Establishment of HPLC fingerprint of Huanglian paste and determination of multi-components

Abstract

The application discloses a method for constructing HPLC fingerprint of Huanglian paste and determining multi-component content, which adopts a Waters Sunfire C 18 (250mm*4.6mm, 5um) chromatographic column, carries out gradient elution with acetonitrile as mobile phase A and 0.3% phosphoric acid solution as mobile phase B, establishes HPLC fingerprint of 16 batches of Huanglian paste samples and content of six main components, carries out similarity evaluation by using traditional Chinese medicine chromatographic fingerprint similarity evaluation software, establishes HPLC fingerprint of Huanglian paste and determines 17 common peaks, and similarity of the 16 batches of samples is greater than 0.9. The HPLC fingerprint method of Huanglian paste established by the method has strong characteristics, the content determination method is accurate and reliable, and has good repeatability, can provide reference for quality evaluation, can reflect information characteristics of overall components of Huanglian paste samples more comprehensively, provides more scientific and reasonable theoretical support for quality control, and can be applied to large-scale industrial production.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine quality detection, and particularly relates to a construction method of HPLC fingerprint of Huanglian Gao and a content determination method. BACKGROUND

[0002] Huanglian Gao is composed of Huanglian, Huangbai, Jianghuang, Danggui (wei) and Shengdihuang, is first recorded in the earliest surgical monograph of traditional Chinese medicine, Liu Danzi Guiyi Fang, and is made according to the principle of clearing damp-heat, and follows the principle of 'the principle of external treatment is the principle of internal treatment', and has drug power that can directly reach the disease site, and can clear heat and remove heat toxin from the skin, and can remove dampness and promote absorption. Huanglian in the prescription can clear heat and remove dampness, Huangbai can clear heat and remove toxin, Jianghuang can break blood and promote qi, Danggui (wei) can activate blood and nourish blood, and Shengdihuang can clear heat, generate fluid, cool blood, nourish yin and moisten dryness. Huanglian Gao is mainly used for clearing heat and resolving toxin, and treating ulcer and trauma. Huanglian Gao has been used for more than 1500 years from ancient times to the present, is an important external preparation in traditional Chinese medicine hospitals all over the country, but the existing Huanglian Gao products have significant differences in color, shape and the like, and cannot guarantee stable and reliable clinical curative effects. Therefore, development of modern quality control technology of Huanglian Gao has important significance for promoting the development of the Huanglian Gao industry.

[0003] Fingerprint technology is widely used as an important analysis method for comprehensive and quantifiable quality control of traditional Chinese medicine, has the characteristics of large information quantity and strong characteristics, and can effectively, comprehensively and scientifically reflect the authenticity, stability and uniformity of the quality of traditional Chinese medicinal materials and traditional Chinese medicinal preparations. Meanwhile, high performance liquid chromatography (HPLC) has the characteristics of high separation efficiency, fast analysis speed, high detection sensitivity and wide application range. Most of the components of traditional Chinese medicines can be analyzed and detected on a high performance liquid chromatograph without being limited by sample volatility and thermal stability. Therefore, high performance liquid chromatography has become the preferred method for constructing a fingerprint of traditional Chinese medicine.

[0004] At present, there are few literatures about quality evaluation of Huanglian Gao, and the quality of the medicine cannot be comprehensively controlled. Therefore, in order to more comprehensively and effectively control the quality of Huanglian Gao and improve the quality control level, it is necessary to establish a modern quality control method that can scientifically and accurately evaluate the quality of Huanglian Gao and meet the requirements of safe and reliable clinical medication. The HPLC fingerprint of Huanglian Gao and the quality control method of multiple components provided by the present application have good accuracy, stability and repeatability, can effectively control the quality of Huanglian Gao, and can be applied to large-scale industrial production. SUMMARY

[0005] The present application aims at the problems in the prior art, and provides a construction method of HPLC fingerprint of Huanglian Gao and a content determination method of multiple components. 18The HPLC fingerprint chromatogram of Huanglian Gao and the content of six main components of 16 batches of Huanglian Gao samples are established by gradient elution with acetonitrile as mobile phase A and 0.3% phosphoric acid solution as mobile phase B, similarity evaluation is carried out by using traditional Chinese medicine chromatographic fingerprint similarity evaluation software, the HPLC fingerprint chromatogram of Huanglian Gao is established and 17 common peaks are determined, the similarity of 16 batches of samples is greater than 0.9, the HPLC fingerprint chromatogram method of Huanglian Gao established by the method has strong characteristics, the content determination method is accurate and reliable, and has good repeatability, can provide reference for quality evaluation, can reflect the information characteristics of the whole component of Huanglian Gao sample, provide more scientific and reasonable theoretical support for quality control, and can be applied to large-scale industrial production.

