CN109085259B

CN109085259B - Fingerprint detection method of traditional Chinese medicine composition

Info

Publication number: CN109085259B
Application number: CN201810770571.1A
Authority: CN
Inventors: 韩赓; 倪健; 王俊宏
Original assignee: Beijing Sanyou Lianheng Technology Co ltd
Current assignee: Zhang Huanyu
Priority date: 2018-07-13
Filing date: 2018-07-13
Publication date: 2021-06-15
Anticipated expiration: 2038-07-13
Also published as: CN109085259A

Abstract

The invention provides a fingerprint detection method of a traditional Chinese medicine composition, which comprises the following steps: preparation of a test solution: preparing a test solution from the traditional Chinese medicine composition; preparation of control solutions: taking schisandrin, and preparing a reference solution; chromatographic conditions are as follows: taking acetonitrile-phosphoric acid aqueous solution as a mobile phase, and performing gradient elution, wherein the concentration of the phosphoric acid aqueous solution is 0.05-0.2%; the determination method comprises the following steps: respectively sucking the reference solution and the test solution, injecting into a liquid chromatograph, and measuring; the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 5-20 parts of radix pseudostellariae, 5-20 parts of prepared rehmannia root, 4-10 parts of wolfberry fruit, 4-10 parts of schisandra fruit, 4-10 parts of polygala root, 4-10 parts of acorus gramineus and 5-20 parts of poria cocos.

Description

Fingerprint detection method of traditional Chinese medicine composition

Technical Field

The invention relates to a fingerprint detection method of a traditional Chinese medicine composition.

Background

The traditional Chinese medicine fingerprint refers to a chromatogram or a spectrogram which can mark chemical characteristics of certain traditional Chinese medicinal materials or traditional Chinese medicine preparations by adopting a certain analysis means after the traditional Chinese medicinal materials or the traditional Chinese medicine preparations are properly processed. The traditional Chinese medicine fingerprint spectrum is a comprehensive and quantifiable identification means, is established on the basis of the systematic research of the chemical components of the traditional Chinese medicine, and is mainly used for evaluating the authenticity, the excellence and the stability of the quality of the semi-finished products of the traditional Chinese medicine and the traditional Chinese medicine preparation. The fingerprint spectrum should have fingerprint properties, namely: (1) the specificity is strong. The finger print prepared should be unique to the traditional Chinese medicine, can be distinguished from other traditional Chinese medicines, and reflects chemical information with high selectivity; (2) the stability is good. The fingerprint spectrum of the traditional Chinese medicine is the commonness induced from a plurality of batches of the traditional Chinese medicine, and the common peak or the characteristic peak in the spectrum is relatively stable; (3) the reproducibility is good. The fingerprint map is prepared to reproduce fingerprint characteristics (such as the number, the size, the position and the like of common peaks) under specified conditions, and the error of the fingerprint map is within an allowable range. Only then, the prepared fingerprint has practical value, and the quality of the medicine can be effectively controlled. Traditional Chinese medicines and preparations thereof are all multi-component complex systems, and the prior art for establishing a fingerprint spectrum of a certain traditional Chinese medicine preparation is not directly taught and needs a great deal of research.

Chinese patent application 201710327985.2 discloses a preparation method of a Chinese medicinal composition for treating attention deficit hyperactivity disorder of children. The traditional Chinese medicine composition is prepared from radix pseudostellariae, prepared rehmannia root, medlar, schisandra chinensis, polygala tenuifolia, rhizoma acori graminei and poria cocos. The patent does not provide a method for quality inspection of the pharmaceutical formulation therein. The invention provides a detection method for the traditional Chinese medicine composition, which effectively controls the quality of the medicine.

