A method for screening and determining the content of active ingredients of five-juice drink

Abstract

The application discloses a method for screening and determining active ingredients of Wuji drink, and a method for determining the content of active ingredients of Wuji drink, which comprises the following steps: adding methanol as an extraction solvent to a Wuji drink dry extract, performing ultrasonic extraction, filtering, and obtaining a test product solution; preparing 3-O-p-coumaroyl quinic acid control product solution, DL-syringaresinol control product solution and methyl ophiopogonanone B control product solution by using methanol; precisely sucking the control product solution and the test product solution respectively; determining the peak area by using UPLC-MS / MS method; and calculating the content of the three components. The application firstly establishes a method for determining the content of three active components, i.e., 3-O-p-coumaroyl quinic acid, DL-syringaresinol and methyl ophiopogonanone B, in a classic medicinal diet Wuji drink dry extract, wherein the 3-O-p-coumaroyl quinic acid is an effective component for determining the content of Wuji drink, and the content determination method meets the relevant regulations for health food declaration.

Description

TECHNICAL FIELD

[0001] The present application relates to the screening and detection of active ingredients of traditional Chinese medicine, in particular to a screening and content determination method of active ingredients of Wuji drink. BACKGROUND

[0002] Wuji drink is from the Qing Dynasty Wu Jutong's 'Dissertation on Warm Diseases', which is a classic Chinese diet, composed of 200g fresh pear juice, 500g fresh lotus root juice, 500g fresh water chestnut juice, 100g reed juice and 50g adenosine juice. The five flavors work together to clear heat and nourish yin, and to produce saliva and stop thirst. Pear, lotus root and water chestnut are fruits, and reed and adenosine are medicinal materials, so Wuji drink is suitable for developing into a health food with auxiliary protection for chemical-induced liver damage.

[0003] According to the health food declaration regulations, the second scheme in 'Evaluation method for auxiliary protection function of chemical-induced liver damage (revised draft)' is selected to evaluate the activity of Wuji drink and its material basis. Wuji drink has 5 raw materials, so there should be 3 or more content determination indexes in the quality control standard, and one of them must be an active or effective ingredient. Reed and adenosus in Wuji drink are both listed in the first volume of 'Chinese Pharmacopoeia' 2020 edition, but there is no content determination method for reed, and the content determination index for adenosus is rusco saponin, which is determined by ultraviolet-visible spectrophotometry. There is no research report on the alcohol-protecting and liver-protecting activity of rusco saponin, and ultraviolet-visible spectrophotometry is not suitable for the analysis of traditional Chinese medicine compound preparations due to its poor specificity. Therefore, there is no quantitative analysis method for Wuji drink dry extract powder at present, and the quality standard of Wuji drink health food cannot be established. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a screening method for active ingredients of Wuji drink, which determines the active ingredients in Wuji drink as content determination indexes, in view of the deficiencies in the prior art.

[0005] The present application also provides a content determination method for active ingredients of Wuji drink, which has good specificity, accuracy, repeatability, stability and feasibility, and the process is simple. It can provide experimental basis for the development of Wuji drink health food and new drugs, and has strong practical industrial development value.

[0006] To solve the above technical problems, the technical scheme adopted by the present application is: a screening method for active ingredients of Wuji drink, characterized in that it comprises the following steps:

[0007] S1. Animal experiments determine that the active ingredient of Wuji drink for treating acute alcoholic liver injury is phenolic acid;

[0008] S2. Find out the chemical components from Wujiyin by using ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry, and calculate the relative peak area of each component;

[0009] S3. Predict the target points and pathways of the chemical components in S2 by using SwissTargetPrediction database, calculate the binding energy of the target proteins according to the chemical components, and determine the active components 3-O-p-coumaroyl quinic acid, DL-syringaresinol, and methyl ophiopogonanone B as the content determination indexes of Wujiyin according to the binding energy and the relative peak area % of the components.

