WO2022022065A1

WO2022022065A1 - Method for establishing fingerprint spectrum of cervicodynia treatment granules, and application thereof

Info

Publication number: WO2022022065A1
Application number: PCT/CN2021/098703
Authority: WO
Inventors: 谭倩; 刘杰; 江玉娟; 潘瑞雪; 高超; 许敏; 王莉
Original assignee: 山东明仁福瑞达制药股份有限公司
Priority date: 2020-07-31
Filing date: 2021-06-07
Publication date: 2022-02-03
Also published as: CN111999423A; CN111999423B

Abstract

A method for establishing a fingerprint spectrum of cervicodynia treatment granules, and the use thereof, which belong to the technical field of medicine testing. On the basis of HPLC technology, a gradient elution method is used to establish a fingerprint spectrum of cervicodynia treatment granules. The method is simple in terms of operation, stable, reliable, and high in precision, and achieves a good degree of separation. The fingerprint spectrum is better in terms of stability and reproducibility and large in terms of information amount, and can fully reflect information of whole chemical components of the cervicodynia treatment granules. By means of the method, one-sidedness in the determination of the overall quality of a preparation due to the fact that only a single chemical component is measured is avoided, and the possibility of manual treatment being required for reaching a quality standard is also reduced; in addition, systematic analysis is performed on a plurality of batches of samples, so that the quality of cervicodynia treatment granules can be evaluated more comprehensively and scientifically, thereby ensuring the quality and the curative effect of a product, and providing a good solution for quality control over cervicodynia treatment granules.

Description

A kind of establishment method of neck pain granule fingerprint and its application

technical field

The invention belongs to the technical field of medical detection, and in particular relates to a method for establishing a fingerprint of neck pain granules and its application.

Background technique

The information disclosed in this Background section is only for enhancement of understanding of the general background of the invention and should not necessarily be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.

Ningtong Granules are made according to the prescription of Professor Sun Shuchun. It is the first national-level new drug for the professional treatment of radiculopathy of cervical spondylosis, an independent innovation product of Shandong Province, and a national key new product. The medicine is made from seven herbs of Panax notoginseng, Chuanxiong, Corydalis, Qianghuo, Baishao, Weilingxian and Pueraria, of which Panax notoginseng, Chuanxiong and Corydalis play the role of promoting blood circulation, dispersing blood stasis and dredging collaterals; Qianghuo, Weilingxian , Puerariae has the effect of dispelling wind, cold and dampness, and relieving neck stiffness; while Baishao focuses on nourishing blood and softening yin, nourishing tendons and meridians, and its main functions are "activating blood and removing blood stasis, promoting qi and relieving pain". It is used for cervical spondylosis of nerve root type, which belongs to the syndrome of blood stasis and qi stagnation and choroid blocking.

At present, the 2015 edition of the Pharmacopoeia of the People's Republic of China (Part I) only uses thin-layer chromatography combined with reference substances such as tetrahydropalmatate, tetrahydropalmatine, paeoniflorin, etc. and reference medicinal material Chuanxiong to identify Jingtong granules, or use high-efficiency liquid The content of ginsenoside Rg ₁ , ginsenoside Rb ₁ and Panax notoginseng saponin R ₁ was determined by liquid chromatography, and the quality control index was single, which could not fully reflect the multi-component and multi-target characteristics of traditional Chinese medicine prescriptions. As mentioned above, traditional Chinese medicine and its compound prescriptions are applied and researched under the guidance of traditional Chinese medicine theory. Their components are complex, and the parts that exert their effects are often not a single component, but a group effect, so any single component cannot respond. The overall efficacy of traditional Chinese medicine and its compounds. Therefore, it will become an inevitable trend to carry out a macroscopic comprehensive analysis of traditional Chinese medicine and its compound prescriptions.

