CN110780008B

CN110780008B - Fingerprint spectrum and component content determination method of Chuanhuang preparation

Info

Publication number: CN110780008B
Application number: CN201911270467.7A
Authority: CN
Inventors: 罗宇东; 覃洁萍; 郭海姣; 谭安蔷; 李芳婵
Original assignee: Guangxi University Of Chinese Medicine Pharmaceutical Factory
Current assignee: Guangxi University Of Traditional Chinese Medicine Bainianle Pharmaceutical Co ltd
Priority date: 2019-12-11
Filing date: 2019-12-11
Publication date: 2022-07-01
Anticipated expiration: 2039-12-11
Also published as: CN110780008A

Abstract

The invention provides a method for establishing a fingerprint spectrum of a Chuanhuang preparation, which comprises the following steps: s1, preparation of a test solution: weighing 1.0g of Chuanhuang preparation powder, precisely weighing, placing in a 25mL volumetric flask, adding 20mL of 70% methanol, performing ultrasonic treatment, cooling, fixing volume to scale, shaking, filtering, collecting filtrate, centrifuging, and collecting supernatant, and filtering with microporous membrane; s2, high performance liquid chromatography detection: sucking the sample solution, injecting into high performance liquid chromatograph for high performance liquid chromatography detection, and recording fingerprint within 65 min. The invention adopts a high performance liquid chromatography combined with an ultraviolet dual-wavelength switching detection method to establish a characteristic fingerprint spectrum which can be used for measuring the Chuanhuang preparation, and the method has the advantages of accuracy, high efficiency, good repeatability and stability, strong fingerprint spectrum characteristic and accurate content measurement result, and can be used as a quality control method of the Chuanhuang preparation.

Description

Fingerprint spectrum and component content determination method of Chuanhuang preparation

Technical Field

The invention relates to the technical field of traditional Chinese medicine detection, and particularly relates to a fingerprint spectrum and component content determination method of a Chuanhuang preparation.

Background

The Chuanhuang preparation is a Chinese patent medicine prepared from two medicinal materials of common andrographis herb and solidago decurrens, is compared with the safest traditional Chinese medicine antibiotic due to wide antimicrobial spectrum. Has effects in clearing away heat and toxic materials, inhibiting bacteria, relieving inflammation, relieving swelling and pain, and clearing away the heat of upper-jiao. The herba Andrographitis in the preparation has effects of clearing heat and detoxicating, diminishing inflammation, and resisting virus, and herba Solidaginis has effects of resisting inflammation, resisting bacteria, dispelling pathogenic wind, clearing heat, removing toxic substance, relieving swelling, and promoting phagocytosis of leukocyte. The two ingredients are used together to treat excessive toxic heat such as acute upper respiratory infection, acute tonsillitis, pharyngolaryngitis, etc. The quality control method of the traditional Chuanhuang preparation not only identifies the thin layer of the andrographis paniculata and the solidago decurrens and measures the content of the dehydroandrographolide, but also only measures the content of the andrographolide and the dehydroandrographolide in the preparation in literature reports. Obviously, the existing quality control method still has certain local effect and cannot achieve the purpose of comprehensively controlling the quality of the Chuanhuang preparation. According to the reports of the literature, the contents of neoandrographolide and deoxyandrographolide in the andrographis paniculata medicinal materials are high besides andrographolide and dehydroandrographolide, and the two components are also effective components of andrographis paniculata with anti-inflammatory and anti-virus effects, so that the andrographis paniculata medicinal materials also need to be included in index components for controlling the quality of the preparations containing andrographis paniculata with jaundice. Research shows that the solidago decurrens contains various caffeoyl quinic acid components such as chlorogenic acid and the like. The components have obvious pharmacological activities of anti-inflammation, antipyresis, antibiosis and antivirus, the anti-inflammation activity of the preparation is stronger than that of common anti-inflammatory aspirin, the antivirus activity and the inhibition effect on respiratory tract infection caused by virus are particularly obvious, and the components are all related to the efficacy of the preparation.

Disclosure of Invention

The invention provides a finger-print and component content determination method for a Chuanhuang preparation, which can accurately and efficiently determine the content of each component in the Chuanhuang preparation with good repeatability and stability and can be used as a quality control method for the Chuanhuang preparation.

The technical scheme of the invention is realized as follows:

the invention provides a method for establishing a fingerprint of a Chuanhuang preparation, which comprises the following steps:

s1, preparation of a test solution: weighing 1.0g of Chuanhuang preparation powder, precisely weighing, placing in a 25mL volumetric flask, adding 70% methanol 20mL, performing ultrasonic treatment for 30min, cooling, fixing volume to scale, shaking, filtering, collecting the filtrate, 13000 r.min^-1Centrifuging, and filtering the supernatant with 0.45 μm microporous membrane;

s2, high performance liquid chromatography detection: sucking the sample solution obtained in the step S1, injecting the sample solution into a high performance liquid chromatograph for high performance liquid chromatography detection, and recording the fingerprint within 65 min;

wherein, the fingerprint chromatogram condition is as follows:

using an Agilent Technologies EC-C18 column, 4.6mm X250 mm, 4 μm, mobile phase: acetonitrile-0.2% aqueous phosphoric acid solution system; adopting a gradient elution mode: 0min → 10min → 15min → 20min → 30min → 40min → 50min → 65 min; acetonitrile 10% → 15% → 21% → 25% → 28% → 34% → 36%; flow rate 1.0 mL/min^-1(ii) a Dual wavelength switching timeAnd (3) sequence sampling: 0-35min, 323 nm; 35-65min, 215 nm; the sample injection amount is 5 mu L; the column temperature is 25 ℃; the analysis time is 65 min; the theoretical plate number is not less than 41000 calculated by isochlorogenic acid C peak.

