A total extract of Cnidium monnieri and its application and map construction method

Abstract

The invention discloses a total extract of Cnidium monnieri containing bergamotin, and also discloses the use of the total extract of Cnidium monnieri in preparing a drug for preventing and / or treating allergic skin diseases, psoriasis or herpes zoster, and a method for constructing a fingerprint of the total extract of Cnidium monnieri. The invention adopts LC-MS technology to establish a fingerprint of the total extract of Cnidium monnieri, effectively improving the quality stability, uniformity and controllability of the total extract of Cnidium monnieri.

Description

Technical Field

[0001] The invention belongs to the technical field of Cnidium monnieri, and particularly relates to a Cnidium monnieri total extract and an application thereof and a map construction method. Background Art

[0002] Traditional Chinese medicine refers to medicinal substances under the guidance of traditional Chinese medicine theory, with a feasible source of innovative materials. The production of Chinese medicine involves multiple links: Chinese medicinal material resources-Chinese medicinal pieces (including processing)-Chinese medicinal prescriptions-Chinese medicinal preparations (Chinese patent medicines). If any link is not well controlled, it will affect the quality of the product and the safety and effectiveness of clinical efficacy. Therefore, quality control of Chinese medicine is one of the key points and difficulties in the development of modern Chinese medicine. At present, in the existing standards in my country, the quality standards of Chinese medicinal materials and Chinese patent medicines are mostly limited to identifying appearance, checking properties, and moisture, and rarely testing the effective ingredients. Even if the component determination standards are established, most of the components measured are only 1 to 2 representative indicator components. The determination of effective ingredients directly related to efficacy is a pain point. Therefore, how to scientifically, reasonably and comprehensively formulate quality control standards for Chinese medicine is of great significance to the quality, safety and effectiveness of Chinese medicine and public health.

[0003] The fingerprint of traditional Chinese medicine is a chromatogram or spectrum that can indicate the chemical characteristics of traditional Chinese medicine or traditional Chinese medicine preparations after proper treatment and certain analytical methods. The fingerprint of traditional Chinese medicine is a comprehensive and quantifiable identification method. It is based on the systematic study of the chemical components of traditional Chinese medicine and is mainly used to evaluate the authenticity, quality and stability of traditional Chinese medicine and traditional Chinese medicine preparations. Its notable characteristics are "holism" and "fuzziness".

[0004] Traditional Chinese medicine fingerprint technology has involved many methods, including chromatographic methods such as thin layer scanning (TLCS), high performance liquid chromatography (HPLC), gas chromatography (GC) and high performance capillary electrophoresis (HPCE), as well as spectroscopic methods such as ultraviolet spectroscopy (UV), infrared spectroscopy (IR), mass spectrometry (MS), nuclear magnetic resonance (NMR) and X-ray diffraction. Among them, chromatographic methods are the mainstream methods, especially HPLC, TLCS and GC have become the three recognized conventional analytical methods. HPLC has the characteristics of high separation efficiency, high selectivity, high detection sensitivity, fast analysis speed and wide application range. Most of the components of traditional Chinese medicine can be analyzed and detected on high performance liquid chromatography, and rich application experience has been accumulated. Therefore, high performance liquid chromatography has become the preferred method of traditional Chinese medicine fingerprint technology. With the application of combined technologies such as HPLC-MS and GC-MS, traditional Chinese medicine fingerprint technology has become more perfect.

[0005] At present, the active ingredients of total extract of Cnidium monnieri include Zanthoxylum toxicol, Zanthoxylum toxin, isoanisole, bergamot lactone, imperatorin, osthole, etc. However, there are still some unknown ingredients that have not been discovered. With the advancement of extraction technology and the development of detection technology, more and more effective ingredients are being gradually extracted and further utilized. Summary of the invention

[0006] The first object of the present invention is a total extract of Cnidium monnieri, which contains bergamotin.

[0007] The present invention also aims to provide the use of the above-mentioned total extract of Cnidium monnieri in the preparation of a medicine for preventing and / or treating allergic skin diseases, psoriasis or herpes zoster.

[0008] The last object of the present invention is to provide a method for constructing the fingerprint of the above-mentioned Cnidium monnieri total extract.

[0009] The first object of the present invention can be achieved through the following technical scheme: a total extract of Cnidium monnieri containing bergamotin.

[0010] The present invention discovers a new ingredient bergamotin in the Cnidium monnieri Chinese medicinal material, the Cnidium monnieri total extract and the Cnidium monnieri preparation for the first time through a method for constructing fingerprints of the Cnidium monnieri Chinese medicinal material, the Cnidium monnieri total extract and the Cnidium monnieri preparation.

[0011] Wherein the total extract of Cnidium monnieri can be a commercially available Cnidium monnieri extract, and can also be prepared by the following method: take Cnidium monnieri Chinese medicinal materials, add 3 to 6 times the total mass of Cnidium monnieri with a volume percentage of 80 to 85% ethanol reflux extraction 1 to 3 times, each time for 2 to 3 hours, filter, combine the filtrate, and concentrate under reduced pressure to obtain an alcohol extract, and then crystallize and dry to obtain a total extract of Cnidium monnieri, i.e., total coumarins of Cnidium monnieri. More preferably, the total extract of Cnidium monnieri is prepared by the following method: take an appropriate amount of Cnidium monnieri medicinal materials, add 3 to 6 times the amount (mass) of 85% (volume percentage) ethanol, heat and reflux extraction 3 times at 75°C, each time for 2 hours, filter, combine the filtrate, and concentrate under reduced pressure to obtain an alcohol extract, and then crystallize and dry under reduced pressure to obtain a total extract of Cnidium monnieri, i.e., total coumarins of Cnidium monnieri.

[0012] Furthermore, the total extract of Cnidium monnieri also contains Zanthoxylum bungeanum, Zanthoxylum bungeanum toxin, isoanisole, bergamot lactone, imperatorin, osthole, hesperidone hydrate and hesperidone.

[0013] In addition, the total extract of Cnidium monnieri in the present application mainly contains total coumarins of Cnidium monnieri.

[0014] The above second object of the present invention can be achieved by the following technical scheme: the use of the above total extract of Cnidium monnieri in the preparation of a drug for preventing and / or treating allergic skin diseases, psoriasis or herpes zoster.

[0015] Preferably, the allergic allergic skin diseases include allergic contact dermatitis, atopic dermatitis, eczema, urticaria and drug eruption.

[0016] Preferably, the medicine comprises the total extract of Cnidium monnieri and a pharmaceutically acceptable carrier or excipient.

[0017] As a preferred embodiment of the present invention, the drug is a Cnidium monnieri ointment, a Cnidium monnieri spray or a Cnidium monnieri liquid containing a total Cnidium monnieri extract.

[0018] That is, the drugs containing total extract of Cnidium monnieri in this application are mainly total coumarin external preparations of Cnidium monnieri, including total coumarin ointment of Cnidium monnieri, total coumarin spray of Cnidium monnieri and total coumarin liquid of Cnidium monnieri, etc., which are all prepared by conventional methods in the art.

[0019] Preferably, the Cnidium monnieri total coumarin ointment is a Cnidium monnieri total coumarin ointment for external use made from Cnidium monnieri total coumarin and conventional pharmaceutical excipients, and the mass percentage of total coumarins in the Cnidium monnieri total coumarin ointment is 10%.

[0020] This application is based on the clinical trials of the Chinese medicine, and according to its applicable subjects and indications, the preparation is preferably an ointment.

[0021] In order to realize the above dosage form, suitable pharmaceutical excipients need to be added when preparing the dosage form, such as solubilizers, bases, moisturizers, etc. Solubilizers include: polysorbate 80, etc., bases include white vaseline, stearic acid, mono- and di-stearate glyceryl, etc., moisturizers include glycerol, etc.

[0022] More preferably, the Cnidium monnieri total coumarin ointment is prepared by the following method: taking the Cnidium monnieri total coumarin raw material and the oil phase made of stearic acid, mono- and di-stearate glyceryl, and vaseline and the water phase made of glycerol, water, and polysorbate 80, and emulsifying and encapsulating to obtain the ointment.

[0023] Preferably, the Cnidium monnieri total coumarin spray is a Cnidium monnieri total coumarin spray for external use prepared by ultrasonically dissolving Cnidium monnieri total coumarin and ethanol, placing, filtering and fixing the volume, and the mass percentage of total coumarin in the Cnidium monnieri total coumarin spray is 1-10%.