Traditional Chinese medicine fingerprints rely on various spectral, spectral and chromatographic techniques to comprehensively reflect the quality information of traditional Chinese medicines and their compounds. However, the inventor found that there is no report on the quality control research based on fingerprints for Ningtong Granules at present, so it is of great significance to provide a method for establishing fingerprints of Ningtong Granules.

SUMMARY OF THE INVENTION

In view of the deficiencies of the prior art, the present invention provides a method for establishing a fingerprint of neck pain granules and a fingerprint thereof. Aiming at the characteristics of Jingtong Granules as a traditional Chinese medicine compound preparation, it is composed of 7 prescription medicinal materials, and the components are complex and diverse, and have not yet been fully clarified. There are many fingerprints that can effectively characterize the quality of the Jingtong granule preparation, thereby completing the present invention.

In order to achieve the purpose, the present invention relates to the following technical solutions:

In a first aspect of the present invention, a method for establishing a fingerprint of Necktong Granules is provided. The method uses Panax notoginseng saponin R1, tetrahydropalmatine and oleanolic acid as mixed standards.

Preferably, the method for establishing the fingerprint spectrum, the steps are as follows:

(1) Preparation of the test solution: take the neck pain granules, add methanol for ultrasonic extraction, and filter to obtain the solution to be tested;

(2) reference substance solution preparation: get notoginseng saponin R1, tetrahydropalmatine and oleanolic acid reference substance dried under reduced pressure to constant weight and add methanol to make mixed reference substance solution;

(3) Determination: Precisely measure the test solution and the reference solution respectively, carry out HPLC analysis, adopt gradient elution, mobile phase A phase: acetonitrile, mobile phase B phase: 0.15% phosphoric acid aqueous solution; The obtained spectrum is analyzed and processed to obtain the fingerprint spectrum of Ningtong Granules.

It can be understood that, there is no sequence between the steps (1) and (2).

The second aspect of the present invention provides the fingerprint of the neck pain granules obtained by the establishment method.

The third aspect of the present invention provides the application of the establishment method and/or fingerprint in the quality control of Jingtong Granules.

A fourth aspect of the present invention provides a quality control method for Ningtong Granules, the method comprising: taking multiple batches of Ningtong Granules to obtain the fingerprints of Ningtong Granules according to the construction method, and obtaining the fingerprints of Ningtong Granules from the multiple batches ( Generally at least 10 batches) the fingerprints of Ningtong Granules are generated according to the mean value method to obtain the control fingerprints;

Take the sample to be detected to prepare the test solution, and obtain the fingerprint of Neck Pain Granules according to the construction method;

The similarity between the fingerprints of the samples to be tested and the control fingerprints of Jingtong Granules was compared.

Preferably, the similarity comparison includes using an included angle cosine method and/or a correlation coefficient method.

Beneficial technical effects of the one or more technical solutions:

The technical scheme adopts the gradient elution method to establish the fingerprint of the neck pain granules. The method is simple to operate, stable and reliable, has high precision, good separation, and the stability and reproducibility of the fingerprint are good, and the amount of information is high. Large, can fully reflect the information of the overall chemical composition of Jingtong Granules;

The technical solution not only avoids the one-sidedness of judging the overall quality of the preparation due to the determination of only a single chemical component, but also reduces the possibility of artificial processing to meet the quality standards. Comprehensive and scientific evaluation of the quality of Ningtong Granules, so as to ensure the quality and efficacy of the products, and provide a good solution for the quality control of Ningtong Granules, so it is of great practical application value.

Description of drawings

The accompanying drawings forming a part of the present invention are used to provide further understanding of the present invention, and the exemplary embodiments of the present invention and their descriptions are used to explain the present invention, and do not constitute an improper limitation of the present invention.

Fig. 1 is the HPLC figure of mixed reference substance in the embodiment of the present invention;

Fig. 2 is the HPLC fingerprints of 14 batches of Neck Pain Granules in the embodiment of the present invention.

detailed description

It should be noted that the following detailed description is exemplary and intended to provide further explanation of the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It should be noted that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly dictates otherwise, the singular is intended to include the plural as well, furthermore, it is to be understood that when the terms "comprising" and/or "including" are used in this specification, it indicates that There are features, steps, operations, devices, components, and/or combinations thereof. It should be understood that the protection scope of the present invention is not limited to the following specific specific embodiments; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments, rather than for limiting the protection scope of the present invention.