As a further improvement of the invention, the preparation method of the Chuanhuang preparation powder comprises the following steps: taking the heat-clearing tablet, removing the film coat, and grinding through a fifth sieve.

As a further improvement of the invention, the preparation method of the Chuanhuang preparation powder comprises the following steps: taking Chuanhuang heat-clearing capsule, pouring out the content, grinding and sieving through a No. five sieve.

The invention further protects the fingerprint of the Chuanhuang preparation established by the method, and the fingerprint comprises characteristic peaks of neochlorogenic acid (1), chlorogenic acid (2), cryptochlorogenic acid (3), isochlorogenic acid B (4), isochlorogenic acid A (5), isochlorogenic acid C (7), andrographolide (8), neoandrographolide (9), deoxyandrographolide (10) and dehydroandrographolide (11), and the retention time of the neoandrographolide (10) and the dehydroandrographolide (11) is 7.1-7.3min, 13.1-13.7min, 15.5-16.2min, 27.8-28.2min, 29.4-29.8min, 33.2-33.8min, 41.5-42.3min, 55.5-56.1min, 60.0-60.8min and 61.6-62.4 min.

As a further improvement of the invention, the fingerprint also comprises a characteristic peak of number (6), and the retention time of the characteristic peak is 32.2-32.8 min.

The invention further provides a method for measuring the component content of the Chuanhuang preparation by using the fingerprint, which comprises the following steps:

s1, preparation of a mixed reference solution: precisely weighing a proper amount of each reference substance, placing into 10 measuring bottles with the volume of 5mL, adding methanol for dissolving, and fixing the volume to obtain reference substance stock solutions of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, andrographolide, neoandrographolide, deoxyandrographolide and dehydroandrographolide; taking appropriate amount of the above reference stock solutions, placing in the same 5mL measuring flask, adding methanol to dilute to scale, shaking to obtain the final product containing the above reference concentrations of 0.184, 0.258, 0.202, 0.260, 0.178, 0.0934, 0.652, 1.034, 0.234, and 1.132 mg/mL^-1Mixed control stock solution of (1). Precise sucking mixed reference substance stock solution0.5mL, placing in a 2mL measuring flask, adding methanol to dilute to scale, shaking, and making into beverage containing neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, andrographolide, neoandrographolide, deoxyandrographolide and dehydroandrographolide with concentrations of 0.0460, 0.0645, 0.0505, 0.0650, 0.0445, 0.0234, 0.163, 0.258, 0.0585, and 0.283 mg/mL^-1Mixing the reference substance solution to obtain the product;

s2, preparation of a test solution: precisely weighing 1.0g of Chuanhuang preparation powder, placing in a 25mL volumetric flask, adding 20mL of 70% methanol, performing ultrasonic treatment for 30min, cooling, fixing volume to scale, shaking up, filtering, collecting filtrate, 13000 r.min^-1Centrifuging, and filtering the supernatant with 0.45 μm microporous membrane;

s3, selecting chromatographic conditions and testing system adaptability: agilent Technologies EC-C18 column, 4.6mm X250 mm, 4 μm, mobile phase: acetonitrile-0.2% aqueous phosphoric acid solution system; adopting a gradient elution mode: 0min → 10min → 15min → 20min → 30min → 40min → 50min → 65 min; acetonitrile 10% → 15% → 21% → 25% → 28% → 34% → 36%; flow rate 1.0 mL/min^-1(ii) a Dual wavelength switching time series sampling: 0-35min, 323 nm; 35-65min, 215 nm; the sample injection amount is 5 mu L; the column temperature is 25 ℃; the analysis time is 65 min; the theoretical plate number is not less than 41000 calculated by isochlorogenic acid C peak;

s4, sucking 1-5 mu L of each of the reference solution and the sample solution, injecting the reference solution and the sample solution into a liquid chromatograph, measuring the corresponding peak areas of the components, and calculating according to an external standard method to obtain the content of 10 main components in the different batch numbers of the Chuanhuang preparation.

As a further improvement of the invention, the preparation method of the Chuanhuang preparation powder comprises the following steps: taking Chuanhuang heat-clearing capsule, pouring out the content, grinding and sieving with a No. five sieve.

As a further improvement of the present invention, the absorption amounts of the control solution and the test solution in step S4 are 5 μ L respectively.