[0006] The method for constructing the HPLC fingerprint chromatogram of Huanglian Gao comprises the following steps:

[0007] a. The Huanglian Gao sample is precisely weighed and placed in a conical flask with a plug, 25ml of methanol is added, the weight is determined, the temperature is 55 DEG C + / - 5 DEG C, ultrasonic treatment is carried out for 30 minutes, cooling is carried out, the weight is determined again, the lost weight is made up with methanol, shaking is carried out, and the sample is placed until the methanol layer is clear, the methanol layer is separated, centrifugation is carried out at a centrifugal force of 15,000g and a temperature of 4 DEG C for 15min, filtration is carried out, and the filtrate is used as the test sample solution;

[0008] b. 5-hydroxymethylfurfural, ferulic acid, berberine hydrochloride, senkyunolide I, bisdemethylcurcumin, demethylcurcumin, curcumin, sesamin, ligustilide and sesamol reference substances are added to methanol to prepare a solution containing 20ug, 42ug, 57ug, 53ug, 10ug, 22ug, 24ug, 80ug, 96ug and 60ug per 1ml respectively, and the first mixed reference solution is obtained;

[0009] c. The test sample solution in step a and the first mixed reference solution in step b are injected into a high performance liquid chromatograph, and the chromatographic conditions are as follows: Waters Sunfire C 18250mmx4.6mm, 5μm as the chromatographic column, with acetonitrile as the mobile phase A, with 0.3% phosphoric acid solution as the mobile phase B, gradient elution according to time 0-5min, mobile phase A 10-20%; 5-23min, mobile phase A 20-25%; 23-26min, mobile phase A 25-50%; 26-46min, mobile phase A 50-60%; 46-51min, mobile phase A 60-65%; 51-56min, mobile phase A 65%; 56-63min mobile phase A 65-70%; 63-70min, mobile phase A 70-80%; column temperature is 25℃; flow rate is 1.0mL / min; injection volume is 10μl, detection wavelength is 260nm, to obtain the HPLC fingerprint of Huanglian paste, with curcumin chromatographic peak as the reference peak S, the HPLC fingerprint of Huanglian paste obtained by detection has 17 common peaks at 260nm.

[0010] The HPLC fingerprint of Huanglian paste is constructed by a multi-component content determination method, which is used for determining the contents of 5-hydroxymethylfurfural, ferulic acid, berberine hydrochloride, senkyunol I, curcumin and ligusticum lactone, and the specific operation is carried out according to the following steps:

[0011] a. Huanglian paste sample is precisely weighed and placed in a conical flask with a plug, 25ml of methanol is added, the weight is determined, the temperature is 55℃±5℃, ultrasonic treatment is carried out for 30 minutes, cooling, the weight is determined again, the lost weight is made up with methanol, shake well, place until the methanol layer is clear, take the methanol layer, centrifugal force is 15,000g, temperature is 4℃, centrifugal for 15min, filter, take the filtrate, which is the test solution;

[0012] b. 5-hydroxymethylfurfural, ferulic acid, berberine hydrochloride, senkyunol I, curcumin and ligusticum lactone control samples are taken and dissolved in methanol to prepare a solution containing 22μg, 2μg, 26μg, 2μg, 70μg and 30μg per 1ml respectively, which is the second mixed control solution;

[0013] c. The test solution and the second mixed control solution are respectively taken and injected into the high performance liquid chromatograph, and the chromatographic conditions are as follows: Waters Sunfire C 18250mm×4.6mm, 5μm as the chromatographic column, acetonitrile as mobile phase A, 0.3% phosphoric acid solution as mobile phase B, gradient elution was carried out according to time 0-5min, mobile phase A 10-20%; 5-23min, mobile phase A 20-25%; 23-26min, mobile phase A 25-50%; 26-46min, mobile phase A 50-60%; 46-51min, mobile phase A 60-65%; 51-56min, mobile phase A 65%; 56-63min, mobile phase A 65-70%; 63-70min, mobile phase A 70-80%; the column temperature was 25 DEG C, the flow rate was 1.0mL / min, the injection volume was 10ul, and the detection wavelength was 280nm for 5-hydroxymethyl furfural and ligustilide I, 260nm for berberine hydrochloride, 321nm for ferulic acid and ligusticum lactone, and 430nm for curcumin;

[0014] d, the content of 5-hydroxymethyl furfural calculated by the linear regression equation is 0.019-0.067mg / g; the content of ligustilide I is 0.008-0.014mg / g; the content of berberine hydrochloride is 0.050-0.149mg / g; the content of ferulic acid is 0.007-0.010mg / g; the content of ligusticum lactone is 0.091-0.141mg / g; and the content of curcumin is 0.071-0.268mg / g.

[0015] The HPLC fingerprint of the Huanglian Gao and the multi-component content determination method have the following beneficial effects:

[0016] 1, the Waters Sunfire C 18 (250mm×4.6mm, 5μm) chromatographic column, acetonitrile as mobile phase A, 0.3% phosphoric acid solution as mobile phase B, gradient elution, establish the HPLC fingerprint of 16 batches of samples and the content of 6 main components, similarity evaluation is carried out by using traditional Chinese medicine chromatographic fingerprint similarity evaluation software, the HPLC fingerprint of Huanglian Gao is established, 17 common peaks are determined, and the similarity of 16 batches of samples is greater than 0.9; the HPLC fingerprint method of Huanglian Gao established has strong characteristics, the peak intensity, number and overall separation degree of common peaks are good, and the precision and repeatability are high, which can provide reference for quality evaluation, can reflect the information characteristics of the whole component of Huanglian Gao sample, provide more scientific and reasonable theoretical support for quality control, and can be applied to large-scale industrial production, is a very good quality control method.