Disclosure of Invention

The invention provides a fingerprint detection method of a traditional Chinese medicine composition, which comprises the following steps:

preparation of a test solution: preparing a test solution from the traditional Chinese medicine composition;

preparation of control solutions: taking schisandrin, and preparing a reference solution;

chromatographic conditions are as follows: taking acetonitrile-phosphoric acid aqueous solution as a mobile phase, and performing gradient elution, wherein the concentration of the phosphoric acid aqueous solution is 0.05-0.2%;

the determination method comprises the following steps: respectively sucking the reference solution and the sample solution, injecting into a liquid chromatograph, and measuring.

In a specific embodiment, the gradient elution procedure is:

preferably, the gradient elution procedure is:

in a specific embodiment, the aqueous phosphoric acid solution has a concentration of 0.1%.

In a specific embodiment, the preparation of the test solution: extracting the Chinese medicinal composition with organic solvent to obtain test solution.

The organic solvent comprises n-butyl alcohol, ethyl acetate, acetone, 50-100% methanol solution or 50-100% ethanol solution; preferably ethyl acetate; the extraction method comprises ultrasonic extraction, reflux extraction, immersion extraction, microwave extraction or percolation extraction; the extraction times are 1-3 times; the solvent amount is 25 to 50 times (g/mL).

In particular embodiments, the preparation of the control solution: dissolving schisandrin in organic solvent to obtain reference solution.

The organic solvent comprises 50-100% methanol solution or 0.1% phosphoric acid water solution-acetonitrile (40: 60).

In a specific embodiment, the column temperature is 30-40 ℃, and the detection wavelength is 210-230 nm.

In a specific embodiment, the detection method comprises the following steps:

preparation of a test solution: taking the traditional Chinese medicine composition, adding 30 times of ethyl acetate, performing ultrasonic extraction for 1 hour, and filtering to obtain a filtrate I; washing the filter residue with ethyl acetate, filtering to obtain filtrate II, combining the filtrate I and II, and recovering ethyl acetate; dissolving the residue with 0.1% phosphoric acid water solution-acetonitrile (40: 60) to obtain test solution;

preparation of control solutions: dissolving schisandrin in 0.1% phosphoric acid water solution-acetonitrile (40: 60) to obtain test solution;

chromatographic conditions are as follows: using acetonitrile-0.1% phosphoric acid water solution as mobile phase, gradient elution, the elution procedure is:

The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 5-20 parts of radix pseudostellariae, 5-20 parts of prepared rehmannia root, 4-10 parts of wolfberry fruit, 4-10 parts of schisandra fruit, 4-10 parts of polygala root, 4-10 parts of rhizoma acori graminei and 5-20 parts of poria cocos;

specifically, the traditional Chinese medicine composition is prepared from the following raw material medicines in parts by weight: 10 parts of radix pseudostellariae, 10 parts of prepared rhizome of rehmannia, 6 parts of medlar, 6 parts of schisandra chinensis, 6 parts of polygala tenuifolia, 6 parts of rhizoma acori graminei and 10 parts of poria cocos.

The traditional Chinese medicine composition is prepared by the following method:

step a, adding a solvent into schisandra chinensis for extraction to obtain an extract A;

step B, extracting volatile oil from rhizoma acori graminei to obtain volatile oil B and water solution C;

step C, mixing radix pseudostellariae, prepared rhizome of rehmannia, medlar, tuckahoe and polygala tenuifolia, extracting with a solvent, mixing the extracting solution with the water solution C obtained in the step b, precipitating with ethanol, taking the filtrate, and concentrating to obtain an extract D;

and D, uniformly mixing the extract A, the volatile oil B and the extract D to obtain the composition.

The extraction solvent in the steps a and c is water or 50-90% ethanol solution;

step b, extracting volatile oil by adopting a steam distillation method, and including the volatile oil with beta-cyclodextrin;

c, the alcohol precipitation concentration is 50% -70%;

the traditional Chinese medicine composition can be further added with conventional preparation auxiliary materials to be prepared into conventional dosage forms such as granules, tablets, capsules, pills, oral liquid and the like.