The invention has the following beneficial effects: the invention adopts a high performance liquid chromatography combined with an ultraviolet dual-wavelength switching detection method to establish a detection method which can determine the characteristic fingerprint of the Chuanhuang preparation and can simultaneously determine 10 main components such as caffeoylquinic acids, andrographolide and the like in the preparation. Test results show that the method is accurate and efficient, has good repeatability and stability, strong fingerprint characteristic and accurate content measurement result, and can be used as a quality control method of the Chuanhuang preparation.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is an overlay of fingerprint spectra of 15 batches of Chuanhuang preparation;

FIG. 2 is a HPLC fingerprint chromatogram common mode chart of Chuanhuang preparation;

FIG. 3 is a diagram showing the common fingerprint pattern of Chuanhuang preparation and the HPLC chromatogram of each reference medicinal material sample;

FIG. 4 a proprietary HPLC chromatogram.

Detailed Description

The invention provides a fingerprint spectrum and a component content measuring method of a Chuanhuang preparation, and the invention is further described in detail below in order to make the purpose, technical scheme and effect of the invention clearer and more clear. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

1 Instrument and reagent

1.1 an Agilent-1260 high performance liquid chromatograph, which comprises a quaternary pump (model: G1311B), an automatic sample injector (model: G1329B), a column oven (model: G1316A), a UV type ultraviolet detector (model G1314F), and an Agilent workstation (Agilent, USA); waters e2695 high performance liquid chromatograph, PDA type ultraviolet Detector (model: 2998PDA Detector); a Misibo super water purifier; electronic analytical balance (one hundred thousand, SQP, sydows scientific instruments ltd); TGL-16G centrifuge (Shanghai' an pavilion scientific instruments factory); model KQ5200B ultrasonic cleaner (ultrasonic instruments ltd, kunshan).

1.2 reagent acetonitrile is chromatographically pure (Fisher company, batch: 186356), water is ultrapure water; other reagents such as methanol and ethanol are analytically pure.

1.3 reference substances of neochlorogenic acid (batch No. 6603), cryptochlorogenic acid (batch No. 3208) and isochlorogenic acid B (batch No. 3089) Shanghai Shidande Standard technology service Co., Ltd; chlorogenic acid (batch number: 110753-201817) China institute for food and drug testing; isochlorogenic acid A (batch No. 180307), isochlorogenic acid C (batch No. 180126) Shanghai Rong medicinal science and technology Co., Ltd; neoandrographolide (batch number: 20171215), deoxyandrographolide (batch number: A2025J08), Tianjin Wanxiang Hengyu technology, Inc.; the purity of the reference substances is more than 98 percent through HPLC content determination in the China pharmaceutical biological product assay institute of andrographolide (batch No. 110797-200307) and dehydroandrographolide (batch No. 110854-200306).

1.4 the medicinal materials Solidago decurrens (batch number: 170301) and Andrographis paniculata (batch number: 180601) are provided by Nanning biogenic Chinese medicine decoction piece Limited liability company and identified by professor Cai Yi of the institute of Chinese medicine identification, university of Guangxi Chinese medicine. Herba Solidago decurrens Thunb of Compositae is dried whole herb of Solidago decurrens Lour, and herba Andrographitis is dried aerial part of Nees of Acanthaceae, Andrographis paniculata (Burm.f.). The above medicinal materials are all in accordance with the relevant regulations of Chinese pharmacopoeia by detection.

1.5 Chuanhuang preparation sample: puncturing yellow tablets: lot number 20171101(0.21 g/tablet, pharmaceutical factory, university of traditional Chinese medicine, Guangxi); chuanhuang capsule: lot numbers 20190408, 20190413, 20190418, 20190421(0.30 g/grain, cantonese university of traditional Chinese medicine pharmaceutical center laboratory); chuanhuang capsule: batch number: 20180101, 20180701, 20180704(0.30 g/pellet, Shanxihuayuan pharmaceutical biotechnology, Inc.); chuanhuang capsule: batch No. 20180331 (0.31 g/pellet, Kangfu pharmaceutical Co., Ltd., Jilin province); chuanhuang capsule: lot numbers 20180504, 20180905 (0.36 g/pellet, Sichuan good doctor pharmaceutical group Co., Ltd.); chuanhuang capsule: batch No. 20171201(0.32 g/pellet, Tianjin and pharmaceutical industries group Co., Ltd.); chuanhuang capsule: lot number 20190201(0.40 g/grain, Jiangxi silver Tao pharmaceutical Co., Ltd.); chuanhuang capsule: lot number 20190101(0.40 g/pellet, Hubei Tanjie pharmaceutical Co., Ltd.); chuanhuang capsule: batch No. 190301 NO. 043(0.41 g/pellet, Chongqing Sanxia Yunyan pharmaceutical Co., Ltd.).