[0017] 2, The application establishes the HPLC fingerprint of Huanglian paste, and multi-component content of 5-hydroxymethylfurfural, ferulic acid, berberine hydrochloride, senkyunol I, curcumin and ligustilide in the Huanglian paste is determined, and the 5-hydroxymethylfurfural content is 0.019 mg / g-0.067 mg / g, the ferulic acid content is 0.007 mg / g-0.010 mg / g, the berberine hydrochloride content is 0.050 mg / g-0.149 mg / g, the senkyunol I content is 0.008 mg / g-0.014 mg / g, the curcumin content is 0.091 mg / g-0.141 mg / g, and the ligustilide content is 0.071 mg / g-0.268 mg / g. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 It is the HPLC fingerprint of Huanglian paste test sample and the first mixed control chromatogram for the application;

[0019] Figure 2 It is the identification diagram of each component in the chromatogram of Huanglian paste control medicinal material;

[0020] Figure 3 It is the HPLC fingerprint (S1-S16) of 16 batches of Huanglian paste samples;

[0021] Figure 4 It is the control chromatogram (R) of Huanglian paste sample. DETAILED DESCRIPTION

[0022] The technical scheme of the application is further described below in combination with the drawings.

[0023] Example 1

[0024] Instruments and materials

[0025] High performance liquid chromatograph: Agilent high performance liquid chromatograph, 1260 type, DAD detector; Electronic balance: Ohaus instruments (Changzhou) Co., Ltd., Scout SE type; Sartorius scientific instruments (Beijing) Co., Ltd., SQP type and BT25S type; Electric heating constant temperature air drying oven: Shanghai Qixin scientific instruments Co., Ltd., DHG-9070A type; Ultrasonic cleaning instrument: Shanghai keda ultrasonic instrument Co., Ltd., SK7210HP type; The chromatographic column is Waters Sunfire C 18 (250mm×4.6mm, 5μm) chromatographic column;

[0026] Methanol, acetonitrile were chromatographic grade, water was ultrapure water, and other reagents were analytical pure. 5-Hydroxymethylfurfural (Shanghai Pure Biological Technology Co., Ltd., batch number: 20011202, content 98.0%), ferulic acid (China Institute for Food and Drug Control, batch number: 110773-201313, content 99.6%), berberine hydrochloride (China Institute for Food and Drug Control, batch number: 110713-201613, content 86.8%), senkyunolide I (Shanghai Pure Biological Technology Co., Ltd., batch number: 21012505, content 98.0%), bisdemethylcurcumin (Shanghai Pure Biological Technology Co., Ltd., batch number: 20031504, content 98.0%), demethylcurcumin (China Institute for Food and Drug Control, batch number: 112003-201501, content 98.5%), curcumin (China Institute for Food and Drug Control, batch number: 110823-201706, content 98.7%), sesamin (Shanghai Pure Biological Technology Co., Ltd., batch number: 21030806, content 98.0%), ligustilide (Shanghai Pure Biological Technology Co., Ltd., batch number: 21031910, content 98.0%), sesamolin (Shanghai Pure Biological Technology Co., Ltd., batch number: 21030203, content 98.0%);

[0027] Huanglian paste sample batch numbers: HLG-1, HLG-2, HLG-3, HLG-4, HLG-5, HLG-6, HLG-7, HLG-8, HLG-9, HLG-10, HLG-11, HLG-12, HLG-13, HLG-14, HLG-15, HLG-16.

[0028] Method for constructing HPLC fingerprint of Huanglian paste:

[0029] a. Take 7 g of Huanglian paste sample, accurately weigh, place in a conical flask with a plug, add 25 ml of methanol, tightly plug, weigh, temperature 55℃±5℃, ultrasonic treatment for 30 minutes, cool, weigh again, make up the weight loss with methanol, shake well, place until the methanol layer is clear, separate the methanol layer, centrifuge at 15,000 g, 4℃ for 15 min, filter, take the filtrate, which is the test solution;

[0030] b. Take 5-hydroxymethylfurfural, ferulic acid, berberine hydrochloride, senkyunolide I, bisdemethylcurcumin, demethylcurcumin, curcumin, sesamin, ligustilide, sesamolin reference substances, add methanol to prepare a solution containing 20 μg, 42 μg, 57 μg, 53 μg, 10 μg, 22 μg, 24 μg, 80 μg, 96 μg, 60 μg per 1 ml respectively, which is the first mixed reference solution;

[0031] c. Inject the test sample solution in step a and the first mixed control sample solution in step b into the high performance liquid chromatograph, respectively, and the chromatographic conditions are as follows: Waters Sunfire C 18 250mmx4.6mm, 5um as the chromatographic column, acetonitrile as the mobile phase A, 0.3% phosphoric acid solution as the mobile phase B, gradient elution according to the time in Table 1: 0-5min, mobile phase A 10-20%; 5-23min, mobile phase A 20-25%; 23-26min, mobile phase A 25-50%; 26-46min, mobile phase A 50-60%; 46-51min, mobile phase A 60-65%; 51-56min, mobile phase A 65%; 56-63min, mobile phase A 65-70%; 63-70min, mobile phase A 70-80%; the column temperature is 25℃; the flow rate is 1.0mL / min; the injection volume is 10ul, and the detection wavelength is 260nm, to obtain the HPLC fingerprint of Huanglian paste, with curcumin as the reference peak S, and 17 common peaks are detected at 260nm, and the theoretical plate number should not be less than 3000 according to the curcumin peak;

