Method for simultaneously determining contents of five components in medicinal and edible beverage and application of method

Abstract

The invention discloses a method for simultaneously determining the contents of five components in a medicinal and edible beverage and application thereof. The method for simultaneously determining the contents of rutin, nuciferine, sinapine, liquiritin and glycyrrhizic acid in the medicinal and edible beverage prepared from mulberry leaves, lotus leaves, mustard seeds and licorice roots under the same chromatographic condition by adopting a high performance liquid chromatography comprises the following steps: firstly preparing a water extract, a mixed reference solution and a test solution, and then detecting under a specific chromatographic condition to realize determination, so that the content of rutin, nuciferine, sinapine, liquiritin and glycyrrhizic acid in the medicinal and edible beverage is determined. The method is simple and easy to operate, strong in specificity, repeatable, good in precision and stability, and capable of comprehensively reflecting the quality of the product. The method can also be used for standardized extraction and preparation of the medicinal and edible beverage. Through simultaneous determination of multiple indexes, an important method guarantee is provided for effective extraction and quality control of the medicinal and edible beverage with lower cost, higher popularity and more flexible operation space, the blank of quality control of multiple components in the medicinal and edible beverage is made up, and a preferable extraction process can be applied to production and has a wide application prospect. The practical application and popularization value is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of beverage quality detection, and in particular to a method for simultaneously determining the contents of five components in a medicine-food homologous beverage and application thereof. BACKGROUND

[0002] Quality by design (QbD) is an advanced quality control concept, which is widely used in the research and development of pharmaceuticals in recent years. It aims to ensure product quality, emphasizes the control of quality and the active design of products in the whole production process, understands product properties and controls processes through the design of scientific experiments, explores the relationship between critical quality attributes (CQAs) and critical process parameters (CPPs) by means of mathematical models, and establishes a robust design space to meet production needs. The extraction process, as the front end of the entire research and development process, significantly affects the subsequent research and development and production processes. The traditional extraction optimization method obtains an optimal point, but in actual production, slight fluctuations in parameters may lead to a decrease in product quality or unqualified products. The poor robustness of the process. Introducing the QbD concept into the extraction process research stage of medicine-food homologous products can further control product quality, convert fixed process parameters into a design space of process parameters, and improve the scientificity and robustness of process parameters, which has important practical significance for improving the quality of subsequent research and development and production and reflecting the value of products.

[0003] Special endowment is a special constitution caused by insufficient congenital endowment or genetic factors in traditional Chinese medicine constitution. It mainly manifests as weak adaptability, easy allergy, and repeated abnormal reactions related to immunity or metabolism. This constitution group is easily affected by external factors such as climate, diet, pollen, and other environmental factors, and has poor physiological function and self-regulation. The reaction to external stimulation is strong. The product is composed of mulberry leaves, lotus leaves, mustard, licorice and other medicine-food homologous traditional Chinese medicines, and is mainly used for conditioning and improving special endowment. Modern pharmacological studies have shown that flavonoids such as rutin in mulberry leaves have antioxidant, hypoglycemic, and cardiovascular protection activities; lotus leaves mainly contain alkaloids such as lotus leaf alkaloids, flavonoids such as rutin, and quercetin, which have the effects of inhibiting lipase activity, antioxidant, anti-inflammatory, and protecting the cardiovascular system; the main components of mustard are glucosinolate mustard alkaloids and volatile oils, which have antioxidant, antitussive, antiasthmatic, and antibacterial effects; the main components of licorice are triterpenoid saponins such as glycyrrhizic acid, flavonoids such as glycyrrhizin, and polysaccharides, which have the effects of anti-allergy, antioxidant, anti-inflammatory, and liver protection. However, there is no standardized extraction process standard and content determination report.

[0004] The present application aims to establish a method for simultaneously determining the contents of five index components (rutin, nuciferine, sinapine, glycyrrhizin and glycyrrhizic acid) by high performance liquid chromatography, covering flavonoids, alkaloids, triterpenoid saponins and other components, which can realize the accurate quantification of the five active components in the product and the comprehensive evaluation and quality control of the product, and the content determination method is applied to the optimization of the extraction process of the product, thereby providing technical support and quality guarantee for the production of the product. SUMMARY

[0005] In view of the above, in order to overcome the shortcomings of the prior art, the present application aims to provide a method for simultaneously determining the contents of five components in a food and medicine beverage for special constitution and application thereof, and the content determination method is applied to the optimization of the production process of the beverage, which is accurate, reproducible, reasonable, stable and feasible, and can be used for the standardized production and quality control of the product.