2 methods and results

2.1 chromatographic conditions Agilent Technologies EC-C18(4.6 mm. times.250 mm, 4 μm); mobile phase acetonitrile (a) -0.2% aqueous phosphoric acid (B); gradient elution (0-10 min, 10% A, 10-15 min, 10% -15% A, 15-20 min, 15% -21% A, 20-30 min, 21% -25% A, 30-40 min, 25% -28% A, 40-50 min, 28% -34% A, 50-65 min, 34% -36% A); flow rate 1.0 ml.min^-1(ii) a Sampling a dual-wavelength switching time sequence (0-35 min, 323 nm; 35-65min, 215 nm); the sample injection amount is 5 mu L; the column temperature was 25 ℃. The theoretical plate number is not less than 41000 calculated by isochlorogenic acid C peak.

2.2 preparation of the solution

2.2.1 preparation of control solution A proper amount of each control is precisely weighed, placed in 10 measuring bottles with 5mL, dissolved by adding methanol and subjected to constant volume to respectively prepare control stock solutions of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, andrographolide, neoandrographolide, deoxyandrographolide and dehydroandrographolide; taking appropriate amount of the above control solutions, placing in the same 5mL measuring flask, adding methanol to dilute to scale, shaking, and making into the product containing the above control solutions with concentrations of 0.184, 0.258, 0.202, 0.260, 0.178, 0.0934, 0.652, 1.034, 0.234, and 1.132 mg/mL^-1Mixed control stock solution of (1). Precisely sucking 0.5mL of mixed control stock solution, placing in a 2mL measuring flask, adding methanol to dilute to scale, shaking, and making into the final product containing neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, andrographolide, neoandrographolide, deoxyandrographolide and dehydroandrographolide with concentrations of 0.0460, 0.0645, 0.0505, 0.0650, 0.0445, 0.0234，0.163，0.258，0.0585，0.283mg·mL^-1Mixing the reference solution to obtain the final product.

2.2.2 preparation of test solutions

The preparation method of the test sample for wearing the yellow heat-clearing tablet comprises the following steps: taking 20 tablets of the product, removing the film coat, and precisely weighing. Grinding, sieving with a five-mesh sieve, weighing about 1.0g, placing in a 25mL volumetric flask, adding 70% methanol 20mL, ultrasonic treating for 30min, cooling, fixing volume to scale, shaking, filtering, collecting filtrate, 13000 r.min^-1Centrifuging, and filtering the supernatant with 0.45 μm microporous membrane.

The preparation method of the Chuanhuang heat-clearing capsule test sample comprises the following steps: taking 10 granules of the product, pouring out the content, precisely weighing, grinding through a five-mesh sieve, taking about 1.0g, precisely weighing, placing in a 25mL volumetric flask, adding 20mL of 70% methanol, performing ultrasonic treatment for 30min, cooling, fixing the volume to the scale, shaking up, filtering, taking the subsequent filtrate, 13000 r.min^-1Centrifuging, and filtering the supernatant with 0.45 μm microporous membrane.

2.2.3 preparation of negative control solution for medicinal materials A part of each of the herba Solidaginis lacking and herba Andrographitis lacking medicinal materials is weighed according to the prescription, and prepared according to the preparation process of the prescription to obtain the control samples of herba Solidaginis lacking and herba Andrographitis lacking medicinal materials respectively. Taking the above control samples, and preparing according to the method under item 2.2.2 to obtain herba Solidaginis negative control sample solution and herba Andrographitis negative control sample solution.

2.3 fingerprint chromatogram research and establishment of Chuanhuang preparation

2.3.1 stability test 1.0g of 20171101 sample was taken, a test solution was prepared by the method under the item "2.2.2", and under the chromatographic condition under the item "2.1", 5. mu.L of sample was injected for analysis in 0, 2, 4, 8, 12, and 24 hours, respectively, to determine the chromatographic peak area. By taking the retention time and the chromatographic peak area of the No. 4 peak (isochlorogenic acid B) as reference, the relative retention time RSD of the other 10 peaks except the isochlorogenic acid B is calculated to be between 0.03 and 0.38 percent, and the relative peak area RSD is between 0.47 and 1.8 percent, which indicates that the test solution of the yellow-through preparation has good stability within 24 hours and meets the related requirements and regulations of fingerprint determination.

2.3.2 precision test sample solution of lot 20171101 was precisely aspirated, and sample introduction was performed continuously for 6 times of assay under the chromatographic condition of "2.1". And (3) calculating to obtain the relative retention time RSD of 10 peaks except the isochlorogenic acid B between 0.05 and 0.25 percent and the relative peak area RSD between 1.0 and 2.0 percent by taking the retention time and the chromatographic peak area of the No. 4 peak (isochlorogenic acid B) as reference, wherein the instrument precision is good and the relative requirements and regulations of fingerprint spectrum determination are met.

2.3.3 repeatability test 6 parts in total of 1.0g of 20171101 lot were weighed out, 6 parts of test solution were prepared by the method under the item "2.2.2", and 5. mu.L of sample was injected under the chromatographic condition of the item "2.1" for assay. And as a result, the retention time of the No. 4 peak (isochlorogenic acid B) and the chromatographic peak area are taken as references, the relative retention time RSD of the other 10 peaks except the isochlorogenic acid B is calculated to be 0.02-0.39%, and the relative peak area RSD is calculated to be 0.90-1.5%, so that the method is good in repeatability and meets the requirement of fingerprint determination.

