Establishment method of mulberry paste fingerprint spectrum and fingerprint spectrum

Abstract

The invention relates to the technical field of traditional Chinese medicine analysis, in particular to an establishment method of a mulberry paste fingerprint spectrum and the fingerprint spectrum. The establishment method of the mulberry paste fingerprint spectrum comprises the following steps: preparing a test solution by taking mulberry paste as a raw material, and respectively preparing reference solutions by taking chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, 5-hydroxymethylfurfural, rutin and isoquercitrin; respectively injecting the test solution and the reference solution into an ultra-high performance liquid chromatograph for determination, recording chromatograms, collecting the chromatograms of the test solutions of multiple batches of mulberry paste, introducing the chromatograms into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, generating a reference map, and calculating the similarity by taking the peak area and retention time as parameters. The method can be used for simultaneously detecting a plurality of components in the mulberry paste, and can be effectively used for evaluating the product quality stability of the mulberry paste.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine analysis, and particularly relates to a method for establishing a fingerprint of mulberry paste and the fingerprint. Background Art

[0002] Mulberry paste is a semi-fluid medicinal plaster prepared by decocting and concentrating mulberries as raw materials. Mulberry paste has the effects of tonifying the liver and kidney, and nourishing essence and blood, and is used for emaciation, weakness of the waist and knees, night sweats, dizziness, thirst and dry throat caused by deficiency of liver and kidney essence and blood. Mulberry paste contains many components such as organic acids, sugars and flavonoids. The origin of the medicinal materials, planting environment, production batches of the medicinal materials, processing technology, etc. may all affect the quality stability of mulberry paste, and ultimately affect the curative effect of the medicinal plaster.

[0003] At present, the pharmacopoeia standard WS3-B-1828-94-2018 of mulberry paste only uses density inspection and thin-layer chromatography for identification. These methods are difficult to characterize the various components of mulberry paste and cannot effectively evaluate the quality stability of mulberry paste. Based on this, it is very necessary to propose a method that can detect the contents of various components in mulberry paste to control the product quality stability of mulberry paste. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a method for establishing a fingerprint of mulberry paste and the fingerprint, which can simultaneously detect multiple components in mulberry paste and can be effectively used to evaluate the product quality stability of mulberry paste.

[0005] In order to achieve the above purpose, the present invention adopts the following technical solutions: The present invention provides a method for establishing a fingerprint of mulberry paste, comprising the following steps: S1. Taking mulberry paste as the raw material to prepare a test solution, and taking chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, 5-hydroxymethylfurfural, rutin and isoquercitrin to prepare reference substance solutions respectively; S2. Injecting the test solution and the reference substance solutions into an ultra-high performance liquid chromatograph for determination respectively, and recording the chromatogram; wherein, the chromatographic conditions adopted by the ultra-high performance liquid chromatograph are: chromatographic column: Waters ACQUITY UPLC HSS T3; mobile phase: acetonitrile-tetrahydrofuran in a ratio of 80-90:10-20 as mobile phase A, and 0.1%-0.3% formic acid water as mobile phase B, with a gradient elution program; S3. Collecting the chromatograms of the test solutions of multiple batches of mulberry paste, importing them into the similarity evaluation system for traditional Chinese medicine chromatographic fingerprints, generating a reference chromatogram, and calculating the similarity with peak area and retention time as parameters.

[0006] Further, in S2, the mobile phase: acetonitrile-tetrahydrofuran in a ratio of 80:20 is used as mobile phase A, and 0.2% formic acid water is used as mobile phase B.

[0007] Further, the gradient elution procedure is as follows: .

[0009] Furthermore, the chromatographic conditions described in S2 also include: detection wavelength 300 nm, flow rate 0.2 mL / min, column temperature 35° C., and injection volume 1 μL.

[0010] Furthermore, the preparation method of the test solution described in S1 is: take 5 g of mulberry paste, put it in a stoppered conical flask, add 25 mL of methanol, and ultrasonically treat it for 30 minutes. The ultrasonic frequency used is 40 kHz and the ultrasonic power is 250 W. Cool, shake well, filter, and take the filtrate as the test solution.

