Detection method of Coptis chinensis fingerprint

Abstract

The invention discloses a detection method for the fingerprint spectrum of the Chinese medicine Coptis chinensis. The establishment of the fingerprint spectrum of Coptis chinensis includes the following steps: step 1, preparation of a test solution of the Chinese medicine Coptis chinensis; step 2, preparation of a mixed reference solution; step 3, respectively accurately aspirating the mixed reference solution and injecting it into a liquid chromatograph to record the chromatogram of the standard product; step 4, respectively accurately aspirating the test solution and injecting it into the liquid chromatograph to record the chromatogram of the sample. The fingerprint spectrum of the Chinese medicine Coptis chinensis provided by the invention can comprehensively and objectively characterize the quality of the Chinese medicine Coptis chinensis. The fingerprint spectrum detection method provided by the invention has the advantages of simple method, stability, high precision, good reproducibility, etc.

Description

Technical Field

[0001] The invention relates to a detection method for traditional Chinese medicine, and in particular to a detection method for a fingerprint spectrum of Coptis chinensis. Background Art

[0002] Traditional Chinese medicine fingerprint is a comprehensive and quantifiable identification method. It is based on the systematic study of the chemical components of traditional Chinese medicine and is mainly used to evaluate the authenticity, quality and stability of traditional Chinese medicine and traditional Chinese medicine preparations. Through the establishment of traditional Chinese medicine fingerprint, the types and quantities of chemical components contained in traditional Chinese medicine can be more comprehensively reflected, especially when the effective ingredients of most traditional Chinese medicines are unclear, it can better indicate the intrinsic quality of traditional Chinese medicine and improve the technical level and scientific and technological content of traditional Chinese medicine quality evaluation.

[0003] Coptis chinensis, Coptis chinensis is the dried whole herb of Corydalis saxicola Bunting, a plant of the Poppy family. It has the effects of clearing away dampness and heat, stopping bleeding and relieving pain. It can be used to treat hepatitis, oral ulcers, swollen and painful gums, leucorrhea, diarrhea, dysentery, hemorrhoidal bleeding, tumors and other diseases.

[0004] In the prior art, there are few reports on the quality research of Coptis chinensis. Based on the prior art, the present invention mainly focuses on the fingerprint of its main components, which can better control the product quality. Summary of the invention

[0005] The purpose of the present invention is to solve the deficiencies of the prior art and provide a fingerprint spectrum detection method for Coptis chinensis, which can objectively, comprehensively and accurately evaluate the quality of Coptis chinensis, and is of great significance for controlling the quality of Coptis chinensis and ensuring clinical efficacy.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] The first object of the present invention is to provide a method for establishing a fingerprint spectrum of Coptis chinensis, comprising the following steps:

[0008] Step 1, preparation of Coptis chinensis test solution:

[0009] Accurately weigh different batches of Coptis chinensis, add 50%-100% ethanol by volume, sonicate, filter with a microporous filter membrane, and take the filtrate to obtain the test solution;

[0010] As a routine operation of ultrasonic treatment, in a special embodiment of the present invention, the specific operation of step 1 is: accurately weigh different batches of Coptis chinensis, place them in a conical flask, add 50%-100% ethanol, weigh them, perform ultrasonic treatment, cool them to room temperature, make up the weight loss with the solvent, filter them with a microporous filter membrane, take the filtrate, and obtain the test solution.

[0011] Step 2: Preparation of mixed standard solution:

[0012] Accurately weigh dehydromethylcavitine, dehydroisoapocavitine, dehydrocavitine, bamipine hydrochloride, and berberine hydrochloride standards, place them in a volumetric flask, dilute to volume with methanol, and shake well to prepare a mixed standard solution;

[0013] Step 3, accurately pipette the mixed standard solution of step 2, inject it into a high performance liquid chromatograph, and record a chromatogram of the mixed standard;

[0014] Step 4, accurately pipette each batch of test sample solution in step 1, inject into high performance liquid chromatograph, and record the chromatogram of the test sample;

[0015] Step 5, importing the test sample chromatogram obtained in step 4 into the traditional Chinese medicine chromatographic fingerprint similarity evaluation system; selecting the chromatographic peaks existing in the chromatograms of different batches of Coptis chinensis test samples as common peaks; generating a reference fingerprint of Coptis chinensis using the average value calculation method, and calculating the relative retention time and relative peak area of ​​each common peak; and marking the chemical composition of the peaks in the reference fingerprint of Coptis chinensis according to the retention time of the chromatogram of the mixed standard solution to obtain the fingerprint of Coptis chinensis.

