A method for establishing a mulberry fruit paste fingerprint and the fingerprint

Abstract

The present application relates to the technical field of traditional Chinese medicine analysis, and particularly relates to a mulberry fruit paste fingerprint spectrum establishment method and fingerprint spectrum. The mulberry fruit paste fingerprint spectrum establishment method comprises the following steps: taking mulberry fruit paste as raw material to prepare a test sample solution, taking chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, 5-hydroxymethyl furfural, rutin and isoquercitrin to prepare reference sample solutions; injecting the test sample solution and the reference sample solutions into an ultra-high performance liquid chromatograph for determination, recording chromatograms, collecting chromatograms of test sample solutions of multiple batches of mulberry fruit paste, importing a traditional Chinese medicine chromatographic fingerprint spectrum similarity evaluation system, generating a control spectrum, and taking peak area and retention time as parameters to calculate similarity. The method can simultaneously detect multiple components in mulberry fruit paste, and can be effectively used to evaluate product quality stability of mulberry fruit paste.

Description

TECHNICAL FIELD

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

[0002] Mulberry fruit paste is a semi-liquid paste prepared from mulberry fruit as raw material through decoction and concentration. The mulberry fruit paste has the effects of tonifying liver and kidney and nourishing essence and blood, and is used for treating body weight loss, soreness of waist and knees, night sweat, dizziness and blurred vision, and dry mouth and throat caused by deficiency of liver and kidney essence and blood. The mulberry fruit paste contains many components such as organic acids, sugars and flavonoids, and the quality stability of the mulberry fruit paste may be affected by the production place, planting environment, production batch and processing technology of the medicinal material, and finally the curative effect of the paste is affected.

[0003] At present, the pharmacopoeia standard WS3-B-1828-94-2018 of the mulberry fruit paste only adopts density inspection and thin layer chromatography for identification, and these methods are difficult to characterize the multiple components of the mulberry fruit paste, and cannot effectively evaluate the quality stability of the mulberry fruit paste. Therefore, it is necessary to provide a method for detecting the content of multiple components in the mulberry fruit paste to control the product quality stability of the mulberry fruit paste. SUMMARY

[0004] In view of the deficiencies of the prior art, the purpose of the present application is to provide a method for establishing a mulberry fruit paste fingerprint and the fingerprint, which can detect multiple components in the mulberry fruit paste at the same time and can be effectively used for evaluating the product quality stability of the mulberry fruit paste.

[0005] In order to achieve the above purpose, the present application adopts the following technical solutions:

[0006] The present application provides a method for establishing a mulberry fruit paste fingerprint, comprising the following steps:

[0007] S1, taking the mulberry fruit paste as a raw material to prepare a test solution, and taking chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, 5-hydroxymethylfurfural, rutin and isoquercitrin to prepare a control solution, respectively;

[0008] S2, injecting the test solution and the control solution into an ultra-high performance liquid chromatograph for determination, and recording the chromatogram; wherein the chromatographic conditions of the ultra-high performance liquid chromatograph are as follows: a chromatographic column: Waters ACQUITY UPLC HSS T3; a mobile phase: acetonitrile-tetrahydrofuran as a mobile phase A in a gradient elution program according to 80-90:10-20, and 0.1%-0.3% formic acid water as a mobile phase B;

[0009] S3, collecting the chromatograms of the test solution of multiple batches of mulberry fruit paste, importing a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, generating a control chromatogram, taking the peak area and the retention time as parameters, and calculating the similarity.

[0010] Further, the mobile phase in S2: acetonitrile-tetrahydrofuran is 80:20 as mobile phase A, and 0.2% formic acid water as mobile phase B.

[0011] Further, the gradient elution program is as follows:

[0012]

[0013] Further, the chromatographic conditions of S2 also include: detection wavelength 300 nm, flow rate 0.2 mL / min, column temperature 35°C, injection volume 1 μL.

[0014] Further, the preparation method of the test solution of S1 is as follows: take mulberry fruit paste 5 g, put it in a stoppered triangular flask, add methanol 25 mL, ultrasonic treatment for 30 min, 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.

[0015] Further, the preparation method of the control solution of S1 is as follows: take chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, 5-hydroxymethyl glycoside, rutin, and isoquercitrin in appropriate amounts, respectively, add methanol to prepare a solution containing 20 μg per 1 mL as the control solution.