[0024] Preferably, the Cnidium monnieri total coumarin liquid is a Cnidium monnieri total coumarin liquid for external use made from Cnidium monnieri total coumarin and a non-pure ethanol solvent, wherein the non-pure ethanol solvent includes benzyl alcohol, propylene glycol and polysorbate 80, and the mass percentage of total coumarins in the Cnidium monnieri total coumarin solution is 0.5% to 5%.

[0025] The third object of the present invention can be achieved by the following technical solution: A method for constructing a fingerprint of a total extract of Cnidium monnieri, comprising the following steps:

[0026] (1) Preparation of mixed reference substance solution: p-coumaric acid, osthole, isoanisole, zanthoxylum phenol, zanthoxylum toxin, bergamot lactone, and imperatorin reference substances are dissolved in ethanol to obtain a mixed reference substance solution;

[0027] (2) Preparation of test solution: Take the total extract of Cnidium monnieri, add ethanol, ultrasonicate, make up the loss with ethanol, mix well, filter, and take the filtrate to obtain the test solution;

[0028] (3) Mix the reference solution and the test solution, inject the sample, perform full scan analysis using liquid chromatography-high resolution mass spectrometry, use software to identify common peaks in the characteristic spectrum of the test sample, extract and confirm the characteristic components, perform structural analysis, attribution identification and confirmation on the unique chemical components of Cnidium monnieri, obtain the chemical components of Cnidium monnieri, and establish a standard fingerprint spectrum of the total extract of Cnidium monnieri.

[0029] In the method for constructing the fingerprint of the Cnidium monnieri extract:

[0030] Preferably, the preparation of the mixed reference solution in step (1) includes: accurately weighing about 1 mg of p-coumaric acid, osthole, isoanisole, zanthoxylum phenol, zanthoxylum toxin, bergamot lactone, and imperatorin reference substances, respectively placing them in 10 mL volumetric bottles, adding ethanol to dissolve and dilute to the scale, shaking well, then accurately measuring 1.0 mL of each of the above solutions, placing them in a 10 mL volumetric bottle, diluting to the scale with 70% by volume ethanol, and shaking well.

[0031] Preferably, the volume percentage of ethanol in step (2) is 70%, the dosage relationship of the total extract of Cnidium monnieri to ethanol is 0.05 g:50 mL, and the ultrasonic treatment time is 60 min.

[0032] Preferably, the parameters of the liquid chromatography in step (3) are as follows: chromatographic conditions: C18 chromatographic column; flow rate: 0.5 mL / min; column temperature: 40° C.; wavelength: 310 nm; injection volume: 10 μL; mobile phase A: 0.1% (volume percentage) acetic acid aqueous solution; mobile phase B: methanol, and the gradient elution conditions are as follows:

[0033] time Mobile phase Mobile phase B 0 95 5 10 65 35 20 55 45 35 55 45 40 53 47 65 53 47 70 30 70 75 10 90 85 10 90 85.1 0 100 120 0 100 120.1 95 5 130 95 5

[0034] Preferably, the chromatographic column in step (3) is Thermo Scientific Acclaim™ 120C18 250×4.6 mm, 5 μm.

[0035] Preferably, the standard fingerprint of the total extract of Cnidium monnieri in step (3) includes the following compounds: Zanthoxylum toxicans, Zanthoxylum toxin, isoanisole, bergamot lactone, imperatorin, osthole, hesperidone hydrate, hesperidone and bergamotrin.

[0036] The present application further establishes a method for constructing fingerprints of the Chinese medicinal material "Cnidium monnieri", its total extract "Cnidium monnieri coumarin" and its preparations such as "Cnidium monnieri total coumarin ointment", which effectively improves the quality stability, consistency and controllability of the Chinese medicinal material "Cnidium monnieri", the total extract "Cnidium monnieri coumarin" and its preparations.

[0037] The fingerprint spectrum determination method in the present application can be used to determine 10 common peaks in the Chinese medicinal material of Cnidium monnieri, and confirm that the compounds of 7 peaks are p-coumaric acid, zanthoxylum toxicol, zanthoxylum toxin, isoanisole, bergamot lactone, imperatorin, and osthole; 9 common peaks in Cnidium monnieri extract and Cnidium monnieri preparation can be determined, and it can be confirmed that the compounds of 7 peaks are zanthoxylum toxicol, zanthoxylum toxin, isoanisole, bergamot lactone, imperatorin, and osthole.

[0038] Specifically, the standard fingerprint of the Chinese medicinal material Cnidium monnieri includes the following compounds: p-coumaric acid, zanthoxylum toxicol, zanthoxylum toxin, isoanisole, bergamotol, imperatorin, osthole, hesperidin hydrate, hesperidin and bergamotrin.

[0039] Specifically, the standard fingerprints of the total extract of Cnidium monnieri and the Cnidium monnieri preparation include the following compounds: Zanthoxylum toxicol, Zanthoxylum toxicol, isoanisole, bergamotolactone, imperatorin, osthole, hesperidone hydrate, hesperidone and bergamotrin.

[0040] The present invention has the following advantages:

[0041] (1) This application obtains a new ingredient, bergamotin, from the total extract of Cnidium monnieri;

[0042] (2) The present application uses LC-MS technology to establish the fingerprint of the total extract of Cnidium monnieri. The fingerprint of the Chinese medicinal material Cnidium monnieri and preparations containing the total extract of Cnidium monnieri, such as Cnidium monnieri total coumarin ointment, can also be further established based on this method, which effectively improves the quality stability, uniformity and controllability of the Cnidium monnieri medicinal material, the total extract of Cnidium monnieri and its preparation products;

[0043] (3) The present application detected the content of each component in the total extract of Cnidium monnieri by optimizing the chromatographic conditions and establishing the detection method, and used modern scientific identification methods (LC-MS, etc.) to confirm the common peaks of the characteristic spectrum, confirm the attribution of each component, establish the fingerprint spectrum of the total extract of Cnidium monnieri, and further establish the fingerprint spectrum of Cnidium monnieri Chinese medicinal materials and Cnidium monnieri total extract preparations, thereby improving the controllability of product quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 is the fingerprint of the mixed reference substance in Example 3;

[0045] Figure 2 is the fingerprint of the reference medicinal material in Example 3, wherein the compounds represented by the peak numbers are peak 1 p-coumaric acid, peak 2 zanthoxylum toxicol, peak 3 hesperidin hydrate, peak 4 zanthoxylum toxin, peak 5 isoanisole, peak 6 hesperidin, peak 7 bergamotol, peak 8 imperatorin, peak 9 osthole, peak 10 bergamotrin;

[0046] Figure 3 is the fingerprint of the Cnidium monnieri medicinal material in Example 3, and peak numbers 1-10 are the same as Figure 2 ;

[0047] Figure 4 It is the fingerprint of the total extract of Cnidium monnieri (i.e., total coumarins of Cnidium monnieri) in Example 3, peak 1 Zanthoxylum toxicol, peak 2 hesperidin hydrate, peak 3 Zanthoxylum toxin, peak 4 isoanisole, peak 5 hesperidin, peak 6 bergamotol, peak 7 imperatorin, peak 8 osthole, peak 9 bergamotrin;

[0048] Figure 5 is the fingerprint of the total coumarin ointment preparation of Cnidium monnieri in Example 3, and peak numbers 1-9 are the same as Figure 4 ;

[0049] Figure 6 It is the fingerprint comparison chromatogram of 15 batches of Chinese medicinal materials samples in Example 3;

[0050] Figure 7 It is the fingerprint comparison chromatogram of 15 batches of samples of Chinese herbal medicine extract in Example 3;

[0051] Figure 8 It is the fingerprint comparison chromatogram of 15 batches of samples of the preparation made from the Chinese medicinal material extract in Example 3;

[0052] Fig. 9 The mass spectrum of the unknown peak No. 10 of the Cnidium monnieri Chinese medicinal material in Example 3;

[0053] Fig.10This is a partially enlarged extracted ion chromatogram of unknown peak No. 10 of the Cnidium monnieri Chinese medicinal material in Example 3;

[0054] Fig.11 The mass spectrum of the unknown peak No. 10 of the Cnidium monnieri Chinese medicinal material in Example 3;

[0055] Fig.12 This is the secondary mass spectrum of unknown peak No. 10 of the Cnidium monnieri Chinese medicinal material in Example 3;

[0056] Fig.13 is the standard mass spectrum of bergamotin in Example 3;

[0057] Fig.14 is the structural diagram of bergamotin in Example 3;

[0058] Fig.15 This is a picture of the effect of Cnidium monnieri total coumarin ointment on herpes zoster in Example 4;

[0059] Fig.16 This is a picture showing the effect of the Cnidium monnieri total coumarin ointment on abdominal psoriasis in Example 4;

[0060] Fig.17 This is a picture showing the effect of the Cnidium monnieri total coumarin ointment on back psoriasis in Example 4;

[0061] Fig.18 This is a picture showing the effect of the Cnidium monnieri total coumarin ointment on atopic dermatitis in the knee fossa in Example 4;

[0062] Fig.19 This is a picture showing the effect of the Cnidium monnieri total coumarin ointment in Example 4 on atopic dermatitis of the ear. DETAILED DESCRIPTION

[0063] The present invention will be further described below in conjunction with specific embodiments so that those skilled in the art can better understand the present invention, but the present invention is not limited thereby. The raw materials used below are all from commercial channels unless otherwise specified.

