phase content determination; therefore, this method is determined as the method for determining ferulic acid, cinnamaldehyde, 6-gingerol and ligustilide in the Chinese medicine composition granules containing angelica and cinnamon of the present invention.

[0222] 3. Preparation of reference sample test solution

[0223] According to the results of the research on the preparation process of the reference sample, the reference sample was stored in the form of a decoction, so the preparation method of the decoction test solution was investigated. Therefore, it was determined that 15g of the decoction was taken for the research.

[0224] 3.1. Investigation of extraction solvent

[0225] Six portions of the decoction were taken to investigate the effects of pure water, 50% methanol and methanol as extraction solvents on the determination of the content of the reference sample of the Chinese medicine composition granules containing angelica and cinnamon bark of the present invention.

[0226] Take 6 portions of the reference sample decoction, about 15g each, weigh accurately, place in a 25ml volumetric bottle, add pure water, 50% methanol, and appropriate amount of methanol, respectively, treat with ultrasound (500W, 40kHz) for 30 minutes, cool, adjust to volume, shake well, centrifuge at 5000rpm / min for 10min, filter the supernatant, and take the filtrate to obtain. The injection results are as follows:

[0227] Table 5 Summary of the content detection information of the Chinese medicine composition particles containing angelica and cinnamon treated with different extraction solvents

[0228]

[0229] Note: Content (mg / g) means the content per gram of decoction, the same below.

[0230] The results show that after the Chinese medicine composition particles containing angelica and cinnamon of the present invention are treated with different extraction solvents, the detected content is higher after being treated with methanol. Therefore, the processing solvent for content determination of the Chinese medicine composition particles containing angelica and cinnamon of the present invention is methanol.

[0231] 3.2. Extraction time investigation

[0232] Six portions of the decoction were taken to investigate the effects of extraction times of 20 minutes, 30 minutes, and 45 minutes on the content determination of the reference sample of the Chinese medicine composition granules containing angelica and cinnamon bark of the present invention.

[0233] Take 6 portions of the reference sample decoction, about 15g each, weigh accurately, place in a 25ml volumetric bottle, add an appropriate amount of methanol, treat with ultrasound (500W, 40kHz) for 20 minutes, 30 minutes, and 45 minutes respectively, cool, adjust to volume, shake well, centrifuge at 5000rpm / min for 10min, filter the supernatant, and take the filtrate to obtain. Injection, the results are as follows:

[0234] Table 6 Summary of content detection information of the Chinese medicine composition granules containing angelica and cinnamon at different extraction times of the present invention

[0235]

[0236] The results show that after the Chinese medicine composition particles containing Angelica sinensis and cinnamon of the present invention are treated with methanol at different extraction times, the detection values ​​are close when the extraction time is 20 to 45 minutes. In view of the fact that the detection value is slightly higher after 30 minutes of ultrasound, the processing time for content determination of the Chinese medicine composition particles containing Angelica sinensis and cinnamon of the present invention is 30 minutes.

[0237] 3.3. Sampling volume investigation

[0238] Six portions of the decoction were taken to investigate the effects of sampling amounts of 10 g, 15 g, and 20 g on the determination of the content of the reference sample of the Chinese medicine composition granules comprising angelica sinensis and cinnamon bark of the present invention.

[0239] Take 6 portions of the reference sample decoction, about 10g, 15g, and 20g each, weigh accurately, place in a 25ml volumetric flask, add appropriate amount of methanol, treat with ultrasound (500W, 40kHz) for 30 minutes, cool, make up to volume, shake well, centrifuge at 5000rpm / min for 10min, filter the supernatant, and take the filtrate to obtain the product.

[0240] The results show that after different sampling amounts of the Chinese medicine composition particles containing angelica and cinnamon of the present invention are treated with methanol, the detection values ​​of the sampling amount of 10 to 15 g are close, considering the peak area size of 6-gingerol and ligustilide; therefore, the sampling amount is determined to be 15 g.

