A method for detecting a traditional Chinese medicine composition

Abstract

The application discloses a method for detecting the content of a traditional Chinese medicine composition, wherein the traditional Chinese medicine composition comprises angelica, radix cyathulae, cistanche, alisma, radix clematidis and fructus aurantii; the method comprises: taking a traditional Chinese medicine composition test sample solution to perform HPLC detection, and the chromatographic conditions comprise: adopting a C18 chromatographic column, taking acetonitrile as mobile phase A, and taking a 0.1% concentration phosphoric acid aqueous solution as mobile phase B; the elution in the chromatographic conditions of the HPLC detection is gradient elution, and the gradient elution program is as follows: 0-35 min, the mobile phase is 12% A, 35-40 min, the mobile phase is 12%-16% A, 40-60 min, the mobile phase is 16%-16.5% A, 60-70 min, the mobile phase is 16.5%-20% A, 70-75 min, the mobile phase is 20% A, 75-76 min, the mobile phase is 20%-90% A, 76-90 min, the mobile phase is 90% A. The content of the chemical components contained in the traditional Chinese medicine composition is detected by the method, a content determination method of the granules is established, and the quality of the granules can be comprehensively reflected, so that the whole preparation quality can be described and evaluated.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of analytical chemistry, in particular to a method for detecting the content of a traditional Chinese medicine composition. BACKGROUND

[0002] The prescription is composed of six medicinal materials, namely, Angelica sinensis, Radix Achyranthis Bidentatae, Cistanche deserticola, Alisma orientalis, Cimicifuga foetida and Fructus Aurantii. Angelica sinensis is the monarch drug, which is good at tonifying blood and moistening dryness, moistening intestines and promoting defecation. Cistanche deserticola and Radix Achyranthis Bidentatae are the ministerial drugs, Cistanche deserticola has the effects of warming kidney and nourishing essence, and Radix Achyranthis Bidentatae can tonify liver and kidney, strengthen waist and knees, and is good at downward movement. Fructus Aurantii can promote defecation by promoting qi and relaxing the bowels, and Alisma orientalis can drain dampness and remove kidney turbidity. A small amount of Cimicifuga foetida is used to promote the upward movement of yang, and the upward movement of yang can promote the downward movement of yin, so as to promote defecation. The above are the auxiliary drugs. The prescription has the effects of tonifying blood and warming kidney to treat the root, and moistening intestines and promoting defecation to treat the branch, and can treat the root and branch at the same time. The prescription has the compatibility characteristics of promoting defecation in tonifying and promoting downward movement in upward movement.

[0003] Traditional Chinese medicines and their preparations are complex multi-component systems, so the quality thereof should be evaluated by a method suitable therefor. Through prediction analysis of the effective material components and potential quality control indexes of the granules of the composition of the present application for treating slow transit constipation, it is indicated that the main effective material components include ferulic acid from the monarch drug Angelica sinensis, verbascoside and pinoresinol diglcoside from the ministerial drug Cistanche deserticola, isoferulic acid from the auxiliary drug Cimicifuga foetida, naringin and neohesperidin from Fructus Aurantii. In addition, through analysis and identification of the blood components in the subsequent research process, the prototype component of isoacteoside with the effects of immunomodulation and antioxidation is also detected. Ligustilide I is a volatile oil component of Angelica sinensis, and the volatile oil components of Angelica sinensis can stimulate the smooth muscle of the stomach and intestines, reduce the residual rate in the stomach, promote gastric emptying, increase the discharge rate of the stomach and intestines, and promote the peristalsis of the stomach and intestines, thereby playing the effect of moistening intestines and promoting defecation.