The detection method is adopted to detect 11 batches of samples, 30 common peaks are identified in total, wherein the signal peak with t being 21.876 is the signal peak of schisandrin, and the chromatographic similarity of all batches of samples is more than 0.99; through methodology investigation, the stability, precision and repeatability of the detection method provided by the invention meet requirements.

Drawings

FIG. 1 is a chromatogram of chromatographic condition one;

FIG. 2 is a chromatogram of chromatography condition two (chromatograms of two batches of samples S1 and S2, respectively);

FIG. 3 is a chromatogram of chromatographic condition three (210 nm, 220nm, 228nm chromatograms, respectively);

FIG. 4 is a chromatogram of chromatographic condition four (chromatograms of three batches of samples S1, S2, and S3, respectively);

FIG. 5 is a chromatogram for system adaptability examination (chromatograms of Chinese medicinal composition-Schisandrin A, Chinese medicinal composition, Schisandrin A, blank group, respectively);

FIG. 6 is a chromatogram fingerprint of 11 batches of the Chinese medicinal composition;

FIG. 7 is a comparative fingerprint of the present invention.

Detailed Description

Example 1

Raw materials: 10g of radix pseudostellariae, 10g of prepared rhizome of rehmannia, 6g of medlar, 6g of schisandra chinensis, 6g of polygala tenuifolia, 6g of rhizoma acori graminei and 10g of poria cocos;

the preparation method comprises the following steps:

adding 10 times of 60% ethanol into fructus Schisandrae, heating and reflux-extracting for 3 times, each for 2 hr, mixing extractive solutions, concentrating, and drying under reduced pressure; adding 8 times of water into rhizoma Acori Graminei, heating and distilling for 8 hr to extract volatile oil, and clathrating the obtained volatile oil with cyclodextrin (clathration process comprises mixing oil and beta-CD at a ratio of 1:8, 40 deg.C, stirring for 2 hr); adding 12 times of water into the radix pseudostellariae, the prepared rhizome of rehmannia, the medlar, the tuckahoe and the polygala tenuifolia, carrying out reflux extraction for 3 times, wherein each time lasts for 1.5 hours, combining the obtained liquid medicine with the water solution of the rhizoma acori graminei, concentrating the liquid medicine until the relative density is 1.15-1.20 (60 ℃), adding ethanol until the ethanol content is 60%, precipitating the ethanol with the ethanol, filtering, concentrating, and drying under reduced pressure; and (4) uniformly taking the dry paste powder of each extract to obtain the dry paste of the traditional Chinese medicine composition.

The fingerprint detection method comprises the following steps:

1. instruments and reagents

1.1 Main Instrument

A chromatographic system: agilent Technologies 1200Series (B1322A Degasser, G1311A Quart Pump, G1320A ALS, G1316A TCC, G1315D DAD); ultrasonic cleaner: 15B065, 250W, Ningbo Xinzhi Biotech GmbH; rotating the evaporator: EYELA N1100;

1.2 reagent

Schizandrol A: DST161117-010, purity not less than 98%, ESITE; dry paste of the Chinese medicinal composition (prepared according to this example);

1.3 reagents

Ethyl acetate: 20101112, purity not less than 99.5%, Suzuki technology development Limited in Tianjin; n-butanol: 20101112, purity not less than 99.0%, Beijing chemical plant; acetone: 20110104, purity is more than or equal to 99.5 percent, Beijing chemical plant; acetonitrile: LOT 175164, purity not less than 99.9%, Fisher; phosphoric acid: 20170616, purity not less than 85%, Beijing chemical plant; purified water: 20180208, Wahaha.