[0064] Example 1 Preparation of Cnidium monnieri Extract

[0065] Preparation method: Take 1 kg of the Chinese medicinal material Cnidium monnieri and grind it into coarse powder, put it in an extractor, add 6 times the mass of 85% (volume percentage) ethanol solvent, heat and reflux for 2 hours, collect the extract, add 3 times the mass of 85% (volume percentage) ethanol solvent and extract twice in the same way, combine the extracts, leave it at room temperature overnight, and concentrate under reduced pressure at 80°C to obtain an extract. After leaving it at room temperature for 24 hours, a green precipitate precipitates, which is filtered out and dried to obtain the total extract of Cnidium monnieri, i.e., total coumarins of Cnidium monnieri.

[0066] Example 2 Preparation of ointment

[0067] Preparation method: The total coumarin of Cnidium monnieri obtained by the preparation method of Example 1 is prepared into a paste-like external ointment with a suitable matrix, that is, an oil-in-water preparation, with the formula: 100g of total coumarin of Cnidium monnieri, 80g of stearic acid, 100g of mono- and distearic glyceryl, 80g of white vaseline, 160g of glycerol, 60g of polysorbate-80, 280g of water, and glycerol: water (4:10) is added to 1000g to obtain a Cnidium monnieri ointment preparation.

[0068] Example 3

[0069] Test example: The following methodological investigation was carried out using Chinese herbal medicine (Cnidium monnieri), the total extract sample of Cnidium monnieri prepared in Example 1, and the Cnidium monnieri ointment preparation sample prepared in Example 2. The method was verified in terms of system suitability, specificity, repeatability, intermediate precision, stability, etc.

[0070] 1. Chromatographic conditions

[0071] 1.1 Instrument: Thermo Fisher Vanquish high performance liquid chromatograph, UV detector;

[0072] 1.2 Chromatographic conditions: Chromatographic column Agilent ZORBAX Eclipse plus C18, 4.6*250mm, 5μm: No.: GB-L-20-02-192;

[0073] The following parameters were set as initial conditions: flow rate: 0.5 mL / min; column temperature: 40°C; wavelength: 310 nm; mobile phase A: 0.1% acetic acid aqueous solution; mobile phase B: methanol; gradient elution. The elution program is as shown in Table 1:

[0074] Table 1 Mobile phase gradient elution program

[0075]

[0076]

[0077] 2. Optimization of chromatographic conditions

[0078] 2.1 Optimization of chromatographic conditions

[0079] The dominant components (osthole, zanthoxylum phenol, zanthoxylum toxin, bergamot lactone, imperatorin, isoanisole, and p-coumaric acid) that can be identified by the reference substance with high content in the Cnidium monnieri medicinal material samples were screened. The basic chromatographic conditions (Table 1) were first used, and the sum of their peak areas was used as an indicator. At the same time, the separation between the components was investigated. The screening of chromatographic columns and the screening of mobile phase ratios were carried out respectively. Finally, the goal was to make the dominant components p-coumaric acid, zanthoxylum phenol, zanthoxylum toxin, bergamot lactone, imperatorin, isoanisole, and osthole be well separated from each other, and the above dominant components and the adjacent components in the test sample can be well separated. The final optimized chromatographic conditions are shown in Table 2.

[0080] Table 2 Optimized chromatographic conditions

[0081]

[0082]

[0083] 3. Extraction condition test

[0084] 3.1 Sample processing method: Take about 0.5g of Cnidium monnieri medicinal material, about 50mg of Cnidium monnieri total coumarin raw material and about 0.1g of Cnidium monnieri total coumarin ointment respectively, and optimize the extraction method through orthogonal test, including the investigation of extraction solvent, extraction solvent concentration, extraction method, extraction time, and extraction solvent volume. The condition settings are shown in Table 3.

[0085] Table 3 Optimization of extraction conditions test

[0086]

[0087] 3.2 Test conclusion:

[0088] (1) When 70% (volume percentage) ethanol is used as the extraction solvent for medicinal materials, its "total peak area / sample weight" value is the largest, that is, the extraction efficiency is the highest; when used as the extraction solvent for raw materials and preparations, the "total peak area / sample weight" of methanol, 70% methanol, ethanol, and 70% ethanol are basically the same. Considering the toxicity and economy of the solvent, 70% ethanol was selected as the extraction solvent.

[0089] (2) The "total peak area / sample weight" of ultrasonic extraction of medicinal materials and preparations was slightly higher than that of heating reflux extraction, while the "total peak area / sample weight" of ultrasonic extraction of raw materials was slightly lower than that of heating reflux extraction. There was no significant difference in the extraction capacity of the two extraction methods. Considering the simplicity of operation, ultrasonic treatment was selected as the extraction method.

[0090] (3) The value of "total peak area / sample weight" of the medicinal material ultrasonic treatment for 60 minutes is the largest, that is, the extraction efficiency is the highest; the total peak area / sample weight of the raw material is basically the same from 10 to 60 minutes; the total peak area / sample weight of the preparation is basically the same from 20 to 60 minutes. Combined with the test results of the medicinal material, raw material and preparation, the final extraction time is selected to be ultrasonic treatment for 60 minutes.

[0091] (4) As the amount of extraction solvent increases, the “total peak area / sample weight” also increases when converted to the same concentration. Considering the response of each characteristic peak, the final extraction solvent volume was selected to be 50 mL.

[0092] 4. Preparation of test samples

[0093] Through the extraction condition test, the preparation method of the test solution is determined as follows:

[0094] The preparation method of the test sample of the characteristic spectrum of Cnidium monnieri medicinal material is as follows: take about 0.5g of Cnidium monnieri medicinal material coarse powder, accurately weigh it, accurately add 50mL of 70% ethanol, weigh the weight, ultrasonically treat it for 60 minutes, weigh it again after the solution is cooled, make up the lost weight with 70% ethanol, shake it well, filter it with a microporous filter membrane, and take the filtrate to obtain it.

[0095] Characteristic spectrum of raw material of total coumarin from Fructus Cnidium monnieri. The preparation method of the test sample is as follows: take about 50 mg of total coumarin from Fructus Cnidium monnieri (i.e., total extract from Fructus Cnidium monnieri), accurately weigh, accurately add 50 mL of 70% ethanol, weigh the weight, ultrasonically treat for 60 minutes, weigh again after the solution is cooled, make up the lost weight with 70% ethanol, shake well, filter with a microporous filter membrane, and take the filtrate to obtain.

[0096] Characteristic spectrum of total coumarin ointment of Cnidium monnieri. The preparation method of the test sample is as follows: take about 0.1 g of total coumarin ointment of Cnidium monnieri, accurately weigh it, accurately add 50 mL of 70% ethanol, weigh the weight, ultrasonically treat it for 60 minutes, weigh it again after the solution is cooled, make up the lost weight with 70% ethanol, shake well, filter it with a microporous filter membrane, and take the filtrate to obtain it.

[0097] 4.1 Reference material and sample source information

[0098] 4.1.1 Reference substances

[0099] Zanthoxylum toxicogenin was purchased from Chengdu Lemeitian Pharmaceutical Technology Co., Ltd., p-coumaric acid, zanthoxylum toxicogenin, isoanisole, bergamotol and imperatorin were purchased from Chengdu Gelipu Biotechnology Co., Ltd., and osthole and fructus cnidii control materials were purchased from China National Institute for the Control of Food and Biological Products (NIPFDA).