[0006] The technical solution solved by the present application is a method for simultaneously determining the contents of five components in a food and medicine beverage, the five components are rutin, nuciferine, sinapine, glycyrrhizin and glycyrrhizic acid, and the method comprises the following steps: S1. Water extract preparation: according to the prescription weight fraction, the dried leaves of mulberry, lotus leaf, mustard, licorice root are weighed and added with water for decoction, filtration, and the filtrate is combined to obtain the extract.

[0007] S2. Preparation of mixed reference solution A certain amount of rutin, nuciferine, sinapine thiocyanate, glycyrrhizin and glycyrrhizic acid monammonium salt reference substances are precisely weighed and placed in a volumetric flask, diluted with methanol to constant volume to prepare a reference stock solution for standby use; when used, the mixed reference solution of different concentrations is prepared by diluting with methanol (the mass of glycyrrhizic acid = the mass of glycyrrhizic acid monammonium salt / 1.0207, and sinapine is calculated based on sinapine thiocyanate); S3. Preparation of test sample solution: the above extract is centrifuged and filtered to obtain the test sample solution; S4. Determination of the contents of the five components: the above mixed reference solution and test sample solution are injected into a high performance liquid chromatograph, the peak areas of each peak are measured under the chromatographic conditions, and the contents of the five components are calculated.

[0008] Application of the method of the present application in the quality detection of food and medicine beverages.

[0009] Application of the method of the present application in the optimization of the extraction process of active components in the production of food and medicine beverages.

[0010] The method is simple, easy to operate, strong in operability and repeatable, and through simultaneous determination of multiple indexes, the method provides important method guarantee for realizing effective extraction and quality control of the medicine-food homologous beverage, simultaneously provides a reproducible technical framework for modern quality evaluation of the medicine-food homologous beverage, promotes the transformation of the industry from "single component control" to "overall quality control", and has actual application and popularization value. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 HPLC chromatogram (first part) of the present application, wherein: A~D are mixed control, test sample, negative sample of lack of mulberry leaf and lotus leaf, and negative sample of lack of licorice, respectively, under 270 nm; 1. glycyrrhizin; 2. rutin; 3. nuciferine; 4. glycyrrhizic acid monomethyl ammonium salt; 5. mustard oil (the same below); Figure 2 HPLC chromatogram (second part) of the present application, wherein: E~G are mixed control, test sample, and negative sample of lack of mustard, respectively, under 326 nm; Figure 3 Box-Behnken test contour line and 3D graph of the present application; Figure 4 Design space graph of the water extraction process of the present application (extraction times = 3 times); Figure 5 Chromatogram of different gradient elution ratios 1 of the present application; Figure 6 Chromatogram of different gradient elution ratios 2 of the present application; Figure 7 Chromatogram of different gradient elution ratios 3 of the present application. DETAILED DESCRIPTION

[0012] The specific embodiments of the present application will be described in detail below in combination with examples and specific cases.

