A Jingli fingerprint spectrum and its construction method and application

Abstract

The present application belongs to the technical field of analysis of components of traditional Chinese medicine, and specifically relates to a Jingli fingerprint spectrum and its construction method and application. The method is to prepare a test solution by using multiple batches of Jingli as samples and methanol as a solvent; select a reference substance and prepare a reference solution by using methanol as a solvent; inject the test solution and the reference solution into a high performance liquid chromatograph respectively for measurement, and obtain chromatograms of the sample and the reference respectively; calibrate the common peaks of the sample and use the reference substance to identify the common peaks, construct a Jingli fingerprint spectrum, and perform similarity calculation. The present invention provides a method for constructing a Jingli fingerprint spectrum, by which Jingli with different processing techniques can be effectively identified and distinguished, providing an effective method for quality control and supervision of Jingli products.

Description

Technical Field

[0001] The present application belongs to the technical field of Chinese medicine component analysis, and specifically relates to a Jingli fingerprint spectrum and a construction method and application thereof. Background Art

[0002] In the preparation process of Jingli, the traditional direct fire processing technology demonstrates its irreplaceable unique advantages, which ensures the stability of the medicinal effect. During the ancient processing method, through the exquisite control of the fire, a complex and precise temperature distribution is formed, which prompts a series of subtle reactions inside Jingli and generates a variety of key effective ingredients, thereby significantly improving its medicinal efficacy.

[0003] In recent years, non-traditional direct-fire processing techniques such as boiling and distillation have rapidly emerged. Although they have greatly improved production efficiency, they have also exposed many drawbacks: On the one hand, the efficacy varies significantly. Compared with the ancient processing methods, these new processes lack fine control over key factors such as temperature and time, which is very likely to lead to the loss of some effective ingredients in Jingli, or change the chemical composition, and ultimately make it difficult to achieve the expected therapeutic effect. On the other hand, the quality has seriously declined. Because of the deviation from the principle of natural leaching cherished by the ancient processing methods, these methods have made Jingli lose its inherent purity, not only greatly reducing the quality, but also making the taste unsatisfactory.

[0004] At present, the research on different processing techniques of Jingli is still in its infancy, and there is no relevant literature that reports it comprehensively and in depth. Therefore, it is urgent to conduct systematic and scientific research on the components of Jingli under different processing techniques, accurately find out the differences between traditional direct-fire processing and non-traditional direct-fire processing, and provide a solid theoretical basis for the quality control, clinical application, and inheritance and development of traditional processing techniques of Jingli. Summary of the invention

[0005] The purpose of the present invention is to solve the deficiencies of the prior art, focusing on the fingerprint spectrum technology of jingli, aiming to distinguish jingli produced by non-traditional direct fire processing technology (boiling and dry distillation), hoping to restore the true meaning of ancient processing through the analysis of these processes, ensure the quality and efficacy of jingli, so that this ancient medical treasure can be revitalized in modern society and continue to play its unique value. To this end, the present invention provides a jingli fingerprint spectrum and its construction method and application, specifically adopting the following technical solutions:

[0006] In a first aspect, the present invention provides a method for constructing a fingerprint of jujube, comprising the following steps:

[0007] S1. Prepare the test solution using multiple batches of jingli as samples and methanol as solvent;

[0008] S2. Select a reference substance and prepare a reference solution using methanol as solvent;

[0009] S3, respectively injecting the test solution and the reference solution into a high performance liquid chromatograph for measurement to obtain a sample chromatogram and a reference chromatogram, respectively;

[0010] S4, selecting the sample chromatogram, using the Chinese medicine chromatographic fingerprint similarity evaluation system, through multi-point correction and Mark peak matching, to obtain a fingerprint common pattern diagram containing 12 common peaks, that is, to construct a Jingli fingerprint;

[0011] The reference substance is one or more of furfural, cryptochlorogenic acid, vecinin-2, schaftoside, vecinin-3, vitexin, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid;

[0012] The chromatographic conditions of the high performance liquid chromatograph include the following:

[0013] Acetonitrile was used as mobile phase A, and 0.1% phosphoric acid was used as mobile phase B;

[0014] The detection wavelengths were: 280 nm for 0 min-13 min and 320 nm for 13 min-50 min;

[0015] The chromatographic column is: Shiseido CAPCELL PAK C18, 25cm×4.6mm×5µm.