2.3.4 establishment of HPLC fingerprint of Chuanhuang preparation A proper amount of Chuanhuang preparation is taken from each batch, a test solution is prepared according to the method under item 2.2.2, and sample injection determination analysis is carried out under the chromatographic condition of item 2.1, and the result is shown in figure 1. The fingerprint similarity evaluation system of traditional Chinese medicine chromatogram (2012.130723 edition) is adopted to analyze the measured fingerprints of each sample, a common mode of HPLC fingerprints of the Chuanhuang preparation is established, 11 common characteristic peaks are formed, and the result is shown in figure 2. Wherein, the 4 peak is isochlorogenic acid B, which has better separation degree, higher chromatographic response value and proper retention time, so that the peak is selected as the reference peak. The results of the similarity calculation of the finger prints of 15 batches of Chuanhuang preparation are shown in Table 1. As can be seen from Table 1, the similarity of the Chuanhuang preparation in each batch is above 0.90, and the similarity is good, which indicates that the consistency of the index components contained in each batch of samples is good.

In FIG. 2, 1 is neochlorogenic acid; 2 is chlorogenic acid; 3 is cryptochlorogenic acid; 4 is isochlorogenic acid B; 5 is isochlorogenic acid A; 7 is isochlorogenic acid C; 8 is andrographolide; 9 is neoandrographolide; 10 is deoxyandrographolide; 11 is dehydroandrographolide.

2.3.5 HPLC fingerprint common peak attribution analysis of Chuanhuang preparation precisely absorbs reference solution of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, andrographolide, neoandrographolide, deoxyandrographolide and dehydroandrographolide, analyzes and determines according to the chromatographic condition under item '2.1', adopts diode array detector to record chromatogram and ultraviolet absorption spectrum of each peak at the same time, and compares with each common characteristic peak in the fingerprint common mode, and shows that the retention time and ultraviolet absorption spectrum of the characteristic peaks No. 1, 2, 3, 4, 5, 7, 8, 9, 10 and 11 in the fingerprint common mode are consistent with those of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, andrographolide, neoandrographolide, andrographolide and dehydroandrographolide reference, therefore, the 1 st characteristic peak in the fingerprint spectrum common mode of the Chuanhuang preparation is determined to be neochlorogenic acid, the 2 nd characteristic peak is chlorogenic acid, the 3 rd characteristic peak is cryptochlorogenic acid, the 4 th characteristic peak is isochlorogenic acid B, the 5 th characteristic peak is isochlorogenic acid A, the 7 th characteristic peak is isochlorogenic acid C, the 8 th characteristic peak is andrographolide, the 9 th characteristic peak is neoandrographolide, the 10 th characteristic peak is deoxyandrographolide, and the 11 th characteristic peak is dehydroandrographolide. Precisely sucking the herba Andrographitis test solution and herba Solidaginis test solution prepared by the same method respectively, analyzing and determining according to the chromatographic condition under item "2.1", and comparing with each common characteristic peak in common fingerprint chromatogram of Chuanhuang preparation, the result is shown in FIG. 3. In fig. 3, a is a test solution spectrum of andrographis paniculata medicinal material; b is a sample solution spectrum of solidago decurrens; c is fingerprint of Chuanhuang preparation.

From the test results, the 1 st peak (neochlorogenic acid), the 2 nd peak (chlorogenic acid), the 3 rd peak (cryptochlorogenic acid), the 4 th peak (isochlorogenic acid B), the 5 th peak (isochlorogenic acid A) and the 7 th peak (isochlorogenic acid C) are belonging to the solidago decurrens medicinal material; peak No. 6 (unknown component), Peak No. 8 (andrographolide), Peak No. 9 (neoandrographolide), Peak No. 10 (deoxyandrographolide), and Peak No. 11 (dehydroandrographolide) were assigned to Andrographis paniculata Nees.

2.4 assay of 10 ingredients in Chuanhuang preparation

2.4.1 the system applicability and specificity test accurately absorbs the mixed reference substance solution, the Chuanhuang tablet and Chuanhuang capsule test substance solution and the medicinal material negative reference sample solution under the item 2.2.1, samples are injected and determined and analyzed under the chromatographic condition under the item 2.1, and the system adaptability is inspected. The result shows that the corresponding positions of the chromatogram of the reference substance and the chromatogram of the test substance have chromatographic peaks with the same retention time, the UV spectrums of the reference substance and the sample are consistent when being detected by a DAD detector, the separation degree of the chromatographic peaks of all the components reaches more than 1.5, and the requirement of content determination is met. Typical chromatograms are detailed in FIG. 4. In FIG. 4, A is the mixed control solution; b is a goldenrod negative control solution; c is andrographis paniculata negative control solution; d is Chuan Huang Capsule (batch No. 20180701); e is Chuanhuang tablet (batch No. 20171101), wherein 1 is neochlorogenic acid; 2 is chlorogenic acid; 3 is cryptochlorogenic acid; 4 is isochlorogenic acid B; 5 is isochlorogenic acid A; 6 is isochlorogenic acid C; 7 is andrographolide; 8 is neoandrographolide; 9 is deoxyandrographolide; 10 is dehydroandrographolide. The theoretical plate number is not less than 41000 calculated by the chromatographic peak of isochlorogenic acid C which is difficult to separate.