[0016] Furthermore, the Coptis chinensis in step 1 is crushed; the volume percentage of ethanol is 50%-80%; the ultrasonic time is not less than 15 minutes; the ratio of the mass M of the Coptis chinensis to the volume V of ethanol is M:V=1g / (100-250)mL; preferably, the Coptis chinensis powder is passed through a No. 3 sieve; preferably, the ultrasonic time is 15-60 minutes.

[0017] Furthermore, the concentrations of the mixed standard solution in step 2 are 100ug / ml of dehydrogenated mecavitine, 40ug / ml of dehydroisoapocavitine, 160ug / ml of dehydrogenated cavitine, 100ug / ml of bamipine hydrochloride, and 20ug / ml of berberine hydrochloride.

[0018] Further, in step 3 and step 4, the liquid chromatography conditions are:

[0019] Chromatographic column: octadecylsilane bonded silica gel chromatographic column;

[0020] Mobile phase: Phase A is a 0.01-0.05 mol / L potassium dihydrogen phosphate aqueous solution, and phase B is acetonitrile;

[0021] Elution procedure: gradient elution with a volume fraction of 30-95% phase A and 5-70% phase B;

[0022] Flow rate: 0.8-1.2mL / min;

[0023] Column temperature: 30-40°C;

[0024] Injection volume: 10-20 μL;

[0025] Detection wavelength: 345±5nm.

[0026] Furthermore, the mobile phase is 0.05 mol / L potassium dihydrogen phosphate aqueous solution (A)-acetonitrile (B).

[0027] Further, the elution procedure is:

[0028]

[0029]

[0030] Furthermore, the flow rate is 1 ml / min, the column temperature is 30° C., the injection volume is 10 μL, and the detection wavelength is 345 nm.

[0031] The second object of the present invention is to provide a fingerprint of Coptis chinensis obtained by the aforementioned establishment method.

[0032] Furthermore, there are 17 peaks in the fingerprint spectrum of Coptis chinensis, and 5 components are identified, among which the retention time of dehydromethylcavitine is 33.925, which is Peak 10; the retention time of dehydroisoapocavitine is 35.200min, which is Peak 11; the retention time of dehydrocavitine is 41.800min, which is Peak 14; the retention time of bamipine hydrochloride is 45.250min, which is Peak 16; the retention time of berberine hydrochloride is 46.650min, which is Peak 17.

[0033] The third object of the present invention is to provide the application of the aforementioned Coptis chinensis fingerprint in the quality control of the traditional Chinese medicine Coptis chinensis.

[0034] Optimization of fingerprint detection conditions:

[0035] 1. Optimization of test sample preparation

[0036] When the applicant used water, methanol, hydrochloric acid water, hydrochloric acid methanol, and different proportions of ethanol as extraction solvents for investigation, it was found that 50%-100% ethanol was able to meet the detection requirements when used as the extraction solvent. In particular, when 50%-80% ethanol was used as the extraction solvent, the peak capacity of Coptis chinensis was larger and the response value was higher. Therefore, 50%-80% ethanol was selected as the extraction solvent.

[0037] Comparison of ultrasonic and reflux extraction methods showed that the peak area of ​​ultrasonic extraction was higher. The extraction time was 15, 30, 45, and 60 minutes, and it was found that the extraction was complete after 15 minutes of ultrasonic extraction.

[0038] 2. Optimization of chromatographic conditions

[0039] The present invention uses a diode array detector to investigate the detection wavelength and performs a full wavelength scan on the test solution. The results show that when the detection wavelength is 345nm, the chromatographic information is relatively rich and the response value is generally high, so 345nm is selected as the detection wavelength;

[0040] The present invention investigates mobile phases of methanol-water, acetonitrile-water, acetonitrile-phosphoric acid water, and acetonitrile-potassium dihydrogen phosphate aqueous solution. The results show that when eluting with potassium dihydrogen phosphate aqueous solution-acetonitrile, the chromatographic peak separation and peak shape are good. Therefore, the mobile phase is determined to be potassium dihydrogen phosphate aqueous solution-acetonitrile, and the concentration of the potassium dihydrogen phosphate aqueous solution is 0.01-0.05 mol / L.