[0032] Table 1: Gradient elution of mobile phase

[0033]

[0034]

[0035] Preparation of control medicinal material solution:

[0036] Each single medicinal material of Huanglian, Huangbai, Jianghuang, Danggui (tail), Shengdihuang was extracted, and 4.2g of each extract was precisely weighed and placed in a conical bottle with a plug, 25ml of methanol was precisely added, the weight was determined, and the temperature was 55℃±5℃, and ultrasonic treatment was performed for 30 minutes. After cooling, the weight was determined again, the lost weight was made up with methanol, and the mixture was shaken and placed until the methanol layer was clear. The methanol layer was separated, centrifuged at 15,000g and 4℃ for 15min, filtered, and the filtrate was collected, to obtain each control medicinal material solution.

[0037] Methodology of fingerprint spectrum:

[0038] Precision investigation:

[0039] Take Huanglian paste sample 7 g (batch number HLG-1), accurately weigh, put into a conical flask with a plug, add methanol 25 ml, tightly plug, weigh, temperature 55℃±5℃, ultrasonic treatment for 30 minutes, cool, weigh again, make up the weight loss with methanol, shake well, place until the methanol layer is clear, separate the methanol layer, centrifuge at 15,000g at 4℃ for 15 min, filter, take the filtrate, i.e. the test sample solution. Take Waters Sunfire C 18 250mm×4.6mm, 5μm as the chromatographic column, with acetonitrile as the mobile phase A and 0.3% phosphoric acid solution as the mobile phase B, gradient elution according to time 0-5min, mobile phase A 10-20%; 5-23min, mobile phase A 20-25%; 23-26min, mobile phase A 25-50%; 26-46min, mobile phase A 50-60%; 46-51min, mobile phase A 60-65%; 51-56min, mobile phase A 65%; 56-63min, mobile phase A 65-70%; 63-70min, mobile phase A 70-80%, continuous sampling determination for 6 times, record the chromatogram; select curcumin chromatographic peak (No. 9 peak) as the reference peak (S), the relative retention time RSD of each common peak is less than 0.3% (as shown in Table 2), and the relative peak area RSD is less than 3.0% (as shown in Table 3), indicating that the precision of the instrument is good;

[0040] Table 2 Precision investigation-the relative retention time RSD of each common peak

[0041]

[0042]

[0043] Table 3 Precision investigation-the relative peak area RSD of each common peak

[0044]

[0045] Repeatability investigation:

[0046] Take Huanglian paste sample 7 g (batch number HLG-1), accurately weigh, put into a conical flask with a plug, add methanol 25 ml, tightly plug, weigh, temperature 55℃±5℃, ultrasonic treatment for 30 minutes, cool, weigh again, make up the weight loss with methanol, shake well, place until the methanol layer is clear, separate the methanol layer, centrifuge at 15,000g at 4℃ for 15 min, filter, take the filtrate, i.e. the test sample solution. Take Waters Sunfire C 18250mm x 4.6mm, 5μm as the chromatographic column, with acetonitrile as mobile phase A, and 0.3% phosphoric acid solution as mobile phase B, gradient elution was carried out according to time 0-5min, mobile phase A 10-20%; 5-23min, mobile phase A 20-25%; 23-26min, mobile phase A 25-50%; 26-46min, mobile phase A 50-60%; 46-51min, mobile phase A 60-65%; 51-56min, mobile phase A 65%; 56-63min, mobile phase A 65-70%; 63-70min, mobile phase A 70-80%, recording chromatogram; select curcumin chromatographic peak (No. 9 peak) as reference peak (S), the relative retention time RSD of each common peak was less than 0.3% (as shown in Table 4), the relative peak area RSD was less than 3.0% (as shown in Table 5), indicating that the repeatability of the method was good;

[0047] Table 4 Reproducibility investigation - relative retention time RSD of each common peak

[0048]

[0049]

[0050] Table 5 Reproducibility investigation - relative peak area RSD of each common peak

[0051]

[0052] Stability investigation:

[0053] Take Huanglian paste sample 7g (batch number HLG-1), accurately weigh, put into a conical flask with plug, add methanol 25ml, seal, weigh, temperature 55℃±5℃, ultrasonic treatment for 30 minutes, cool, weigh again, make up the weight loss with methanol, shake well, place until the methanol layer is clear, separate the methanol layer, centrifuge at 15,000g and 4℃ for 15min, filter, take the filtrate, i.e. test solution. Take the test solution and the reference solution to Waters Sunfire C 18A 250 mm × 4.6 mm, 5 μm column was used. Acetonitrile was used as mobile phase A, and 0.3% phosphoric acid solution was used as mobile phase B. The concentrations were as follows: 0-5 min, mobile phase A 10-20%; 5-23 min, mobile phase A 20-25%; 23-26 min, mobile phase A 25-50%; 26-46 min, mobile phase A 50-60%; 46-51 min, mobile phase A 60-65%; 51-56 min, mobile phase A 65%; 56-63 min, mobile phase A 65-70%; 63-70 min, mobile phase A... Gradient elution was performed at 70-80% of the sample, and the chromatograms were recorded at 0h, 3h, 6h, 9h, 12h, and 24h. The curcumin chromatographic peak (peak 9) was selected as the reference peak (S). The relative retention time RSD of each common peak was less than 0.3% (as shown in Table 6), and the relative peak area RSD was less than 3.0% (as shown in Table 7), indicating that the Coptis chinensis extract test solution had good stability within 24h.