[0241] 3.4. Confirmation of the preparation method of the reference sample solution

[0242] In summary, the preparation method of the reference sample test piece is determined as follows: take about 15 g of the reference sample decoction, accurately weigh it, put it in a 25 ml volumetric flask, add an appropriate amount of methanol, ultrasonically (500 W, 40 kHz) for 30 minutes, cool, make up to volume, shake well, centrifuge at 5000 rpm / min for 10 minutes, filter the supernatant, and take the filtrate to obtain the result.

[0243] 4. Methodological Validation

[0244] 4.1. Exclusivity

[0245] The blank solvent, the reference solution, the negative solutions of each medicinal flavor of the benchmark sample of the Chinese medicinal composition granules containing Angelica sinensis and cinnamon bark of the present invention, and the test solution of the benchmark sample of the Chinese medicinal composition granules containing Angelica sinensis and cinnamon bark of the present invention (2024082904-L) are injected for analysis. At the peak positions of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide, the negative and blank solvents shall not have interference. Figure 1 The present invention is a chromatogram of the content of a reference sample solution of a traditional Chinese medicine composition comprising angelica and cinnamon bark.

[0246] The blank solvent, reference solution, negative control solutions of each medicinal flavor and the reference sample solution of the Chinese medicine composition granules containing angelica and cinnamon of the present invention were sampled and analyzed. At the peak positions of ferulic acid, cinnamaldehyde, 6-gingerol and ligustilide, the blank solvent and the negative control solution had no interference and had strong specificity.

[0247] 4.2 Precision

[0248] 4.2.1 Repeatability

[0249] According to the above method for preparing the test solution (2024082904-L), 6 test solutions were prepared, the samples were tested, the chromatograms were recorded, and the contents of ferulic acid, cinnamaldehyde, 6-gingerol and ligustilide were calculated; if the RSD of the contents of ferulic acid, cinnamaldehyde, 6-gingerol and ligustilide was ≤6%, it indicated that the method had good repeatability.

[0250] The results showed that the RSD of ferulic acid, cinnamaldehyde, 6-gingerol and ligustilide contents were ≤6%, which indicated that the method had good repeatability according to the 9101 analytical method validation guidelines of the 2020 edition of the Chinese Pharmacopoeia.

[0251] 4.2.2 Intermediate precision

[0252] Two personnel prepared 6 test solutions (2024082904-L) according to the above test solution preparation method, and tested them on Agilent HPLC and Waters HPLC, respectively, and calculated the contents of ferulic acid, cinnamaldehyde, 6-gingerol and ligustilide. The RSD of the contents of ferulic acid, cinnamaldehyde, 6-gingerol and ligustilide was ≤6%, which indicated that the intermediate precision of the method was good.

[0253] The results showed that the RSD of ferulic acid, cinnamaldehyde, 6-gingerol and ligustilide in the injection results of Agilent HPLC and Waters HPLC was ≤6%; the intermediate precision of the content detection method was good.

[0254] 4.2.3 Instrument precision

[0255] Prepare a test solution (2024082904-L) according to the above test solution preparation method, inject 6 times continuously, record the chromatogram, calculate the retention time and peak area RSD values ​​of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide peaks; if the RSD of the relative retention time is ≤1%, and the RSD of the relative peak area is ≤3%, it means that the instrument precision is good.

[0256] The RSD of retention time of ferulic acid, cinnamaldehyde, 6-gingerol and ligustilide peaks was ≤1%, and the RSD of peak area was ≤3%, indicating good instrument precision.

[0257] 4.3 Accuracy

[0258] Take 9 portions of this product solution (2024082904-L), each portion is about 7.5g, accurately weighed, placed in 25ml volumetric flasks, add 1ml, 2ml, and 3ml of four reference solutions of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide respectively; prepare 3 portions in parallel; calculate the recovery rate of the reference solution; the recovery rate RSD of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide should be ≤6%, and the recovery rate is 80% to 115%, which means that the method has good accuracy. The concentration of ferulic acid reference solution is: 0.1141mg / ml; the concentration of cinnamaldehyde reference solution is: 0.1485mg / ml; the concentration of 6-gingerol reference solution is: 0.0840mg / ml; the concentration of ligustilide reference solution is: 0.1195mg / ml.