[0100] 4.1.2 Samples

[0101] p-Coumaric acid (99.97%, purity, the same below), Zanthoxylum bungeanum (99.88%), isoanisole (99.97%), bergamotol (99.85%), and imperatorin (98.24%) were purchased from Chengdu Lemeitian Pharmaceutical Technology Co., Ltd., Zanthoxylum bungeanum (≥98.0%) was purchased from Chengdu Lemeitian Pharmaceutical Technology Co., Ltd., and osthole (99.5) and fructus cnidii control medicinal materials (NA) were purchased from China National Institute for the Control of Food and Biological Products (NIPDE).

[0102] 4.1.2 Samples

[0103] The samples of Cnidium monnieri, Cnidium monnieri total coumarin raw material, and Cnidium monnieri total coumarin ointment used for method development were from Guangdong Haisaite Pharmaceutical Co., Ltd.

[0104] The samples of Cnidium monnieri for methodological validation and fingerprint determination were purchased from Anhui and Guangzhou, and the Cnidium monnieri total coumarin raw material, Cnidium monnieri total coumarin ointment and Cnidium monnieri total coumarin blank ointment were purchased from Guangdong Haisaite Pharmaceutical Co., Ltd.

[0105] The Cnidium monnieri total coumarin raw material and Cnidium monnieri total coumarin ointment can also be prepared according to the method in Example 1-2.

[0106] 4.2 Preparation of reference substances and samples

[0107] 4.2.1 Preparation of mixed reference substances

[0108] Accurately weigh about 1 mg of p-coumaric acid, osthole, isoanisole, physalis phenol, physalis toxin, bergamot lactone, and imperatorin reference substances, respectively, and place them in 10 mL volumetric bottles, add ethanol to dissolve and dilute to the scale, shake well, and then accurately measure 1.0 mL of each of the above solutions, place them in 10 mL volumetric bottles, dilute to the scale with 70% ethanol, shake well, and obtain. The spectrum measured by the method of the present invention is shown in Figure 1 .

[0109] 4.2.2 Sample preparation

[0110] 4.2.2.1 Preparation of medicinal material samples: Take about 0.5g of medicinal material, accurately add 50mL of 70% ethanol, weigh it, ultrasonicate it for 60min, wait for the solution to cool to room temperature and weigh it again, add 70% ethanol to make up the lost weight, shake well, filter it with a microporous filter membrane, and take the filtrate. The spectrum determined by the method of the present invention is shown in Figure 2 (Cnidium monnieri reference material) and Figure 3 (Cnidium monnieri medicinal material).

[0111] 4.2.2.2 Preparation of extract samples Weigh about 50 mg of total coumarin raw material from Cnidium monnieri accurately, add 50 mL of 70% ethanol accurately, weigh the weight, ultrasonicate for 60 minutes, wait for the solution to cool to room temperature, weigh it again, add 70% ethanol to make up the loss, shake well, filter with a microporous filter membrane, and take the filtrate to obtain the result. The spectrum determined by the method of the present invention is shown in Figure 4 .

[0112] 4.2.2.3 Preparation of ointment sample Weigh about 0.1g of Cnidium monnieri total coumarin ointment accurately, add 50mL of 70% ethanol accurately, weigh the weight, ultrasonicate for 60min, wait for the solution to cool to room temperature and weigh it again, add 70% ethanol to make up the lost weight, shake well, filter with a microporous filter membrane, and take the filtrate to obtain the result. The spectrum determined by the method of the present invention is shown in Figure 5 .

[0113] 5. Methodological Validation

[0114] 5.1 Specificity test

[0115] 5.1.1 Specificity of medicinal materials

[0116] Prepare mixed reference solution and sample solution according to the methods in 4.2.1 and 4.2.2 respectively. At the same time, prepare blank solution and negative sample of ointment preparation (without extract). Measure according to the determined chromatographic conditions and record the chromatogram.

[0117] 5.1.2 Test conclusion

[0118] (1) The blank solvent had no interference on the detection of 10 characteristic peaks of Cnidium monnieri, and the retention time was consistent with that of the reference peaks. The retention time of p-coumaric acid, zanthoxylum toxicol, zanthoxylum toxin, isoanisole, bergamotol, imperatorin, and osthole was consistent with the corresponding peaks in the mixed reference solution. This showed that the method had good specificity for the determination of the fingerprint of Cnidium monnieri.

[0119] (2) The blank solvent had no interference in the detection of the 9 characteristic peaks of the total coumarin raw material of Cnidium monnieri, among which the retention times of p-coumaric acid, tannic acid phenol, tannic acid toxin, isoanisole, bergamotol, imperatorin, and osthole were consistent with those of the corresponding peaks in the mixed reference solution. This indicates that this method has good specificity for the determination of the fingerprint spectrum of the total coumarin raw material of Cnidium monnieri.

[0120] (3) The blank solvent and negative sample had no interference in the detection of 9 characteristic peaks in the total coumarin ointment of Cnidium monnieri, among which the retention times of p-coumaric acid, fennel, fennel toxin, isoanisole, bergamotol, imperatorin, and osthole were consistent with those of the corresponding peaks in the mixed reference solution. This indicates that this method has good specificity for the fingerprint determination of the total coumarin ointment of Cnidium monnieri.

[0121] 5.2 Precision test

[0122] 5.2.1 Test methods

[0123] Take the mixed reference solution, take osthole as reference, measure according to the selected chromatographic conditions, record the chromatogram, and calculate the relative retention time and relative peak area. The results are shown in Tables 4-5.

[0124] Table 4 Precision investigation results of reference solution (relative peak area)

[0125]

[0126] Table 5 Precision investigation results of reference solution (relative retention time)

[0127]

[0128] 5.2.2 Test conclusion

[0129] The relative retention time RSD of each characteristic peak in the reference solution was in the range of 0.0% to 0.2%, and the relative peak area RSD was in the range of 0.0% to 0.2%, indicating that the instrument had good precision.

[0130] 5.3 Repeatability test

[0131] 5.3.1 Operation method

[0132] Take the same portion of medicinal materials, extracts and ointment preparations and prepare 6 portions of test sample solutions in parallel according to the method under 4.2 Reference and Sample Preparation. Take osthole as the reference and determine according to the selected chromatographic conditions. Record the chromatograms, calculate the relative retention time and relative peak area, and examine the RSD. The results are shown in Tables 6 to 11.

[0133] Table 6 Repeatability test results of Cnidium monnieri medicinal materials (relative peak area)

[0134] Sample name Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Peak 10 Repeatability 1 0.00861 0.0162 0.0790 0.0275 0.0408 0.0292 0.0571 0.274 1.000 0.00872 Repeatability 2 0.00824 0.0152 0.0709 0.0277 0.0405 0.0274 0.0578 0.278 1.000 0.00858 Repeatability 3 0.00809 0.0151 0.0698 0.0294 0.0411 0.0275 0.0576 0.280 1.000 0.00867 Repeatability 4 0.00841 0.0163 0.0789 0.0277 0.0402 0.0286 0.0570 0.277 1.000 0.00870 Repeatability 5 0.00829 0.0158 0.0738 0.0281 0.0418 0.0278 0.0583 0.276 1.000 0.00878 Repeatability 6 0.0084 0.0161 0.0739 0.0289 0.0415 0.0288 0.0572 0.278 1.000 0.00861 RSD(%) 2.2 3.3 5.3 2.8 1.5 2.7 0.9 0.8 0.0 0.9

[0135] Table 7 Repeatability test results of Cnidium monnieri medicinal materials (relative retention time)

[0136] Sample name Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Peak 10 Repeatability 1 0.306 0.379 0.512 0.540 0.684 0.707 0.730 0.979 1.000 1.045 Repeatability 2 0.306 0.379 0.512 0.540 0.683 0.707 0.730 0.979 1.000 1.045 Repeatability 3 0.306 0.379 0.512 0.540 0.683 0.707 0.729 0.979 1.000 1.045 Repeatability 4 0.306 0.379 0.512 0.540 0.683 0.706 0.729 0.979 1.000 1.045 Repeatability 5 0.306 0.378 0.512 0.539 0.683 0.706 0.729 0.979 1.000 1.045 Repeatability 6 0.306 0.378 0.512 0.539 0.683 0.706 0.729 0.979 1.000 1.045 RSD(%) 0.0 0.2 0.0 0.1 0.1 0.1 0.1 0.0 0.0 0.0