[0054] Table 6 Stability Study - Relative Retention Time (RSD) of Common Peaks

[0055]

[0056] Table 7 Stability Study - Relative Peak Area RSD of Each Common Peak

[0057]

[0058] Fingerprint mapping and similarity evaluation:

[0059] Identification of characteristic peaks and assignment of common peaks: Based on the chromatographic analysis results of the Coptis chinensis extract test solution and the first mixed reference solution, peak 2 was identified as 5-hydroxymethylfurfural, peak 3 as ferulic acid, peak 4 as berberine hydrochloride, peak 5 as ligustrazine lactone I, peak 7 as bis(demethoxy)curcumin, peak 8 as demethoxy)curcumin, peak 9 as curcumin, peak 10 as sesamin, peak 11 as ligustilide, and peak 12 as sesamin. Comparison of each reference herb solution with the Coptis chinensis extract test solution revealed that peaks 1, 2, 3, 5, and 11 originated from Angelica sinensis (tail); peaks 1 and 2 originated from Rehmannia glutinosa; peak 4 originated from Coptis chinensis and Phellodendron chinense; peaks 7, 8, 9, 14, 15, 16, and 17 originated from Curcuma longa; and peaks 6, 10, 11, 12, and 13 originated from the extraction solvent sesame oil. The results are shown in the table below. Figure 1 and Figure 2 ;

[0060] Establishment of the fingerprint chromatogram for Coptis chinensis extract: Sixteen batches of Coptis chinensis extract sample solutions (S1-S16) were prepared, and the fingerprints of each batch were determined. The corresponding chromatograms were recorded. The results are shown in the figure below. Figure 3 ;

[0061] Fingerprint chromatogram similarity evaluation: The fingerprint chromatograms of the 16 batches of Coptis chinensis extract samples were imported into the 2012 version of the Chinese Pharmacopoeia Commission's official software for evaluating the similarity of chromatographic fingerprint chromatograms of traditional Chinese medicine, and reference chromatograms were obtained. Figure 4 Fingerprint patterns of 16 batches of Coptis chinensis extract samples Figure 3 Comparison map with generated map Figure 4 The similarity scores among the samples were 0.978, 0.984, 0.970, 0.980, 0.975, 0.970, 0.987, 0.990, 0.977, 0.988, 0.958, 0.987, 0.948, 0.976, 0.977, and 0.989, respectively, all greater than 0.9, indicating that the 16 batches of Coptis chinensis extract samples had stable quality and no significant fluctuations in chemical composition.

[0062] Example 2

[0063] Methods for determining the content of multiple components in Coptis chinensis extract:

[0064] The method for determining the content of multiple components in Coptis chinensis extract using HPLC fingerprinting is described above. This method is used to determine the content of 5-hydroxymethylfurfural, ferulic acid, berberine hydrochloride, ligustilide I, curcumin, and ligustilide in Coptis chinensis extract. The specific operation is as follows:

[0065] a. Take 7g of Coptis chinensis extract sample, accurately weigh it, place it in a stoppered conical flask, add 25ml of methanol, seal tightly, weigh it, sonicate at 55℃±5℃ for 30 minutes, cool it, weigh it again, replenish the lost weight with methanol, shake well, let it stand until the methanol layer is clear, separate the methanol layer, centrifuge at 15,000g and 4℃ for 15 minutes, filter it, and take the filtrate to obtain the test solution.

[0066] b. Take 5-hydroxymethylfurfural, ferulic acid, berberine hydrochloride, ligustilide I, curcumin, and ligustilide reference standards, and add methanol to prepare solutions containing 22 μg of 5-hydroxymethylfurfural, 2 μg of ferulic acid, 26 μg of berberine hydrochloride, 2 μg of ligustilide I, 70 μg of curcumin, and 30 μg of ligustilide per 1 ml, respectively, to obtain the second mixed reference solution;

[0067] c. Inject the test solution from step a and the second mixed reference solution from step b into the high-performance liquid chromatograph (HPLC) using Waters Sunfire C. 18250mm×4.6mm, 5μm as the chromatographic column, acetonitrile as mobile phase A, 0.3% phosphoric acid solution as mobile phase B, gradient elution according to time 0-5min, mobile phase A 10-20%; 5-23min, mobile phase A 20-25%; 23-26min, mobile phase A 25-50%; 26-46min, mobile phase A 50-60%; 46-51min, mobile phase A 60-65%; 51-56min, mobile phase A 65%; 56-63min, mobile phase A 65-70%; 63-70min, mobile phase A 70-80%; column temperature was 25℃, flow rate was 1.0mL / min, injection volume was 10μl, detection wavelength was 280nm for 5-hydroxymethylfurfural and ligustilide I, 260nm for berberine hydrochloride, 321nm for ferulic acid and ligusticum lactone, 430nm for curcumin;