[0259] The results showed that the recovery rates of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide were RSD ≤ 6%, and the recoveries were all in the range of 80% to 115%; according to the 9101 Analytical Method Validation Guidelines of the 2020 edition of the "Chinese Pharmacopoeia", this method has good accuracy.

[0260] 4.4. Sensitivity (Limit of Quantitation and Limit of Detection)

[0261] The detection limit refers to the minimum content (concentration) in a sample that can be detected qualitatively but does not require accurate quantification. In chromatographic analysis, the signal-to-noise ratio (S / N) is generally used to define the detection limit. The signal-to-noise ratio is the ratio of the response value of the target in the sample to the baseline noise. It is generally believed that a signal-to-noise ratio greater than or equal to 3 can be used for qualitative analysis, and a signal-to-noise ratio greater than or equal to 10 can be used for quantitative analysis (RSD≤10.0%).

[0262] The results showed that the limits of quantification of ferulic acid were 1.3960μg / ml, the limits of quantification of cinnamaldehyde were 0.2200μg / ml, the limits of quantification of 6-gingerolide were 3818μg / ml, and the limits of quantification of ligustilide were 0.5961μg / ml.

[0263] The detection limits of ferulic acid were 0.4189 μg / ml, and those of cinnamaldehyde were 0.06601 μg / ml. The detection limits of 6-gingerol and ligustilide were 0.1146 g / ml and 0.1788 μg / ml, respectively.

[0264] 4.5. Linear Relationship Investigation

[0265] Prepare 6 different concentration levels of reference solution for determination. Plot the measured response signal as a function of the concentration of the substance to observe whether it is linear, and then use the least squares method for linear regression. The correlation coefficient r is not less than 0.9990, indicating that the method has good linearity.

[0266] The results showed that the linear correlation coefficients r of the contents of ferulic acid, cinnamaldehyde, 6-gingerol and ligustilide were all greater than 0.9990, indicating that this method had good linearity.

[0267] 4.6 Durability test

[0268] 4.6.1. Flow rate durability

[0269] Prepare 2 test solutions according to the above test solution (2024082904-L) preparation method, set the mobile phase flow rate to 0.9ml / min, 1.0ml / min, and 1.1ml / min, respectively, perform sample injection detection, record the chromatogram, and calculate the contents of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide; the relative average deviation of the contents of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide should be ≤6%, indicating that the flow rate durability is good.

[0270] The results showed that at flow rates of 0.9 ml / min, 1.0 ml / min and 1.1 ml / min, the relative average deviations of ferulic acid, cinnamaldehyde, 6-gingerol and ligustilide contents were all ≤6%, indicating that this content detection method has good flow rate durability.

[0271] 4.6.2 Column temperature durability

[0272] Prepare 2 test solutions according to the above test solution (2024082904-L) preparation method, set the column temperature to 20℃, 25℃, and 30℃ respectively, inject and detect, record the chromatogram, and calculate the contents of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide; the relative average deviation of the contents of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide should be ≤6%, which indicates that the column temperature durability is good.

[0273] The results showed that under different column temperature conditions, the relative average deviation of ferulic acid, cinnamaldehyde, 6-gingerol and ligustilide contents was ≤6%, indicating that the column temperature had good durability.

[0274] 4.6.3 Durability at different acid concentrations

[0275] According to the preparation method of the test solution (2024082904-L) above, two test solutions were prepared, and phosphoric acid solutions with concentrations of 0.09%, 0.10% and 0.11% were prepared respectively. The samples were injected for detection, and the chromatograms were recorded. The contents of ferulic acid, cinnamaldehyde, 6-gingerol and ligustilide were calculated; the relative average deviation of the contents of ferulic acid, cinnamaldehyde, 6-gingerol and ligustilide should be ≤6%, indicating that the durability of different acid concentrations is good.

[0276] The results showed that the relative average deviation of ferulic acid, cinnamaldehyde, 6-gingerol and ligustilide contents was ≤6% under different acid concentrations, indicating good durability under different acid concentrations.