In a specific embodiment of the present invention, a method for establishing a fingerprint of Ningtong Granules is provided, and the method uses notoginsenoside R1, tetrahydropalmatine and oleanolic acid as mixed standards.

In yet another specific embodiment of the present invention, the method for establishing a fingerprint spectrum includes the following steps:

It can be understood that, there is no sequence between the steps (1) and (2).

In another specific embodiment of the present invention, in the step (1), a certain amount of Neck Pain Granules is weighed and added to methanol and then ultrasonicated for 10-30 min (preferably 20 min), wherein the ratio of Neck Pain Granules: methanol is (80- 120) mg: (4-6) mL (preferably 100 mg: 5 mL); after ultrasonic extraction, shake well and filter, and the filtrate is filtered through a 0.22 μm microporous membrane to obtain the test solution;

In yet another specific embodiment of the present invention, the preparation method of the mixed reference solution in the step (2) is as follows: Precisely weigh 0.0100 g of notoginsenoside R1 standard product, 0.0099 g of tetrahydropalmatine standard product and oleanolic acid Place 0.0099g of the standard in a 10mL volumetric flask, add methanol to dissolve and dilute to the mark, and prepare a mixed standard solution.

In another specific embodiment of the present invention, in the step (3), the chromatographic column is CAPCELL PAK C18 MGII (4.6mm×250mm, 5μm); the flow rate of the mobile phase is 1ml/min; the column temperature is 30°C; the detection wavelength is 300nm, the injection volume is 10μL.

In another specific embodiment of the present invention, the gradient elution mode in step (3) is: gradient elution: 0-15min, 2%A→15%A; 15-30min, 15%A→30%A; 30 ～45min, 30%A→50%A; 45～60min, 50%A→65%A; 60～75min, 65%A→100%A. It should be noted that the chromatogram of 2h was recorded when the elution time of the fingerprint was selected. The results showed that there were basically no obvious chromatographic peaks after 65min. At the same time, in order to take into account the differences of each batch of samples and ensure that the characteristic peaks of all batches of samples could be detected, 75min was selected as the analysis time. The gradient elution conditions were optimized, the retention time of each chromatographic peak was moderate, the resolution of the chromatogram was improved, the tailing phenomenon of the chromatogram was avoided, and the detection and analysis of the chromatographic fingerprint was facilitated.

In yet another specific embodiment of the present invention, in the fingerprint of Ningtong Granules obtained by the method for constructing the fingerprint of Ningtong Granules as described above, there are 22 common peaks in the fingerprint.

In another specific embodiment of the present invention, the fingerprint of the neck pain granules obtained by the establishment method is provided.

In yet another specific embodiment of the present invention, the application of the establishment method and/or the fingerprint in the quality control of Jingtong Granules is provided.

In another specific embodiment of the present invention, a method for quality control of Ningtong Granules is provided, the method comprising: taking multiple batches of Ningtong Granules to obtain the fingerprint of Ningtong Granules according to the construction method, and obtaining the fingerprints of Ningtong Granules from the multiple batches The fingerprints of Ningtong Granules are generated every time (generally at least 10 batches) according to the mean value method to obtain the control fingerprints;

Take the sample to be tested to prepare the test solution, and obtain the fingerprint of Ningtong Granules according to the construction method.

The present invention is further explained and illustrated by the following examples, but it does not constitute a limitation of the present invention. It should be understood that these examples are only intended to illustrate the present invention and not to limit the scope of the present invention.