2. Chromatographic conditions

2.1 chromatographic conditions one

Stationary phase: agilent Zorbox SB-C18, 4.6X 250mm, 5 μm;

mobile phase: methanol-water system, gradient elution procedure as in table 1:

TABLE 1 chromatographic conditions one

t/min	A (water)/%)	B (methanol)/%
			0	95	5
10	95	5
			20	80	20
40	80	20
			50	5	95
60	5	95
			70	95	5
80	95	5

Preparation of a test solution: taking 5g of the dry extract powder of the traditional Chinese medicine composition, placing the dry extract powder in a 100mL conical flask, adding 50mL of methanol, carrying out ultrasonic extraction for 20min, standing for 10min, filtering with a 0.45-micron pinhole filter membrane, taking the subsequent filtrate as a sample to be detected, refrigerating and storing for later use, and placing the sample to room temperature during detection.

Detection wavelength: 228 nm; the sample volume is 10 mu L; the flow rate of fluidity was 1.0mL/min, and the column temperature was 35 ℃.

Taking the sample, analyzing under chromatographic condition, and recording the chromatographic information. The results are shown in FIG. 1.

As can be seen from fig. 1, when a sample is analyzed under a high-water-phase mobile phase condition, the content of a large polar substance is very high when methanol is used for extraction, and signal peaks are rich, the large polar component cannot be effectively separated and identified by using an Agilent ZorBox SB-C18 chromatographic column, and the high-water-phase mobile phase is not suitable for analyzing for a long time by using an SB-C18 chromatographic column, so the adoption of the chromatographic column is not recommended.

2.2 chromatographic conditions two

Stationary phase: agilent Zorbox SB-Aq C18, 4.6X 250mm, 5 μm;

mobile phase: methanol-0.1% aqueous phosphoric acid system, gradient elution procedure as in table 2;

TABLE 2 chromatographic conditions II

Preparing a test solution: taking 5g of the dry paste powder of the traditional Chinese medicine composition, placing the dry paste powder in a 100mL conical flask, adding 50mL of ethyl acetate, carrying out ultrasonic extraction for 60min, extracting for 2 times, filtering with a 0.45-micron pinhole filter membrane, taking the subsequent filtrate, recovering the solvent under reduced pressure, adding a proper amount of methanol to dissolve, diluting with methanol to a constant volume of 10mL, filtering with the 0.45-micron pinhole filter membrane, taking the subsequent filtrate as a sample to be detected, refrigerating for later use, and placing to room temperature during detection.

Two batches of the test sample (S1 and S2) are taken and analyzed under chromatographic conditions, and the chromatographic information is recorded. The results are shown in FIG. 2.

As can be seen from FIG. 2, when the chromatographic column is replaced by Agilent Zorbox SB-Aq C18, and the methanol in sample preparation is changed into ethyl acetate, the signal peak of the large polar substance (retention time is less than or equal to 10min) is obviously reduced and lowered, but the solvent peak and the signal of the miscellaneous peak of the methanol are more, and the interference to sample analysis is larger, therefore, acetonitrile is supposed to be used as the organic phase in the mobile phase, and the analysis conditions are further investigated.

2.3 chromatographic conditions III

Stationary phase: agilent Zorbox SB-Aq C18, 4.6X 250mm, 5 μm;

mobile phase: acetonitrile-0.1% aqueous phosphoric acid system, gradient elution procedure as in table 3:

TABLE 3 chromatographic conditions III

Preparation of a test solution: taking 1g of the dry paste powder of the traditional Chinese medicine composition, placing the dry paste powder in a 100mL conical flask, adding 50mL of ethyl acetate, carrying out ultrasonic extraction for 60min, filtering with a 0.45-micron pinhole filter membrane, taking the subsequent filtrate, recovering the solvent under reduced pressure, precisely adding 2.5mL of 0.1% phosphoric acid-acetonitrile mixed solution (40-60) for dissolving, filtering with the 0.45-micron pinhole filter membrane, taking the subsequent filtrate as a sample to be detected, refrigerating for later use, and placing the sample to room temperature during detection.