[0068] Methodology investigation:

[0069] Linear range investigation:

[0070] 5-hydroxymethylfurfural, ligustilide I, berberine hydrochloride, ferulic acid, ligusticum lactone, curcumin concentration of 136μg / ml, 81μg / ml, 166μg / ml, 75μg / ml, 804μg / ml, 95μg / ml mixed control stock solution, sequentially diluted by multiple, add methanol to the scale to prepare a series of control solutions; with Waters Sunfire C 18 250mm×4.6mm, 5μm as the chromatographic column, acetonitrile as mobile phase A, 0.3% phosphoric acid solution as mobile phase B, gradient elution according to time 0-5min, mobile phase A 10-20%; 5-23min, mobile phase A 20-25%; 23-26min, mobile phase A 25-50%; 26-46min, mobile phase A 50-60%; 46-51min, mobile phase A 60-65%; 51-56min, mobile phase A 65%; 56-63min, mobile phase A 65-70%; 63-70min, mobile phase A 70-80%; column temperature was 25℃, flow rate was 1.0mL / min, injection volume was 10μl, detection wavelength was 280nm for 5-hydroxymethylfurfural and ligustilide I, 260nm for berberine hydrochloride, 321nm for ferulic acid and ligusticum lactone, 430nm for curcumin;

[0071] The peak area (Y) as the ordinate, the concentration of the control (X) as the abscissa, the standard curve was drawn, the standard curve equation and the correlation coefficient (R) of 5-hydroxymethylfurfural, ligustilide I, berberine hydrochloride, ferulic acid, ligusticum lactone, curcumin were shown in Table 8, the results showed that each component had good linear relationship in the corresponding concentration range;

[0072] Table 8 Results of linear relationship investigation

[0073]

[0074] Precision investigation:

[0075] Take 5-hydroxymethyl furfural, ligustilide I, berberine hydrochloride, ferulic acid, ligusticum lactone, curcumin concentration of 34.00 μg / ml, 10.13 μg / ml, 41.50 μg / ml, 9.38 μg / ml, 100.50 μg / ml, 47.50 μg / ml mixed reference solution, Waters Sunfire C 18 250mm×4.6mm, 5μm as chromatographic column, with acetonitrile as mobile phase A, with 0.3% phosphoric acid solution as mobile phase B, according to time 0-5min, mobile phase A 10-20%; 5-23min, mobile phase A 20-25%; 23-26min, mobile phase A 25-50%; 26-46min, mobile phase A 50-60%; 46-51min, mobile phase A 60-65%; 51-56min, mobile phase A 65%; 56-63min, mobile phase A 65-70%; 63-70min, mobile phase A 70-80% gradient elution; column temperature is 25℃, flow rate is 1.0mL / min, injection volume is 10μl, detection wavelength is 5-hydroxymethyl furfural and ligustilide I 280nm, berberine hydrochloride 260nm, ferulic acid and ligusticum lactone 321nm, curcumin 430nm, continuous injection determination for 6 times, record chromatogram; the results show that the RSD value of peak area of each component is less than 0.5% (as shown in Table 9), indicating that the precision of the instrument is good;

[0076] Table 9 Results of precision investigation

[0077]

[0078]

[0079] Repeatability investigation:

[0080] Take 7g of Huanglian paste sample, accurately weigh, put it in a conical flask with a plug, add 25ml of methanol, seal tightly, weigh, temperature 55℃±5℃, ultrasonic treatment for 30 minutes, cool, weigh again, make up the weight loss with methanol, shake well, place until the methanol layer is clear, take the methanol layer, centrifuge at 15,000g, 4℃ for 15min, filter, take the filtrate, which is the test solution. Prepare 6 test solutions in parallel; take Waters Sunfire C 18A 250 mm × 4.6 mm, 5 μm column was used. Acetonitrile was used as mobile phase A, and 0.3% phosphoric acid solution was used as mobile phase B. Gradient elution was performed at the following times: 0-5 min, mobile phase A 10-20%; 5-23 min, mobile phase A 20-25%; 23-26 min, mobile phase A 25-50%; 26-46 min, mobile phase A 50-60%; 46-51 min, mobile phase A 60-65%; 51-56 min, mobile phase A 65%; 56-63 min, mobile phase A 65-70%; 63-70 min, mobile phase A 70-80%. The column temperature was 25℃, the flow rate was 1.0 mL / min, the injection volume was 10 μl, and the detection wavelengths were 5-hydroxymethylfurfural and ligustilide I. The concentrations of berberine hydrochloride were 280 nm, berberine hydrochloride 260 nm, ferulic acid and ligustilide 321 nm, and curcumin 430 nm. The results showed that the RSD values ​​of each component were less than 6% (as shown in Table 10), indicating that the method had good reproducibility.