[0041] After optimizing the best mobile phase composition, since Coptis chinensis mainly contains alkaloids, such as coptisine, berberine, methyl coptisine, jatrorrhizine, etc., the isocratic elution method cannot achieve good separation of the components, so the present invention continues to screen the gradient elution method, and the best gradient elution method is screened as follows:

[0042]

[0043] Beneficial effects:

[0044] 1. According to the structural properties of the active ingredients contained in Coptis chinensis, the present invention screens out the optimal mobile phase composition, gradient elution program, detection wavelength, chromatographic column, column temperature and other analytical conditions through a large number of experiments. Multiple experimental verifications have shown that the Coptis chinensis fingerprint spectrum detection method provided by the present invention can comprehensively, objectively and accurately detect and evaluate the quality of the traditional Chinese medicine Coptis chinensis, which is of great significance for ensuring the clinical efficacy of the traditional Chinese medicine Coptis chinensis.

[0045] 2. The Rhizoma Coptidis fingerprint established by the method provided by the present invention can effectively characterize the quality of the traditional Chinese medicine Rhizoma Coptidis, objectively reflect the sequence and mutual relationship of each characteristic peak that constitutes the fingerprint, and focus on the overall appearance characteristics. It can avoid the one-sidedness of judging the quality of the traditional Chinese medicine Rhizoma Coptidis due to the determination of individual chemical components, and reduce the possibility of artificial processing to meet quality standards.

[0046] 3. The detection method of the Coptis chinensis fingerprint provided by the present invention has the advantages of simple method, good stability, high precision and good reproducibility, in which the common peaks account for more than 90% of the peak area of ​​the whole spectrum, and the identification peak area accounts for more than 60% of the peak area of ​​the whole spectrum. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 It is the chromatogram of the mixed standard of the present invention.

[0048] Figure 2This is the reference fingerprint of the test sample of the Coptis chinensis sample of the present invention.

[0049] Figure 3 The fingerprints of 16 batches of Coptis chinensis of the present invention are shown in FIG. DETAILED DESCRIPTION

[0050] The embodiments of the present invention are described in detail below with reference to the examples. If no specific conditions are specified in the examples, the reaction is carried out according to conventional conditions or conditions recommended by the manufacturer. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0051] The instruments and reagents used in the embodiments are as follows:

[0052] Experimental equipment

[0053] 1.1 Instrument

[0054] Shimadzu dual-wavelength scanning high-performance liquid chromatography system, including fully automatic online degassing system, fully automatic injection system SIL-20A, UV detector SPD-20A and automatic temperature control column oven CTD-20AC; RODI-220A1 pure water machine (Rui Sijie Water Purification Technology Co., Ltd.); 1 / 10,000 electronic balance, 1 / 100,000 electronic balance (Mettler Toledo, Switzerland); Chinese medicine chromatographic fingerprint similarity evaluation system (2012.130723 version): National Pharmacopoeia Committee.

[0055] 1.2 Drugs and reagents

[0056] 16 batches of Coptis chinensis samples (see Table 1 for sample information) were collected by Nanjing Agricultural University and identified as the dried whole herb of Corydalis saxicola Bunting, a plant of the Poppy family, by Professor Guo Qiaosheng of Nanjing Agricultural University; standard products dehydrocavitin, bamipine hydrochloride, and berberine hydrochloride were purchased from the China Food and Drug Administration, and dehydromethylcavitin and dehydroisoapocavitin were homemade standards. Acetonitrile was purchased from Anhui Tiandi High Purity Solvent Co., Ltd.; potassium dihydrogen phosphate was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; and RODI-220A1 ultrapure water.

[0057] Table 1 Information on samples of Coptis chinensis

[0058]

[0059] Embodiment 1 A method for detecting a fingerprint of Coptis chinensis, comprising the following steps:

[0060] Step 1, preparation of Coptis chinensis test solution:

[0061] Take 0.2 g of the Coptis chinensis powder (passed through No. 3 sieve) from the above 16 batches, accurately weigh it, place it in a stoppered conical flask, accurately add 50 mL of 75% ethanol, weigh it, ultrasonicate for 15 minutes, take it out, cool it, weigh it again, make up the lost weight with 75% ethanol, shake it well, filter it with a 0.22 μm microporous filter membrane, and take the filtrate to obtain it.

[0062] Step 2: Preparation of mixed standard solution:

[0063] Take 5 mg of dehydrogenated mecavitine, 2 mg of dehydroisoapocavitine, 8 mg of dehydrogenated cavitine, 5 mg of bamipine hydrochloride, and 1 mg of berberine hydrochloride, accurately weigh, put into a 50 ml volumetric flask, dissolve in methanol, sonicate, cool, and make up to volume to prepare mixed standard solutions with concentrations of 100 ug / ml, 40 ug / ml, 160 ug / ml, 100 ug / ml, and 20 ug / ml, respectively.