2.4.2 precision test 5. mu.L of the mixed control solution was precisely aspirated, and sample introduction was performed 6 times under the condition of "2.1" chromatography, and the area of each chromatographic peak was measured. The results show that the peak areas RSD of 6 times of determination of each component of the neochlorogenic acid, the chlorogenic acid, the cryptochlorogenic acid, the isochlorogenic acid B, the isochlorogenic acid A, the isochlorogenic acid C, the andrographolide, the neoandrographolide, the deoxyandrographolide and the dehydroandrographolide are respectively 0.89%, 0.85%, 0.78%, 0.82%, 0.83%, 0.80%, 0.83%, 0.84% and 0.84%, which indicates that the precision of the instrument is good.

2.4.3 stability test 5. mu.L of test solution of lot 20171101 was extracted precisely, and the sample was analyzed at 0, 2, 4, 8, 12, and 24h under the chromatographic conditions of item "2.1" to determine the area of the chromatographic peak. The results show that the RSD of the peak areas of each component of the neochlorogenic acid, the chlorogenic acid, the cryptochlorogenic acid, the isochlorogenic acid B, the isochlorogenic acid A, the isochlorogenic acid C, the andrographolide, the neoandrographolide, the deoxyandrographolide and the dehydroandrographolide is respectively 1.7%, 0.11%, 0.46%, 0.45%, 1.8%, 1.4%, 0.55%, 0.65%, 0.08% and 0.30%. The test solution is stable within 24 h.

2.4.4 Linear relationship examination respectively and precisely sucking 0.1, 0.3, 0.5, 0.7, 0.9 and 1.1mL of mixed reference stock solution prepared under item 2.2.1, placing in 62 mL measuring bottles, adding methanol to dilute to scale, shaking uniformly, and respectively making into beverage containing neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, andrographolide, neoandrographolide, deoxyandrographolide and dehydroandrographolide at concentration of 0.0092-0.101 mg/mL^-1，0.0129～0.142mg·mL^-1，0.0101～0.111mg·mL^-1， 0.0130～0.143mg·mL^-1，0.00890～0.0979mg·mL^-1，0.00467～0.0514mg·mL^-1， 0.0326～0.359mg·mL^-1，0.0517～0.569mg·mL^-1，0.0117～0.129mg·mL^-1，0.0566～ 0.623mg·mL^-1And (3) accurately sucking 5 mu L of each mixed reference substance solution from 6 mixed reference substance solutions within the range, injecting the solution into an HPLC chromatograph, performing sample injection analysis under the condition of '2.1' item chromatography, and measuring corresponding peak areas. Peak area is taken as ordinate (y), concentration (mg.mL)^-1) Regression calculation is performed for the abscissa (x) to obtain a linear equation, and the results show that the peak areas and the concentrations of the components in the above concentration ranges have good linear relations, and the results are shown in table 2.

2.4.5 repeatability test 6 samples with lot number 20171101 each 1.0g were weighed precisely, 6 test solutions were prepared according to the method under item "2.2.2", 5 μ L of sample was injected under the chromatographic condition of item "2.1" for analysis, the corresponding peak areas were determined, the content of each component in each sample was calculated, and the average content of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, andrographolide, neoandrographolide, deoxyandrographolide and dehydroandrographolide in the samples was 1.07, 1.42, 1.20, 1.68, 1.12, 0.518, 1.12, 6.46, 1.37, 5.80mg · g, respectively^-1RSD is respectively 1.6%, 2.7%, 2.9%, 2.1%, 2.0%,1.6%, 1.5%, 1.3%, 1.4%, indicating that the method is highly reproducible.

2.4.6 sample application recovery test 6 parts in total of 0.5g of 20171101 lot sample are precisely weighed, and each part is precisely added with chlorogenic acid (0.574 mg. mL)^-1) Chlorogenic acid (0.806 mg. mL)^-1) Cryptochlorogenic acid (0.629 mg. mL)^-1) Isochlorogenic acid B (0.814 mg. multidot.mL)^-1) Isochlorogenic acid A (0.556 mg. multidot.mL)^-1) Isochlorogenic acid C (0.292 mg. multidot.mL)^-1) Andrographolide (0.588 mg. mL)^-1) Neoandrographolide (3.23 mg. mL)^-1) Deoxyandrographolide (0.732 mg/mL)^-1) And dehydroandrographolide (3.535 mg/mL)^-1) The mixed reference solution (1.00 mL) was subjected to sample injection (5. mu.L) under the chromatographic condition of "2.1" for analysis, and the results are shown in Table 3. Test results show that the method has good accuracy.