[0081] Table 10 Results of Repeatability Testing

[0082]

[0083] Stability assessment:

[0084] Accurately weigh 7g of Coptis chinensis extract sample and place it in a stoppered conical flask. Add 25ml of methanol, seal tightly, and weigh. Sonicate at 55℃±5℃ for 30 minutes, cool, and weigh again. Make up the lost weight with methanol, shake well, and let stand until the methanol layer is clear. Separate the methanol layer, centrifuge at 15,000g at 4℃ for 15 minutes, filter, and collect the filtrate to obtain the test solution. (Waters Sunfire C) 18250mm x 4.6mm, 5μm as chromatographic column, with acetonitrile as mobile phase A, with 0.3% phosphoric acid solution as mobile phase B, according to time 0-5min, mobile phase A 10-20%; 5-23min, mobile phase A 20-25%; 23-26min, mobile phase A 25-50%; 26-46min, mobile phase A 50-60%; 46-51min, mobile phase A 60-65%; 51-56min, mobile phase A 65%; 56-63min, mobile phase A 65-70%; 63-70min, mobile phase A 70-80% for gradient elution, respectively at 0h, 3h, 6h, 9h, 12h, 24h for determination, column temperature is 25℃, flow rate is 1.0mL / min, injection volume is 10μl, detection wavelength is 280nm for 5-hydroxymethylfurfural and senkyunol I, 260nm for berberine hydrochloride, 321nm for ferulic acid and ligusticum lactone, 430nm for curcumin, the results show that the RSD of peak area of each component is less than 3.0%(as shown in Table 11), which indicates that the Huanglian paste test sample solution has good stability within 24h;

[0085] Table 11 Stability test results

[0086]

[0087] Recovery rate investigation:

[0088] Take the known content of Huanglian paste sample 3.5g (batch number HLG-1), accurately weigh, add 5-hydroxymethylfurfural, senkyunol I, berberine hydrochloride, ferulic acid, ligusticum lactone, curcumin reference substance at the ratio of 1:1, place in a stoppered conical flask, add 25ml of methanol, seal tightly, weigh, temperature 55℃±5℃, ultrasonic treatment for 30 minutes, cool, weigh again, make up the weight loss with methanol, shake well, place until the methanol layer is clear, take the methanol layer, centrifuge at 15,000g, 4℃ for 15min, filter, take the filtrate, namely the test sample solution; prepare 6 test sample solutions in parallel, use Waters Sunfire C 18250mm x 4.6mm, 5μm as chromatographic column, with acetonitrile as mobile phase A, with 0.3% phosphoric acid solution as mobile phase B, according to time 0-5min, mobile phase A 10-20%; 5-23min, mobile phase A 20-25%; 23-26min, mobile phase A 25-50%; 26-46min, mobile phase A 50-60%; 46-51min, mobile phase A 60-65%; 51-56min, mobile phase A 65%; 56-63min, mobile phase A 65-70%; 63-70min, mobile phase A 70-80% gradient elution, column temperature is 25℃, flow rate is 1.0mL / min, injection volume is 10μl, detection wavelength is 280nm for 5-hydroxymethylfurfural and ligustilide I, 260nm for berberine hydrochloride, 321nm for ferulic acid and ligusticum lactone, 430nm for curcumin, the results show that the average recovery of each component is 101.49%, 95.68%, 92.55%, 90.11%, 94.80%, 100.77% respectively, and the RSD value is 4.33%, 3.50%, 5.26%, 4.86%, 5.13%, 5.94% respectively (as shown in Table 12), the accuracy of the method is good;

[0089] Table 12: Recovery rate test results

[0090]

[0091]

[0092] Determination of multiple components in Huanglian paste:

[0093] Take 7g of 16 batches of Huanglian paste samples, accurately weigh, place in a conical flask with a plug, add 25ml of methanol, seal tightly, weigh, temperature 55℃±5℃, ultrasonic treatment for 30 minutes, cool, weigh again, make up the weight loss with methanol, shake well, place until the methanol layer is clear, take the methanol layer, centrifuge at 15,000g and 4℃ for 15min, filter, take the filtrate, i.e. 16 batches of Huanglian paste sample test solution;

[0094] Take 5-hydroxymethylfurfural, ferulic acid, berberine hydrochloride, ligustilide I, curcumin, ligusticum lactone control, add methanol to prepare a solution containing 5-hydroxymethylfurfural 22μg, ferulic acid 2μg, berberine hydrochloride 26μg, ligustilide I 2μg, curcumin 70μg, ligusticum lactone 30μg per 1ml, i.e. second mixed control solution;

[0095] Take the test solution and the second mixed control solution and inject them into the high performance liquid chromatograph, with Waters Sunfire C 18250mm x 4.6mm, 5μm as chromatographic column, with acetonitrile as mobile phase A, with 0.3% phosphoric acid solution as mobile phase B, according to time 0-5min, mobile phase A 10-20%; 5-23min, mobile phase A 20-25%; 23-26min, mobile phase A 25-50%; 26-46min, mobile phase A 50-60%; 46-51min, mobile phase A 60-65%; 51-56min, mobile phase A 65%; 56-63min, mobile phase A 65-70%; 63-70min, mobile phase A 70-80% gradient elution; column temperature is 25℃, flow rate is 1.0mL / min, injection volume is 10μl, detection wavelength is 280nm for 5-hydroxymethyl furfural and ligustilide I, 260nm for berberine hydrochloride, 321nm for ferulic acid and ligusticum lactone, 430nm for curcumin;

[0096] Table 13 Content determination results of 16 batches of huangliancao samples

[0097]

[0098]

[0099] As can be seen from Table 13, the content of 5-hydroxymethyl furfural is 0.019mg / g-0.067mg / g; the content of ligustilide I is 0.008mg / g-0.014mg / g; the content of berberine hydrochloride is 0.050mg / g-0.149mg / g; the content of ferulic acid is 0.007mg / g-0.010mg / g; the content of ligusticum lactone is 0.091mg / g-0.141mg / g; the content of curcumin is 0.071mg / g-0.268mg / g.

