阅读说明：本技术 热炎宁制剂指纹特征图谱的建立方法及应用 (Method for establishing fingerprint characteristic spectrum of Reyanning preparation and application thereof ) 是由 杜成强 于 2020-07-13 设计创作，主要内容包括：本发明公开了一种热炎宁制剂指纹特征图谱的建立方法及应用。该建立方法包括以下步骤：分别配制热炎宁制剂对照品溶液及供试品溶液；分别配制不含有虎杖的热炎宁合剂的第一阴性对照溶液、不含有北败酱的热炎宁合剂的第二阴性对照溶液、不含有半枝莲的热炎宁合剂的第三阴性对照溶液、不含有蒲公英的热炎宁合剂的第四阴性对照溶液；吸取对照品溶液、供试品溶液、第一阴性对照溶液、第二阴性对照溶液、第三阴性对照溶液及第四阴性对照溶液注入高效液相色谱仪,按照高效液相色谱法检测,得到热炎宁制剂指纹特征图谱。应用本发明的技术方案,热炎宁制剂指纹特征图谱具有信息量大,重复性、稳定性良好,精密度高、特征性强,能整体控制多种组分特性。(The invention discloses a method for establishing a fingerprint characteristic spectrum of a Reyanning preparation and application thereof. The establishing method comprises the following steps: respectively preparing a control solution and a test solution of the Reyanning preparation; respectively preparing a first negative control solution without the hotanning mixture of giant knotweed, a second negative control solution without the hotanning mixture of patrinia scabiosaefolia, a third negative control solution without the hotanning mixture of barbed skullcap herb and a fourth negative control solution without the hotanning mixture of dandelion; and (3) sucking the reference substance solution, the test sample solution, the first negative control solution, the second negative control solution, the third negative control solution and the fourth negative control solution, injecting into a high performance liquid chromatograph, and detecting according to the high performance liquid chromatography to obtain the fingerprint characteristic spectrum of the feixianning preparation. By applying the technical scheme of the invention, the fingerprint characteristic spectrum of the Reyanning preparation has the advantages of large information amount, good repeatability and stability, high precision and strong characteristics, and can integrally control the characteristics of various components.)

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

respectively preparing a control solution and a test solution of the Reyanning preparation;

respectively preparing a first negative control solution without the hotanning mixture of giant knotweed, a second negative control solution without the hotanning mixture of patrinia scabiosaefolia, a third negative control solution without the hotanning mixture of barbed skullcap herb and a fourth negative control solution without the hotanning mixture of dandelion;

and (3) sucking the reference substance solution, the test article solution, the first negative reference solution, the second negative reference solution, the third negative reference solution and the fourth negative reference solution, injecting into a high performance liquid chromatograph, and detecting according to the high performance liquid chromatography to obtain the fingerprint characteristic spectrum of the feixianing preparation.

2. The establishing method according to claim 1, wherein the chromatographic column used in the high performance liquid chromatograph is C₁₈A reverse phase chromatography column.

3. The establishing method of claim 1, wherein the high performance liquid chromatography detection adopts gradient elution, and the flow rate of the gradient elution is 0.6 ml/min-1.4 ml/min; the mobile phase is acetonitrile: 0.1 to 0.4 percent of phosphoric acid solution; the column temperature is 25-35 ℃.

4. The establishing method of claim 3, wherein the gradient elution time in the high performance liquid chromatography detection is 0-60 min.

5. The method as set forth in claim 3, wherein the detection wavelength in the HPLC detection is 300-320 nm.

6. The method of building according to any one of claims 1 to 5, wherein the control solution is prepared by: accurately weighing emodin reference substance, polydatin reference substance, luteolin reference substance, chlorogenic acid reference substance and caffeic acid reference substance, and adding 50-100% methanol to prepare a solution containing 60-100 μ g of emodin, 280-440 μ g of polydatin, 40-80 μ g of luteolin, 70-110 μ g of chlorogenic acid and 60-100 μ g of caffeic acid per 1mL as reference substance solution;

preferably, 50-100% methanol is added to prepare a solution containing 80 μ g of emodin, 350 μ g of polydatin, 60 μ g of luteolin, 90 μ g of chlorogenic acid and 80 μ g of caffeic acid per 1mL, and the solution is used as a control solution.