2.4.7 content determination 1.0g of Chuanhuang preparation of different lot numbers is precisely weighed and prepared into test solution according to the method under item 2.2.2. Under the condition of '2.1' item of chromatogram, respectively and precisely sucking 5 microlitres of mixed reference substance solution and each batch of Chuanhuang preparation sample solution, injecting into a high performance liquid chromatograph for analysis, measuring the corresponding peak areas of each component, and calculating according to an external standard method to obtain the content of 10 main components in Chuanhuang preparations of different batches. The results are shown in Table 4.

Discussion of 3

3.1 selection of chromatographic conditions

This trialThe experiment examined the separation of 3 different chromatographic columns, and examined the separation degree, peak shape and column efficiency of 3 different chromatographic columns of Agilent Technologies EC-C18 (4.6X 250mm, 4 μm), Agilent ZORBAX SB-C18(4.6mm X150 mm, 5.0 μm) and Elite Supersil ODS2(250mm X4.6 mm, 5 μm), and the result showed that Agilent Technologies EC-C18 (4.6X 250mm, 4 μm) was the best; 4 mobile phases of methanol-water, acetonitrile-0.1% phosphoric acid solution and acetonitrile-0.2% phosphoric acid solution are respectively considered for gradient elution, and the results show that the acetonitrile-0.2% phosphoric acid solution is used as the mobile phase, the separation degree of each peak is good, the peak-out time is moderate, and the peak type is good under the condition of gradient elution; by inspecting the spectra at different column temperatures (25, 30 and 35 ℃), the result is that the separation degree and the peak shape at the column temperature of 25 ℃ are better; for 0.8 mL/min^-1、 1.0mL·min^-1、1.2mL·min^-1The flow rates were examined at 3 different flow rates, using 1.0mL min^-1The flow rate elution effect is good; performing full-wavelength scanning on neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, andrographolide, neoandrographolide, deoxyandrographolide and dehydroandrographolide at the wavelength of 190-400 nm by using an ultraviolet diode array detector of a high performance liquid chromatograph, and obtaining the result that the optimal absorption wavelength of the neochlorogenic acid, the chlorogenic acid, the cryptochlorogenic acid, the isochlorogenic acid B, the isochlorogenic acid A and the isochlorogenic acid C is 323nm, and the optimal absorption wavelength of the andrographolide, the neoandrographolide and the deoxyandrographolide is 215 nm; in order to obtain the highest possible measurement sensitivity of each target component, the test adopts a dual-wavelength switching time sequence sampling mode for detection, namely 0-35min, 323 nm; 35-65min, 215 nm. Test results show that under the detection condition, each target component has better measurement sensitivity and better peak type, and can be well separated from coexisting components.

3.2 selection of extraction conditions

Tests investigate different preparation methods (cold soaking, ultrasonic extraction and reflux) of the test sample, and the results show that the efficiency of ultrasonic extraction and reflux extraction is obviously superior to that of cold soaking extraction, but the results measured by ultrasonic extraction and reflux extraction are not much different, so that a simpler, more convenient and faster ultrasonic extraction method is selected in the tests; the test also considers and compares the influence of different extraction solvents, different solvent dosage, different extraction times and extraction time on the content determination result, and the result shows that 25mL of 70% methanol is used for ultrasonic extraction for 30min, and all components are basically and completely extracted after 1 ultrasonic extraction, so the test sample preparation method is selected as the test sample preparation method in the test.

3.3 analysis of results the experiment determined the HPLC fingerprint of 15 preparations from different sources and the content of 10 main efficacy-related components. Test results show that the similarity of HPLC fingerprint spectra of 15 Chuanhuang preparations from different sources reaches above 0.9, which indicates that the components of Chuanhuang preparation products produced by different enterprises are basically consistent; however, the content determination result shows that there is a certain difference in the content of 10 main functional components of Chuanhuang preparation from different sources, for example, in Chuanhuang preparation produced by enterprises such as shanxi, tianjin and Hubei, the content of caffeoylquinic acid components is lower than that of products of enterprises such as Guangxi, Jiangxi, Sichuan and Chongqing, which may be related to factors such as the harvesting and producing area, harvesting time and maturity of medicinal materials.

3.4 summary of

The test adopts high performance liquid chromatography, combines a detection mode of ultraviolet double-wavelength switching, and establishes a detection method which can be simultaneously used for measuring the fingerprint spectrum of the Chuanhuang preparation and the content of 10 main components for the first time. The test result shows that the method is simple and rapid to operate, the measured fingerprint spectrum has strong characteristics, the repeatability and the accuracy of the content measurement result are good, and the method can be simultaneously used for qualitative and quantitative analysis of the preparation for treating jaundice. The method can control quality of rhizoma Dioscoreae Septemlobae preparation more completely.