[0013] The present application can be given by the following examples in specific implementation: A method for simultaneously determining the contents of five components in a medicine-food homologous beverage, the five components being rutin, nuciferine, mustard oil, glycyrrhizin and glycyrrhizic acid, the determination method comprising the following steps: S1, take 30 parts of mulberry leaves, 30 parts of lotus leaves, 15 parts of mustard and 15 parts of licorice, add 9.5~12 times the amount of water and extract 3 times, each time for 1~1.7 h, combine the extract, and obtain the water extract. S2. Preparation of mixed control solution: accurately weigh rutin, nuciferine, mustard alkaloid thiocyanate, glycyrrhizin, and glycyrrhizic acid monoammonium salt control samples, and place them in a volumetric flask. Dilute to the mark with methanol to prepare control stock solutions with concentrations of 0.2432 mg / mL of rutin, 0.0644 mg / mL of nuciferine, 0.1268 mg / mL of mustard alkaloid, 0.0632 mg / mL of glycyrrhizin, and 0.1220 mg / mL of glycyrrhizic acid; accurately take 0.5, 1, 2, 3, 4, and 5 mL of the above solution, and dilute with methanol to obtain mixed control solutions with different concentrations; S3. Preparation of test solution: take the above extract and centrifuge it in a centrifuge at 12000 r / min for 10 min. Take the supernatant and filter it through a 0.22 μm microporous filter membrane. Take the filtrate to obtain the test solution; S4. Determination of the contents of the five components: take the above mixed control solution and test solution, and inject them into a high-performance liquid chromatograph. Determine the peak areas of each peak under specific chromatographic conditions and gradient elution program, and calculate to simultaneously determine the five components; The specific chromatographic conditions are as follows: the chromatographic column is an Agilent ZORBAX SB-C18 column with a specification of 4.6*250 mm and a particle size of 5 μm. Gradient elution is performed with acetonitrile (A)-0.1% phosphoric acid (B) as the mobile phase, the injection volume is 10 μL, the flow rate is 1 mL / min, the column temperature is 30°C, the detection wavelength is 326 nm for mustard alkaloid, and the detection wavelength is 270 nm for rutin, nuciferine, glycyrrhizin, and glycyrrhizic acid. The theoretical plate number calculated according to the rutin peak should not be less than 5000; The gradient elution program is as follows: from 0 to 15 minutes, the flow rate of acetonitrile (A) is 10.5%; from 15 to 45 minutes, the flow rate of acetonitrile (A) is 10.5→13%; from 45 to 46 minutes, the flow rate of acetonitrile (A) is 13→22%; from 46 to 60 minutes, the flow rate of acetonitrile (A) is 22→23%; from 60 to 61 minutes, the flow rate of acetonitrile (A) is 23→32%; and from 61 to 80 minutes, the flow rate of acetonitrile (A) is 32→40%.

[0014] Application of the method in the quality detection of the food and medicine beverage.

[0015] Application of the method in the process optimization of producing the food and medicine beverage to extract active ingredients.

[0016] The above embodiments are only used to illustrate the specific implementation of the present application, but not used to limit the protection scope of the present application. The technical core of the present application is the determination method and application. Any transformation made by using equivalent alternatives, which is the same in nature as the technical solution of the present application, belongs to the protection scope of the present application.

[0017] The method is simple, easy to operate, strong operability and repeatable, and through simultaneous determination of multiple indexes, lower cost, higher popularity and more flexible operation space are provided for realizing effective extraction and quality control of the medicine-food homologous beverage, and a reproducible technical framework is provided for modern quality evaluation of the medicine-food homologous beverage, and the effect is very good through experiments, and the relevant experimental data are as follows: 1. Experimental materials 1.1. Instruments

[0018] 1.2. Reagents and medicinal materials

[0019] Acetonitrile, methanol, chromatographically pure, Germany Merck Company; water is ultrapure water.

[0020] 2. Experimental methods and results 2.1. Determination of the contents of rutin, nuciferine, sinapine, glycyrrhizin and glycyrrhizic acid 2.1.1. Preparation of solutions 2.1.1.1. Preparation of mixed control solution Accurately take the rutin, nuciferine, sinapine thiocyanate, glycyrrhizin and glycyrrhizic acid monammonium salt control substances into a 10 mL volumetric flask, add methanol to constant volume, and obtain the mixed control stock solution with the concentrations of rutin 0.2432 mg / mL, nuciferine 0.0644 mg / mL, sinapine 0.1268 mg / mL, glycyrrhizin 0.0632 mg / mL and glycyrrhizic acid 0.1220 mg / mL, respectively, for standby use. When used, dilute the mixed control stock solution with methanol to obtain mixed control solutions with different concentrations (the mass of glycyrrhizic acid = the mass of glycyrrhizic acid monammonium salt / 1.0207, and sinapine is calculated based on sinapine thiocyanate).

[0021] 2.1.1.2. Preparation of test solution Take the extract and centrifuge it in a centrifuge at 12000 r / min for 10 min, filter the supernatant through a 0.22 μm microporous filter membrane, and take the filtrate to obtain the test solution.

[0022] 2.1.1.3. Preparation of negative sample solution Prepare the double negative samples of lack of mulberry leaves and lotus leaves, lack of mustard, and lack of licorice according to the prescription proportion, and prepare the negative sample solutions of lack of mulberry leaves and lotus leaves, lack of mustard, and lack of licorice according to the method of “2.2.2”.

[0023] 2.1.2. Determination method: accurately pipette 10 μL of the mixed control solution and the test solution respectively, inject them into the liquid chromatograph, determine, record the peak area, and calculate.