[0064] Step 3: Accurately pipette the mixed standard solution from step 2, inject it into the HPLC, and record the chromatogram ( Figure 1 );

[0065] Step 4, accurately pipette 16 batches of Coptis chinensis test solution from step 1, inject into high performance liquid chromatograph, and record the chromatogram;

[0066] The liquid chromatography conditions were:

[0067] Chromatographic column: Waters CSHTM C18 (250×4.6mm, 5μm);

[0068] Mobile phase: 0.05 mol / L potassium dihydrogen phosphate (A)-acetonitrile (B),

[0069] Gradient elution;

[0070] Flow rate: 1ml / min;

[0071] Detection wavelength: 345nm;

[0072] Column temperature: 30°C;

[0073] Injection volume: 10 μL,

[0074] The specific gradients are shown in the table below:

[0075]

[0076] Step 5, import the sample chromatogram obtained in step 4 into the Chinese medicine chromatographic fingerprint similarity evaluation system (2012.130723 version); select the chromatographic peaks existing in the chromatograms of different batches of Coptis chinensis sample as common peaks; generate the reference fingerprint of Coptis chinensis by the average value calculation method, calculate the relative retention time and relative peak area of ​​each common peak; and mark the chemical composition of the peak in the reference fingerprint of Coptis chinensis according to the retention time of the chromatogram of the mixed standard solution to obtain the fingerprint of Coptis chinensis. Results There are 17 common peaks in Coptis chinensis, and the reference fingerprint of Coptis chinensis is shown in Figure 2 , 16 batches of fingerprints are shown in Figure 3 Five common peaks were identified based on the standard, among which the retention time of dehydromethylcavitine was 33.925, which was peak 10; the retention time of dehydroisoapocavitine was 35.200min, which was peak 11; the retention time of dehydrocavitine was 41.800min, which was peak 14; the retention time of bamipine hydrochloride was 45.250min, which was peak 16; the retention time of berberine hydrochloride was 46.650min, which was peak 17.

[0077] The similarity of 16 batches of Coptis chinensis is as follows:

[0078]

[0079] Example 2 Legal research on fingerprint detection method:

[0080] 1. Precision research

[0081] The sample numbered S1 test solution prepared according to the method of Example 1 was analyzed according to the detection method of Example 1, and parallel injections were performed 6 times. Peak No. 14 (dehydrocarvidine) was taken as the S peak. The peak area and retention time of the common peaks of the sample HPLC fingerprint were analyzed, and the RSD value was calculated. The "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012.130723 Version)" was used for fingerprint comparison and data analysis. The RSD of the relative retention time of each common peak was calculated to be less than 0.05%, the RSD of the relative peak area was less than 0.58%, and the similarity was greater than 0.98. The results showed that the parallel injection precision of the equipment was good.

[0082] 2. Stability study

[0083] The sample numbered S1 test solution prepared according to the method of Example 1 was analyzed according to the detection method of Example 1, and the sample was injected and analyzed at different times of 0h, 2h, 4h, 8h, 10h, 12h, and 24h. Peak 14 (dehydrocarvidine) was taken as the S peak. The peak area and retention time of the common peaks of the HPLC fingerprint of the sample were analyzed and the RSD value was calculated. The "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012.130723 Version)" was used for fingerprint comparison and data analysis. The RSD of the relative retention time of each common peak was calculated to be less than 0.06%, and the RSD of the relative peak area was less than 0.81%. The similarity was greater than 0.99, indicating that the chromatographic peaks of the Coptis chinensis test solution within 24h had almost no change, and the stability was very good.

[0084] 3. Reproducible studies

[0085] Six samples numbered as S1 were accurately weighed in parallel, and 6 identical test solutions were made according to the method of Example 1. The samples were analyzed according to the detection method of Example 1, and the samples were injected in parallel 6 times. Peak No. 14 (dehydrocarvidine) was taken as the S peak. The peak area and retention time of the common peaks of the HPLC fingerprint of the samples were analyzed and the RSD value was calculated. The "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012.130723 Version)" was used for fingerprint comparison and data analysis. The RSD of the relative retention time of each common peak was calculated to be less than 0.05%, and the RSD of the relative peak area was less than 2.78%, and the similarity was greater than 0.99. The results showed that the sample chromatographic peak had good reproducibility and the method had good repeatability.

[0086] The above experimental results show that the Coptis chinensis fingerprint detection method provided by the present invention has good stability, high precision, good repeatability, and can comprehensively and objectively evaluate the quality of the traditional Chinese medicine Coptis chinensis, which is of great significance for ensuring clinical efficacy.