[0016] Further, the mulberry fruit paste of S1 is prepared by the following method:

[0017] (1) Extraction: take mulberry medicinal materials, add 4-6 times the amount of water, heat and boil for 1.5-2.5 h, filter hot after the first decoction, add 4-6 times the water to the filter residue and decoct in the same way, combine the two water decoctions, and centrifuge at high speed for standby;

[0018] (2) Concentration: vacuum concentrate the extracted liquid after centrifugation, and concentrate to obtain a clear paste with a relative density of about 1.30-1.33, and then heat and centrifuge at high speed for standby;

[0019] (3) Sugar refining: take 30%-40% of the weight of the mulberry medicinal materials as sucrose, add water of half the amount of sucrose, heat and boil for half an hour, filter the syrup hot, and standby;

[0020] (4) Paste collection: mix the clear paste after centrifugation and the refined sugar, stir for 15-20 min, and mix evenly to obtain the paste.

[0021] Further, the mulberry fruit paste of S3 is collected in 18 batches.

[0022] Further, the fingerprint spectrum has 11 fingerprint peaks, and the 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.

[0023] The application further provides the fingerprint spectrum of mulberry serum prepared by the method.

[0024] Compared with the prior art, the application has the beneficial effects that: the method for establishing the fingerprint spectrum of mulberry serum provided by the application uses chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, 5-hydroxymethylfurfural, rutin and isoquercitrin to prepare the reference solution, uses 18 batches of different mulberry serum to prepare the sample solution, and obtains the standard fingerprint spectrum of mulberry serum by optimizing the chromatographic conditions. The established fingerprint spectrum has 11 fingerprint peaks, can monitor various components of mulberry serum, and can be effectively used to evaluate the quality stability of mulberry serum products. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The chromatograms of the sample solution (Y) and different reference 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 sample solution, No. 1 peak is 5-hydroxymethylfurfural, No. 2 peak is neochlorogenic acid, No. 3 peak is chlorogenic acid, No. 4 peak is cryptochlorogenic acid, No. 5 peak is rutin, and No. 6 peak is isoquercitrin;

[0026] Figure 2 The chromatogram of the sample solution under different flow rates;

[0027] Figure 3 The chromatogram of the sample solution under different mobile phases;

[0028] Figure 4 The fingerprint spectrum of mulberry serum of different batches. DETAILED DESCRIPTION

[0029] The technical solutions of the application will be described clearly and completely below in combination with the embodiments of the application. The described embodiments are only some of the embodiments of the application, rather than all the embodiments. Based on the embodiments of the application, all other embodiments obtained by those skilled in the art without creative efforts fall within the protection scope of the application.

[0030] The method is a conventional method unless otherwise specified, and the raw material can be obtained from a public commercial channel unless otherwise specified.

[0031] Example 1: Chromatographic condition establishment

[0032] 1. Instruments and reagents

[0033] 1.1 Instruments: ultra-high performance liquid chromatograph Waters H-Class (Waters Corporation, USA); analytical balance EG204, ME204, XSR105 (Mettler-Toledo Instrument Co., Ltd., Switzerland); electric heating water bath HWS-26 (Shanghai Yiheng Scientific Instrument Co., Ltd.); electric heating air drying oven BO-120FL (Shanghai Yiheng Scientific Instrument Co., Ltd.); digital ultrasonic cleaner KQ-250DE (Kunshan Ultrasonic Instrument Co., Ltd.); pure water instrument MILLI-Q-7005 (Merck Life Science Technology Co., Ltd.).

[0034] 1.2 Reagents: acetonitrile and methanol were chromatographically pure, purchased from Merck Life Science Technology Co., Ltd.; formic acid and tetrahydrofuran were chromatographically pure, purchased from Shanghai Maikelin Biochemical Technology Co., Ltd.; pure water was prepared in the laboratory.

[0035] 1.3 Control information: chlorogenic acid (batch number: YRL0098200419, purchased from Baoji Yirui Biological Technology Co., Ltd.); neochlorogenic acid (batch number: CR-069200117, purchased from Baoji Yirui Biological Technology Co., Ltd.); cryptochlorogenic acid (batch number: YRY0026210420, purchased from Baoji Yirui Biological Technology Co., Ltd.); isoquercitrin (batch number: 111809-201804, purchased from China Institute for Food and Drug Control); 5-hydroxymethylfurfural (batch number: 111626-201912, purchased from China Institute for Food and Drug Control); rutin (batch number: 100080-202012, purchased from China Institute for Food and Drug Control).