[0024] 2.1.3, Selection of chromatographic conditions Agilent ZORBAX SB-C18 column (4.6 x 250 mm, 5 μm) was used as the chromatographic column, and gradient elution was performed with acetonitrile (A) - 0.1% phosphoric acid water (B) as the mobile phase at a flow rate of 1 mL / min, the column temperature was 30℃, and the detection wavelength was 326 nm (sinapine), 270 nm (rutin, nuciferine, glycyrrhizin and glycyrrhizic acid), and the injection volume was 10 μL. The theoretical plate number calculated according to the rutin peak should not be less than 5000.

[0025] 2.1.3.1 Selection of mobile phase There are four kinds of mobile phases in the chromatographic conditions under the determination of the contents of rutin, nuciferine, sinapine, glycyrrhizin and glycyrrhizic acid in the 2025 edition of Chinese Pharmacopoeia, which are methanol-0.5% phosphoric acid gradient elution, acetonitrile-water-triethylamine-glacial acetic acid, acetonitrile-0.08 mol / L potassium dihydrogen phosphate solution, acetonitrile-0.05% phosphoric acid gradient elution, the compositions and proportions of the mobile phases used are completely different, the operation is cumbersome, time-consuming and high-cost. In this study, a mobile phase of acetonitrile-0.1% phosphoric acid water was used, and through different elution conditions, one-time detection can be realized, and the operation is simple, fast and economical.

[0026] 2.1.3.2 Selection of mobile phase elution proportion The present application carries out related exploration and optimization for different elution conditions of the mobile phase acetonitrile-0.1% phosphoric acid water.

[0027] Comparative Example 1: octadecylsilane-bonded silica gel was used as the filler, acetonitrile was used as the mobile phase A, and 0.1% phosphoric acid solution was used as the mobile phase B, and gradient elution was performed according to the table below: Table 1 Gradient elution proportion 1

[0028] Comparative Example 2: octadecylsilane-bonded silica gel was used as the filler, acetonitrile was used as the mobile phase A, and 0.1% phosphoric acid solution was used as the mobile phase B, and gradient elution was performed according to the table below: Table 2 Gradient elution proportion 2

[0029] Comparative Example 3: octadecylsilane-bonded silica gel was used as the filler, acetonitrile was used as the mobile phase A, and 0.1% phosphoric acid solution was used as the mobile phase B, and gradient elution was performed according to the table below: Table 3 Gradient elution proportion 3

[0030] Compared with the chromatographic conditions in Table 4, the separation of rutin, nuciferine, mustard oil, glycyrrhizin and glycyrrhizic acid in the chromatographic conditions of Comparative Example 1, Comparative Example 2 and Comparative Example 3 was poor; while the chromatographic peaks of the 5 components were sharp and well separated by using the gradient elution program of the chromatographic conditions, which met the requirements and could realize simultaneous determination of multiple components.

[0031] Table 4 Gradient elution table

[0032] Optimal chromatographic conditions: Agilent ZORBAX SB-C18 column (4.6 x 250 mm, 5 μm); gradient elution was performed with acetonitrile (A)-0.1% phosphoric acid (B) as the mobile phase, the gradient elution program was as follows: 0-15 min, 10.5% acetonitrile (A) in the mobile phase; 15-45 min, 10.5→13% acetonitrile (A) in the mobile phase; 45-46 min, 13→22% acetonitrile (A) in the mobile phase; 46-60 min, 22→23% acetonitrile (A) in the mobile phase; 60-61 min, 23→32% acetonitrile (A) in the mobile phase; 61-80 min, 32→40% acetonitrile (A) in the mobile phase; flow rate 1 mL / min, column temperature 30°C, detection wavelength 326 nm (mustard oil), 270 nm (rutin, nuciferine, glycyrrhizin and glycyrrhizic acid), injection volume 10 μL. The theoretical plate number should not be less than 5000 calculated by the rutin peak.

[0033] 2.2, Investigation of content determination method 2.2.1, Specificity test Take the solution under item “2.1.1” and inject 10 μL of each under the above-mentioned “2.1.3” chromatographic conditions, record the chromatogram for analysis, see Figure 1 . The results show that the mustard oil chromatographic peak in the chromatogram of the test sample is well separated from the adjacent chromatographic peaks at 326 nm (separation degree >1.5), and there is no interfering peak at the corresponding position of the chromatogram of the mustard oil thiocyanate reference substance; the rutin, nuciferine, glycyrrhizin and glycyrrhizic acid chromatographic peaks in the chromatogram of the test sample are well separated from the adjacent chromatographic peaks at 270 nm (separation degree >1.5), and there is no interfering peak in the chromatogram of each negative sample at the corresponding position of the chromatogram of the reference substance, indicating that the method has good specificity.