Example 1

1. Instruments and materials

Agilent (agilent1200) high performance liquid chromatograph; Sartorius 1/100,000 electronic analytical balance, fingerprint similarity evaluation software "Chinese medicine chromatographic fingerprint similarity evaluation system" (National Pharmacopoeia Commission 2004A version); KQ-1000DE type CNC Ultrasound.

Chromatographic column: Japan Shiseido CAPCELL PAK C18 MGII (4.6mm×250mm, 5μm), notoginseng saponin R1, tetrahydropalmatine and oleanolic acid standard products were purchased from Shanghai Chunyou Biotechnology Co., Ltd.

Phosphoric acid, methanol (chromatographically pure, Shandong Chuangyi Chemical Co., Ltd.); acetonitrile (chromatographically pure, Fisher, USA); experimental water was ultrapure water (18MΩ); 14 batches (S1-S14) of neck pain granules (Mingren ) samples were taken from Shandong Mingren Freda Pharmaceutical Co., Ltd.

2. Experimental method

2.1 Preparation of the test solution

Take 0.5 g of Neck Pain Granules, accurately weigh them, put them in a stoppered conical flask, add 10 mL of chromatographic methanol, extract by ultrasonic for 20 min (extraction power 300 W, frequency 40 kHz), shake well, filter, and filter the filtrate through a 0.22 μm microporous membrane Afterwards, it was used as the test solution for use.

2.2 Preparation of mixed reference solution

Precisely weigh 0.0100 g of notoginsenoside R1 standard product, 0.0099 g of tetrahydropalmatine standard product and 0.0099 g of oleanolic acid standard product into a 10 mL volumetric flask, add chromatographic methanol to dissolve and dilute to the mark, and prepare a mixed standard solution.

2.3 Chromatographic conditions

Japan Shiseido CAPCELL PAK C18 MGII (4.6mm×250mm, 5μm); mobile phase: acetonitrile (A)-0.15% phosphoric acid aqueous solution (B). Gradient elution: 0～15min, 2%A→15%A; 15～30min, 15%A→30%A; 30～45min, 30%A→50%A; 45～60min, 50%A→65% A; 60～75min, 65%A→100%A. The flow rate of the mobile phase was 1 ml/min; the column temperature was 30 °C; the detection wavelength was 300 nm, and the injection volume was 10 μL.

3. Results and discussion

3.1 Comparison of fingerprints

The obtained spectrum was analyzed and processed with fingerprint spectrum software to obtain the fingerprint spectrum of each batch of Ningtong Granules (as shown in Figure 2). There are 22 common characteristic peaks in the spectrum. The fingerprint of the mixed reference substance is shown in Figure 1. By comparing with the chromatogram of the reference substance, it is confirmed that the No. 4 peak is oleanolic acid, the No. 6 peak is notoginsenoside R1, and the No. 21 peak is tetrahydropalmatine.

The measured spectra of 14 batches of Ningtong Granules were imported into the software of "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" for analysis, and chromatographic peak matching was performed. Taking S1 as the reference spectrum, the generated control fingerprint spectrum is R (using the average method, see Figure 1). The fingerprints of each batch of samples and the control fingerprints and the similarity results of the fingerprints between the samples are shown in Table 1.

Table 1 Similarity values of 14 batches of fingerprints

It can be seen from Table 1 that the similarity between each batch of samples and R is more than 0.900, indicating that the chemical composition of each batch of Jingtong Granules is generally similar.

3.2 Methodological investigation

The methodological investigation mainly examines the precision, stability and repeatability. The investigation methods and results are as follows: (1) The same batch of Ningtong Granules samples were taken in the precision experiment. , and the results are shown in Table 2. It can be seen from Table 2 that the relative retention time of each common peak and the RSD of the relative peak are both less than 3%, indicating that the precision of the instrument is good.

(2) Stability experiment Take the same batch of Jingtong granules samples, under the above chromatographic conditions, inject samples for analysis at 0h, 2h, 4h, 8h, 12h and 24h respectively, record the chromatogram, and the results are shown in Table 2. It can be seen from Table 2 that the relative retention time of each common peak and the RSD of the relative peak are both less than 3%, indicating that the components of the test solution are stable within 24 hours.