[0004] However, there is no method for detecting the above components in the prior art. In order to better control the quality of the preparation, a method for simultaneously determining the contents of pinoresinol diglcoside, ferulic acid, isoferulic acid, verbascoside, ligustilide I, isoacteoside, naringin and neohesperidin is established by combining the prescription process, blood component research and main index components of each medicinal material of the prescription, thereby providing a reference for comprehensively evaluating the quality of the preparation. SUMMARY

[0005] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0006] A method for detecting the content of a traditional Chinese medicine composition, characterized by taking a traditional Chinese medicine composition test sample solution for HPLC detection, and the chromatographic conditions of HPLC detection include:

[0007] The HPLC detection chromatographic conditions are gradient elution, the gradient elution program is: 0-35 min, the mobile phase is 12% A, 35-40 min, the mobile phase is 12%-16% A, 40-60 min, the mobile phase is 16%-16.5% A, 60-70 min, the mobile phase is 16.5%-20% A, 70-75 min, the mobile phase is 20% A, 75-76 min, the mobile phase is 20%-90% A, 76-90 min, the mobile phase is 90% A.

[0008] Specifically, the traditional Chinese medicine composition comprises angelica, radix astragali, cistanche, alisma, and citri fructus.

[0009] Specifically, the traditional Chinese medicine composition can be solid, liquid or external preparation; for example: decoction, granules, capsules, tablets, pills, oral liquid, tincture, syrup, suppository, gel, spray, injection, etc.

[0010] Further, when the traditional Chinese medicine composition is granules, the preparation method comprises: angelica 1066g, radix astragali 533g, cistanche 666g, citri fructus 266g, alisma 400g, and cimicifuga 186g, 8 times the amount of water is added, reflux extraction is performed for 40 minutes, filtration is performed, the filtrate is concentrated to a relative density of 1.05-1.10 (60-70°C) of extract, spray drying is performed, and the total mixture is pulverized into fine powder, 5g stevioside is added, and granulation is performed.

[0011] Alternatively, the preparation of the test solution is as follows: about 1g of the product is finely ground, accurately weighed, placed in a conical flask with a plug, accurately added with 50ml of 25% methanol, tightly sealed, weighed, ultrasonically treated (40KHz, 500W) for 30 minutes, cooled, weighed again, supplemented with the lost weight with 25% methanol, shaken uniformly, centrifuged, and the supernatant is obtained.

[0012] Further, the chromatographic conditions comprise: a flow rate of 0.9-1.05mL / min, a column temperature of 28-32°C, and a detection wavelength of 280-285nm.

[0013] Further, the chromatographic conditions comprise: a flow rate of 1.0mL / min, a column temperature of 30°C, and a detection wavelength of 283nm.

[0014] Specifically, the C18 chromatographic column is selected from Waters Symmetry C 18 (4.6×250mm, 5μm), Agilent ZORBAX SB-C 18 (4.6×250mm, 5μm).

[0015] Further, the detection method further comprises detection of the control samples including ponicidin, ferulic acid, isoferulic acid, verbascoside, senkyunol I, epipachymarin, naringin, neohesperidin.

[0016] Specifically, the control samples are prepared into a solution for detection, and the method for preparing the control samples into a solution comprises: taking ponicidin, ferulic acid, isoferulic acid, verbascoside, naringin and neohesperidin, and adding 50% methanol to obtain a single-label or mixed-label solution containing 200 μg, 10 μg, 7 μg, 30 μg, 15 μg, 20 μg, 100 μg, and 70 μg of ponicidin, ferulic acid, isoferulic acid, verbascoside, senkyunol I, epipachymarin, naringin, and neohesperidin per 1 ml.

[0017] By establishing a standard curve of one or more control samples, the composition ingredient information or ingredient content information is ultimately obtained.

[0018] The present application provides a method for detecting the ingredient content of a composition comprising angelica, radix astragali, cistanche, alisma, and citri fructus, and the detection method of the present application can accurately determine the ingredient information or ingredient content information therein. After systematic methodological investigation and verification, it is shown that the method has strong specificity, good durability, repeatability and accuracy, and can accurately determine the ingredient content, so as to objectively evaluate the quality of the preparation. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Chromatogram for investigating gradient elution procedure for detecting ingredient content of traditional Chinese medicine composition;

[0020] Figure 2 Chromatogram for investigating specificity of traditional Chinese medicine composition content detection.