Detection wavelength: 210nm, 220nm, 228 nm; the sample volume is 10 mu L; the flow rate of fluidity was 1.0mL/min, and the column temperature was 35 ℃.

Taking the sample, analyzing under chromatographic condition, and recording the chromatographic information. The results are shown in FIG. 3.

After the organic phase of the mobile phase is changed into acetonitrile, and signal peaks under different wavelengths of 210nm, 220nm and 228nm are compared, the solvent baseline under 228nm is found to be stable, the sample signal peaks are rich, the separation degree is better, and the influence of the solvent peaks and impurities is less, so that the analysis condition can be optimized on the basis at the later stage.

2.4 chromatographic conditions IV

Stationary phase: zorbax SB-Aq C-184.6 mm ID × 250mm (5 μm);

mobile phase: acetonitrile-0.1% phosphoric acid system, gradient program as in table 4:

TABLE 4 chromatographic conditions IV

t/min	Acetonitrile/%)	0.1% aqueous phosphoric acid solution/%)
			0	30	70
5	35	65
			65	55	45
70	60	60
			75	95	5
78	95	5
			83	30	70
90	30	70；

Detection wavelength: 228 nm; the sample injection volume is 10 mu l; flow rate of mobile phase: 1 ml/min; column temperature: at 40 ℃.

Three batches of the test sample (S1, S2 and S3) were taken, analyzed under chromatographic conditions, and the chromatographic information was recorded. The results are shown in FIG. 4.

As can be seen in fig. 4, the separation was best under this chromatographic condition. The chromatographic conditions are selected for detection.

3. Preparation of test solution and control solution

3.1 examination of extraction solvent

The n-butyl alcohol, the ethyl acetate and the acetone are respectively used as extraction solvents, the extraction effect is inspected, and the result shows that: when the extraction solvent is n-butyl alcohol, the peak signal is the least, and the signal intensity is weak; when the extraction solvent is acetone, the peak signal is the most and the signal intensity is the greatest, but the acetone is difficult to recover due to high boiling point in the decompression recovery process; when the solvent is ethyl acetate, the peak signal is rich, the signal intensity is moderate, and the solvent is easy to recover (reduced pressure is 40-50 ℃), so that the extraction solvent is determined to be ethyl acetate.

3.2 the preparation method of the test solution determines that: weighing 0.3g of dry paste powder, placing the dry paste powder in a 100ml conical flask, adding 9ml of ethyl acetate, extracting for 1 time, performing ultrasonic extraction for 1 hour each time, standing to room temperature, and filtering (a glass syringe and a 0.45 mu m organic pinhole filter membrane) to obtain a filtrate I, and storing for later use; respectively taking 9ml of ethyl acetate, washing filter residues for 2-3 times, filtering (a glass syringe and a 0.45 mu m organic pinhole filter membrane) to obtain a filtrate II, combining the filtrates I and II, and recovering the ethyl acetate under reduced pressure in a water bath at 50 ℃; dissolving the residue with 10ml 0.1% phosphoric acid-acetonitrile (40-60), filtering with 0.22 μm filter membrane, collecting the filtrate as sample, storing at 4 deg.C for testing, taking out, and standing at room temperature before testing.

3.3 preparation of control solutions: weighing 10mg of schizandrol A, adding 0.1% phosphoric acid-acetonitrile (40-60) for dissolving to prepare 1mg/ml of schizandrol A reference solution mother liquor, precisely transferring 0.1ml of the mother liquor, placing in a 10ml volumetric flask, adding 0.1% phosphoric acid-already (40-60) for dissolving, diluting, fixing the volume, shaking up, filtering with a 0.22 mu m filter membrane, taking the subsequent filtrate as a test sample, storing at 4 ℃ for testing, taking out and placing to room temperature before testing.