[0016] As a further preferred embodiment, the chromatographic conditions of the above-mentioned high performance liquid chromatograph also include the following:

[0017] Gradient elution conditions were: 0 min-10 min-30 min-45 min-50 min; mobile phase A: 5%-12%-20%-35%-45%;

[0018] The injection volume was 5 µL;

[0019] The column temperature was 30°C;

[0020] The flow rate was 1.0 mL / min.

[0021] As a further preferred embodiment, the preparation of the test solution specifically comprises the following steps:

[0022] Measure 10 mL of Jingli and add methanol to make the volume to 20 mL, shake well, filter, and take the filtrate to obtain the test solution.

[0023] As a further preferred embodiment, the concentrations of the reference solutions are as follows:

[0024] The concentrations of the reference solutions prepared with furfural, cryptochlorogenic acid, vecin-2, schaftoside, vecin-3, vitexin, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid as reference substances were 59.58 µg / mL, 19.88 µg / mL, 58.62 µg / mL, 150.35 µg / mL, 12.23 µg / mL, 57.59 µg / mL, 74.88 µg / mL, 37.29 µg / mL, and 79.78 µg / mL, respectively.

[0025] As a further preferred embodiment, among the 12 common peaks of the above fingerprint, peak 1 is furfural, peak 4 is cryptochlorogenic acid, peak 5 is vecin-2, peak 6 is schaftoside, peak 8 is vecin-3, peak 9 is vitexin, peak 10 is 3,4-O-dicaffeoylquinic acid, peak 11 is 3,5-O-dicaffeoylquinic acid, and peak 12 is 4,5-O-dicaffeoylquinic acid.

[0026] In the second aspect, the present invention provides the application of the Jingli fingerprint map constructed by the above-mentioned construction method in distinguishing Jingli products prepared by different processing techniques.

[0027] As a further preferred embodiment, the fingerprints of the jingli samples prepared by different processing techniques are compared with the jingli fingerprint;

[0028] If the fingerprint of the Jingli sample shows 12 peaks corresponding to the Jingli fingerprint, and the similarity is greater than 0.85, it is determined that the processing technology of the Jingli sample is direct-fired processing;

[0029] If the fingerprint spectrum of the Jingli sample does not show the 12 peaks corresponding to the Jingli fingerprint spectrum, and there is any missing peak, it is determined that the processing technology of the Jingli sample is a non-direct fire processing product.

[0030] As a further preferred embodiment, the above-mentioned non-direct fire artillery products include dry distillation products or water-boiled products.

[0031] In a third aspect, the present invention provides a Jingli fingerprint map constructed by the above-mentioned construction method.

[0032] The beneficial effects of the present invention are:

[0033] The present invention provides a method for constructing a Jingli fingerprint spectrum, by which Jingli with different processing techniques can be effectively identified and distinguished, providing an effective method for the quality control and supervision of Jingli products. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0035] Figure 1 It is the Jingli fingerprint provided in Example 1 of the present application;

[0036] Figure 2 It is an overlay of 22 batches of direct-fire process samples provided in Example 1 of the present application;

[0037] Figure 3 This is the spectrum of the direct-fire process test product (batch number: Y20240817) provided in Example 1 of the present application;

[0038] Figure 4 This is the atlas of the water boiling process test product (batch number: Y20241101) provided in Example 1 of the present application;

[0039] Figure 5 This is the spectrum of the dry distillation process sample (batch number: Y20240501) provided in Example 1 of the present application;

[0040] Figure 6 It is the 9 mixed reference sample spectra provided in Example 1 of the present application;

[0041] Figure 7 It is the furfural reference substance spectrum provided in Example 1 of the present application;

[0042] Figure 8 It is the spectrum of the cryptochlorogenic acid reference substance provided in Example 1 of the present application;

[0043] Fig. 9 It is the spectrum of the reference substance of Weicaining-2 provided in Example 1 of the present application;

[0044] Fig.10 It is the schaftoside reference substance spectrum provided in Example 1 of the present application;

[0045] Fig.11 It is the spectrum of the reference substance of Weicaining-3 provided in Example 1 of the present application;

[0046] Fig.12 It is the spectrum of the vitexin reference substance provided in Example 1 of the present application;