[0021] 1. ponicidin, 2. ferulic acid, 3. isoferulic acid, 4. verbascoside, 5. senkyunol I, 6. epipachymarin, 7. naringin, and 8. neohesperidin. DETAILED DESCRIPTION

[0022] The present application discloses a method for detecting the ingredient content of a composition comprising angelica, radix astragali, cistanche, alisma, and citri fructus, and those skilled in the art can refer to the content herein and appropriately improve the process parameters for implementation. It is particularly important to note that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The method and application of the present application have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0023] Unless otherwise specified, the reagents, instruments used in the detection method provided by the present application can be purchased from the market:

[0024] Instruments and reagents

[0025] Mettler Toledo AL204 electronic analytical balance (Mettler Toledo, Switzerland);

[0026] Mettler Toledo XP6 electronic analytical balance (Mettler Toledo, Switzerland);

[0027] KQ-500DB digital ultrasonic cleaning instrument (Kunshan Ultrasonic Instrument Co., Ltd.);

[0028] Agilent 1200 high performance liquid chromatograph (Agilent Technologies Co., Ltd.);

[0029] Waters Arc-2998 high performance liquid chromatograph (Waters Co., Ltd.);

[0030] Thermo Ultimate 3000 high performance liquid chromatograph (Thermo Fisher Scientific Co., Ltd.);

[0031] Milli-Q ultrapure water instrument (Millipore, USA);

[0032] Sample: composition sample (produced by Jiangsu Kangle Pharmaceutical Co., Ltd., batch number 210401);

[0033] Reference substance: matricarynoside reference substance (China Institute for Drug Control, batch number 111670-201907, purity 91.8%);

[0034] Ferulic acid reference substance (China Institute for Drug Control, batch number 110773-201915, purity 99.4%);

[0035] Isoferulic acid reference substance (China Institute for Drug Control, batch number 111698-201904, purity 99.3%);

[0036] Mucin flower glycoside reference substance (China Institute for Drug Control, batch number 111530-201914, purity 95.2%);

[0037] Ligustilide I reference substance (China Institute for Drug Control, batch number 112071-202101, purity 99.2%);

[0038] Isovistin reference substance (Chengdu Man Sit Biological Technology Co., Ltd., batch number MUST-12081001);

[0039] Naringin reference standard (China National Institutes for Food and Drug Control, batch number 110722-201815, purity 91.7%);

[0040] Neohesperidin (China National Institutes for Food and Drug Control, batch number 111857-201804, purity 99.4%).

[0041] Reagents: Methanol and acetonitrile were of chromatographic grade (Tianhex Corporation, USA); water was ultrapure water; all other reagents were of analytical grade.

[0042] 1. Chromatographic conditions

[0043] 1.1 Selection of detection wavelength

[0044] By performing full-wavelength scanning on reference solutions of echinacoside, ferulic acid, isoflavonic acid, verbascoside, ligusticin I, isocitric acid, naringin, and neohesperidin, the absorption wavelength for multi-component determination was determined to be 283 nm based on the maximum absorption wavelength of each component.

[0045] 1.2 Selection of Chromatographic System

[0046] Different mobile phase compositions and elution programs were investigated. Under the acetonitrile-0.1% phosphoric acid system, the chromatographic peaks of echinacoside, ferulic acid, isoflavone, verbascoside, ligustilide I, isocitric acid, naringin, and neohesperidin showed good separation. Therefore, acetonitrile-0.1% phosphoric acid was selected as the elution system, with gradient elution. Other chromatographic conditions: the flow rate for HPLC detection was 1.0 mL / min, and an Agilent ZORBAX SB C14 micrometer was used. 18 The column temperature was 30℃, and the injection volume was 10 μl. Results are shown in Tables 1-4. Figure 1 (1. Echinacoside, 2. Ferulic acid, 3. Isofructic acid, 4. Verbascoside, 5. Ligusticin I, 6. Iso-cicadin, 7. Naringin, 8. Neohesperidin).

[0047] Table 1 Gradient elution program 1

[0048]

[0049] Result: Peak 4, verbascoside, merged with its adjacent peak, resulting in low peak purity. The method was inappropriate.

[0050] Table 2 Gradient elution procedure 2.