[0034] 2.2.2, Investigation of linearity Precisely take 0.5, 1, 2, 3, 4, 5 mL of the mixed reference substance stock solution under item “2.1.2.1” and place them in 5 mL volumetric flasks, add methanol to constant volume, thus obtaining a series of reference substance solutions with different mass concentrations, which are injected and analyzed under the chromatographic conditions in item “2.1.3”. Take the mass concentration of the reference substance solution as the abscissa ( X ), and the peak area as the ordinate ( Y), linear regression analysis was performed, see Table 5, the correlation coefficients of the five components were all greater than 0.999, the linear ranges of rutin, nuciferine, sinapine, glycyrrhizin and glycyrrhizic acid were 0.0243~0.2432mg / mL, 0.0064~0.0644mg / mL, 0.0127~0.1268mg / mL, 0.0063~0.0632mg / mL, 0.0122~0.1220mg / mL respectively, the results showed that the linear relationships of the five components were all good within their concentration ranges.

[0035] Table 5 Linear relationship determination results of each index component

[0036] 2.2.3, Precision test The same test sample solution was taken, the chromatographic conditions under item “2.1.3” were continuously used for sampling for 6 times, the peak areas of rutin, nuciferine, sinapine, glycyrrhizin and glycyrrhizic acid were recorded, and the RSD values were calculated, the results were shown in Table 6. The RSD values of the peak areas of each component were 1.35%, 1.71%, 0.49%, 1.94% and 1.80% respectively, which indicated that the precision of the instrument was good.

[0037] Table 6 Precision test results

[0038] 2.2.4, Stability test The test sample solution was taken, the chromatographic conditions under item “2.1.3” were used for sampling analysis at 0, 2, 4, 6, 8 and 24h after preparation respectively, the peak areas of rutin, nuciferine, sinapine, glycyrrhizin and glycyrrhizic acid were recorded, and the RSD values were calculated, the results were shown in Table 7. The RSD values of the peak areas of each component were 1.88%, 1.75%, 1.14%, 1.70% and 1.72% respectively, which indicated that the stability of the test sample solution was good within 24h.

[0039] Table 7 Stability test results

[0040] 2.2.5, Reproducibility test According to the method under item “2.1.2.2”, 6 test sample solutions were prepared in parallel, the chromatographic conditions under item “2.1.3” were used for sampling, and the peak areas of each chromatographic peak were recorded. The content and RSD value of each component in the test sample were calculated. The RSD values of the content of rutin, nuciferine, sinapine, glycyrrhizin and glycyrrhizic acid were 1.92%, 1.55%, 1.41%, 1.45% and 1.82% respectively, which indicated that the reproducibility of the determination method was good, see Table 8.

[0041] Table 8 Reproducibility test results

[0042] 2.2.6, Sample loading recovery test Preparation of control solution: accurately weigh rutin, nuciferine, mustard alkaloid thiocyanate, glycyrrhizin, glycyrrhizic acid monoammonium salt control substances into a volumetric flask, dilute and dissolve with methanol, respectively, to obtain control solution, the concentration is: rutin 0.418 mg / mL, nuciferine 0.240 mg / mL, mustard alkaloid 0.507 mg / mL, glycyrrhizin 0.391 mg / mL, glycyrrhizic acid 0.324 mg / mL, ready for use.

[0043] Accurately take 2.5 mL of the extract solution into a 5 mL volumetric flask, and add 0.505 mL, 0.488 mL, 0.195 mL, 0.182 mL, and 0.506 mL of the above rutin, nuciferine, mustard alkaloid, glycyrrhizin, and glycyrrhizic acid control solution, respectively, shake well, prepare the sample according to the method under item “2.1.2.2”, 6 groups in parallel, inject according to the chromatographic conditions under item “2.1.3”, record the peak area of each chromatographic peak. Calculate the content of rutin, nuciferine, mustard alkaloid, glycyrrhizin, and glycyrrhizic acid in the test sample, and calculate the recovery rate and RSD value, the results are shown in Table 9. The average sample loading recovery rates of each component are: 100.8%, 100.0%, 97.25%, 102.3%, and 99.47%, respectively, and the RSD values are: 3.34%, 1.88%, 3.54%, 1.78%, and 1.38%, respectively, all meet the requirements, indicating that the determination method is accurate and reliable.