4 methodological investigation

4.1 System Adaptation

Weighing 0.3g of Chinese medicinal composition dry extract powder, preparing test solution under item 3.2, storing at 4 deg.C for testing, taking out, and standing to room temperature before testing;

measuring 10ml of ethyl acetate, preparing a solvent blank solution under the item of 3.2, storing at 4 ℃ to be tested, taking out before testing, and standing to room temperature;

weighing 10mg of schizandrol A, preparing a reference solution according to item 3.2, storing at 4 ℃ to be tested, taking out before testing, and standing to room temperature;

weighing 0.3g of Chinese medicinal composition dry extract powder, preparing test solution under item 3.2, adding 0.1ml of schizandrol A mother liquor, storing at 4 deg.C, taking out, and standing to room temperature before measurement.

Taking the above test sample, analyzing under the item of '2.4 chromatographic condition four', observing chromatographic information, and analyzing the system adaptability of chromatographic condition with reference to liquid chromatographic parameters.

The chromatogram is shown in FIG. 5.

By comparing three groups of signals of schisandrin A, the traditional Chinese medicine composition and the traditional Chinese medicine composition-schisandrin A, a signal peak with t being 21.848min is schisandrin A, the separation degree of the chromatographic peak and the front and back chromatographic peaks is 2.32, the theoretical plate number is 40197, the selectivity is 1.05, and the requirements are met;

the information is integrated to show that the analysis method has good system adaptability and specificity and meets the requirement of fingerprint spectrum research.

4.2 stability

Sampling a test solution and a reference solution every 3h, wherein the retention time RSD values of the chromatographic peaks in the sample and the reference are less than 1%; the RSD values of the peak areas of the reference substances are all less than 0.5 percent; most RSD values of the peak areas of the samples are less than 5%. The test solution and the control solution were stable within 24 hours.

4.3 precision

Taking a sample solution, and continuously injecting samples for 6 times according to determined chromatographic conditions, wherein the retention time RSD of each chromatographic peak is less than 0.1%; the peak area RSD of the main chromatographic peak is less than 2 percent, which indicates that the precision of chromatographic analysis conditions is good.

4.4 repeatability

The method comprises the steps of taking 6 parts of the traditional Chinese medicine composition, preparing according to a determined preparation method of a test solution, injecting the prepared traditional Chinese medicine composition into a liquid chromatograph for detection, and determining that the RSD of each main chromatographic peak area is less than 10 percent, which indicates that the method has good repeatability.

5 sample determination

Taking 11 batches of samples of the traditional Chinese medicine composition, preparing a test solution under the item of '3.2', carrying out sample injection analysis under the chromatographic condition to obtain the fingerprints of the 11 batches of the traditional Chinese medicine composition, introducing the fingerprints into similarity software to carry out fitting to generate a comparison fingerprint, wherein the results are shown in a detailed graph 6 and a detailed graph 7, and the similarity results are shown in a table 5.

TABLE 511 evaluation of similarity of Chinese medicinal compositions

The analysis result of the similarity software shows that 30 common peaks are identified in 11 batches of samples, wherein the signal peak of t ═ 21.876 is the signal peak of schizandrol A, and the chromatographic similarity of all batches of samples is more than 0.99.

Example 2

Preparation of a test solution: weighing 0.3g of dry paste powder, placing the dry paste powder in a 100ml conical flask, adding 9ml of acetone, extracting for 1 time, performing ultrasonic extraction for 1 hour each time, standing to room temperature, and filtering (a glass syringe and a 0.45 mu m organic pinhole filter membrane) to obtain a filtrate I, and storing for later use; respectively taking 9ml of acetone, washing filter residues for 2-3 times, filtering (a glass syringe and a 0.45 mu m organic pinhole filter membrane) to obtain a filtrate II, combining the filtrates I and II, and recovering ethyl acetate in a water bath at 50 ℃ under reduced pressure; dissolving the residue with 10ml 0.1% phosphoric acid-acetonitrile (40-60), filtering with 0.22 μm filter membrane, collecting the filtrate as sample, storing at 4 deg.C for testing, taking out, and standing at room temperature before testing.