[0137] Table 8 Repeatability test results of total coumarin raw materials of Fructus Cnidium monnieri (relative peak area)

[0138] Sample name Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Repeatability 1 0.000151 0.000288 0.000280 0.0000940 0.00150 0.00144 0.0131 1.000 0.000124 Repeatability 2 0.000140 0.000255 0.000276 0.0000892 0.00137 0.00128 0.0128 1.000 0.000128 Repeatability 3 0.000145 0.000264 0.000281 0.0000938 0.00141 0.00134 0.0132 1.000 0.000130 Repeatability 4 0.000144 0.000263 0.000273 0.0000963 0.00141 0.00130 0.0129 1.000 0.000125 Repeatability 5 0.000149 0.000274 0.000273 0.0000961 0.00145 0.00133 0.0128 1.000 0.000126 Repeatability 6 0.000165 0.000316 0.000278 0.0001053 0.00160 0.00144 0.0124 1.000 0.000115 RSD(%) 5.9 8.1 1.3 5.6 5.7 5.2 2.2 0.0 4.2

[0139] Table 9 Repeatability test results of total coumarin raw materials of Fructus Cnidium monnieri (relative retention time)

[0140]

[0141]

[0142] Table 10 Results of repeatability test of total coumarin ointment of Cnidium monnieri (relative retention time)

[0143] Sample name Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Repeatability 1 0.378 0.512 0.540 0.684 0.706 0.729 0.979 1.00 1.045 Repeatability 2 0.378 0.512 0.540 0.684 0.706 0.730 0.979 1.00 1.045 Repeatability 3 0.378 0.512 0.540 0.684 0.706 0.729 0.979 1.00 1.045 Repeatability 4 0.378 0.512 0.540 0.683 0.706 0.730 0.979 1.00 1.045 Repeatability 5 0.379 0.513 0.541 0.685 0.707 0.732 0.979 1.00 1.045 Repeatability 6 0.379 0.513 0.541 0.685 0.708 0.732 0.979 1.00 1.045 RSD(%) 0.2 0.2 0.1 0.2 0.2 0.2 0.0 0.0 0.0

[0144] Table 11 Results of repeatability test of total coumarin ointment of Cnidium monnieri (relative peak area)

[0145] Sample name Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Repeatability 1 0.000459 0.000223 0.00175 0.000450 0.00118 0.00398 0.0761 1.000 0.00146 Repeatability 2 0.000463 0.000269 0.00174 0.000446 0.00119 0.00402 0.0781 1.000 0.00147 Repeatability 3 0.000473 0.000269 0.00175 0.000446 0.00119 0.00396 0.0764 1.000 0.00148 Repeatability 4 0.000463 0.000252 0.00177 0.000438 0.00119 0.00400 0.0758 1.000 0.00147 Repeatability 5 0.000462 0.000268 0.00175 0.000456 0.00117 0.00392 0.0777 1.000 0.00145 Repeatability 6 0.000457 0.000193 0.00171 0.000450 0.00115 0.00386 0.0754 1.000 0.00146 RSD(%) 1.2 12.8 1.2 1.4 1.4 1.5 1.5 0.0 0.8

[0146] 5.3.2 Test conclusion

[0147] (1) Six samples of Cnidium monnieri were repeatedly measured, and the relative peak area RSD of each characteristic peak was in the range of 0.8% to 5.3%, and the relative retention time RSD was in the range of 0.0% to 0.2%, indicating that the characteristic spectrum analysis method has good repeatability.

[0148] (2) Six samples of total coumarin from Fructus Cnidium monnieri were repeatedly measured, and the relative retention time RSD of each characteristic peak was in the range of 0.0% to 0.1%, and the relative peak area RSD was in the range of 1.3% to 8.1%, indicating that the characteristic spectrum analysis method has good repeatability.

[0149] (3) Six samples of Cnidium monnieri total coumarin ointment were repeatedly measured, and the relative retention time RSD of each characteristic peak was in the range of 0.0% to 0.2%, and the relative peak area RSD was in the range of 0.8% to 12.8%, indicating that the characteristic spectrum analysis method has good repeatability.

[0150] 5.4 Intermediate precision

[0151] 5.4.1 Operation method

[0152] Other analysts operated on different dates, different chromatographs, and different batches of chromatographic columns. They took samples of Cnidium monnieri, extracts, and ointment from the same batch, and prepared 6 test sample solutions in parallel according to the method in 4.2 Reference and Sample Preparation. Using Cnidium monnieri as a reference, the samples were determined under the selected chromatographic conditions, the chromatograms were recorded, and the relative retention times and relative peak areas were calculated. The experimental results are shown in Tables 12 to 17.

[0153] Table 12 Results of intermediate precision study of Cnidium monnieri medicinal materials (relative peak area)

[0154] Sample name Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Peak 10 Intermediate precision 1 0.0115 0.0160 0.0710 0.0306 0.0409 0.0279 0.0542 0.265 1.0000 0.00753 Intermediate precision 2 0.0119 0.0157 0.0680 0.0299 0.0404 0.0268 0.0542 0.266 1.0000 0.00750 Intermediate precision 3 0.0114 0.0160 0.0691 0.0306 0.0412 0.0277 0.0532 0.266 1.0000 0.00741 Intermediate precision 4 0.0116 0.0158 0.0696 0.0310 0.0410 0.0279 0.0539 0.266 1.0000 0.00752 Intermediate precision 5 0.0103 0.0127 0.0535 0.031O 0.0423 0.0226 0.0577 0.265 1.0000 0.00704 Intermediate precision 6 0.011O 0.0143 0.0594 0.0302 0.0412 0.0247 0.0561 0.266 1.0000 0.00724 RSD(%) 5.0 8.9 10.8 1.5 1.6 8.3 3.1 0.2 0.0 2.7 Repeatability RsD (%) 16.2 6.7 10.4 4.6 1.5 6.8 3.2 2.3 0.0 8.7

[0155] Table 13 Results of intermediate precision study of Cnidium monnieri medicinal materials (relative retention time)

[0156] Sample name Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Peak 10 Intermediate precision 1 0.308 0.386 0.532 0.560 0.717 0.746 0.766 0.979 1.000 1.049 Intermediate precision 2 0.308 0.386 0.531 0.560 0.716 0.745 0.765 0.979 1.000 1.049 Intermediate precision 3 0.308 0.386 0.531 0.560 0.716 0.745 0.766 0.979 1.000 1.049 Intermediate precision 4 0.308 0.386 0.531 0.560 0.716 0.745 0.765 0.979 1.000 1.049 Intermediate precision 5 0.308 0.386 0.531 0.560 0.716 0.745 0.765 0.979 1.000 1.049 Intermediate precision 6 0.308 0.386 0.531 0.559 0.716 0.745 0.765 0.979 1.000 1.049 RSD(%) 0.0 0.1 0.1 0.1 0.1 0.1 0.1 0.0 0.0 0.1 Repeatability RSD (%) 0.4 1.1 2.0 2.0 2.5 2.8 2.6 0.1 0.0 0.2

[0157] Table 14 Intermediate precision investigation results of total coumarin raw materials of Fructus Cnidium monnieri (relative peak area)

[0158] Sample name Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Intermediate precision 1 0.000226 0.000334 0.000372 0.0000625 0.00193 0.00151 0.0174 1.0000 0.000170 Intermediate precision 2 0.000226 0.000334 0.000371 0.0000570 0.00195 0.00156 0.0175 1.0000 0.000171 Intermediate precision 3 0.000237 0.000361 0.000372 0.0000560 0.00200 0.00159 0.0180 1.0000 0.000173 Intermediate precision 4 0.000232 0.000338 0.000364 0.0000620 0.00196 0.00155 0.0176 1.0000 0.000173 Intermediate precision 5 0.000230 0.000339 0.000361 0.0000510 0.00197 0.00155 0.0177 1.0000 0.000163 Intermediate precision 6 0.000250 0.000360 0.000388 0.0000620 0.00211 0.00166 0.0190 1.0000 0.000187 RSD(%) 3.9 3.7 2.6 7.9 3.3 3.3 3.4 0.0 4.6 Repeatability RSD% 23.6 13.0 16.0 26.4 16.6 8.6 17.3 0.0 17.5