[0044] Table 9: Results of sample loading recovery test

[0045] 2.3, Application of content determination method in extraction process optimization 2.3.1, Determination of key quality attributes (CQAs) in extraction process This product is composed of mulberry leaves, lotus leaves, mustard seeds, and licorice, among other medicinal and edible herbs, primarily used for the conditioning and improvement of individuals with allergic constitutions. Modern pharmacological research shows that rutin, a flavonoid component in mulberry leaves, possesses antioxidant, hypoglycemic, and cardiovascular protective activities; lotus leaves mainly contain alkaloids such as nuciferine, flavonoids such as rutin and quercetin, which have effects such as inhibiting lipase activity, antioxidant, anti-inflammatory, and cardiovascular protective effects; mustard seeds mainly contain glucosinolates such as sinigrin and volatile oils, which have antioxidant, antitussive, antiasthmatic, and antibacterial effects; licorice mainly contains triterpenoid saponins such as glycyrrhizic acid, flavonoids such as glycyrrhizin, and glycyrrhizin polysaccharides, which have anti-allergic, antioxidant, anti-inflammatory, and liver-protective effects. The yield of the extract, as a measure of the amount of decoction, is an important basis for subsequent molding process research. Therefore, the contents of rutin, nuciferine, sinigrin, glycyrrhizin, and glycyrrhizic acid, as well as the yield, are selected as the CQAs for the water extraction process.

[0046] 2.3.2 Extraction Process Design Based on relevant literature and practical production experience, the water addition amount ( A Extraction time () B ) and number of extractions ( C The key process parameters (CPPs) for water extraction were used as evaluation indicators. The yield and the contents of rutin, lotus leaf alkaloids, sinigrin, glycyrrhizin, and glycyrrhizic acid were used as evaluation indices. A comprehensive evaluation was conducted using the AHP-entropy weighted mixed weighting method. A Box-Behnken response surface design was employed for the experiment. The factors and levels are shown in Table 10. Seventeen portions of mulberry leaves, lotus leaves, mustard seeds, and licorice slices were weighed according to the weight percentages. Water was added for reflux extraction according to the experimental scheme in Table 9. The extracts were filtered, and the filtrates were combined to obtain the extracts. The volumes of the extracts were recorded. The experimental scheme and results are shown in Table 11.

[0047] Content determination of the five components: Refer to the content determination method under "2.1" above, take an appropriate amount of each extract, prepare the test solution, inject the sample for determination, record the peak area, and calculate the content of the five components in the test sample.

[0048] Determination of extract yield: Accurately measure 25 mL of each aqueous extract, place it in an evaporating dish that has been dried to constant weight, evaporate to dryness in a water bath, then dry in a drying oven at 105℃ for 3 h, cool in a desiccator for 30 min, and weigh quickly to determine the extract yield. Extract yield = (W3-W2)V / 25W1×100%; where W1, W2, and W3 are the mass of the aqueous extract slices, the mass of the constant-weight empty evaporating dish, and the total mass of the dried extract and the evaporating dish, respectively, and V is the total volume of the aqueous extract.

[0049] Table 10 Factors and Levels of Extraction Process

[0050] Table 11 Box-Behnken Test Protocol and Results

[0051] 2.3.3, AHP-entropy weight mixed weighting method comprehensive evaluation 2.3.3.1, AHP method According to the prescription compatibility rule and the content of the components, the priority order of the indexes can be determined as rutin > nuciferine > sinapine > glycyrrhizin > glycyrrhizic acid > the yield of extract, the judgment priority matrix is constructed, see Table 12. The weight coefficients W1 of rutin, nuciferine, sinapine, glycyrrhizin, glycyrrhizic acid and the yield of extract are 46.275%, 24.338%, 11.297%, 6.903%, 6.903% and 4.284% respectively; the consistency check result shows that CR = 0.022 < 0.1, the AHP weighting is reasonable.

[0052] Table 12 AHP judgment priority matrix

[0053] 2.3.3.2, entropy weight method The test results are processed by the standard deviation method, the evaluation index matrix is established, which is converted into a probability matrix, and the objective weight coefficients W2 of rutin, nuciferine, sinapine, glycyrrhizin, glycyrrhizic acid and the yield of extract are calculated as 19.328%, 16.282%, 15.442%, 18.405%, 17.465% and 13.077% respectively.