[0051]

[0052]

[0053] Results: Peak 3 of isoferulic acid was combined with the following chromatographic peak, peaks 5 and 6 could not be distinguished, and the purity of the chromatographic peak was low. The method was not suitable.

[0054] Table 3 Gradient elution program 3

[0055]

[0056] Results: The separation degree of each chromatographic peak was good, the purity was high, and the elution gradient was ideal.

[0057] Table 4 Gradient elution program 4

[0058]

[0059] Results: Peak 4 had poor separation degree with the adjacent chromatographic peak after it, and peak 7 of naringin had poor separation degree with the adjacent chromatographic peak before it. The method was not suitable.

[0060] By investigating different elution gradients, the elution program of Table 3 was the best. The separation degree of echinacoside, ferulic acid, isoferulic acid, verbascoside, senkyunolide I, epipachymoside, naringin and neohesperidin in the test sample chromatogram was greater than 1.5, which reached baseline separation, and the symmetry factor was 1.0. Therefore, gradient elution program 3 was selected.

[0061] The finally established detection condition was as follows: acetonitrile was used as mobile phase A, and 0.1% concentration of phosphoric acid aqueous solution was used as mobile phase B. The elution in the chromatographic condition of the HPLC detection was gradient elution. The gradient elution program was as follows: 0-35 min, the mobile phase was 12% A, 35-40 min, the mobile phase was 12%-16% A, 40-60 min, the mobile phase was 16%-16.5% A, 60-70 min, the mobile phase was 16.5%-20% A, 70-75 min, the mobile phase was 20% A, 75-76 min, the mobile phase was 20%-90% A, 76-90 min, the mobile phase was 90% A. The flow rate of the HPLC detection was 1.0 mL / min, the column temperature was 30°C, the injection amount was 10 μl, and the wavelength was 283 nm. 18

[0062] 1.3 System suitability test

[0063] 1.3.1 Preparation of the control solution

[0064] ​Take Ginkgo biloba L. flavonoid glycosides, ferulic acid, isoferulic acid, verbascoside, chuanxiong lactone I, isoacteoside, naringin and neohesperidin control a proper amount, accurately weigh, add 50% methanol to prepare 1 ml containing Ginkgo biloba L. flavonoid glycosides, ferulic acid, isoferulic acid, verbascoside, chuanxiong lactone I, isoacteoside, naringin and neohesperidin 200 μg, 10 μg, 7 μg, 30 μg, 15 μg, 20 μg, 100 μg, 70 μg respectively.

[0065] 1.3.2 Preparation of test solution

[0066] Take sample particles 1 g, accurately weigh, put into a conical flask with a plug, accurately add 25% methanol 50 ml, tightly plug, weigh, ultrasonic treatment (40 KHz, 500 W) for 30 minutes, cool, weigh again, make up the weight loss with 25% methanol, shake well, centrifuge, take the supernatant.

[0067] 1.3.3 Determination

[0068] Accurately take 10 μl of Ginkgo biloba L. flavonoid glycosides, ferulic acid, isoferulic acid, verbascoside, chuanxiong lactone I, isoacteoside, naringin and neohesperidin control solution, inject into high performance liquid chromatograph, continuously repeat injection for 6 times, calculate the relative standard deviation of peak area measurement value; accurately take 10 μl of test solution, inject into high performance liquid chromatograph, detect according to the chromatographic conditions screened before, continuously repeat injection for 6 times, calculate the theoretical plate number, separation degree and tailing factor of the components to be measured.

[0069] 1.3.4 Results

[0070] Under the above chromatographic conditions, the separation degree of Ginkgo biloba L. flavonoid glycosides, ferulic acid, isoferulic acid, verbascoside, chuanxiong lactone I, isoacteoside, naringin and neohesperidin in the test sample chromatogram and the adjacent chromatographic peaks is greater than 1.5, reaching baseline separation, the symmetry factor is 1.0, the relative standard deviation of the chromatographic system repeatability is less than 1%, the theoretical plate number calculated by Ginkgo biloba L. flavonoid glycosides peak is greater than 10000, in order to ensure the accuracy of quantitative analysis, it is specified that the theoretical plate number calculated by Ginkgo biloba L. flavonoid glycosides peak should not be less than 5000.