[0159] Table 15 Intermediate precision investigation results of total coumarin raw materials of Cnidium monnieri (relative retention time)

[0160] Sample name Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Intermediate precision 1 0.3855 0.5322 0.5602 0.7174 0.7462 0.7698 0.9795 1.0000 1.0486 Intermediate precision 2 0.3854 0.5323 0.5603 0.7168 0.7461 0.7698 0.9795 1.0000 1.0485 Intermediate precision 3 0.3854 0.5320 0.5599 0.7160 0.7457 0.7695 0.9795 1.0000 1.0485 Intermediate precision 4 0.3854 0.5319 0.5601 0.7161 0.7453 0.7690 0.9794 1.0000 1.0485 Intermediate precision 5 0.3855 0.5321 0.5600 0.7164 0.7457 0.7689 0.9793 1.0000 1.0485 Intermediate precision 6 0.3857 0.5321 0.5602 0.7178 0.7456 0.7692 0.9794 1.0000 1.0486 RSD(%) 0.1 0.1 0.1 0.2 0.1 0.1 0.1 0.0 0.1 Repeatability RsD (%) 1.0 2.1 1.9 2.5 2.9 2.9 0.1 0.0 0.2

[0161] Table 16 Intermediate precision test results of Cnidium monnieri total coumarin ointment (relative peak area)

[0162] Sample name Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Intermediate precision 1 0.000388 0.000259 0.00182 0.000442 0.00120 0.00409 0.0762 1.0000 0.00136 Intermediate precision 2 0.000415 0.000254 0.00185 0.000461 0.00118 0.00430 0.0783 1.0000 0.00137 Intermediate precision 3 0.000415 0.000249 0.00177 0.000478 0.00132 0.00434 0.0743 1.0000 0.00135 Intermediate precision 4 0.000403 0.000284 0.00182 0.000447 0.00123 0.00422 0.0790 1.0000 0.00144 Intermediate precision 5 0.000389 0.000248 0.00175 0.000453 0.00123 0.00424 0.0787 1.0000 0.00144 Intermediate precision 6 0.000385 0.000194 0.00176 0.000439 0.00123 0.00425 0.0755 1.0000 0.00134 RSD(%) 3.5 12.0 2.3 3.2 3.9 2.1 2.6 0.0 3.3 Repeatability RSD% 8.1 16.5 2.3 6.3 7.1 7.1 2.0 0.0 3.8

[0163] Table 17 Results of intermediate precision test of total coumarin ointment of Cnidium monnieri (relative retention time)

[0164] Sample name Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Intermediate precision 1 0.386 0.533 0.561 0.717 0.747 0.768 0.979 1.000 1.048 Intermediate precision 2 0.386 0.532 0.561 0.717 0.747 0.768 0.979 1.000 1.049 Intermediate precision 3 0.386 0.532 0.560 0.717 0.747 0.768 0.979 1.000 1.048 Intermediate precision 4 0.386 0.532 0.560 0.718 0.747 0.768 0.979 1.000 1.048 Intermediate precision 5 0.386 0.532 0.560 0.717 0.746 0.767 0.979 1.000 1.048 Intermediate precision 6 0.386 0.532 0.560 0.716 0.747 0.768 0.979 1.000 1.048 RSD(%) 0.1 0.1 0.1 0.1 0.1 0.1 0.0 0.0 0.1 Repeatability RSD (%) 1.1 2.0 1.9 2.5 2.9 2.7 0.1 0.0 0.2

[0165] 5.4.2 Test conclusion

[0166] (1) Different analysts operated on different dates, different chromatographs, and different batches of chromatographic columns. The RSD of the relative retention time of the 10 common characteristic peaks was in the range of 0.0% to 0.1%, which was in the range of 0.1% to 2.8% compared with the RSD of the 6 data in the repeatability test. The RSD of the relative peak area of ​​the 10 common characteristic peaks was in the range of 0.2% to 10.8%, which was in the range of 1.5% to 16.2% compared with the RSD of the 6 data in the repeatability test. The intermediate precision of this characteristic spectrum analysis method met the requirements.

[0167] (2) Different analysts operated on different dates, different chromatographs, and different batches of chromatographic columns. The RSD of the relative retention time of the 9 common characteristic peaks in the total coumarin raw material of Cnidium monnieri was in the range of 0.1% to 0.2%, which was in the range of 0.1% to 2.9% compared with the RSD of the 6 data in the repeatability test. The RSD of the relative peak area of ​​the 9 common characteristic peaks was in the range of 2.6% to 7.9%, which was in the range of 8.6% to 26.4% compared with the RSD of the 6 data in the repeatability test. The intermediate precision of this characteristic spectrum analysis method met the requirements.

[0168] (3) Different analysts operated on different dates, different chromatographs, and different batches of chromatographic columns. The RSD of the relative retention time of the 9 common characteristic peaks in the Cnidium monnieri total coumarin ointment was in the range of 0.0% to 0.1%, which was in the range of 0.1% to 2.9% compared with the RSD of the 6 data in the repeatability test. The RSD of the relative peak areas of the 9 common characteristic peaks was in the range of 2.1% to 12.0%, which was in the range of 2.0% to 16.5% compared with the RSD of the 6 data in the repeatability test. The intermediate precision of this characteristic spectrum analysis method met the requirements.

[0169] 5.5 Stability test

[0170] 5.5.1 Test methods

[0171] According to the method in 4.2, the drug, extract and ointment test solutions were prepared, placed at room temperature, and injected at 0, 21, 40, 60, 87, and 94 hours. The results are shown in Tables 18 to 23.

[0172] Table 18 Results of stability study on Cnidium monnieri medicinal material solution (relative peak area)

[0173] time Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Peak 10 0h 0.00862 0.0161 0.0758 0.0273 0.0404 0.0286 0.0566 0.276 1.00 0.00882 21h 0.00850 0.0164 0.0757 0.0271 0.0404 0.0286 0.0566 0.277 1.00 0.00882 40h 0.00853 0.0164 0.0758 0.0270 0.0402 0.0286 0.0566 0.276 1.00 0.00878 60h 0.00858 0.0164 0.0758 0.0272 0.0403 0.0286 0.0565 0.276 1.00 0.00884 87h 0.00823 0.0164 0.0764 0.0271 0.0396 0.0284 0.0561 0.269 1.00 0.00861 94h 0.00817 0.0165 0.0766 0.0273 0.0395 0.0283 0.0559 0.269 1.00 0.00857 RSD(%) 2.3 0.9 0.6 0.5 1.1 0.5 0.6 1.4 0.0 1.4

[0174] Table 19 Results of stability study on the solution of Cnidium monnieri medicinal material (relative retention time)

[0175] time Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Peak 10 0h 0.306 0.379 0.513 0.541 0.685 0.709 0.732 0.979 1.00 1.045 21h 0.306 0.379 0.512 0.540 0.684 0.707 0.730 0.979 1.00 1.045 40h 0.306 0.378 0.512 0.540 0.683 0.706 0.729 0.979 1.00 1.045 60h 0.306 0.379 0.513 0.541 0.685 0.708 0.731 0.979 1.00 1.045 87h 0.305 0.378 0.511 0.539 0.681 0.705 0.727 0.979 1.00 1.045 94h 0.305 0.378 0.511 0.538 0.681 0.705 0.727 0.979 1.00 1.045 RSD(%) 0.2 0.2 0.2 0.3 0.3 0.3 0.3 0.0 0.0 0.0

[0176] Table 20 Results of stability study of total coumarin raw materials from Fructus Cnidium monnieri (relative peak area)

[0177] time Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 0h 0.000118 0.000211 0.000284 0.0000789 0.00116 0.00101 0.0139 1.000 0.000148 21h 0.000119 0.000203 0.000271 0.0000835 0.00117 0.00102 0.0139 1.000 0.000145 40h 0.000121 0.000197 0.000278 0.0000830 0.00118 0.00102 0.0138 1.000 0.000147 60h 0.000121 0.000204 0.000272 0.0000813 0.00117 0.00100 0.0137 1.000 0.000145 87h 0.000120 0.000209 0.000268 0.0000819 0.00118 0.00102 0.0134 1.000 0.000153 94h 0.000123 0.000204 0.000279 0.0000850 0.00118 0.00102 0.0135 1.000 0.000148 RSD(%) 1.5 2.5 2.2 2.6 0.7 0.9 1.6 0.0 2.0