[0277] 4.6.4. Durability of different chromatographic columns

[0278] Two test solutions (2024082904-L) were prepared according to the above test solution preparation method. Three types of chromatographic columns, Agilent 5TC-C18(2)250×4.6mm, were used for sample injection and detection, and the contents of ferulic acid, cinnamaldehyde, 6-gingerol and ligustilide were calculated. The relative average deviation of the contents of ferulic acid, cinnamaldehyde, 6-gingerol and ligustilide should be ≤6%, indicating that the durability of different chromatographic columns is good.

[0279] The results showed that the relative average deviation of the contents of ferulic acid, cinnamaldehyde, 6-gingerol and ligustilide in different chromatographic columns should be ≤6%, indicating that the durability of different chromatographic columns was good.

[0280] 4.7. Stability of test solution

[0281] Prepare 1 sample solution (2024082904-L) according to the above sample solution preparation method, and inject and test at 0, 3, 6, 10, 15, 18, 24, 36, 48, and 62 hours respectively. Record the chromatogram and calculate the content of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide; the RSD of the content of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide should be ≤6%, indicating that the stability of the sample solution is good.

[0282] According to the above experimental results, the RSD of the contents of ferulic acid, cinnamaldehyde, 6-gingerol and ligustilide in the test solution within 62 hours is ≤6%, and the test solution has good stability.

[0283] 5. Determination of the content determination method of ferulic acid, cinnamaldehyde, 6-gingerol and ligustilide in the benchmark samples

[0284] Based on the above research results, according to the 2020 edition of the General Chapter 9101 Analytical Method Validation Guidance of the Chinese Pharmacopoeia, the content determination indicators of this method are determined to be ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide. Through methodological validation, the specificity, accuracy, precision, sensitivity, linearity, column temperature, flow rate, different acid concentrations, different chromatographic columns, and stability of the test solution of this content detection method all meet the requirements; therefore, this detection method is better, as follows.

[0285] Determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 General Rules 0512)

[0286] Chromatographic conditions and system suitability test: Octadecyl bonded silica gel was used as filler (Agilent 5TC-C18 4.6×250mm 5μm); acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in Table 4; the flow rate was 1 ml per minute; the column temperature was 25°C; the detection wavelength was 316 nm from 0 to 60 min, 290 nm from 60 to 77 min, and 210 nm from 77 to 100 min. The theoretical plate number calculated based on the ferulic acid peak should be no less than 5000.

[0287] Preparation of reference solution: Take appropriate amounts of ferulic acid, paeoniflorin, cinnamaldehyde, 6-gingerol, and ligustilide reference substances, weigh accurately, and add methanol to prepare a mixed solution containing 18 μg of ferulic acid, 13 μg of cinnamaldehyde, 6 μg of 6-gingerol, and 18 μg of ligustilide per 1 ml, as the reference solution.

[0288] Preparation of test solution: Take about 15 g of the reference sample decoction, weigh it accurately, put it in a 25 ml volumetric flask, add an appropriate amount of methanol, sonicate (500 W, 40 kHz) for 30 minutes, cool, make up to volume, shake well, centrifuge at 5000 rpm / min for 10 minutes, filter the supernatant, and take the filtrate to obtain the test solution.

[0289] Determination method: Accurately pipette 10μl of reference solution and test solution, inject into liquid chromatograph, and determine.

[0290] The embodiments of the present invention are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, changes or deformations made by those skilled in the art based on the ideas of the present invention, the specific implementation methods and application scope of the present invention, all belong to the scope of protection of the present invention. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A method for determining the content of a Chinese medicine composition containing angelica and cinnamon, characterized in that: The determination method comprises the following steps: Take the test solution and the reference solution of the Chinese medicine composition for testing. The chromatographic conditions of the detection are as follows: a chromatographic column with octadecyl bonded silica as filler is used, mobile phase A is selected from one or more of acetonitrile, methanol and tetrahydrofuran, mobile phase B is an acid aqueous solution, an alkaline aqueous solution and / or a buffered saline solution, and the gradient elution program is as follows: 0-10 min, 95% B→94% B; 10-12 min, 94% B→88% B; 12-20 min, 88% B; 20-21 min, 88% B→84% B; 21-38 min, 84% B; 38-48 min, 84% B→80% B; 48-52 min, 84% B→80% B; in, 80% B→75% B; 52~58min, 75% B→71% B; 58~62min, 71% B→68% B; 62~80min, 68% B→44% B; 80~83min, 44% B→26% B; 83~88min, 26% B→15% B; 88~89min, 15% B→95% B; 89~100min, 95% B; flow rate is 0.5~1.5ml / min, column temperature is 10~40℃, detection wavelength is 180~360nm, injection volume is 1~20μl; According to the test results, obtaining the content information of the reference substance of the Chinese medicine composition; The Chinese medicine composition includes angelica, cinnamon bark, white peony root, ginger, jujube and licorice, and the reference substances are ferulic acid, cinnamaldehyde, 6-gingerol and ligustilide.