[0071] 1.4 Durability test

[0072] According to the selected conditions, the detection wavelength, flow rate, column temperature and other factors in the chromatographic conditions were investigated, the results showed that slight changes in detection wavelength, flow rate and column temperature would not have a significant impact on the analysis results of Ginkgo biloba L. flavonoid glycosides, ferulic acid, isoferulic acid, verbascoside, chuanxiong lactone I, isoacteoside, naringin and neohesperidin, the test results showed that the content determination method had good durability. The results are shown in Table 5.

[0073] Table 5 Results of chromatographic condition durability test

[0074]

[0075] The same batch of granules was determined and calculated by using two kinds of chromatographic columns. The results showed that different brands of chromatographic columns had no significant effect on the determination results of pinyelin, ferulic acid, isoferulic acid, verbascoside, senkyunol I, isoacteoside, naringin and neohesperidin. The results are shown in Table 6.

[0076] Table 6 Test results of column durability

[0077]

[0078] The same batch of granules was determined and calculated by using two kinds of chromatographic columns. The results showed that different brands of chromatographic columns had no significant effect on the determination results of pinyelin, ferulic acid, isoferulic acid, verbascoside, senkyunol I, isoacteoside, naringin and neohesperidin. The results are shown in Table 6.

[0079] Table 7 Test results of instrument durability

[0080]

[0081] 1.6 Specificity test

[0082] 1.6.1 Preparation of reference solution

[0083] An appropriate amount of pinyelin, ferulic acid, isoferulic acid, verbascoside, senkyunol I, isoacteoside, naringin and neohesperidin reference substances were precisely weighed and dissolved in 50% methanol to prepare a mixed reference solution containing 200 μg of pinyelin, 10 μg of ferulic acid, 7 μg of isoferulic acid, 15 μg of verbascoside, 20 μg of senkyunol I, 30 μg of isoacteoside, 100 μg of naringin and 70 μg of neohesperidin per 1 ml.

[0084] 1.6.2 Preparation of test solution

[0085] 1 g of the composition granules was precisely weighed and placed in a conical flask with a stopper. 50 ml of 25% methanol was precisely added, tightly sealed, weighed, and treated with ultrasound (40 KHz, 500 W) for 30 minutes. After cooling, the weight was re-weighed, and the lost weight was made up with 25% methanol. After shaking and centrifugation, the supernatant was obtained.

[0086] 1.6.3 Preparation of negative test solution

[0087] 1.6.3.1 Preparation of negative test solution of Cistanche

[0088] Take angelica, cow knee, jujube, alisma, cimicifuga, according to the composition of granules preparation process into negative preparation, again according to the test solution preparation method with law prepared Cistanche negative test solution.

[0089] 1.6.3.2 Jujube negative test solution preparation

[0090] Take Cistanche, angelica, cow knee, alisma, cimicifuga, according to the composition of granules preparation process into negative preparation, again according to the test solution preparation method with law prepared Jujube negative test solution.

[0091] 1.6.3.3 Cimicifuga negative test solution preparation

[0092] Take Cistanche, angelica, cow knee, jujube, alisma, according to the composition of granules preparation process into negative preparation, again according to the test solution preparation method with law prepared Cimicifuga negative test solution.

[0093] 1.6.3.4 Angelica negative test solution preparation

[0094] Take Cistanche, cow knee, jujube, alisma, cimicifuga, according to the composition of granules preparation process into negative preparation, again according to the test solution preparation method with law prepared Angelica negative test solution.

[0095] 1.6.3.5 Cimicifuga and Angelica negative test solution preparation

[0096] Take Cistanche, cow knee, jujube, alisma, according to the composition of granules preparation process into negative preparation, again according to the test solution preparation method with law prepared Angelica and Cimicifuga negative test solution.

[0097] 1.6.4 determination

[0098] Precise suction of control solution, test solution, negative test solution each 10 μl, inject high performance liquid chromatograph, determination, the results show that the negative test solution has no interference on Echinacoside, Ferulic acid, Isoferulic acid, Verbascoside, I, Isocimifugoside, Naringin and Neohesperidin. Results see attached Figure 2 .