Compared with the prior art, the invention adopts a high performance liquid chromatography combined with an ultraviolet dual-wavelength switching detection method to establish a detection method which can determine the characteristic fingerprint of the Chuanhuang preparation and can simultaneously determine 10 main components such as caffeoylquinic acids, andrographolide and the like in the preparation. Test results show that the method is accurate and efficient, has good repeatability and stability, strong fingerprint characteristic and accurate content measurement result, and can be used as a quality control method of the Chuanhuang preparation.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A method for establishing a fingerprint spectrum of a Chuanhuang preparation is characterized by comprising the following steps:

s1, preparation of a test solution: precisely weighing 1.0g of Chuanhuang preparation powder, placing in a 25mL volumetric flask, adding 20mL of 70% methanol, performing ultrasonic treatment for 30min, cooling, fixing volume to scale, shaking up, filtering, collecting filtrate, 13000 r.min^-1Centrifuging, and filtering the supernatant with 0.45 μm microporous membrane;

wherein, the fingerprint chromatogram condition is as follows:

using an Agilent Technologies EC-C18 column, 4.6mm X250 mm, 4 μm, mobile phase: acetonitrile-0.2% aqueous phosphoric acid solution system; adopting a gradient elution mode: 0min → 10min → 15min → 20min → 30min → 40min → 50min → 65 min; acetonitrile 10% → 15% → 21% → 25% → 28% → 34% → 36%; flow rate 1.0 mL/min^-1(ii) a Dual wavelength switching time series sampling: 0-35min, 323 nm; 35-65min, 215 nm; the sample injection amount is 5 mu L; the column temperature is 25 ℃; the analysis time is 65 min; the theoretical plate number is not less than 41000 calculated by isochlorogenic acid C peak.

2. The method for establishing the fingerprint spectrum of the Chuanhuang preparation according to claim 1, wherein the Chuanhuang preparation powder is prepared by the following steps: taking the heat-clearing tablet, removing the film coat, and grinding through a fifth sieve.

3. The method for establishing the fingerprint spectrum of the Chuanhuang preparation according to claim 1, wherein the preparation method of the Chuanhuang preparation powder is as follows: taking Chuanhuang heat-clearing capsule, pouring out the content, grinding and sieving with a No. five sieve.

4. A method for measuring the content of the components of the preparation for eliminating jaundice by fingerprint spectrum according to any one of claims 1 to 3, which comprises the following steps:

s1, preparation of a mixed reference solution: precisely weighing a proper amount of each reference substance, placing into 10 measuring bottles with the volume of 5mL, adding methanol for dissolving, and fixing the volume to obtain reference substance stock solutions of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, andrographolide, neoandrographolide, deoxyandrographolide and dehydroandrographolide; taking appropriate amount of the above reference stock solutions, placing in the same 5mL measuring flask, adding methanol to dilute to scale, shaking to obtain the final product containing the above reference concentrations of 0.184, 0.258, 0.202, 0.260, 0.178, 0.0934, 0.652, 1.034, 0.234, and 1.132 mg/mL^-1The mixed reference stock solution is precisely sucked by 0.5mL of the mixed reference stock solution, is placed in a 2mL measuring flask, is diluted to scale by adding methanol, is shaken evenly, and is prepared into the mixed reference stock solution containing neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, andrographolide, neoandrographolide, deoxyandrographolide and dehydroandrographolide with the concentrations of 0.0460, 0.0645, 0.0505, 0.0650, 0.0445, 0.0234, 0.163, 0.258, 0.0585 and 0.283 mg/mL respectively^-1Mixing the reference substance solution to obtain the product;

s2, preparation of a test solution: weighing 1.0g of Chuanhuang preparation powder, precisely weighing, placing in a 25mL volumetric flask, adding 70% methanol 20mL, performing ultrasonic treatment for 30min, cooling, fixing volume to scale, shaking, filtering, collecting the filtrate, 13000 r.min^-1Centrifuging, and filtering the supernatant with 0.45 μm microporous membrane;

s3, selecting chromatographic conditions and testing system adaptability: using an Agilent Technologies EC-C18 column, 4.6mm X250 mm, 4 μm, mobile phase: acetonitrile-0.2% aqueous phosphoric acid solution system; adopting a gradient elution mode: 0min → 10min → 15min → 20min → 30min → 40min → 50min → 65 min; acetonitrile 10% → 15% → 21% → 25% → 28% → 34% → 36%; flow rate 1.0 mL/min^-1(ii) a Dual wavelength switching time series sampling: 0 to 35min, 323 nm; 35-65min, 215 nm; the sample injection amount is 5 mu L; the column temperature is 25 ℃; the analysis time is 65 min; the theoretical plate number is not less than 41000 calculated by isochlorogenic acid C peak;

5. The assay method of claim 4, wherein the preparation method of the Chuanhuang preparation powder is as follows: taking the Chuanhuang Qingre tablet, removing the film coat, grinding and sieving with a No. five sieve.

6. The assay method of claim 4, wherein the preparation method of the Chuanhuang preparation powder is as follows: taking Chuanhuang heat-clearing capsule, pouring out the content, grinding and sieving through a No. five sieve.

7. The method according to claim 4, wherein the amounts of the control solution and the sample solution drawn in step S4 are 5. mu.L, respectively.