Preparation of control solutions: weighing 10mg of schizandrol A, adding 0.1% phosphoric acid-acetonitrile (40-60) for dissolving to prepare 1mg/ml of schizandrol A reference solution mother liquor, precisely transferring 0.1ml of the mother liquor, placing in a 10ml volumetric flask, adding 0.1% phosphoric acid-already (40-60) for dissolving, diluting, fixing the volume, shaking up, filtering with a 0.22 mu m filter membrane, taking the subsequent filtrate as a test sample, storing at 4 ℃ for testing, taking out and placing to room temperature before testing.

Chromatographic conditions

Stationary phase: zorbax SB-Aq C-184.6 mm ID X250 mm (5 μm)

Mobile phase: acetonitrile-0.2% phosphoric acid system, gradient program as follows:

column temperature: 40 ℃;

flow rate of mobile phase: 1 ml/min;

the injection volume was 20. mu.l.

The determination method comprises the following steps: precisely sucking 20 μ L of reference solution and sample solution respectively, injecting into ultra high liquid chromatograph, measuring, and recording chromatogram.

Claims

1. A fingerprint detection method of a traditional Chinese medicine composition is characterized by comprising the following steps:

preparation of a test solution: extracting the Chinese medicinal composition with organic solvent to obtain test solution; the organic solvent is ethyl acetate or acetone;

chromatographic conditions are as follows: taking acetonitrile-phosphoric acid aqueous solution as a mobile phase, and performing gradient elution, wherein the concentration of the phosphoric acid aqueous solution is 0.05-0.2%; the chromatographic column is as follows: AgilentZorbox SB-Aq C18, 4.6X 250mm, 5 μm;

the determination method comprises the following steps: respectively sucking the reference solution and the sample solution, injecting into a liquid chromatograph, and measuring;

the gradient elution procedure was:

2. the detection method of claim 1, wherein the gradient elution procedure is:

3. the detection method according to claim 1, wherein the concentration of the phosphoric acid aqueous solution is 0.1%.

4. The assay of claim 1, wherein the control solution is prepared by: dissolving schisandrin in organic solvent to obtain reference solution; the organic solvent comprises 50-100% of methanol solution or 40: 60 of 0.1% aqueous phosphoric acid solution-acetonitrile.

5. The detection method according to claim 1, wherein the column temperature is 30 ℃ to 40 ℃ and the detection wavelength is 210nm to 230 nm.

6. The detection method according to claim 1, characterized in that it comprises the steps of:

preparation of a test solution: adding ethyl acetate into the traditional Chinese medicine composition, carrying out ultrasonic extraction, and filtering to obtain a filtrate I; washing the filter residue with ethyl acetate, filtering to obtain filtrate II, combining the filtrate I and II, and recovering ethyl acetate; dissolving the residue with 0.1% phosphoric acid water solution-acetonitrile to obtain test solution;

preparation of control solutions: dissolving schisandrin in 0.1% phosphoric acid water solution-acetonitrile to obtain reference solution;

7. The detection method according to any one of claims 1 to 6, wherein the Chinese medicinal composition is prepared from the following raw material medicines in parts by weight: 10 parts of radix pseudostellariae, 10 parts of prepared rhizome of rehmannia, 6 parts of medlar, 6 parts of schisandra chinensis, 6 parts of polygala tenuifolia, 6 parts of rhizoma acori graminei and 10 parts of poria cocos.

8. The assay method of any one of claims 1-6, wherein the traditional Chinese medicine composition is prepared by the following method:

step C, mixing the radix pseudostellariae, the prepared rhizome of rehmannia, the medlar, the tuckahoe and the polygala root, extracting by adding a solvent, mixing an extracting solution and the water solution C obtained in the step b, precipitating by alcohol, taking filtrate and concentrating to obtain an extract D;