[0010] The application adopts ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-MS / MS) to determine the contents of 3-O-p-coumaroyl quinic acid, DL-syringaresinol, and methyl ophiopogonanone B in the Wujiyin dry extract powder.

[0011] In a preferred embodiment of the application, the active components in Wujiyin for treating acute alcoholic liver injury are mainly 3-O-p-coumaroyl quinic acid, which is determined by the rat experiment in S1.

[0012] In a preferred embodiment of the application, the target protein in S3 is JNK.

[0013] The application further discloses a method for determining the content of active components in Wujiyin, which comprises the following steps:

[0014] Step one, preparation of test solution: add an extraction solvent to the Wujiyin dry extract for reaction and filtration, and obtain the test solution;

[0015] Step two, preparation of control solution: prepare 3-O-p-coumaroyl quinic acid control solution, DL-syringaresinol control solution, methyl ophiopogonanone B control solution, and extraction solvent solution by using an extraction solvent, and filter, and obtain the control solution;

[0016] Step three, precisely pipette the control solution and the test solution, determine the peak area by using UPLC-MS / MS method, and calculate the content, and obtain the content of active components in Wujiyin.

[0017] The chromatographic conditions of the UPLC-MS / MS method are as follows: methanol (A) and 0.1% formic acid aqueous solution (B) are used as the mobile phase, the gradient elution conditions are as follows: 0-10 min, 10% A; 10-45 min, 10%-90% A; 45-49 min, 90% A; 49-50 min, 90-10% A; 50-60 min, 10% A, the flow rate is 0.2-0.4 mL / min, and the column temperature is 30-40℃.

[0018] The application firstly establishes a method for determining the contents of three active ingredients, 3-O-p-coumaroyl quinic acid, DL-syringaresinol and methyl ophiopogonanone B, in the classical medicinal diet recipe Wuji Drink, wherein 3-O-p-coumaroyl quinic acid has the activity of treating acute alcoholic liver injury (proven by rat experiments) and is the effective ingredient for determining the content of Wuji Drink, and the content determination method meets the relevant regulations for health food declaration.

[0019] In the screening test, it is found that the chromatographic peak shape separated by the chromatographic column is more slender after 0.1% and 0.2% formic acid is added to the mobile phase (B), and the resolution is all greater than 2.0, so the method selects 0.1% formic acid aqueous solution as the mobile phase (B) for analysis and measurement.

[0020] In a preferred embodiment of the application, the MS conditions are: detection by a triple quadrupole mass spectrometer; an electrospray ion source (ESI), multiple ion scanning in the multiple reaction monitoring (MRM) mode for each compound, positive ion mode: injection voltage (EP), collision chamber outlet voltage (CXP) is 10, the parent ion (m / z) and daughter ion (m / z) of each component, declustering voltage (DP), collision voltage (CE) are shown in the following table:

[0021]

[0022] Under the conditions, the signal response value is the highest, so the MRM conditions of UPLC-MS / MS in Table 1 are selected by the application for analysis and measurement.

[0023] In a preferred embodiment of the application, the extraction solvent in step one is methanol. When the extraction solvent is methanol, the sum of the extraction rates of the three components is the highest.

[0024] In a preferred embodiment of the application, the ratio of Wuji Drink dry extract powder to extraction solvent in step one is 2.0-3.0 g: 50 ml; preferably, the ratio of Wuji Drink dry extract powder to extraction solvent in step one is 2.0 g: 50 ml. When the ratio of Wuji Drink dry extract powder to extraction solvent in step one is 2.0-3.0 g: 50 ml, the RSD% meets the relevant provisions of quantitative analysis. Preferably, when the ratio of Wuji Drink dry extract powder to extraction solvent in step one is 2.0-3.0 g: 50 ml, the extraction rates of the three components are higher.