7. The method of establishing according to any one of claims 1 to 5, wherein the test solution is prepared by: measuring 5-15 mL of thermite mixture in a centrifuge tube, centrifuging for 3-10 minutes at 3000-5000 r/min, precisely measuring 1-3 mL of supernatant, placing the supernatant in a 100mL volumetric flask, adding 80-95 mL of 50-100% methanol, performing ultrasonic treatment for 20-40 min, adding methanol to scale after the supernatant is cooled to room temperature, shaking up, filtering with a 0.22 mu m filter membrane, and taking a subsequent filtrate to obtain the sample solution;

preferably, 10mL of the mixture for treating the enteritis is measured in a centrifuge tube, the mixture is centrifuged for 5 minutes at 4000r/min, 2mL of supernatant is precisely measured and placed in a 100mL volumetric flask, 90mL of methanol is added, the mixture is subjected to ultrasonic treatment for 30min, the mixture is added with the methanol to the scale after the temperature is reduced to the room temperature, the mixture is shaken up, the mixture is filtered by a 0.22-micrometer filter membrane, and the subsequent filtrate is taken to obtain the sample solution.

8. The establishing method according to any one of claims 1 to 5, wherein the first negative control solution, the second negative control solution, the third negative control solution and the fourth negative control solution are prepared by referring to the preparation method of Reyanning mixture extraction of Chinese pharmacopoeia 2015 edition.

9. Use of the method for establishing the fingerprint of the thermionic preparation according to any one of claims 1 to 8 in the quality detection of the thermionic preparation.

10. A quality detection method of a Reyanning preparation is characterized by comprising the following steps:

establishing a thermitis preparation fingerprint according to the establishing method of the thermitis preparation fingerprint characteristic map of any one of claims 1 to 9;

and detecting the quality of the antipyretic preparation according to the fingerprint characteristic spectrum of the antipyretic preparation.

Technical Field

The invention relates to the technical field of medicines, in particular to a method for establishing a fingerprint characteristic spectrum of a Reyanning preparation and application thereof.

Background

The Reyanning preparation is a variety of Chinese pharmacopoeia and consists of 4 kinds of Chinese medicinal materials including dandelion, giant knotweed, patrinia scabiosaefolia link and barbed skullcap herb. Can be used for treating wind-heat type common cold, fever, sore throat, bitter taste of mouth, dry throat, cough with yellow phlegm, yellow urine, and constipation due to affection of exogenous wind-heat and internal stagnation of fire; suppurative tonsillitis, acute pharyngitis, acute bronchitis, and simple pneumonia. The product has better effects on treating respiratory diseases such as upper respiratory tract infection, cold and fever, acute pharyngitis, pneumonia and the like, is more reported in the aspect of treating hand-foot-and-mouth disease, and shows obvious antiviral effect. The medicine is determined as Chinese medicine for preventing and treating key infectious diseases in Shaanxi province, and the medicine for preventing and treating novel coronavirus in Shaanxi province is specified.

The medicine effect of the Reyanning preparation as a Chinese patent medicine is the result of the combined action of various active ingredients. However, the existing quality standard of the existing preparation for treating the enteritis is only measured singly, other effective components are not detected, certain limitation exists, and the quality level of the preparation for treating the enteritis cannot be comprehensively reflected.

Disclosure of Invention

The invention aims to provide a method for establishing a fingerprint characteristic spectrum of a Reyanning preparation and application thereof, so as to solve the technical problem that the quality level of the Reyanning preparation cannot be comprehensively reflected by quality detection in the prior art.

In order to achieve the above object, according to one aspect of the present invention, a method for establishing a fingerprint of a thermite preparation is provided. The establishing method comprises the following steps: respectively preparing a control solution and a test solution of the Reyanning preparation; respectively preparing a first negative control solution without the hotanning mixture of giant knotweed, a second negative control solution without the hotanning mixture of patrinia scabiosaefolia, a third negative control solution without the hotanning mixture of barbed skullcap herb and a fourth negative control solution without the hotanning mixture of dandelion; and (3) sucking the reference substance solution, the test sample solution, the first negative control solution, the second negative control solution, the third negative control solution and the fourth negative control solution, injecting into a high performance liquid chromatograph, and detecting according to the high performance liquid chromatography to obtain the fingerprint characteristic spectrum of the feixianning preparation.

Further, the chromatographic column used in the high performance liquid chromatograph is C₁₈A reverse phase chromatography column.

Furthermore, gradient elution is adopted in the high performance liquid chromatography detection, and the flow rate of the gradient elution is 0.6ml/min to 1.4 ml/min; the mobile phase is acetonitrile: 0.1 to 0.4 percent of phosphoric acid solution; the column temperature is 25-35 ℃.