[0099] 1.7 peak purity check

[0100] The purity of Echinacoside, Ferulic acid, Isoferulic acid, Verbascoside, I, Isocimifugoside, Naringin and Neohesperidin in the test solution chromatogram was checked by diode array detector. The purity of each chromatographic peak in the test solution chromatogram met the requirements.

[0101] 1.8 linear relationship investigation

[0102] Take Ginkgo biloba Linn. glycoside, ferulic acid, isoferulic acid, verbascoside, senkyunolide I, isoacteoside, naringin and neohesperidin control substance appropriate amount, precision weighing, respectively add 50% methanol to prepare containing Ginkgo biloba Linn. glycoside 2101.76 μg / ml, ferulic acid 82.90 μg / ml, isoferulic acid 60.92 μg / ml, verbascoside 251.99 μg / ml, senkyunolide I 59.34 μg / ml; isoacteoside 73.55 μg / ml, naringin 1039.88 μg / ml, neohesperidin 608.33 μg / ml control substance stock solution. Dilute in proportion, precision pipette control substance stock solution and dilution solution each 10 μl, inject high performance liquid chromatograph, record chromatographic peak area. With concentration (μg / ml) as abscissa (X), peak area as ordinate (Y), draw standard curve, calculate regression equation and correlation coefficient: Ginkgo biloba Linn. Y=14816X+71869 (r=1), ferulic acid Y=51790X+13950 (r=0.9999), isoferulic acid Y=45796X+1395.6 (r=0.9999), verbascoside Y=18192X+10537 (r=1), senkyunolide I Y=45609X+1353.7 (r=1), isoacteoside Y=51369X+122.76 (r=1), naringin Y=17368X+46503 (r=1) and neohesperidin Y=18624X+29359 (r=1); The results show that Ginkgo biloba Linn. has a good linear relationship between the concentration of 32.84-2101.76 μg / ml; Ferulic acid has a good linear relationship between the concentration of 1.30-41.45 μg / ml; Isoferulic acid has a good linear relationship between the concentration of 1.90-30.46 μg / ml; Verbascoside has a good linear relationship between the concentration of 3.94-126.00 μg / ml; Senkyunolide I has a good linear relationship between the concentration of 1.85-29.67 μg / ml; Isoacteoside has a good linear relationship between the concentration of 2.30-36.78 μg / ml; Naringin has a good linear relationship between the concentration of 32.50-519.94 μg / ml; Neohesperidin has a good linear relationship between the concentration of 19.01-304.16 μg / ml.

[0103] 1.9 Recovery test

[0104] Take the known content of the sample (11.853 mg / g of ginsenoside, 0.463 mg / g of ferulic acid, 0.304 mg / g of isoferulic acid, 1.407 mg / g of verbascoside, 0.205 mg / g of senkyunol I, 0.407 mg / g of isoacteoside, 6.027 mg / g of naringin, and 3.135 mg / g of neohesperidin) about 0.5 g, accurately weigh 9 portions, place in a conical flask with a plug, 3 portions in a group, add 0.5 ml, 1.0 ml, and 1.5 ml of the mixed reference solution (6.406 mg / ml of ginsenoside, 0.245 mg / ml of ferulic acid, 0.161 mg / ml of isoferulic acid, 0.772 mg / ml of verbascoside, 0.107 mg / ml of senkyunol I, 0.211 mg / ml of isoacteoside, 3.259 mg / ml of naringin, and 1.691 mg / ml of neohesperidin), respectively, and then determine according to the preparation method of the test sample solution. The average recovery of ginsenoside is 99.9%, and the RSD is 0.70%; the average recovery of ferulic acid is 100.7%, and the RSD is 0.78%; the average recovery of isoferulic acid is 94.9%, and the RSD is 2.96%; the average recovery of verbascoside is 103.1%, and the RSD is 1.03%; the average recovery of senkyunol I is 95.2%, and the RSD is 2.43%; the average recovery of isoacteoside is 97.3%, and the RSD is 1.66%; the average recovery of naringin is 102.0%, and the RSD is 0.70%; and the average recovery of neohesperidin is 102.0%, and the RSD is 1.14%. The test results show that the method has good accuracy.