[0047] Fig.13 It is the 3,4-O-dicaffeoylquinic acid reference substance spectrum provided in Example 1 of the present application;

[0048] Fig.14It is the 3,5-O-dicaffeoylquinic acid reference substance spectrum provided in Example 1 of the present application;

[0049] Fig.15 It is the 4,5-O-dicaffeoylquinic acid reference substance spectrum provided in Example 1 of the present application;

[0050] Fig.16 This is a different preparation process diagram for determination by changing the mobile phase gradient provided in Comparative Example 1 of the present application;

[0051] Fig.17 This is a different preparation process diagram for determination of changing the mobile phase composition provided in Comparative Example 2 of the present application;

[0052] Fig.18 This is a graph of different processing techniques measured by a single wavelength provided in Comparative Example 3 of the present application;

[0053] Fig.19 It is the different processing process spectra determined by GC-MS method provided in Comparative Example 4 of the present application; DETAILED DESCRIPTION

[0054] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0055] The instruments, reagents and reference substances used in the specific embodiments of the present invention are as follows:

[0056] Instruments and equipment: Ultimate 3000 high performance liquid chromatograph, DAD detector; Sartorius Secura125-1CN electronic balance (one hundred thousandth balance); similarity calculation software is "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (V2.0), (issued by the National Pharmacopoeia Committee).

[0057] Reagents: Acetonitrile and phosphoric acid were of chromatographic grade, and water was purified water prepared by Milli-Q; other reagents were of analytical grade.

[0058] Standard: Furfural (Shanghai MacLean Biochemical Technology Co., Ltd., batch number: C15583883, content is 99.5%)

[0059] Chlorogenic acid reference substance (Sichuan Jingcui Tiancheng Pharmaceutical Technology Co., Ltd., batch number: TC0388-231125, content is 98.0%)

[0060] Weicaining-2 reference substance (Sichuan Jingcui Tiancheng Pharmaceutical Technology Co., Ltd., batch number: TC2090-230129, content is 98.0%)

[0061] Schaftoside reference substance (China Food and Drug Administration, batch number: 111912-202204, content is 94.9%)

[0062] Weicaining-3 reference substance (Shanghai Shidande Standard Technology Service Co., Ltd., batch number: 11060-G241001, content is 98.0%)

[0063] Vitexin reference substance (CERTIFICATE OF ANALYSIS, batch number: 24Y228-D5, content is 99.04%)

[0064] 3,4-O-dicaffeoylquinic acid reference substance (Chem Faces, batch number: CFS202302, content is 98.0%)

[0065] 3,5-O-dicaffeoylquinic acid reference substance (Chem Faces, batch number: CFS202302, content is 99.0%)

[0066] 4,5-O-dicaffeoylquinic acid reference substance (China Food and Drug Administration, batch number: 111894-202205, content is 94.9%)

[0067] Example 1

[0068] A method for constructing a Jingli fingerprint map comprises the following steps:

[0069] (1) Preparation of test solution: Take 10 mL of the jingli prepared by different processes, place it in a 20 mL volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate to obtain the solution;

[0070] (2) Preparation of reference solution: Take furfural, cryptochlorogenic acid, vecin-2, schaftoside, vecin-3, vitexin, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid as reference substances and place them in a 100 mL volumetric flask, add methanol to dissolve, and make a constant volume to make a solution containing 59.58 µg, 19.88 µg, 58.62 µg, 150.35 µg, 12.23 µg, 57.59 µg, 74.88 µg, 37.29 µg, and 79.78 µg per 1 mL, respectively;

[0071] (3) Inject the test solution and the reference solution into a high performance liquid chromatograph for measurement to obtain the chromatograms of the test sample and the reference solution, respectively;

[0072] The chromatographic conditions of the HPLC include:

[0073] Chromatographic column: Shiseido CAPCELL PAK C18 column, 25cm×4.6mm×5µm;

[0074] Inject 5 µL;

[0075] The column temperature was 30°C;

[0076] Flow rate: 1.0 ml per minute

[0077] Acetonitrile was used as mobile phase A, and 0.1% phosphoric acid was used as mobile phase B;

[0078] The gradient elution conditions are shown in Table 1 , and the detection wavelength conditions are shown in Table 2 ;