[0036] 1.4 The origin and batch number information of mulberry fruit are shown in Table 1 as follows:

[0037] Table 1: Information table of mulberry fruit

[0038]

[0039] 2. Preparation of solutions

[0040] 2.1 Preparation of test solution

[0041] Take 5 g of mulberry paste and place it in a stoppered triangular flask. Add 25 mL of methanol and ultrasonically treat for 30 min. The ultrasonic frequency used is 40 kHz and the ultrasonic power is 250 W. Let it cool, shake well, filter, and take the filtrate as the test solution.

[0042] Among them, the preparation method of mulberry paste is as follows:

[0043] 1) Extraction: Take mulberry fruit medicinal material 500 g, add water 2.5 L, heat and boil, start timing after boiling, extract for 2 h, record the liquid level height, add water every half hour to supplement the liquid level height, filter with 200 mesh non-woven filter cloth after the first decoction, add five times water to the filter residue and decoct in the same way, combine the two water decoctions and centrifuge for standby.

[0044] 2) Concentration: Transfer the centrifuged extract to a rotary evaporator (parameter settings: water bath temperature 75℃, 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 then centrifuge at high speed while hot for standby.

[0045] 3) Sugar refining: Take sucrose 150 g, add half the amount of water, heat and boil for half an hour, filter the sugar syrup with 200 mesh non-woven filter cloth while hot for standby.

[0046] 4) Paste collection: Mix the centrifuged clear paste and the refined sugar filtered with 200 mesh filter cloth, stir for 15-20 min, and mix evenly to obtain.

[0047] 2.3 Preparation of control solution

[0048] Take chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, 5-hydroxymethyl sugar aldehyde, rutin, isoquercitrin, and add methanol to prepare a solution containing 20 μg per 1 mL as the control solution.

[0049] 3. Chromatographic conditions

[0050] 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 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℃; injection volume 1 μL.

[0051] Table 2 Gradient elution conditions

[0052]

[0053] Precisely take 1 μL of the control solution and the test solution respectively, inject into the ultra-high performance liquid chromatograph, sample according to 3. Chromatographic conditions, record the chromatogram;

[0054] The chromatograms of the test solution and different control solution (A-F) are as follows Figure 1As shown in the figure, Y is the test sample solution, A is the chlorogenic acid control solution, B is the neochlorogenic acid control solution, C is the cryptochlorogenic acid control solution, D is the 5-hydroxymethylfurfural control solution, E is the rutin control solution, and F is the isoquercitrin control solution. In the test sample 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.

[0055] 4. Flow rate optimization

[0056] The flow rates were 0.15 mL / min, 0.2 mL / min, and 0.3 mL / min, respectively, and the results are shown in Figure 2 . The results show that when the flow rate is 0.15 mL / min, the peak time is delayed, and the separation of two peaks is poor (as indicated by the arrows); when the flow rate is 0.3 mL / min, the peak time is advanced, but the separation of two peaks is poor (as indicated by the arrows); when the flow rate is 0.2 mL / min, the separation of each peak is good, so the flow rate of 0.2 mL / min is used.

[0057] 5. Mobile phase optimization

[0058] According to 3. Chromatographic conditions, acetonitrile was used as mobile phase A, and 0.2% formic acid water was used as mobile phase B as the mobile phase, and the results are shown in Figure 3 . Using the mobile phase system of the present application (acetonitrile-tetrahydrofuran (v:v 80:20) as mobile phase A, and 0.2% formic acid water as mobile phase B), the results are shown in Figure 3 . The results show that using acetonitrile as mobile phase A and 0.2% formic acid water as mobile phase B, the chromatogram shows that the separation of two peaks is poor (as indicated by the arrows), and the number of chromatographic peaks is small. Using the mobile phase of the present application, the separation of each peak is good, and the number of chromatographic peaks is large.

[0059] Example 2: Fingerprint spectrum and similarity determination method of mulberry paste

[0060] 1 μL of the control solution and the test sample solution was precisely taken and injected into the ultra-high performance liquid chromatograph, and the sample was determined according to the sample injection in 3. Chromatographic conditions in Example 1, and the chromatogram was recorded. The fingerprint spectrum data of 18 batches of mulberry paste were introduced into the traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) to generate a control spectrum (R). There were 11 common peaks matched, and the retention times were 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 spectra of different batches of mulberry paste are shown in Figure 4 .