[0011] Furthermore, the preparation method of the reference solution described in S1 is: take appropriate amounts of chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, 5-hydroxymethylfurfural, rutin, and isoquercetin, and add methanol to prepare a solution containing 20 μg per 1 mL as the reference solution.

[0012] Furthermore, the mulberry paste described in S1 is prepared by the following method: (1) Extraction: Take mulberry fruit, add 4 to 6 times the amount of water, heat and boil for 1.5 to 2.5 hours, filter while hot after the first decoction, add 4 to 6 times the amount of water to the residue and decoct it again in the same way, combine the two decoctions, and centrifuge at high speed for later use; (2) Concentration: The extract after centrifugation is concentrated by vacuum concentration to obtain a clear paste with a relative density of about 1.30 to 1.33. The clear paste is centrifuged at high speed while hot and set aside; (3) Sugar refining: Take 30% to 40% of the weight of mulberry medicinal materials sucrose, add half the amount of water, heat and boil for half an hour, filter the syrup while it is hot and set aside; (4) Collecting the paste: Mix the clear paste after centrifugation with refined sugar to obtain the paste, stir for 15 to 20 minutes, and mix well.

[0013] Furthermore, the mulberry paste described in S3 was collected in 18 batches.

[0014] Furthermore, the fingerprint spectrum has 11 fingerprint peaks, and the retention times are 4.884min, 5.848min, 9.044min, 9.565min, 12.654min, 13.439min, 14.558min, 16.005min, 16.703min, 17.238min and 21.400min, respectively.

[0015] The present invention also provides a fingerprint of mulberry paste constructed by the above method.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: The method for establishing the fingerprint of mulberry paste provided by this application uses chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, 5-hydroxymethylfurfural, rutin and isoquercitrin to prepare a reference substance solution. Using 18 batches of different mulberry pastes to prepare a test solution, by optimizing the chromatographic conditions, a standard fingerprint of mulberry paste is obtained. The established fingerprint has 11 fingerprint peaks, which can monitor various components of mulberry paste and can be effectively used to evaluate the quality stability of mulberry paste products. Description of the Drawings

[0017] Figure 1 Chromatograms of the test solution (Y) and different reference substance solutions (A - F); in the figure, A is chlorogenic acid, B is neochlorogenic acid, C is cryptochlorogenic acid, D is 5-hydroxymethylfurfural, E is rutin, and F is isoquercitrin; in the chromatogram of the test solution, peak 1 is 5-hydroxymethylfurfural, peak 2 is neochlorogenic acid, peak 3 is chlorogenic acid, peak 4 is cryptochlorogenic acid, peak 5 is rutin, and peak 6 is isoquercitrin; Figure 2 Chromatograms of the test solution at different flow rates; Figure 3 Chromatograms of the test solution with different mobile phases; Figure 4 Fingerprints of different batches of mulberry paste. Detailed Embodiments

[0018] Next, in combination with the embodiments of the present invention, the technical solutions of the present invention will be clearly and completely described. The described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0019] Unless otherwise specified, the methods are all conventional methods, and the raw materials can all be obtained from public commercial channels unless otherwise specified.

[0020] Example 1: Establishment of Chromatographic Conditions 1. Instruments and Reagents 1.1 Instruments: Ultra-high performance liquid chromatograph Waters H-Class (Waters Corporation, USA); Analytical balances EG204, ME204, XSR105 (Mettler Toledo Instruments Co., Ltd., Switzerland); Electric water bath HWS-26 (Shanghai Yiheng Scientific Instrument Co., Ltd.); Electrothermal forced air drying oven BO-120FL (Shanghai Yiheng Scientific Instrument Co., Ltd.); Numerical control ultrasonic cleaner KQ-250DE (Kunshan Ultrasonic Instruments Co., Ltd.); Pure water instrument MILLI-Q-7005 (Merck Life Science Technology Co., Ltd.).

[0021] 1.2 Reagents: Acetonitrile and methanol are both chromatographically pure and purchased from Merck Life Science Technology Co., Ltd.; formic acid and tetrahydrofuran are both chromatographically pure and purchased from Shanghai Macklin Biochemical Technology Co., Ltd.; pure water is prepared in the laboratory.