2. The measuring method according to claim 1, characterized in that The licorice is stir-fried licorice.

3. The measuring method according to claim 1, characterized in that The information is the content of the reference substance in the Chinese medicine composition calculated according to the external standard method based on the corresponding peak areas in the recorded chromatogram of the test solution of the Chinese medicine composition and the chromatogram of the reference substance solution; Preferably, the linear equation of ferulic acid is y=47.474x+18.878, R 2 =1.0000; Preferably, the linear equation of cinnamaldehyde is y=90.332x+44.58, R 2 =1.0000; Preferably, the linear equation of 6-gingerol is y=25.992x+8.2874, R 2 =0.9999; Preferably, the linear equation of ligustilide is y=10.805x+10.113, R 2 =0.9999.

4. The measuring method according to claim 1, characterized in that The mass ratio of the angelica sinensis, the cinnamon bark, the white peony root, the ginger, the jujube and the liquorice is (45-60):(30-50):(70-90):(30-50):(25-45):(20-40); Preferably, the mass ratio of the angelica sinensis, the cinnamon bark, the white peony root, the ginger, the jujube and the liquorice is about 55.2: about 41.4: about 82.8: about 41.4: about 36: about 27.6; The preparation method of the Chinese medicine composition test solution comprises: taking an appropriate amount of a decoction of the Chinese medicine composition containing angelica and cinnamon, placing the decoction in a container, adding a solvent, ultrasonically treating the decoction for a period of time, fixing the volume, shaking, centrifuging, filtering the supernatant, and taking the filtrate to obtain the Chinese medicine composition test solution; Preferably, the container is a volumetric flask; Preferably, the solvent is an alcohol, such as methanol; Preferably, the mass volume ratio between the decoction of the traditional Chinese medicine composition and the solvent is 0.4 to 0.8, such as about 0.6, in units of g / ml; Preferably, the mass of the decoction of the Chinese medicine composition is 5 to 30 g, for example, about 15 g; More preferably, the power of the ultrasonic treatment is 400-600W, for example about 500W; More preferably, the frequency of the ultrasonic treatment is 30 to 50 kHz, such as about 40 kHz; More preferably, the ultrasonic treatment time is 20 to 40 min, for example about 30 min; Preferably, the centrifugal speed is 4000-6000 rpm / min, for example, about 5000 rpm / min; Preferably, the centrifugation time is 5 to 15 minutes, for example, about 10 minutes; Particularly preferably, the preparation method of the decoction of the Chinese medicine composition containing angelica and cinnamon comprises: weighing appropriate amounts of angelica, cinnamon, white peony root, ginger, jujube and liquorice, crushing them into coarse particles, placing them in a container, soaking them in water, boiling them until boiling for a period of time, then keeping them slightly boiling for a period of time, filtering the filtrate with a filter while it is hot to obtain the decoction of the Chinese medicine composition containing angelica and cinnamon; Particularly preferably, the volume / mass ratio of the water to the Chinese medicinal composition comprising angelica and cinnamon is 5 to 10, for example, about 7; Particularly preferably, the container is a ceramic medicine decoction pot or an electric ceramic stove; Particularly preferably, the soaking time is 10 to 100 minutes, for example, about 45 minutes; Particularly preferably, the decocting is covered when boiling; Particularly preferably, the power of the container during boiling is 1000-3000W, for example about 2000W; Particularly preferably, the boiling time is 5 to 15 minutes, for example about 10 minutes; Particularly preferably, the power of the container at a slight boiling point is 500 to 1500 W, for example about 900 W; Particularly preferably, the slight boiling time is 10 to 100 minutes, for example, about 60 minutes; Particularly preferably, the volume / volume ratio of the decoction of the Chinese medicinal composition comprising angelica and cinnamon to the water is 0.1 to 0.5, for example, about 0.3; Particularly preferably, the filter is a 200-mesh nylon filter.