Further, the gradient elution time in the detection of the high performance liquid chromatography is 0-60 min.

Furthermore, the detection wavelength in the detection of the high performance liquid chromatography is 300-320 nm.

Further, the control solution was prepared by the following steps: accurately weighing emodin reference substance, polydatin reference substance, luteolin reference substance, chlorogenic acid reference substance and caffeic acid reference substance, and adding 50-100% methanol to prepare a solution containing 60-100 μ g of emodin, 280-440 μ g of polydatin, 40-80 μ g of luteolin, 70-110 μ g of chlorogenic acid and 60-100 μ g of caffeic acid per 1mL as reference substance solution; preferably, 50-100% methanol is added to prepare a solution containing 80 μ g of emodin, 350 μ g of polydatin, 60 μ g of luteolin, 90 μ g of chlorogenic acid and 80 μ g of caffeic acid per 1mL, and the solution is used as a control solution.

Further, the test solution is prepared by the following steps: measuring 5-15 mL of thermite mixture in a centrifuge tube, centrifuging for 3-10 minutes at 3000-5000 r/min, precisely measuring 1-3 mL of supernatant, placing the supernatant in a 100mL volumetric flask, adding 80-95 mL of 50-100% methanol, performing ultrasonic treatment for 20-40 min, adding methanol to scale after the supernatant is cooled to room temperature, shaking up, filtering with a 0.22 mu m filter membrane, and taking a subsequent filtrate to obtain the sample solution; (ii) a Preferably, 10mL of the mixture for treating the enteritis is measured in a centrifuge tube, the mixture is centrifuged for 5 minutes at 4000r/min, 2mL of supernatant is precisely measured and placed in a 100mL volumetric flask, 90mL of methanol is added, the mixture is subjected to ultrasonic treatment for 30min, the mixture is added with the methanol to the scale after the temperature is reduced to the room temperature, the mixture is shaken up, the mixture is filtered by a 0.22-micrometer filter membrane, and the subsequent filtrate is taken to obtain the solution of the sample.

Further, the first negative control solution, the second negative control solution, the third negative control solution and the fourth negative control solution are prepared according to a preparation method for extracting the mixture of Reyanning in the 'Chinese pharmacopoeia' of 2015 edition.

According to another aspect of the invention, the application of the establishing method of the fingerprint characteristic spectrum of the Yanning preparation in the quality detection of the Yanning preparation is provided.

According to still another aspect of the present invention, a method for quality control of a Reyanning preparation is provided. The quality detection method comprises the following steps: establishing a fingerprint characteristic spectrum of the Reyanning preparation according to the establishing method of the fingerprint characteristic spectrum of the Reyanning preparation; and detecting the quality of the antipyretic preparation according to the fingerprint characteristic spectrum of the antipyretic preparation.

By applying the technical scheme of the invention, the fingerprint characteristic spectrum of the thermitis preparation has the advantages of large information amount, good repeatability and stability, high precision and strong characteristics, and can integrally control the characteristics of various components, so that the fingerprint characteristic spectrum of the thermitis preparation can comprehensively reflect the quality level of the thermitis preparation when being applied to quality detection.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 shows HPLC fingerprints of 10 batches of the mixture of Reyanning in example 1;

FIG. 2 shows a control fingerprint of example 1;

FIG. 3 shows the consensus peak assignment of example 1, wherein S1 is a mixture of thermite, S2 is a polygonum cuspidatum negative sample, S3 is a sculellaria barbata negative sample, S4 is a dandelion negative sample, and S5 is a North Patrinia negative sample;

FIG. 4A shows a control HPLC profile of example 1; and

figure 4B shows the sample HPLC profile of example 1.

Detailed Description

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail below with reference to the embodiments with reference to the attached drawings.

Aiming at the technical problem that the quality level of the existing preparation of the existing REYANNING can not be fully reflected, the invention controls the quality of the mixture of the existing preparation of the existing REYANNING by establishing HPLC fingerprint spectra taking chemical components in dandelion, giant knotweed rhizome, herba patriniae scabiosaefolia and barbed skullcap herb as main indexes and sharing peak attributi.