[0025] In a preferred embodiment of the application, the injection volume of the reference solution and the test solution in step three is 1-2 μL. When the injection volume exceeds 2 μL, the chromatographic peak of 3-O-p-coumaroyl quinic acid and other components is tailing, so the injection volume of the reference solution and the test solution in step three is 1-2 μL.

[0026] In a preferred embodiment of the present invention, in step two, the concentration of the 3-O-coumaroylquinic acid reference solution is 2-2.5 μg / ml, the concentration of the DL-eugenol reference solution is 1-1.5 μg / ml, and the concentration of the methyl ophiopogon flavanone B reference solution is 2-2.7 μg / ml.

[0027] In a preferred embodiment of the present invention, extraction is performed using ultrasound during the preparation of the test solution in step one. The ultrasound method requires less time and is simpler to operate.

[0028] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention uses ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-MS / MS) to determine the content of three components—3-O-p-coumaryl quinic acid, DL-eugenol, and methyl ophiopogon flavanone B—in the dry extract powder of Wuzhiyin (Five-Juice Drink). This invention innovatively establishes a method for determining the content of these three components in the dry extract powder of Wuzhiyin using UPLC-MS / MS. 3-O-p-coumaryl quinic acid has activity in treating acute alcoholic liver injury (proven by rat experiments) and is an effective component for content determination in Wuzhiyin. This content determination method complies with the relevant regulations for health food application. The method is reasonable, feasible, and simple, and can provide experimental basis for the development of Wuzhiyin health food and new drugs, possessing strong practical industrial development value. Attached Figure Description

[0029] Figure 1 To analyze the components of each test solution of the Five Juices Drink using UPLC-Q-TOF-MS.

[0030] Figure 2 The components from reed rhizome and ophiopogon japonicus in the Five-Juice Drink are identified as targets in the signaling pathways of alcoholic liver injury.

[0031] Figure 3 The UPLC-MS / MS MRM chromatograms (338.0 / 191.0, 417.3 / 180.9, 328.1 / 207.0 m / z channels) of the three component reference standards obtained in Example 1 of this invention are shown.

[0032] Figure 4 The MRM chromatograms of the dry extract powders of reed rhizome, ophiopogon japonicus, fruit, and the whole formula of Five Juice Drink (excluding reed rhizome, ophiopogon japonicus, fruit, and the whole formula of Five Juice Drink) obtained in Example 1 of the present invention are shown in UPLC-MS / MS (338.0 / 191.0, 417.3 / 180.9, 328.1 / 207.0 m / z channels). Detailed Implementation

[0033] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These descriptions are illustrative rather than limiting, and will be understood by those skilled in the art. In the description of the present invention, it should be noted that unless specific conditions are specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Instruments or reagents whose manufacturers are not specified are all commercially available conventional products.

[0034] The main instruments, equipment, materials, and reagents used in the examples are briefly described below:

[0035] The American AB Sciex Exion LC AD ultra-high performance liquid chromatograph, TripleQuad 3500 triple quadrupole mass spectrometer, and X-500R time-of-flight quadrupole mass spectrometer;

[0036] Acetonitrile (mass spectrometry grade, 18100306LM01) and methanol (mass spectrometry grade, 17111204LM01) were purchased from OCEAN PAK; acetonitrile (chromatographic grade, 19100619G202) and methanol (chromatographic grade, 18110927G203) were purchased from Tedia Company; water was ultrapure water (18.25 M ohms); formic acid (88.0%, 176131) was purchased from Fisher Chemical.

[0037] 3-O-p-coumaroylquinic acid (97.0%, M31HB182895), DL-eugenol (95.0%, J19HB185430), and methyl ophiopogon flavanone B (98.0%, M29N11S132592) were all purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0038] The reed rhizomes used in this study were all fresh reed rhizomes (20210101). They, along with fresh snow pears (20210101), fresh lotus roots (20210101), fresh water chestnuts (20210101), and Ophiopogon japonicus (20210101), were all provided by Hunan Yinshuang Biotechnology Co., Ltd. The fresh reed rhizomes and Ophiopogon japonicus were identified by Associate Professor Wang Zhi of the Department of Medicinal Plant Identification of Hunan University of Traditional Chinese Medicine as fresh rhizomes of Phragmites communis Trin. (Poaceae) and dried tuberous roots of Ophiopogon japonicus (Lf) Ker-Gawl. (Liliaceae).