(3) Repeatability experiment Take the same batch of Neck Pain Granules samples, under the above chromatographic conditions, inject and analyze respectively, record the chromatogram, the results are shown in Table 2, the relative retention time of each common peak and the RSD of the relative peak are both less than 3 %, indicating that the experimental method has better repeatability.

Table 2 Methodological verification results of neck pain granules fingerprints

It should be noted that the above examples are only used to illustrate the technical solutions of the present invention but not to limit them. Although the present invention has been described in detail with reference to the given examples, those skilled in the art can modify or equivalently replace the technical solutions of the present invention as required without departing from the spirit and scope of the technical solutions of the present invention.

Claims

A method for establishing a fingerprint of Ningtong Granules, characterized in that the method uses notoginsenoside R1, tetrahydropalmatine and oleanolic acid as mixed standards.
The method of claim 1, wherein the steps are as follows:

(1) Preparation of the test solution: take the neck pain granules, add methanol for ultrasonic extraction, and filter to obtain the solution to be tested;

(2) reference substance solution preparation: get notoginseng saponin R1, tetrahydropalmatine and oleanolic acid reference substance dried under reduced pressure to constant weight and add methanol to make mixed reference substance solution;

(3) Determination: Precisely measure the test solution and the reference solution respectively, carry out HPLC analysis, adopt gradient elution, mobile phase A phase: acetonitrile, mobile phase B phase: 0.15% phosphoric acid aqueous solution; The obtained spectrum is analyzed and processed to obtain the fingerprint spectrum of Ningtong Granules;

There is no order of precedence between the steps (1) and (2).
The method of claim 2, wherein in the step (1), the Neck Pain Granules are weighed and added to methanol for 10-30 min (preferably 20 min), wherein the ratio of Neck Pain Granules: methanol is (450- 550) mg: (8-12) mL (preferably 500 mg: 10 mL); after ultrasonic extraction, shake well and filter, and pass the filtrate through a 0.22 μm microporous membrane to obtain the test solution.
The method according to claim 2, characterized in that, in the step (2), the compounding method of the mixed reference solution is as follows: accurately weigh 0.0100 g of notoginsenoside R1 standard product, 0.0099 g of tetrahydropalmatine standard product and q. Put 0.0099g of leanolic acid standard in a 10mL volumetric flask, add methanol to dissolve and dilute to the mark, and prepare a mixed standard solution.
The method according to claim 2, characterized in that, in the step (3), the chromatographic column is CAPCELL PAK C18 MGII, 4.6 mm×250 mm, 5 μm; the flow rate of the mobile phase is 1 ml/min; the column temperature is 30°C; The wavelength was 300 nm, and the injection volume was 10 μL.
The method according to claim 2, wherein the gradient elution mode in step (3) is: 0～15min, 2%A→15%A; 15～30min, 15%A→30%A; 30～30min 45min, 30%A→50%A; 45～60min, 50%A→65%A; 60～75min, 65%A→100%A.
The fingerprint of the neck pain granules obtained by the method of any one of claims 1-6.
Application of the method according to any one of claims 1 to 6 and/or the fingerprint according to claim 7 in the quality control of Jingtong granules.
A quality control method for Ningtong Granules, characterized in that, the method comprises: taking multiple batches of Ningtong Granules to obtain a fingerprint of Ningtong Granules according to the method according to any one of claims 1-6, The fingerprints of Jingtong Granules were generated according to the mean value method to obtain the control fingerprints;

Get the sample to be detected to prepare the test solution, and obtain the fingerprint of Neck Pain Granules according to the method described in any one of claims 1-6;

The similarity between the fingerprints of the samples to be tested and the control fingerprints of Jingtong Granules was compared.
The quality control method according to claim 9, wherein the similarity comparison comprises using an included angle cosine method and/or a correlation coefficient method.