[0079] Table 1 Gradient elution table

[0080]

[0081] Table 2 Detection wavelength switching table

[0082]

[0083] (4) Generate a reference fingerprint (Jingli fingerprint)

[0084] The 2012 version of the "Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System" was used, with a time window width of 0.1, and through multi-point correction and Mark peak matching, a fingerprint spectrum common pattern diagram containing 12 common peaks was obtained (i.e., the Jingli product processed by direct fire). The spectrum is as follows Figure 1 As shown, among the 12 common peaks, peak 1: furfural, peak 4: cryptochlorogenic acid (4-O-caffeoylquinic acid), peak 5: vecin-2, peak 6: shafota glycoside, peak 8: vecin-3, peak 9: vitexin (vitex glycoside), peak 10: cryptochlorogenic acid B (3,4-O-dicaffeoylquinic acid), peak 11: 3,5-O-dicaffeoylquinic acid, peak 12: 4,5-O-dicaffeoylquinic acid. Its Jingli fingerprint is as follows Figure 1 As shown; 22 batches of direct fire process test samples are shown Figure 2 As shown; 9 mixed reference samples are shown Figure 6 As shown; the spectra of furfural, cryptochlorogenic acid, vecin-2, schaftoside, vecin-3, vitexin, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid reference substances are shown respectively Figure 7-Figure 15 shown.

[0085] The method for identifying the Jingli products with different processing techniques by using the above fingerprint spectrum specifically comprises the following steps:

[0086] (1) Take 10 mL of the Jingli samples prepared under different processing methods, place them in a 20 mL volumetric flask, add methanol to the scale, shake well, filter, and take the filtrate to obtain the Jingli samples with different processing methods;

[0087] (2) injecting the samples of the Chinese calamus prepared in step (1) into a high performance liquid chromatograph (the chromatographic conditions are the same as those described above) for measurement, and obtaining spectra of the samples of the Chinese calamus prepared by different processing techniques;

[0088] (3) Perform similarity analysis on the Jingli sample spectrum obtained in step (3) and the Jingli fingerprint spectrum, and identify and distinguish products with different preparation processes based on the similarity analysis results. The Jingli samples prepared by non-traditional direct fire processing (boiling and dry distillation) processes were accurately distinguished, and the results are as follows: Figure 3-Figure 5 As shown, Figure 3 It is a direct fire processing sample. Figure 4 For the boiling process samples; Figure 5 This is a sample of the dry distillation process.

[0089] Comparative Example 1

[0090] The method for identifying the Jingli products with different processing techniques has the same operating steps as Example 1, except that the mobile phase A is changed to acetonitrile: 0.1% phosphoric acid = 45:55 for determination.

[0091] This method cannot construct the fingerprint of Jingli. Fig.16 As shown, no distinction can be made.

[0092] Comparative Example 2

[0093] The method for identifying the products of jingli with different processing techniques is similar to that of Example 1, except that the mobile phase A, acetonitrile, is replaced with methanol for determination:

[0094] Methanol was used as mobile phase A, and 0.1% phosphoric acid was used as mobile phase B;

[0095] The gradient elution conditions are shown in Table 3;

[0096] Table 3 Gradient elution table

[0097]

[0098] This method cannot construct the fingerprint of Jingli. Fig.17 As shown, no distinction can be made.

[0099] Comparative Example 3

[0100] The method for identifying Jingli products with different processing techniques has the same operating steps as Example 1, with the only difference being that the wavelength is not switched at 13 minutes and a single 280nm wavelength is used for measurement.

[0101] This method cannot construct the fingerprint of Jingli. Fig.18 As shown, no distinction can be made.

[0102] Comparative Example 4

[0103] The method for identifying the Jingli products with different processing techniques by using different determination methods (GC-MS method) specifically includes the following steps:

[0104] Chromatographic column: DB-5MS, 30 m × 0.25 mm × 0.25 μm;

[0105] Inject 1 µL, splitless;

[0106] Inlet temperature: 250°C;

[0107] The specific temperature program was as follows: 60°C for 0.5 min, then 5°C / min to 110°C, then 2°C / min to 230°C for 5 min, and then 280°C for 5 min.

[0108] Except for the above special instructions, other conditions of this embodiment are the same as those of Embodiment 1.