[0022] 1.3 Reference substance information: Chlorogenic acid (batch number: YRL0098200419, purchased from Baoji Yirui Biotechnology Co., Ltd.); neochlorogenic acid (batch number: CR - 069200117, purchased from Baoji Yirui Biotechnology Co., Ltd.); cryptochlorogenic acid (batch number: YRY0026210420, purchased from Baoji Yirui Biotechnology Co., Ltd.); isoquercitrin (batch number: 111809 - 201804, purchased from National Institutes for Food and Drug Control); 5 - hydroxymethylfurfural (batch number: 111626 - 201912, purchased from National Institutes for Food and Drug Control); rutin (batch number: 100080 - 202012, purchased from National Institutes for Food and Drug Control).

[0023] 1.4 The origin and batch number information of the mulberry medicinal materials are shown in Table 1 below: Table 1: Information Table of Mulberry Medicinal Materials

[0024] 2. Preparation of solutions 2.1 Preparation of test solution Take 5 g of mulberry paste, place it in a stoppered triangular flask, add 25 mL of methanol, ultrasonically treat for 30 min, the ultrasonic frequency used is 40 kHz, the ultrasonic power is 250 W, let it cool, shake well, filter, and take the subsequent filtrate as the test solution.

[0025] Among them, the preparation method of the mulberry paste is as follows: 1) Extraction: Take 500 g of mulberry medicinal materials, add 2.5 L of water, heat to boiling, start timing after boiling, extract for 2 h, record the liquid level height of the extract, add water every half hour to supplement the liquid level height difference, after the first decoction, filter while it is hot with a 200 - mesh non - woven filter cloth, add five times the amount of water to the filter residue and decoct in the same way for the second time, combine the two water decoctions, and centrifuge at high speed for standby.

[0026] 2) Concentration: Transfer the centrifuged extract to a rotary evaporator (parameter settings: water bath temperature 75 °C, pressure - 0.1 MPa, rotation speed 100 rpm), concentrate to obtain a clear paste with a relative density of about 1.30 - 1.33, and after high - speed centrifugation of the clear paste while it is hot, set aside for standby.

[0027] 3) Sugar refining: Take 150 g of sucrose, add half the amount of water, heat to boiling for half an hour, filter the syrup while it is hot with a 200 - mesh non - woven filter cloth, and set aside for standby.

[0028] 4) Concentrating the extract: Mix the centrifuged clear extract and refined sugar filtered through a 200-mesh filter cloth for concentrating the extract, stir for 15 - 20 min, and obtain the product after uniform mixing.

[0029] 2.3 Preparation of reference substance solution Weigh appropriate amounts of chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, 5-hydroxymethylfurfural, rutin, and isoquercitrin, and dissolve them in methanol to prepare solutions containing 20 μg per 1 mL as reference substance solutions.

[0030] 3. Chromatographic conditions Chromatographic column: Waters ACQUITY UPLC HSS T3 (1.8 μm, 2.1 mm * 100 mm); Mobile phase: Acetonitrile - tetrahydrofuran (v:v = 80:20) as mobile phase A, 0.2% formic acid in water as mobile phase B; Gradient elution conditions are shown in Table 2; Detection wavelength: 300 nm; Flow rate: 0.2 mL / min, Column temperature: 35 °C; Injection volume: 1 μL.

[0031] Table 2 Gradient elution conditions

[0032] Precisely pipette 1 μL each of the reference substance solution and the test solution, inject them into an ultra-high performance liquid chromatograph, and perform injection and determination according to the chromatographic conditions in 3., and record the chromatogram; The chromatograms of the test solution and different reference substance solutions (A - F) are as Figure 1 shown, where Y is the test solution, A is the chlorogenic acid reference substance solution, B is the neochlorogenic acid reference substance solution, C is the cryptochlorogenic acid reference substance solution, D is the 5-hydroxymethylfurfural reference substance solution, E is the rutin reference substance solution, and F is the isoquercitrin reference substance solution; in the test solution, peak 1 is 5-hydroxymethylfurfural, peak 2 is neochlorogenic acid, peak 3 is chlorogenic acid, peak 4 is cryptochlorogenic acid, peak 5 is rutin, and peak 6 is isoquercitrin.