[0054] 2.3.3.3, comprehensive score According to the formula , the mixed weighting weight coefficients W of rutin, nuciferine, sinapine, glycyrrhizin, glycyrrhizic acid and the yield of extract are calculated as 50.57%, 22.40%, 9.86%, 7.18%, 6.82% and 3.17% respectively. Therefore, the comprehensive score = (rutin content / maximum value of rutin content x 50.57% + nuciferine content / maximum value of nuciferine content x 22.40% + sinapine content / maximum value of sinapine content x 9.86% + glycyrrhizin content / maximum value of glycyrrhizin content x 7.18% + glycyrrhizic acid content / maximum value of glycyrrhizic acid content x 6.82% + the yield of extract / the maximum value of the yield of extract x 3.17%) x 100.

[0055] 2.3.4, Box-Behnken response surface experiment data analysis The comprehensive score calculated by AHP-entropy weight mixed weighting method is taken as the response value, the Design-Expert 11 software is used to fit the mathematical model of CPPs and CQAs, and the regression equation is obtained: Y = 78.23 + 8.89 A -2.28 B + 21.68C -0.1075 AB +2.30 AC -2.95 BC -3.16 A 2 -2.98 B 2 -9.23 C 2 The results of the analysis of variance are shown in Table 13. The contour lines and 3D plots of the comprehensive score are shown in Table 13. Figure 2 The model has significant variance ( P <0.01), the lack-of-fit term is not significant ( P A correlation coefficient > 0.05 indicates that the model is statistically significant and can map the functional relationship between CPPs and the overall score in water extraction processes; the fitting correlation coefficient R0 2 The adjusted coefficient of determination is 99.22%, Radj. 2 The accuracy rate was 98.22%, indicating a good fit between the actual and predicted values ​​of the model, making it suitable for predicting the overall score. The degree of influence of each factor on the extraction process is as follows: C > B > A ,factor A , C It has a highly significant impact on the water extraction process. P <0.01), factor B Significant impact on water extraction process ( P <0.05); interaction factors BC of P A value less than 0.05 indicates a significant interaction between extraction time and the number of extractions on the water extraction process; quadratic term A 2 , B 2 and C 2 All have significant effects ( P <0.05), while other values ​​had no significant effect.

[0056] Table 13 Analysis of Variance of the Box-Behnken Experiment

[0057] 2.3.5 Establishment of Design Space According to the Box-Behnken experimental results, set the comprehensive score ≥ 88 as the optimization goal, and establish the design space by means of DesignExpert 13 software. Since there is uncertainty in the boundary of the design space, therefore, 95% confidence interval is added in the design space for optimization. At the same time, combined with the controllability of production, the regular region in the design space is selected to constitute the operation space to facilitate the actual production, and finally the operation space is obtained as follows: water addition amount 9.5~12 times, extraction time 1~1.7 h, extraction times 3 times. The Overlay plot result of the design space of water extraction process is shown in Figure 3 , the light yellow area is the optimized design space, the rectangular area in it is the operation space; the dark yellow area is the risk area, there is 5% probability that cannot meet the target; the gray area cannot reach the optimization target.

[0058] 2.3.6 Process verification Randomly select 3 different points in the design space for extraction process verification, according to the process conditions, add water and reflux extraction, and measure each evaluation index according to the above method, and calculate the comprehensive score according to the AHP-entropy weight mixed weighting method, the results are shown in Table 14. The results show that the comprehensive scores of the points in the design space are all greater than 82.30, while the comprehensive scores of the points outside the design space are less than 82.30, indicating that the operation in the design space can guarantee the stability of the water extraction process and the quality of the water extract; at the same time, the comprehensive score is not much different from the predicted value of the model, indicating that the prediction effect of the design space is good, and the process parameters are stable and feasible.

[0059] Table 14 Process verification results (n=3)

[0060] Third, the beneficial effects of the present application 3.1, content determination The present application establishes a high performance liquid chromatography method for simultaneously determining the contents of representative components in the drug-food homologous beverage formula, including flavonoids, alkaloids, triterpenoid saponins and other types of components, and the quality control covers a wide range. According to the characteristics of active ingredients rutin, norephedrine, mustard oil, glycyrrhizin and glycyrrhizic acid, the chromatographic conditions are screened and optimized, and the methodology is verified. The method is simple and easy to operate, the sample solution is simple and convenient to process, each characteristic chromatographic peak is well separated, the method is specific, accurate, precise and reproducible. The method can realize accurate quantification of five active ingredients in the product and comprehensive evaluation and quality control of the product. In addition, compared with individual analysis of each component, the liquid phase gradient elution method can effectively reduce the number of sample injections and analysis time, shorten the experimental time, improve the detection efficiency, save reagent consumables and labor cost, reduce cost and increase efficiency, save energy and protect the environment. At the same time, the feasibility and accuracy of the method are further verified in the Box-Behnken response surface design optimization of the extraction process of the formula.