[0039] The research group has previously conducted studies on the extraction, filtration, concentration, drying, formulation, and material basis of the protective effect of Wuzhiyin (Five-Juice Drink) against acute alcoholic liver injury, determining the optimal preparation process for its dry extract powder and granules. They found that 10.80 kg of Wuzhiyin raw materials can produce 490.3 g of dry extract powder. Of this, 43.54 g comes from reed rhizome and 202.8 g from Ophiopogon japonicus. The 43.54 g reed rhizome extract contains 25.02 g of reed rhizome polysaccharide extract and 0.0380 g of reed rhizome extract A (reed rhizome phenolic acid extract); the 202.8 g Ophiopogon japonicus extract contains 168.6 g of Ophiopogon japonicus polysaccharide, 0.0355 g of Ophiopogon japonicus phenolic acid extract, and 6.102 mg of 3-O-p-coumaryl quinic acid. Rat experiments with acute alcoholic liver injury showed that reed rhizome and Ophiopogon japonicus polysaccharides... The extracts showed weak protective effects against acute alcoholic liver injury, while the extracts of reed rhizome, ophiopogon japonicus, and 3-O-p-coumaryl quinic acid showed stronger effects. In particular, 3-O-p-coumaryl quinic acid had a very strong hepatoprotective effect at a low dosage (2.5 mg / kg·d), indicating that the material basis for the protective effect of the Five-Juice Drink against chemical liver injury is not sugary components, but may come from phenolic acid components in reed rhizome and ophiopogon japonicus, with 3-O-p-coumaryl quinic acid being the main active ingredient. Therefore, when establishing a method for determining the content of the Five-Juice Drink, it is necessary to select phenolic acid components such as 3-O-p-coumaryl quinic acid as the content determination index.

[0040] Previous studies used ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS) to identify 110 chemical components from the complete formula, negative formula, and single-herb dry extract powder of Wuzhiyin. The results showed that these 110 components were mainly phenolic acids, flavonoids, and saponins, with 14 derived from reed rhizome and 9 derived solely from Ophiopogon japonicus. Specific results are shown below. Figure 1 Using the SwissTargetPrediction database, 110 components were predicted to act on 359 targets. The top 20 targets by intensity were selected, and the KEGG database was used to predict their action on 36 pathways. Components from reed rhizome (14 types) and ophiopogon japonicus (9 types) can regulate signaling pathways in alcoholic liver disease targeting JNK, p38, etc. Figure 2As shown in Table 1, the binding energies of components from 14 species of reed rhizome and 9 species of ophiopogon japonicus were calculated using JNK(6zr5) target proteins. Based on the binding energy and relative peak area percentage of the components, and combined with the five principles of specificity, measurability, traceability, efficacy, and prescription compatibility in traditional Chinese medicine quality markers, the research group found that 3-O-coumaroylquinic acid, DL-eugenol, and methyl ophiopogon flavanone B are suitable as indicators for the content determination of Wuzhiyin (Five-Juice Decoction). Since the content of these three components in the dry extract of Wuzhiyin is low, high performance liquid chromatography (HPLC) is not suitable for content determination. Therefore, ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-MS / MS) is suitable for establishing a content determination method for these three components.

[0041] Table 1. Binding energy of components from reed rhizome and ophiopogon japonicus in Five-Juice Drink to their target sites.