[0178] Table 21 Results of stability study of total coumarin raw materials from Fructus Cnidium monnieri (relative retention time)

[0179]

[0180]

[0181] Table 22 Results of stability test of total coumarin ointment of Fructus Cnidii (relative peak area)

[0182] time Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 0h 0.000467 0.000264 0.00184 0.000460 0.00122 0.00418 0.0752 1.000 0.00148 21h 0.000482 0.000253 0.00179 0.000477 0.00123 0.00420 0.0752 1.000 0.00144 40h 0.000466 0.000226 0.00178 0.000486 0.00125 0.00417 0.0753 1.000 0.00145 60h 0.000493 0.000259 0.00181 0.000489 0.00126 0.00449 0.0752 1.000 0.00147 87h 0.000465 0.000272 0.00177 0.000481 0.00124 0.00418 0.0733 1.000 0.00141 94h 0.000399 0.000274 0.00174 0.000458 0.00126 0.00419 0.0731 1.000 0.00142 RSD(%) 7.2 6.8 2.0 2.8 1.4 3.0 1.5 0.0 1.9

[0183] Table 23 Cnidium monnieri total coumarin ointment stability test results (relative retention time)

[0184] time Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 0h 0.379 0.513 0.541 0.685 0.708 0.732 0.979 1.00 1.045 21h 0.378 0.513 0.540 0.684 0.706 0.730 0.979 1.00 1.045 40h 0.378 0.512 0.540 0.683 0.706 0.729 0.979 1.00 1.045 60h 0.379 0.513 0.541 0.684 0.707 0.731 0.979 1.00 1.045 87h 0.377 0.511 0.538 0.681 0.704 0.727 0.979 1.00 1.045 94h 0.377 0.510 0.538 0.680 0.703 0.726 0.979 1.00 1.045 RSD(%) 0.3 0.3 0.3 0.3 0.3 0.4 0.0 0.0 0.0

[0185] 5.5.2 Test conclusion

[0186] (1) Within 94 hours at room temperature, the relative retention time RSD of the 10 common characteristic peaks in the Cnidium monnieri medicinal material test solution was in the range of 0.0% to 0.3%, and the relative peak area RSD was in the range of 0.5% to 2.3%, indicating that the test solution had good stability within 94 hours at room temperature.

[0187] (2) Within 94 hours at room temperature, the relative retention time RSD of the 9 common characteristic peaks of the total coumarin raw material test solution of Cnidium monnieri was in the range of 0.0% to 0.3%, and the relative peak area RSD was in the range of 0.7% to 2.6%, indicating that the test solution had good stability within 94 hours at room temperature.

[0188] (3) Within 94 hours at room temperature, the relative retention time RSD of the nine common characteristic peaks of the Cnidium monnieri total coumarin ointment test solution was in the range of 0.0% to 0.4%, and the relative peak area RSD was in the range of 1.4% to 7.2%, indicating that the test solution had good stability within 94 hours at room temperature.

[0189] 6. Establishment and analysis of sample fingerprint

[0190] 6.1 Fingerprint establishment and similarity evaluation

[0191] The fingerprint analysis was performed using the “Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)”, and multiple batches of sample data (no less than 15 batches) were summarized. Automatic matching was performed after multi-point correction, and the median method was used to generate a control fingerprint.

[0192] 6.2 Identification and attribution of common peaks in fingerprint spectra

[0193] (1) Full-scan data acquisition using liquid chromatography-high-resolution mass spectrometry: Prepare Cnidium monnieri medicinal material samples, Cnidium monnieri total extract samples, Cnidium monnieri preparation samples, and Cnidium monnieri preparation negative control samples (i.e., take the remaining excipients except Cnidium monnieri), and inject the samples using the liquid phase method developed for the above fingerprint spectrum, and perform full-scan analysis using liquid chromatography-high-resolution mass spectrometry. The "Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation Software" was used to identify the common peaks of the characteristic spectra of 15 batches of Cnidium monnieri medicinal materials, with osthole as the reference peak (i.e., S peak), and select the common peaks with relatively stable relative retention time and relative peak area as characteristic peaks.

[0194] (2) Component confirmation: Combined with the source data of the sample, the characteristic components obtained in the above software are extracted and confirmed.

[0195] (3) Speculation and confirmation of chemical structure of components: Using a proprietary database of traditional Chinese medicine components and software, the unique chemical components of Cnidium monnieri in Cnidium monnieri (herbal medicines, extracts, preparations) are analyzed, identified, and confirmed.

[0196] (4) Prepare a summary report on the chemical composition of Cnidium monnieri (herbal medicine, extract, preparation) and establish a standard fingerprint.

[0197] Conclusion: The Chinese medicinal material Cnidium monnieri and the control medicinal material in the present invention meet the quality standards of the current edition of the Chinese Pharmacopoeia. The preparation and inspection of the Cnidium monnieri total coumarin raw material and the Cnidium monnieri total coumarin ointment are carried out in accordance with the national drug production GMP. In order to investigate the stability of the product and improve the quality standard of the product, the present invention studies the fingerprint spectra of the Chinese medicinal material Cnidium monnieri, the Chinese medicinal material Cnidium monnieri total extract (Cnidium monnieri total coumarins) and the Cnidium monnieri total coumarin ointment of the present invention, and extracts and analyzes 15 samples from different batches according to the method established after optimization. Seven representative common peaks were screened out from the fingerprint spectra and identified, and the separation between the peaks was good. The similarity of the fingerprint spectra between the 15 batches was greater than 90%, indicating that the product is stable and controllable. The characteristic spectra (fingerprint spectra) of the 15 batches of medicinal materials are shown in Figure 6 ; The characteristic spectrum (fingerprint) of 15 batches of Cnidium monnieri medicinal material extracts (Cnidium monnieri total coumarins) is shown in Figure 7 ; 15 batches of preparations (Cnidium monnieri total coumarin ointment) made from Cnidium monnieri medicinal material extracts (Cnidium monnieri total coumarin) characteristic spectra (fingerprints) see Figure 8 For the identification of the unknown peak (peak 10) of Cnidium monnieri, see Figures 9 to 14 .

[0198] (1) Through high-resolution mass spectrometry and comparison with the common characteristic peaks in the characteristic spectrum of Cnidium monnieri, multiple reference substances were selected for positioning and identification, and it was determined that in the characteristic spectrum of Cnidium monnieri, peak 1: p-coumaric acid, peak 2: Zanthoxylum toxicol, peak 3: hesperidin hydrate, peak 4: Zanthoxylum toxin, peak 5: isoanisole, peak 6: hesperidin, peak 7: bergamotol, peak 8: imperatorin, peak 9: osthole, and peak 10: bergamotrin were identified.

[0199] (2) Through high-resolution mass spectrometry and comparison with the common characteristic peaks in the characteristic spectrum of the total coumarin raw material of Cnidium monnieri, and the use of multiple reference substances for positioning and identification, it was determined that in the characteristic spectrum of the total coumarin raw material of Cnidium monnieri, peak 1: Zanthoxylum toxicol, peak 2: hesperidin hydrate, peak 3: Zanthoxylum toxicol, peak 4: isoanisole, peak 5: hesperidin, peak 6: bergamotol, peak 7: imperatorin, peak 8: osthole, and peak 9: bergamotrin were

[0200] (3) Through high-resolution mass spectrometry and comparison with the common characteristic peaks in the characteristic spectrum of total coumarin ointment of Cnidium monnieri, and the use of multiple reference substances for positioning and identification, it was determined that in the characteristic spectrum of total coumarin ointment of Cnidium monnieri, peak 1: Zanthoxylum toxicol, peak 2: hesperidin hydrate, peak 3: Zanthoxylum toxicol, peak 4: isoanisole, peak 5: hesperidin, peak 6: bergamotol, peak 7: imperatorin, peak 8: osthole, and peak 9: bergamotrin were identified.

[0201] (4) The unknown chromatographic peak No. 10 with a retention time of 86.49 minutes was identified by high-resolution mass spectrometry and comparison with the common characteristic peaks in the characteristic spectrum of Cnidium monnieri. In its UV chromatogram, the corresponding primary mass spectrum and extracted ion chromatogram were taken, as shown in Fig.11 and Fig.12 The mass-to-charge ratios of the primary mass spectrometry of the chromatographic peak are 361.14060, 339.15885 and 203.03382, of which ions 339 and 361 are the [M+H] and [M+Na] ions of the compound, respectively, and 203 is its main fragment ion. The molecular formula of the compound is calculated to be C 21 H 22 O4. Its mass spectrum is shown in 11. Extract the secondary mass spectrum of the corresponding compound, see Fig.12 .