[0033] 4. Flow rate optimization Flow rates of 0.15 mL / min, 0.2 mL / min, and 0.3 mL / min were respectively used, and the results are as Figure 2 shown. The results show that when the flow rate is 0.15 mL / min, the peak elution time is delayed, and the separation effect of two peaks is poor (as indicated by the arrow); when the flow rate is 0.3 mL / min, the peak elution time is advanced, but the separation effect of two peaks is poor (as indicated by the arrow); when the flow rate is 0.2 mL / min, the separation effect of each peak is good, so a flow rate of 0.2 mL / min is adopted.

[0034] 5. Mobile phase optimization According to the chromatographic conditions in 3., using acetonitrile as mobile phase A and 0.2% formic acid in water as mobile phase B as the mobile phase, the results are asFigure 3 As shown in A, using the mobile phase system of the present application (acetonitrile - tetrahydrofuran (v:v = 80:20) as mobile phase A, 0.2% formic acid water as mobile phase B), the results are as follows Figure 3 As shown in B, the results indicate that for the chromatogram with acetonitrile as mobile phase A and 0.2% formic acid water as mobile phase B, the separation effect of the two peaks is poor (as indicated by the arrow position), and the number of chromatographic peaks is small. With the mobile phase of the present application, the separation of each peak is good and the number of chromatographic peaks is large.

[0035] Example 2: Fingerprint and similarity determination method of mulberry paste Precisely pipette 1 μL each of the reference substance solution and the test sample solution, and inject them into the ultra - high performance liquid chromatograph respectively. Inject samples for determination according to the chromatographic conditions in Example 1. Record the chromatogram. Import the fingerprint data of 18 batches of mulberry paste into the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 version) to generate a reference chromatogram (R). There are 11 common peaks in total, and their retention times are 4.884 min, 5.848 min, 9.044 min, 9.565 min, 12.654 min, 13.439 min, 14.558 min, 16.005 min, 16.703 min, 17.238 min, and 21.400 min respectively. The fingerprint chromatograms of different batches of mulberry paste are as follows Figure 4 shown.

[0036] Taking the peak area and retention time as parameters, calculate the similarity between different batches of mulberry paste. The similarity test results are shown in Table 4 below: Table 4: Similarity table of fingerprint chromatograms of different batches of mulberry paste

[0037] From the calculation results of the similarity of 18 batches of fingerprint chromatograms, it is found that the similarity between the mulberry paste prepared from different batches of mulberry medicinal materials from Xinjiang Uygur Autonomous Region (S - 1 to S - 7) and the reference chromatogram (R) is greater than 0.9, indicating that there is no obvious difference in the characteristic chromatograms of the mulberry paste prepared from the mulberry medicinal materials of batches S - 1 to S - 7 in Xinjiang Uygur Autonomous Region. While the origins of S - 10 to S - 17 are Xinjiang Uygur Autonomous Region, Sichuan, Hebei, Jiangsu, Anhui, etc. The calculated similarity results of S - 10 to S - 17 and R range from 0.6 to 0.9. Among them, the similarity results between S - 13 from Fuyang, Anhui and the characteristic chromatograms of S - 1 to S - 10 from Turpan, Xinjiang Uygur Autonomous Region range from 0.6 to 0.9. The similarity between S - 13 from Fuyang, Anhui and the characteristic chromatograms of mulberry from S - 16 and S - 17 in Cangzhou, Hebei is 0.936 and 0.946 respectively. In summary, the characteristic chromatograms of mulberry paste prepared from mulberry medicinal materials of different origins and different batches are significantly different, and the method of the present application can be used to evaluate the quality stability of mulberry paste.

[0038] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application and are not intended to limit them. Although the present application has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that modifications can still be made to the specific implementation manners of the present application or equivalent replacements can be made to some technical features, and all of them should be covered within the scope of the technical solutions claimed in the present application.