[0087] The above embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention. The protection scope of the present invention is defined by the claims. Those skilled in the art may make various modifications or equivalent substitutions to the present invention within the essence and protection scope of the present invention, and such modifications or equivalent substitutions shall also be deemed to fall within the protection scope of the present invention.

Claims

1. A method for establishing a fingerprint spectrum of Coptis chinensis, characterized in that: The following steps are involved: Step 1, preparation of Coptis chinensis test solution: Accurately weigh different batches of Coptis chinensis, add 50%-100% ethanol by volume, sonicate, filter with a microporous filter membrane, and take the filtrate to obtain the test solution; Step 2: Preparation of mixed standard solution: Accurately weigh dehydromethylcavitine, dehydroisoapocavitine, dehydrocavitine, bamipine hydrochloride, and berberine hydrochloride standards, place them in a volumetric flask, dilute to volume with methanol, and shake well to prepare a mixed standard solution; Step 3, accurately pipette the mixed standard solution of step 2, inject it into a high performance liquid chromatograph, and record a chromatogram of the mixed standard; Step 4, accurately pipette each batch of test sample solution in step 1, inject into high performance liquid chromatograph, and record the chromatogram of the test sample; Step 5, importing the test sample chromatogram obtained in step 4 into the Chinese medicine chromatographic fingerprint similarity evaluation system; selecting the chromatographic peaks existing in the chromatograms of different batches of Coptis chinensis test samples as common peaks; generating a reference fingerprint of Coptis chinensis by using the average value calculation method, calculating the relative retention time and relative peak product of each common peak; and marking the chemical composition of the peak in the reference fingerprint of Coptis chinensis according to the retention time of the chromatogram of the mixed standard solution to obtain the fingerprint of Coptis chinensis; In step 3 and step 4, the liquid chromatography conditions are: Chromatographic column: octadecylsilane bonded silica gel chromatographic column; Mobile phase: Phase A is a 0.01-0.05 mol / L potassium dihydrogen phosphate aqueous solution, and phase B is acetonitrile; Elution procedure: gradient elution with a volume fraction of 30-95% phase A and 5-70% phase B; Flow rate: 0.8-1.2mL / min; Column temperature: 30-40°C; Injection volume: 10-20 μL; Detection wavelength: 345±5nm; The elution procedure is:

2. The method for establishing the fingerprint of Coptis chinensis according to claim 1, characterized in that: The Coptis chinensis in step 1 is crushed; the volume percentage of the ethanol is 50%-80%; the ultrasonic time is not less than 15 minutes; the ratio of the mass M of the Coptis chinensis to the volume V of the ethanol is M:V=1g / (100-250)mL.

3. The method for establishing the fingerprint of Coptis chinensis according to claim 2, characterized in that: The Coptis chinensis powder described in step 1 is passed through a No. 3 sieve.

4. The method for establishing the fingerprint of Coptis chinensis according to claim 2, characterized in that: The ultrasonic time in step 1 is 15-60 min.

5. The method for establishing the fingerprint of Coptis chinensis according to claim 1, characterized in that: The concentrations of the mixed standard solution in step 2 are 100ug / ml of dehydrogenated mecavitine, 40ug / ml of dehydroisoapocavitine, 160ug / ml of dehydrogenated mecavitine, 100ug / ml of bamipine hydrochloride, and 20ug / ml of berberine hydrochloride.

6. The method for establishing the fingerprint of Coptis chinensis according to claim 1, characterized in that: The mobile phase A is a 0.05 mol / L potassium dihydrogen phosphate aqueous solution.

7. The method for establishing the fingerprint of Coptis chinensis according to claim 1, characterized in that: The flow rate was 1 ml / min, the column temperature was 30° C., the injection volume was 10 μL, and the detection wavelength was 345 nm.

8. The method for establishing the fingerprint of Coptis chinensis according to claim 1, characterized in that: There are 17 peaks in the fingerprint spectrum of Coptis chinensis, and 5 components are identified, among which the retention time of dehydromethylcavitine is 33.925, which is peak No. 10; the retention time of dehydroisoapocavitine is 35.200min, which is peak No. 11; the retention time of dehydrocavitine is 41.800min, which is peak No. 14; the retention time of bamipine hydrochloride is 45.250min, which is peak No. 16; the retention time of berberine hydrochloride is 46.650min, which is peak No.

17.

9. Application of the Coptis chinensis fingerprint spectrum described in claim 8 in the quality control of the traditional Chinese medicine Coptis chinensis.

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