According to an exemplary embodiment of the present invention, a method for establishing a fingerprint of a Reyanning preparation is provided. The establishing method comprises the following steps: respectively preparing a control solution and a test solution of the Reyanning preparation; respectively preparing a first negative control solution without the hotanning mixture of giant knotweed, a second negative control solution without the hotanning mixture of patrinia scabiosaefolia, a third negative control solution without the hotanning mixture of barbed skullcap herb and a fourth negative control solution without the hotanning mixture of dandelion; and (3) sucking the reference substance solution, the test sample solution, the first negative control solution, the second negative control solution, the third negative control solution and the fourth negative control solution, injecting into a high performance liquid chromatograph, and detecting according to the high performance liquid chromatography to obtain the fingerprint characteristic spectrum of the feixianning preparation.

By applying the technical scheme of the invention, the fingerprint characteristic spectrum of the thermitis preparation has the advantages of large information amount, good repeatability and stability, high precision and strong characteristics, and can integrally control the characteristics of various components, so that the fingerprint characteristic spectrum of the thermitis preparation can comprehensively reflect the quality level of the thermitis preparation when being applied to quality detection.

Preferably, the chromatographic column used in the high performance liquid chromatograph is C₁₈A reverse phase chromatography column.

Preferably, the high performance liquid chromatography detection adopts gradient elution, and the flow rate of the gradient elution is 0.6ml/min to 1.4 ml/min; the mobile phase is acetonitrile: 0.1 to 0.4 percent of phosphoric acid solution; the column temperature is 25-35 ℃.

Preferably, the gradient elution time in the detection of the high performance liquid chromatography is 0-60 min.

Preferably, the detection wavelength in the detection of the high performance liquid chromatography is 300-320 nm.

According to an exemplary embodiment of the present invention, the control solution is prepared by the following steps: accurately weighing emodin reference substance, polydatin reference substance, luteolin reference substance, chlorogenic acid reference substance and caffeic acid reference substance, and adding 50-100% methanol to prepare a solution containing 60-100 μ g of emodin, 280-440 μ g of polydatin, 40-80 μ g of luteolin, 70-110 μ g of chlorogenic acid and 60-100 μ g of caffeic acid per 1mL as reference substance solution; preferably, 50-100% methanol is added to prepare a solution containing 80 μ g of emodin, 350 μ g of polydatin, 60 μ g of luteolin, 90 μ g of chlorogenic acid and 80 μ g of caffeic acid per 1mL, and the solution is used as a control solution.

According to an exemplary embodiment of the present invention, a test solution is prepared by: measuring 5-15 mL of thermite mixture in a centrifuge tube, centrifuging for 3-10 minutes at 3000-5000 r/min, precisely measuring 1-3 mL of supernatant, placing the supernatant in a 100mL volumetric flask, adding 80-95 mL of 50-100% methanol, performing ultrasonic treatment for 20-40 min, adding methanol to scale after the supernatant is cooled to room temperature, shaking up, filtering with a 0.22 mu m filter membrane, and taking a subsequent filtrate to obtain the sample solution; (ii) a Preferably, 10mL of the mixture for treating the enteritis is measured in a centrifuge tube, the mixture is centrifuged for 5 minutes at 4000r/min, 2mL of supernatant is precisely measured and placed in a 100mL volumetric flask, 90mL of methanol is added, the mixture is subjected to ultrasonic treatment for 30min, the mixture is added with the methanol to the scale after the temperature is reduced to the room temperature, the mixture is shaken up, the mixture is filtered by a 0.22-micrometer filter membrane, and the subsequent filtrate is taken to obtain the sample solution.

According to a typical embodiment of the present invention, the first negative control solution, the second negative control solution, the third negative control solution and the fourth negative control solution are prepared by referring to the preparation method of extraction of thermite mixture in "chinese pharmacopoeia" of 2015 edition.

According to an exemplary embodiment of the present invention, there is provided an application of the above-mentioned method for establishing a fingerprint of a antipyretic preparation in quality detection of the antipyretic preparation.

According to an exemplary embodiment of the present invention, a method for quality testing of a thermite preparation is provided. The quality detection method comprises the following steps: establishing a fingerprint characteristic spectrum of the Reyanning preparation according to the establishing method of the fingerprint characteristic spectrum of the Reyanning preparation; and detecting the quality of the antipyretic preparation according to the fingerprint characteristic spectrum of the antipyretic preparation.

By applying the technical scheme of the invention, the fingerprint characteristic spectrum of the thermitis preparation has the advantages of large information amount, good repeatability and stability, high precision and strong characteristics, and can integrally control the characteristics of various components, so that the fingerprint characteristic spectrum of the thermitis preparation can comprehensively reflect the quality level of the thermitis preparation when being applied to quality detection.

The following examples are provided to further illustrate the advantageous effects of the present invention.