[0042]

[0043] Example 1

[0044] This embodiment provides a method for determining the content of three components—3-O-p-coumaryl quinic acid, DL-eugenol, and methyl ophiopogon flavanone B—in the dry extract powder of Five-Juice Drink using UPLC-MS / MS, including the following steps:

[0045] (1) 3-O-p-coumaroylquinic acid with concentrations of 0.854, 1.707, 2.134, 3.414, and 4.268 μg / ml was prepared from methanol; DL-eugenol with concentrations of 0.513, 1.026, 1.539, 2.052, and 2.565 μg / ml was prepared from methanol; and methyl ophiopogon flavanone B with concentrations of 1.548, 3.097, 4.645, 6.194, and 7.742 μg / ml was prepared from methanol. Solvent blank control solutions were prepared simultaneously using the same method.

[0046] (2) Juice fresh pears, lotus roots, and water chestnuts to obtain fruit juice. Decoction of fresh reed rhizome and ophiopogon japonicus, filtered, and concentrated at low temperature to obtain medicinal juice. Mix the medicinal juice with the fruit juice, concentrate at low temperature, and spray dry or freeze dry under reduced pressure to obtain the dry extract powder of the Five Juice Drink formula. Prepare dry extract powders of reed rhizome, ophiopogon japonicus, fruit, Five Juice Drink without reed rhizome, Five Juice Drink without ophiopogon japonicus, and Five Juice Drink without fruit using the same method (fruit refers to fresh pears, lotus roots, and water chestnuts mixed according to the formula amount). Negative means dry extract powder lacking the corresponding raw material.

[0047] (3) Take about 2.0g each of reed rhizome, ophiopogon japonicus, fruit, and the whole formula dry extract powder of the Five Juice Drink (excluding reed rhizome, ophiopogon japonicus, fruit, and the Five Juice Drink formula), accurately weigh them, add 50ml of methanol, weigh them, sonicate (power 250W, frequency 50kHz) for 1 hour, make up for the weight loss with methanol, filter with a 0.22μm microporous membrane, and you will get the product.

[0048] (4) Accurately pipette 1 μl of each of the above-mentioned series of 3-O-p-coumaryl quinic acid, DL-eugenol, and methyl ophiopogon flavanone B reference solutions (1), and inject them into the high-performance liquid chromatography-mass spectrometry (UPLC-MS / MS) instrument. Measure the peak area according to the UPLC-MS / MS method. Plot a standard curve with peak area as the ordinate and injection concentration as the abscissa. Separately, accurately pipette 1 μl of the above-mentioned test solution and inject it into the high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) instrument. Acquire the MRM chromatogram of the UPLC-MS / MS sample, as shown below. Figure 3 , 4 As shown, the concentrations of 3-O-p-coumaroylquinic acid, DL-eugenol, and methyl ophiopogoninone B in the liquid chromatography sample are read from the standard curve based on the peak area of ​​the chromatographic peaks, and then the concentrations are calculated.

[0049] (5) In (4), the analytical conditions for UPLC-MS / MS were as follows: WATERS ACQUITY UPLC@BEH C18 column (2.1×100mm, 1.7μm); mobile phase methanol (A)-0.1% formic acid (B), gradient elution (0-10min, 10%A; 10-45min, 10%-90%A; 45-49min, 90%A; 49-50min, 90-10%A; 50-60min, 10%A); flow rate 0.3mL / min; column temperature 35℃. The triple quadrupole mass spectrometer was used for detection: electrospray ionization (ESI) source. Multiple ion detection (MRM) mode was used for multiple ion scanning of each compound. In positive ion mode, the injection voltage (EP) and collision chamber exit voltage (CXP) were 10. The parent ion (m / z), daughter ion (m / z), declustering voltage (DP), and collision voltage (CE) of each analyte are shown in Table 2.

[0050] Table 2. MRM conditions for UPLC-MS / MS of the three active ingredients.

[0051]

[0052] (6) Inject each reference solution, and calculate the linear regression equation and linear range with the concentration of each component as the abscissa (X) and the peak area as the ordinate (Y). The results are shown in Table 3.