[0105] Preparation Example

[0106] Angelica 1066 g, Radix Hedysari 533 g, Cistanche 666 g, Fructus Aurantii 266 g, Alisma 400 g, and Radix Coptidis 186 g are added with 8 times the amount of water, reflux extracted for 40 minutes, filtered, and the filtrate is concentrated to an extract with a relative density of 1.05-1.10 (60-70 °C), spray dried, and pulverized into fine powder. The spray-dried fine powder is added with an appropriate amount of dextrin and 5 g of steviol glycoside, granulated, and 1000 g is obtained.

[0107] The above is only a preferred embodiment of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered within the protection scope of the present application.

Claims

1. A method for detecting the content of a traditional Chinese medicine composition, characterized in that, The test sample solution of the traditional Chinese medicine composition is detected by HPLC, and the HPLC detection chromatographic conditions include: The C18 chromatographic column has a specification of 250 mm in length, 4.6 mm in inner diameter and 5 μm in particle size; acetonitrile is used as the mobile phase A, and the aqueous solution containing 0.1% phosphoric acid is used as the mobile phase B; the elution in the HPLC detection chromatographic conditions is gradient elution, and the gradient elution procedure is as follows: 0-35 min, the mobile phase is 12% A, 35-40 min, the mobile phase is 12%-16% A, 40-60 min, the mobile phase is 16%-16.5% A, 60-70 min, the mobile phase is 16.5%-20% A, 70-75 min, the mobile phase is 20% A, 75-76 min, the mobile phase is 20%-90% A, 76-90 min, the mobile phase is 90% A; the detection wavelength is 280-285 nm; The method further includes detection of the control sample, and the control sample includes echinacoside, ferulic acid, isoferulic acid, verbascoside, senkyunol I, isoacteoside, naringin and neohesperidin. The traditional Chinese medicine composition is a granule, and the preparation method includes the following steps: 1066 g of angelica, 533 g of radix cyathulae, 666 g of cistanche deserticola, 266 g of fructus aurantii, 400 g of alisma orientalis, and 186 g of radix clematidis are added into 8 times the amount of water, and reflux extraction is performed for 40 minutes; filtration is performed, the filtrate is concentrated to a relative density of 1.05-1.10 of the extract, spray drying is performed, and the spray-dried fine powder is crushed into fine powder; total mixing is performed; 5 g of stevioside is added; and granulation is performed. The preparation of the test sample solution includes the following steps: the product is taken and added into 50 ml of 25% methanol; the weight is determined; ultrasonic treatment is performed at 40 KHz and 500 W for 30 minutes; the weight is determined again after cooling; the lost weight is made up with 25% methanol; shaking is performed; centrifugation is performed; and the supernatant is obtained.

2. The method of claim 1, wherein, The chromatographic conditions include that the flow rate is 0.9-1.05 mL / min, and the column temperature is 28-32 ℃.

3. The method of claim 2, wherein, The chromatographic conditions include that the flow rate is 1.0 mL / min, the column temperature is 30 ℃, and the detection wavelength is 283 nm.

4. The method of claim 1, wherein, The C18 chromatographic column is selected from a Waters Symmetry C18 with a specification of 4.6*250 mm and 5 μm or an Agilent ZORBAX SB-C18 with a specification of 4.6*250 mm and 5 μm.

5. The method of claim 1, wherein, The method for preparing the control sample into a solution includes the following steps: echinacoside, ferulic acid, isoferulic acid, verbascoside, senkyunol I, isoacteoside, naringin and neohesperidin are taken and added into 50% methanol to prepare a single-label or mixed-label solution containing 200 μg of echinacoside, 10 μg of ferulic acid, 7 μg of isoferulic acid, 30 μg of verbascoside, 15 μg of senkyunol I, 20 μg of isoacteoside, 100 μg of naringin and 70 μg of neohesperidin per 1 ml.

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