[0202] Comparing the secondary spectrum with the standard database, the secondary mass spectrum of unknown substance No. 10 (retention time 86.49 minutes) is consistent with the standard secondary spectrum of bergamotrin. The spectrum link and standard spectrum of bergamotrin in the standard database are shown in Fig.13 Therefore, it is speculated that the compound is bergamotin, and its structural formula is shown in Fig.14 .

[0203] The information of bergamot compound is as follows:

[0204] Chinese name: Bergamot

[0205] English name: Bergamottin

[0206] Molecular formula: C 21 H 22 O4

[0207] Molecular weight: 338.40

[0208] CAS number: 7380-40-7.

[0209] (5) Through the above high-resolution mass spectrometry and comparison with the common characteristic peaks of the characteristic spectra of Cnidium monnieri medicinal materials, Cnidium monnieri total coumarins and Cnidium monnieri total coumarin ointment, it was confirmed that Peak 10 in Cnidium monnieri medicinal materials, Peak 9 of Cnidium monnieri total coumarins and Peak 9 of Cnidium monnieri total coumarin ointment were bergamotrin. This is also the first time that the presence of bergamotrin has been identified in Cnidium monnieri medicinal materials.

[0210] Example 4

[0211] Cnidium monnieri was first recorded in Shennong's Herbal Classic and was listed as a top-grade product. It tastes bitter and pungent, is warm in nature, and enters the spleen and kidney meridians. Its functions include warming the kidney and strengthening yang, drying dampness, dispelling wind, and killing insects. It is commonly used clinically for impotence, uterine coldness and infertility, cold and dampness leucorrhea, and dampness and arthralgia; it is also used externally for vulvar eczema, skin itching, vaginal itching in women, and trichomonas vaginitis. According to modern pharmacological studies, Cnidium monnieri has anti-histamine, anti-fungal, and anti-tumor effects.

[0212] The following clinical trials are used to illustrate the therapeutic effects of the Cnidium monnieri total coumarin ointment in Example 2 of the present application on herpes zoster, psoriasis, and atopic dermatitis:

[0213] Patient 1, male, 64 years old, complained of local skin pain (flattening), itching, and burning sensation on the inner side of the left thigh (single skin area at L3) for 5 days on January 26, 2021, and rash and blisters for 2 days.

[0214] Physical examination: a single skin area of ​​left L3 with red or dark red papulovesicles, some blisters clustered, umbilicus, and crusting. The local skin was flattened, and two lymph nodes in the left groin were enlarged, with smooth surface, no adhesion, and mild tenderness.

[0215] Clinical diagnosis: herpes zoster.

[0216] Treatment: I used Pikang Cream for 2 days, followed by Piyanping for 3 days, but the symptoms did not improve and even worsened. After applying Cnidium monnieri Total Coumarin Ointment for 3 days, twice a day, the symptoms were significantly relieved; I continued to use the medicine: once a day for 4 days, and once every other day for 8 days.

[0217] No recurrence has been observed since recovery (see Fig.15 There were no adverse reactions during the treatment.

[0218] Patient 2, male, 54 years old, had suffered from psoriasis for 23 years and had little effect after various treatments. He was cured by topical application of Cnidium monnieri total coumarin ointment and has not had any recurrence since then (see Figure 16-17 ,in Fig.16 For the abdomen, Fig.17 There were no adverse reactions during the treatment.

[0219] Patient 3, female, 5 years old, suffered from atopic dermatitis for 3 years. After external application of Cnidium monnieri total coumarin ointment, there has been no recurrence so far (see Figure 18-19,in Fig.18 For the knee fossa, Fig.19 There were no adverse reactions during the treatment.

[0220] In addition, the total coumarin of Cnidium monnieri and the total coumarin preparation of Cnidium monnieri containing bergamot in the present application have a significant inhibitory effect on the mitosis of mouse vaginal epithelial cells; can significantly increase the number of scales formed by the granular layer in the skin scales of the rat tail; have a strong inhibitory effect on the passive skin allergic reaction of rats; have a significant inhibitory effect on the swelling of mouse ears caused by croton oil or the swelling of rat feet caused by egg white; can significantly increase the itch threshold of guinea pigs caused by histamine phosphate, indicating that it has a significant antipruritic effect (its effect can be referred to the invention The earlier patent applications of the inventors (200410079250.5 and 02114903.8) further illustrate that the total coumarin from Cnidium monnieri containing bergamotin and the total coumarin preparation from Cnidium monnieri in the present application have the efficacy of the common total extract from Cnidium monnieri, and have the effects on the pathological mechanism characteristics of allergic and hypersensitive skin diseases (including eczema, atopic dermatitis, etc.), psoriasis, and herpes zoster, change the pathological changes of such diseases, improve clinical symptoms, have fast onset of action, high efficacy, and few adverse reactions, and at the same time have a local adjustment effect on the human body.

[0221] To sum up, the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention are all covered by the protection scope of the present invention.

Claims

1. Use of total extract of Cnidium monnieri as the sole active ingredient in the preparation of a drug for preventing and / or treating herpes zoster; The total extract of Cnidium monnieri contains bergamotin, and also contains Zanthoxylum toxicol, Zanthoxylum toxin, isoanisole, bergamot lactone, imperatorin, osthole, hesperidone hydrate and hesperidone; The preparation method of the Cnidium monnieri total extract comprises the following steps: Take the Chinese medicinal material of Cnidium monnieri, add 80-85% ethanol with a volume percentage of 3-6 times the total mass of Cnidium monnieri, reflux extract 1-3 times, each time for 2-3 hours, filter, combine the filtrate, concentrate under reduced pressure to obtain an alcohol extract, and then crystallize and dry to obtain a total extract of Cnidium monnieri, i.e., total coumarins of Cnidium monnieri.

2. The use according to claim 1, characterized in that: The medicine is prepared by adding the total extract of Cnidium monnieri and pharmaceutically acceptable carriers or auxiliary materials.

3. The use according to claim 1, characterized in that: The medicine is cnidium monnieri ointment, cnidium monnieri spray or cnidium monnieri liquid containing cnidium monnieri total extract.

4. A method for constructing a fingerprint of a total extract of Cnidium monnieri, characterized in that The following steps are involved: (1) Preparation of mixed reference substance solution: p-coumaric acid, osthole, isoanisole, zanthoxylum phenol, zanthoxylum toxin, bergamot lactone, and imperatorin reference substances are dissolved in ethanol to obtain a mixed reference substance solution; (2) Preparation of test solution: Take the total extract of Cnidium monnieri, add ethanol, ultrasonicate, make up the loss with ethanol, mix well, filter, and take the filtrate to obtain the test solution; (3) taking a mixed reference solution and a test solution, injecting the sample, and performing full scan analysis using liquid chromatography-high resolution mass spectrometry, using software to identify common peaks in the characteristic spectrum of the test sample, extracting and confirming the characteristic components, performing structural analysis, attribution identification and confirmation on the unique chemical components of Cnidium monnieri, obtaining the chemical components of Cnidium monnieri, and establishing a standard fingerprint spectrum of the total extract of Cnidium monnieri; The standard fingerprint of the Cnidium monnieri extract in step (3) includes the following compounds: Zanthoxylum toxicogenin, Zanthoxylum toxicogenin, isoanisole, bergamot lactone, imperatorin, osthole, hesperidone hydrate, hesperidone and bergamotrin.

5. The method for constructing the fingerprint of the total extract of Cnidium monnieri according to claim 4, characterized in that: The volume percentage of ethanol in step (2) is 70%, the dosage of the total Cnidium monnieri extract and ethanol is 0.05 g:50 mL, and the ultrasonic treatment time is 60 min.

6. The method for constructing the fingerprint of the total extract of Cnidium monnieri according to claim 4, characterized in that: The parameters of liquid chromatography in step (3) are as follows: chromatographic conditions: C18 chromatographic column; flow rate: 0.5 mL / min; column temperature: 40° C.; Wavelength: 310nm; injection volume: 10μL; mobile phase A: 0.1% (volume percentage) acetic acid aqueous solution; mobile phase B: methanol, gradient elution conditions are as follows: 。

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