[0053] Table 3. Results of linear relationship investigation of three active ingredients (injection volume 1 μL)

[0054]

[0055] (7) The sample solution of the five-juice drink dry extract powder was injected 6 times consecutively, and the RSD% of the content of each component was calculated to examine the precision. The results are shown in Table 4.

[0056] (8) Six samples of the dry extract powder of Wuzhiyin were prepared in parallel according to the method under (3), and were injected for determination. The RSD% of the contents of each component was calculated to investigate the repeatability. The results are shown in Table 4.

[0057] (9) The sample solution of the dry extract powder of Wuzhiyin was injected at 0.0, 1.5, 3.0, 4.5, 6.0, 12, 18, 24, and 48 h after preparation, and the RSD% of the contents of each component was calculated to investigate the stability. The results are shown in Table 4.

[0058] (10) Six samples of 1.0 g of the dry extract powder of Wuzhiyin with known contents of each component were accurately weighed. A total of 40 mL of the reference substance solution with known concentration and methanol solution were added, and the spiked recovery test sample solution was prepared according to the method under (3), and was injected for determination. The spiked recovery rate and RSD% of each component were calculated to investigate the spiked recovery rate. The results are shown in Table 4.

[0059] Table 4 Results of the investigation on the precision, repeatability, stability, and (spiked) recovery rate of three active components

[0060]

[0061] (11) The test samples of the dry extract powders of Rhizoma Phragmitis, Radix Ophiopogonis, fruits, Wuzhiyin without Rhizoma Phragmitis, Wuzhiyin without Radix Ophiopogonis, Wuzhiyin without fruits, and the whole formula of Wuzhiyin prepared by the method under (3) were injected for determination, and the contents of each component were calculated to investigate the specificity. The results are shown in Table 5.

[0062] Table 5 Results of the determination of the contents of three active components in Wuzhiyin by UPLC-MS / MS

[0063]

[0064]

[0065] Note in Table 5: The raw materials refer to fresh Sydney pears, fresh lotus roots, fresh water chestnuts, fresh Rhizoma Phragmitis, and Radix Ophiopogonis required for preparing the dry extract powder.

[0066] (12) Investigation of the extraction solvent of the test sample

[0067] In this study, the extraction effects of water, methanol, and ethanol as extraction solvents on three components, 3-O-p-coumaroylquinic acid, DL-syringaresinol, and methylophiopogonanone B, in the dry extract powder of Wuzhiyin were investigated. The results showed that when the extraction solvent was methanol, the sum of the extraction rates of the three components was the highest. Therefore, methanol was selected as the extraction solvent.

[0068] (13) Investigation of the extraction method of the test sample

[0069] For the preparation of test samples for the determination of the content of components in Chinese medicinal materials, the methods usually adopted are ultrasonic extraction, reflux extraction, etc. according to the first part of Chinese Pharmacopoeia (2020 Edition). In this study, the methanol extraction effects of ultrasonic extraction (250W, 50K Hz, 60min) and reflux extraction (80℃, 120min) on three components, namely 3-O-p-coumaroylquinic acid, DL-syringaresinol, and methylophiopogonone B in the dry extract powder of Wuzhiyin, were investigated. The results showed that there was no significant difference in the extraction rates between the ultrasonic method and the reflux method (P < 0.05). Since the ultrasonic method requires less time and is easier to operate, the ultrasonic method was selected.

[0070] (14) Optimization of the material-liquid ratio for sample extraction

[0071] In this study, when the sampling amounts of the dry extract powder of Wuzhiyin were 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 g, and 50 ml of methanol was added, the extraction effects on three components, namely 3-O-p-coumaroylquinic acid, DL-syringaresinol, and methylophiopogonone B, were investigated. The results showed that when the sampling amounts were 0.5, 1.0, and 1.5, the RSD% of the repeatability investigation results was too large. When the sampling amount was 2.0, the RSD% of the repeatability investigation results met the relevant regulations for quantitative analysis. When the sampling amounts were 2.5 and 3.0, the extraction rates of the three components were lower than those when the sampling amount was 2.0, and the difference was significant (P < 0.05). Therefore, the optimized material-liquid ratio was: sampling amount 2.0 g, adding 50 ml of methanol for extraction.