[0109] This method cannot construct the fingerprint of Jingli. Fig.19 As shown, no distinction can be made.

[0110] The embodiments of the present application are described above in conjunction with the accompanying drawings. Specific examples are used herein to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the core idea of ​​the present application, but the present application is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the inspiration of the present application, ordinary technicians in this field can also make many forms without departing from the scope of protection of the purpose of the present application and the claims, all of which are within the protection of the present application.

Claims

1. A method for constructing a fingerprint of Lepidium japonicum, characterized in that: The following steps are involved: S1. Use multiple batches of direct-fired jingli as samples and methanol as solvent to prepare the test solution; S2. Select a reference substance and prepare a reference solution using methanol as solvent; S3, respectively injecting the test solution and the reference solution into a high performance liquid chromatograph for measurement to obtain a sample chromatogram and a reference chromatogram, respectively; S4, selecting the sample chromatogram, using the Chinese medicine chromatographic fingerprint similarity evaluation system, through multi-point correction and Mark peak matching, to obtain a fingerprint common pattern diagram containing 12 common peaks, that is, to construct a Jingli fingerprint; The reference substance is one or more of furfural, cryptochlorogenic acid, vecinin-2, schaftoside, vecinin-3, vitexin, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid; The chromatographic conditions of the high performance liquid chromatograph include the following: Acetonitrile was used as mobile phase A, and 0.1% phosphoric acid was used as mobile phase B; The detection wavelength is: 280nm for 0min-13min and 320nm for 13min-50min. The chromatographic column was: Shiseido CAPCELL PAK C18, 25 cm × 4.6 mm × 5 µm; The gradient elution conditions are shown in Table 1: Table 1 The injection volume was 5 µL; The column temperature was 30°C; Flow rate: 1.0 mL / min; The retention times of the 12 common peaks in the Jingli fingerprint spectrum are as follows: Peak 1: 11.27min, peak 2: 15.23min, peak 3: 16.79min, peak 4: 17.36min, peak 5: 21.31min, peak 6: 25.39min, peak 7: 27.20min, peak 8: 27.97min, peak 9: 30.59min, peak 10: 36.12min, peak 11: 37.54min, peak 12: 39.41min.

2. The construction method according to claim 1, characterized in that: The specific steps of using multiple batches of jujube leaves processed by direct fire as samples and methanol as solvent to prepare the test solution include: Measure 10 mL of Jingli and add methanol to make the volume to 20 mL, shake well, filter, and take the filtrate to obtain the test solution.

3. The construction method according to claim 1, characterized in that: The concentrations of the reference solutions are as follows: The concentrations of the reference solutions prepared with furfural, cryptochlorogenic acid, vecin-2, schaftoside, vecin-3, vitexin, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid as reference substances were 59.58 µg / mL, 19.88 µg / mL, 58.62 µg / mL, 150.35 µg / mL, 12.23 µg / mL, 57.59 µg / mL, 74.88 µg / mL, 37.29 µg / mL, and 79.78 µg / mL, respectively.

4. The construction method according to claim 1, characterized in that: Among the 12 common peaks in the fingerprint, peak 1 is furfural, peak 4 is cryptochlorogenic acid, peak 5 is vecin-2, peak 6 is schaftoside, peak 8 is vecin-3, peak 9 is vitexin, peak 10 is 3,4-O-dicaffeoylquinic acid, peak 11 is 3,5-O-dicaffeoylquinic acid, and peak 12 is 4,5-O-dicaffeoylquinic acid.

5. An application of a Jingli fingerprint constructed by the construction method described in any one of claims 1 to 4 in distinguishing Jingli products prepared by different processing techniques.

6. The use according to claim 5, characterized in that: The fingerprints of the samples of jingli prepared by different processing techniques are compared with the jingli fingerprint; If the fingerprint of the Jingli sample shows 12 peaks corresponding to the Jingli fingerprint, and the similarity is greater than 0.85, it is determined that the processing technology of the Jingli sample is direct-fired processing; If the fingerprint spectrum of the Jingli sample does not show the 12 peaks corresponding to the Jingli fingerprint spectrum, and there is any missing peak, it is determined that the processing technology of the Jingli sample is a non-direct fire processing product.

7. The use according to claim 6, characterized in that: The non-direct fire artillery products include dry distillation products or water boiling products.

Images