Claims

1. A method for constructing an HPLC fingerprint of Coptis chinensis extract, characterized in that, Follow these steps: a. Accurately weigh the Coptis chinensis extract sample, place it in a stoppered conical flask, add 25 ml of methanol, seal tightly, weigh, sonicate at 55℃±5℃ for 30 minutes, cool, weigh again, replenish the lost weight with methanol, shake well, let stand until the methanol layer is clear, separate the methanol layer, centrifuge at 15,000 g at 4℃ for 15 minutes, filter, and take the filtrate to obtain the test solution. b. Take 5-hydroxymethylfurfural, ferulic acid, berberine hydrochloride, ligustrazine lactone I, bis(demethoxycurcumin), demethoxycurcumin, curcumin, sesamin, ligustilide, and sesamolin reference standards, and add methanol to prepare solutions containing 20 μg, 42 μg, 57 μg, 53 μg, 10 μg, 22 μg, 24 μg, 80 μg, 96 μg, and 60 μg per ml, respectively, to obtain the first mixed reference solution; c. Inject the test solution from step a and the first mixed reference solution from step b into the high-performance liquid chromatograph. Chromatographic conditions: Waters Sunfire C 18 A 250mm × 4.6mm, 5μm column was used. Acetonitrile was used as mobile phase A, and 0.3% phosphoric acid solution was used as mobile phase B. The mobile phase concentrations were as follows: 0-5 min, mobile phase A 10-20%; 5-23 min, mobile phase A 20-25%; 23-26 min, mobile phase A 25-50%; 26-46 min, mobile phase A 50-60%; 46-51 min, mobile phase A 60-65%; 51-56 min, mobile phase A 65%; 56-63 min, mobile phase A 65-70%; 63-70 min, mobile phase A... Gradient elution was performed at 70-80%; column temperature was 25℃; flow rate was 1.0 mL / min; injection volume was 10 μl; detection wavelength was 260 nm. The HPLC fingerprint of Coptis chinensis extract was obtained. With curcumin chromatographic peak as reference peak S, 17 common peaks were detected in the HPLC fingerprint of Coptis chinensis extract at 260 nm.

2. A method for determining the content of multiple components by constructing an HPLC fingerprint of Coptis chinensis extract as described in claim 1, characterized in that, This method is used to determine the content of 5-hydroxymethylfurfural, ferulic acid, berberine hydrochloride, ligustilide I, curcumin, and ligustilide in Coptis chinensis extract. The specific operation is carried out according to the following steps: a. Accurately weigh the Coptis chinensis extract sample, place it in a stoppered conical flask, add 25 ml of methanol, seal tightly, weigh, sonicate at 55℃±5℃ for 30 minutes, cool, weigh again, replenish the lost weight with methanol, shake well, let stand until the methanol layer is clear, separate the methanol layer, centrifuge at 15,000 g at 4℃ for 15 min, filter, and take the filtrate to obtain the test solution. b. Take 5-hydroxymethylfurfural, ferulic acid, berberine hydrochloride, ligustilide I, curcumin, and ligustilide reference standards, and add methanol to prepare solutions containing 22 μg, 2 μg, 26 μg, 2 μg, 70 μg, and 30 μg per ml, respectively, to obtain the second mixed reference solution. c. Inject the test solution and the second mixed reference solution separately into the high-performance liquid chromatograph. Chromatographic conditions: Waters Sunfire C 18 A 250mm × 4.6mm, 5μm column was used. Acetonitrile was used as mobile phase A, and 0.3% phosphoric acid solution was used as mobile phase B. The mobile phase concentrations were as follows: 0-5 min, mobile phase A 10-20%; 5-23 min, mobile phase A 20-25%; 23-26 min, mobile phase A 25-50%; 26-46 min, mobile phase A 50-60%; 46-51 min, mobile phase A 60-65%; 51-56 min, mobile phase A 65%; 56-63 min, mobile phase A 65-70%; 63-70 min, mobile phase A... 70-80% were eluted using a gradient; the column temperature was 25℃, the flow rate was 1.0 mL / min, the injection volume was 10 μl, and the detection wavelengths were 5-hydroxymethylfurfural and ligusticil I 280 nm, berberine hydrochloride 260 nm, ferulic acid and ligustilide 321 nm, and curcumin 430 nm. d. The contents of 5-hydroxymethylfurfural were calculated to be 0.019-0.067 mg / g, ligustilide I to be 0.008-0.014 mg / g, berberine hydrochloride to be 0.050-0.149 mg / g, ferulic acid to be 0.007-0.010 mg / g, ligustilide to be 0.091-0.141 mg / g, and curcumin to be 0.071-0.268 mg / g, respectively, by linear regression equation.

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