[0072] (15) Investigation of the stationary phase

[0073] In this study, the separation effects of three components, namely 3-O-p-coumaroylquinic acid, DL-syringaresinol, and methylophiopogonone B, were investigated using the ACQUITY UPLC@BEH C18 column (1.7um, 2.1×100mmColumn) of WATERS company, the Luna-Omega 1.6um C18 column (100A, LCColumn 100×2.1mm) of Guangzhou Phenomenex Scientific Instruments Co., Ltd., and the SinoChrom ODS2 column (1.8um, 2.1*100mm) of Dalian Elite Analytical Instruments Co., Ltd. It was found that the chromatographic peaks separated by the three columns were sharp and thin, and the resolution was all > 2.0. There was no significant difference in the determination results. Therefore, this method can be used for analysis and detection with octadecylsilyl silica gel as the filler.

[0074] (16) Investigation of adding acid to the mobile phase

[0075] In this study, methanol was used as the mobile phase (A) and pure water was used as the mobile phase (B). The separation effect of mobile phase (B) on the chromatographic peaks of three components, namely 3-O-p-coumarylquinic acid, DL-eugenol, and methyl ophiopogon flavanone B, was investigated under the conditions of no formic acid, 0.1%, and 0.2% formic acid. It was found that after adding 0.1% and 0.2% formic acid, the chromatographic peaks separated by the column were finer, and the resolution was >2.0 for both mobile phases. Therefore, 0.1% formic acid aqueous solution was selected as the mobile phase (B) for analysis and detection.

[0076] (17) Analysis of injection volume

[0077] This study investigated the separation effect of chromatographic peaks of three components, namely 3-O-p-coumarylquinic acid, DL-eugenol, and methyl ophiopogon flavanone B, at injection volumes of 1, 2, 5, and 10 μL. It was found that when the injection volume exceeded 2 μL, the chromatographic peaks of components such as 3-O-p-coumarylquinic acid showed tailing, and when the injection volume exceeded 5 μL, the chromatographic peaks of components such as 3-O-p-coumarylquinic acid showed cracking. Therefore, this method selected an injection volume of 1 μL for analysis and detection.

[0078] (18) Optimization of mass spectrometry MRM conditions

[0079] This study optimized the MRM mass spectrometry conditions for UPLC-MS / MS of three components: 3-O-coumaroylquinic acid, DL-eugenol, and methyl ophiopogon flavanone B. The results showed that 3-O-coumaroylquinic acid exhibited the highest signal response at a parent ion of 338.0 m / z, a daughter ion of 191.0 m / z, a declustering voltage of -20 eV, and a collision voltage of -20 eV. DL-eugenol showed the highest signal response at a parent ion of 417.3 m / z, a daughter ion of 180.9 m / z, a declustering voltage of -86 eV, and a collision voltage of -26 eV. Methyl ophiopogon flavanone B showed the highest signal response at a parent ion of 328.1 m / z, a daughter ion of 207.0 m / z, a declustering voltage of -103 eV, and a collision voltage of -32.7 eV. The signal response value is highest under the condition of V, so the above MRM conditions of UPLC-MS / MS are selected for analysis and measurement.

[0080] This invention provides an analytical method for determining the content of three components—3-O-p-coumaryl quinic acid, DL-eugenol, and methyl ophiopogon flavanone B—in the dry extract powder of Wuzhiyin (Five-Juice Decoction). The contents of 3-O-p-coumaryl quinic acid and methyl ophiopogon flavanone B in the negative control sample lacking Ophiopogon japonicus accounted for 0.018% and 2.710% of the total sample, respectively. The content of DL-eugenol in the negative control sample lacking Phragmites communis accounted for 0.171% of the total sample, indicating that the analytical method for determining the content of these three components in the dry extract powder of Wuzhiyin has good specificity. As shown in Table 4, the precision, repeatability, stability within 48 hours, and recovery rate of this analytical method all meet the relevant requirements for quantitative analysis.