[0061] The similarity between different batches of mulberry paste was calculated with peak area and retention time as parameters, and the similarity test results are shown in Table 3 below:

[0062] Table 3: Similarity table of fingerprint of different batches of mulberry paste

[0063]

[0064] From the similarity calculation results of the 18 batches of fingerprint, it was found that the similarity of mulberry paste prepared from mulberry medicinal materials of different batches S-1 to S-7 in Turpan, Xinjiang Uygur Autonomous Region to the control fingerprint (R) was greater than 0.9, indicating that there was no obvious difference in the characteristic spectrum of mulberry paste prepared from mulberry medicinal materials of S-1 to S-7 batches in Turpan, Xinjiang Uygur Autonomous Region. The production places of S-10 to S-17 were Turpan in Xinjiang Uygur Autonomous Region, Sichuan, Hebei, Jiangsu, Anhui and other places, and the similarity calculation results of S-10 to S-17 to R were 0.6 to 0.9, among which the similarity results of characteristic spectrum of S-13 in Fuyang, Anhui to S-1 to S-10 in Turpan, Xinjiang Uygur Autonomous Region were 0.6 to 0.9, and the similarity of characteristic spectrum of mulberry in Fuyang, Anhui S-13 to mulberry in Cangzhou, Hebei S-16 and S-17 was 0.936 and 0.946. In summary, there was obvious difference in the characteristic spectrum of mulberry paste prepared from mulberry medicinal materials of different production places and different batches, and the method of the present application can be used to evaluate the quality stability of mulberry paste.

[0065] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the specific embodiments of the present application can be modified or some technical features can be replaced by equivalent ones, which should be covered in the technical solution range claimed by the present application.

Claims

1. A method for establishing a fingerprint spectrum of mulberry extract, characterized in that, Includes the following steps: S1. Prepare the test solution using mulberry extract as raw material, and prepare reference solutions using chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, 5-hydroxymethylfurfural, rutin and isoquercitrin respectively. S2. Inject the test solution and reference solution separately into an ultra-high performance liquid chromatograph (UHPLC) for determination and record the chromatograms. The chromatographic conditions used in the UHPLC are as follows: column: Waters ACQUITY UPLC HSS T3; mobile phase: acetonitrile-tetrahydrofuran at a ratio of 80–90:10–20 as mobile phase A, 0.1%–0.3% formic acid water as mobile phase B; detection wavelength: 300 nm; gradient elution program; the gradient elution program is as follows: S3. Collect chromatograms of test solutions from multiple batches of mulberry extract, import them into the Chinese herbal chromatographic fingerprint similarity evaluation system, generate a reference chromatogram, and calculate the similarity using peak area and retention time as parameters. The preparation method of the test solution described in S1 is as follows: Take 5g of mulberry paste, place it in a stoppered triangular flask, add 25mL of methanol, sonicate for 30min, the ultrasonic frequency used is 40kHz, the ultrasonic power is 250W, cool, shake well, filter, and take the filtrate as the test solution. The mulberry paste described in S1 is prepared using the following method: (1) Extraction: Take mulberry medicinal material, add 4 to 6 times the amount of water, heat and boil to extract for 1.5 to 2.5 hours. After the first decoction, filter while hot. Add 4 to 6 times the amount of water to the filter residue and decoct again in the same way. Combine the two decoctions and centrifuge at high speed for later use. (2) Concentration: The centrifuged extract is concentrated under vacuum to obtain a clear paste with a relative density of 1.30 to 1.

33. The clear paste is centrifuged at high speed while hot and then set aside. (3) Sugar refining: Take 30% to 40% of the weight of mulberry medicinal material in sucrose, add half the amount of water in the sucrose, heat and boil for half an hour, filter the syrup while it is hot and set aside; (4) Concentration: Mix the centrifuged clear paste and refined sugar together and stir for 15 min to 20 min until evenly mixed.

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

3. The method for establishing a fingerprint spectrum of mulberry extract according to claim 1, characterized in that, The chromatographic conditions described in S2 also include: flow rate 0.2 mL / min, column temperature 35 °C, and injection volume 1 μL.

4. The method for establishing a fingerprint spectrum of mulberry extract according to claim 1, characterized in that, The method for preparing the reference solution described in S1 is as follows: Take appropriate amounts of chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, 5-hydroxymethylglucuronide, rutin, and isoquercitrin, and add them to methanol to prepare solutions containing 20 μg per 1 mL, which are used as reference solutions.

5. The method for establishing a fingerprint spectrum of mulberry extract according to claim 1, characterized in that, The mulberry extract described in S3 was collected in 18 batches.

6. The method for establishing a fingerprint spectrum of mulberry extract according to claim 1, characterized in that, The fingerprint spectrum has 11 fingerprint peaks, with retention times of 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.

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