Claims

1. A method for establishing a fingerprint of mulberry paste, characterized in that It includes the following steps: S1. Take mulberry paste as the raw material to prepare the test solution, and take chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, 5-hydroxymethylfurfural, rutin and isoquercitrin respectively to prepare the reference substance solutions; S2. Inject the test solution and the reference substance solutions into an ultra-high performance liquid chromatograph for determination respectively, and record the chromatograms; among them, the chromatographic conditions adopted by the ultra-high performance liquid chromatograph are as follows: chromatographic column: Waters ACQUITY UPLC HSS T3; mobile phase: acetonitrile-tetrahydrofuran in a ratio of 80-90:10-20 is mobile phase A, and 0.1%-0.3% formic acid water is mobile phase B, with a gradient elution program; S3. Collect the chromatograms of the test solutions of multiple batches of mulberry paste, import them into the similarity evaluation system for traditional Chinese medicine chromatographic fingerprints, generate a reference chromatogram, and calculate the similarity with peak area and retention time as parameters.

2. The method for establishing a fingerprint spectrum of mulberry paste according to claim 1, characterized in that, In S2, the mobile phase: acetonitrile-tetrahydrofuran in a ratio of 80:20 is mobile phase A, and 0.2% formic acid water is mobile phase B.

3. The method for establishing the fingerprint spectrum of mulberry paste according to claim 1 or 2, characterized in that, The gradient elution program is as follows: 。 4. The method for establishing a fingerprint of mulberry paste according to claim 1, characterized in that, The chromatographic conditions in S2 also include: detection wavelength 300 nm, flow rate 0.2 mL / min, column temperature 35 °C, injection volume 1 μL.

5. The method for establishing a fingerprint spectrum of mulberry paste according to claim 1, wherein The preparation method of the test solution in S1 is: take 5 g of mulberry paste, place it in a stoppered triangular flask, add 25 mL of methanol, ultrasonically treat for 30 min, the ultrasonic frequency used is 40 kHz, the ultrasonic power is 250 W, let it cool, shake well, filter, and take the subsequent filtrate as the test solution.

6. The method for establishing the fingerprint spectrum of mulberry paste according to claim 1, characterized in that, The preparation method of the reference substance solutions in S1 is: take appropriate amounts of chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, 5-hydroxymethylfurfural, rutin, and isoquercitrin, and add methanol to make solutions containing 20 μg per 1 mL respectively as the reference substance solutions.

7. The method for establishing the fingerprint of mulberry paste according to claim 1, characterized in that, The mulberry paste in S1 is prepared by the following method: (1) Extraction: Take mulberry medicinal materials, add 4-6 times the amount of water, heat to boiling and extract for 1.5-2.5 h. Filter while it is hot after the first decoction. Add 4-6 times the amount of water to the filter residue and decoct in the same way for the second time. Combine the two water decoctions and centrifuge at high speed for standby; (2) Concentration: Vacuum concentrate the centrifuged extract to obtain a clear paste with a relative density of about 1.30-1.

33. After centrifuging the clear paste at high speed while it is hot, set it aside for standby; (3) Sugar refining: Take 30%-40% of the weight of the mulberry medicinal materials as sucrose, add half the amount of water of the sucrose, heat to boiling for half an hour, filter the syrup while it is hot, and set it aside for standby; (4) Paste collection: Mix and collect the centrifuged clear paste and the refined sugar, stir for 15 min-20 min, and mix evenly to obtain.

8. The method for establishing the fingerprint spectrum of mulberry paste according to claim 1, characterized in that, The collection batches of the mulberry paste in S3 are 18 batches.

9. The method for establishing a fingerprint spectrum of mulberry paste according to claim 1, characterized in that, The fingerprint chromatogram has 11 fingerprint peaks, and their retention times are 4.884 min, 5.848 min, 9.044 min, 9.565 min, 12.654 min, 13.439 min, 14.558 min, 16.005 min, 16.703 min, 17.238 min and 21.400 min respectively.

10. The fingerprint chromatogram of the mulberry paste constructed by the method described in any one of claims 1-9.

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