[0081] The analytical method of this invention is scientific, reasonable, stable, and feasible, and the process is simple. Among them, 3-O-p-coumaryl quinic acid has the activity of treating acute alcoholic liver injury (proven by rat experiments) and is an effective component for the content determination of Wuzhiyin. This analytical method complies with the relevant regulations for the application of health food and can provide experimental basis for the development of Wuzhiyin health food and new drugs, and has strong practical industrial development value.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

[0083] Furthermore, those skilled in the art will understand that although some embodiments herein include certain features included in other embodiments but not others, combinations of features from different embodiments are intended to be within the scope of the invention and form different embodiments. For example, in the foregoing claims, any of the claimed embodiments can be used in any combination. The information disclosed in this background section is intended only to enhance the understanding of the general background of the invention and should not be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

Claims

1. A method for determining the content of active ingredients in a five-juice beverage, characterized in that... Includes the following steps: Step 1: Preparation of test solution: Add extraction solvent to the dry extract powder of Five-Juice Drink, extract by ultrasonication, and filter to obtain the test solution; The solvent extracted in step one is methanol; In step one, the ratio of the dry extract powder of the Five-Juice Drink to the extraction solvent is 2.0~3.0g: 50ml; The preparation method of the Five Juice Drink Dry Extract Powder includes juicing fresh snow pear, fresh lotus root, and fresh water chestnut to obtain fruit juice, decocting fresh reed rhizome and ophiopogon japonicus in water, filtering, concentrating at low temperature to obtain medicinal juice, mixing the medicinal juice with the fruit juice, concentrating at low temperature, and spray drying or freeze-drying under reduced pressure at low temperature. Step 2, Preparation of reference solutions: Prepare 3-O-p-coumaroylquinic acid reference solution, DL-eugenol reference solution, methyl ophiopogon flavanone B reference solution and extraction solvent solution respectively using extraction solvent, and filter; Step 3: Accurately pipette the reference solution and the test solution separately, determine the peak area using UPLC-MS / MS, and calculate the content; The chromatographic conditions for UPLC-MS / MS were as follows: using methanol A-0.1% formic acid aqueous solution B as the mobile phase, the gradient elution conditions were: 0~10 min, 10% A; 10~45 min, 10%~90% A; 45~49 min, 90% A; 49~50 min, 90~10% A; 50~60 min, 10% A; flow rate 0.2~0.4 mL / min; column temperature 30~40℃; and the chromatographic column was packed with octadecylsilane-bonded silica gel. Mass spectrometry conditions were as follows: triple quadrupole mass spectrometer; electrospray ionization (ESI); multiple ion detection (MRM) mode for each compound; positive ion mode: injection voltage EP and collision chamber exit voltage CXP were 10. The m / z of the parent ion, m / z of the daughter ion, declustering voltage DP, and collision voltage CE for each component are shown in the table below. 。 2. The method for determining the content of active ingredients in the five-juice beverage according to claim 1, characterized in that, In step one, the ratio of the dry extract powder of the Five-Juice Drink to the extraction solvent is 2.0g:50ml.

3. The method for determining the content of active ingredients in the five-juice beverage according to claim 1, characterized in that, In step three, the injection volume of the reference solution and the test solution is 1~2µL.

4. The method for determining the content of active ingredients in the five-juice beverage according to claim 1, characterized in that, In step two, the concentration of 3-O-coumaroylquinic acid reference solution is 2~2.5µg / ml, the concentration of DL-eugenol reference solution is 1~1.5µg / ml, and the concentration of methyl ophiopogon flavanone B reference solution is 2~2.7µg / ml.

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