5. The measuring method according to claim 1, characterized in that The preparation method of the reference solution comprises: weighing appropriate amounts of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide reference substances, and adding methanol to prepare the reference solution with the concentrations of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide being 1 to 100 μg / ml respectively; Preferably, the concentration of ferulic acid in the reference solution is about 18 μg / ml; Preferably, the concentration of cinnamaldehyde in the reference solution is about 13 μg / ml; Preferably, the concentration of 6-gingerol in the reference solution is about 6 μg / ml; Preferably, the concentration of ligustilide in the control solution is about 18 μg / ml.

6. The measuring method according to claim 1, characterized in that The flow rate of the HPLC detection is 0.8 to 1.2 ml / min, for example, about 1.0 ml / min; Preferably, the column temperature is 20-30°C, such as about 25°C; Preferably, between the 0th minute and the 60th minute, the detection wavelength is 280 to 340 nm, for example, about 316 nm; Preferably, between the 60th minute and the 77th minute, the detection wavelength is 260 to 320 nm, for example, about 290 nm; Preferably, between the 77th minute and the 100th minute, the detection wavelength is 190 to 230 nm, for example, about 210 nm; Preferably, the injection volume is 5 to 15 μl, for example, about 10 μl; More preferably, the theoretical plate number of the chromatographic peak corresponding to the ferulic acid is not less than 5000; Preferably, the chromatographic column is an Agilent 5TC-C18 chromatographic column; Even more preferably, the specifications of the chromatographic column are: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm.

7. The measuring method according to claim 1, characterized in that Mobile phase A was acetonitrile; Preferably, the acid aqueous solution, base aqueous solution and / or buffered saline solution is selected from one or more of weak acids and their salts, weak bases and their salts of different concentrations; Preferably, the acid aqueous solution, alkali aqueous solution and / or buffered saline solution is selected from formic acid, glacial acetic acid, phosphoric acid, trifluoroacetic acid, formic acid and ammonium formate, acetic acid and sodium acetate, acetic acid and ammonium acetate, disodium hydrogen phosphate and sodium dihydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and citric acid, citric acid and sodium citrate, glycine and hydrochloric acid, or phthalic acid and hydrochloric acid in different concentrations; More preferably, the acid aqueous solution is a 0.01% to 1% acid aqueous solution; More preferably, the acid aqueous solution is a 0.01% to 1% phosphoric acid aqueous solution; More preferably, the aqueous acid solution is an aqueous solution of about 0.1% phosphoric acid; Also preferably, the buffered saline solution is an acetate solution and / or an acetate solution; Also preferably, the pH value of the buffered saline solution is not greater than 7.

0.

8. The measuring method according to claim 1, characterized in that The detection limit of ferulic acid is about 0.4189 μg / ml; Preferably, the detection limit of cinnamaldehyde is about 0.06601 μg / ml; Preferably, the detection limit of 6-gingerol is about 0.1146 μg / ml; Preferably, the detection limit of ligustilide is about 0.1788 μg / ml.

9. The measuring method according to claim 1, characterized in that The limit of quantification of the ferulic acid is about 1.3960 μg / ml; Preferably, the limit of quantification of cinnamaldehyde is about 0.2200 μg / ml; Preferably, the limit of quantification of 6-gingerol is about 0.3818 μg / ml; Preferably, the limit of quantification of ligustilide is about 0.5961 μg / ml.

10. Use of the determination method according to any one of claims 1 to 9 in quality detection and / or quality evaluation and / or quality control of a traditional Chinese medicine composition comprising angelica sinensis and cinnamon bark.

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