[0061] 3.2, extraction process optimization The present application introduces the concept of "quality by design" into the extraction process development of the drug-food homologous product, establishes a complete research method, and obtains an operable and robust parameter range: water addition amount 9.5~12 times, extraction time 1~1.7 h, extraction times 3 times. As long as the operation is within this region, the product meeting the predetermined quality standard can be continuously and stably produced. Compared with the traditional optimization method of single "optimal parameter point", it is more scientific, stable and efficient, and the process operation is more flexible, which is suitable for large-scale factory production. Through process verification comparison, the contents of rutin, norephedrine, mustard oil, glycyrrhizin and glycyrrhizic acid and the extract yield can meet the production requirements. The process is reasonable, stable and feasible, and can be used for standardized production and quality control of the product.

[0062] In summary, the extraction process and the simultaneous determination method of five components in the design of the present application are operable and repeatable. Through multi-index simultaneous determination, the method provides important method guarantee for realizing effective extraction and quality control of the drug-food homologous beverage, and provides a reproducible technical framework for modern quality evaluation of drug-food homologous beverage, promotes the transformation from "single component control" to "overall quality control" in the industry, and has practical popularization and application value.

[0063] It is to be explained that the above given is only an embodiment, is used to explain the specific implementation of the present application, and is not used to limit the protection scope of the present application, any equivalent replacement means is made in essence with the same technical solution of the present application, and the present application can also be used for determination of other solid beverage ingredients. As long as the method of the present application, or similar method, belongs to the protection scope of the application.

Claims

1. A method for simultaneously determining the content of five components in a food-medicine homology beverage, characterized in that, The five components are rutin, lotus leaf alkaloid, sinigrin, glycyrrhizin, and glycyrrhizic acid. The determination method includes the following steps: S1. Take 30 parts of mulberry leaves, 30 parts of lotus leaves, 15 parts of mustard seeds and 15 parts of licorice, add 9.5 to 12 times the amount of water and extract 3 times, each time for 1 to 1.7 hours. Combine the extracts to obtain the aqueous extract. S2. Preparation of mixed reference solutions: Accurately weigh appropriate amounts of rutin, nuciferine, sinigrin thiocyanate, glycyrrhizin, and glycyrrhizic acid monoammonium salt reference standards, place them in volumetric flasks, and dilute to volume with methanol to obtain stock solutions with concentrations of: rutin 0.2432 mg / mL, nuciferine 0.0644 mg / mL, sinigrin 0.1268 mg / mL, glycyrrhizin 0.0632 mg / mL, and glycyrrhizic acid 0.1220 mg / mL; accurately measure 0.5, 1, 2, 3, 4, and 5 mL of the above solutions, dilute with methanol, and obtain mixed reference solutions of different concentrations; S3. Preparation of the test solution: Take the above extract, centrifuge at 12000 r / min for 10 min, take the supernatant and filter it through a 0.22 μm microporous membrane, take the filtrate, and the test solution is obtained. S4, Determination of the content of five components: Take the above mixed reference solution and test solution, inject them into the high performance liquid chromatograph, and determine the peak area of ​​each peak under specific chromatographic conditions and gradient elution program. Calculate the results to achieve simultaneous determination of the five components. The specific chromatographic conditions are as follows: the column is an Agilent ZORBAX SB-C18 column with dimensions of 4.6 × 250 mm and 5 μm; gradient elution is performed using acetonitrile (A) - 0.1% phosphoric acid water (B) as the mobile phase; the injection volume is 10 μL; the flow rate is 1 mL / min; the column temperature is 30℃; the detection wavelength is 326 nm for sinigrin; and the detection wavelength is 270 nm for rutin, nuciferine, glycyrrhizin, and glycyrrhizic acid. The theoretical plate number, calculated based on the rutin peak, should not be less than 5000. The gradient elution program is as follows: 0-15 minutes, mobile phase acetonitrile (A) 10.5%; 15-45 minutes, mobile phase acetonitrile (A) 10.5 → 13%; 45-46 minutes, mobile phase acetonitrile (A) 13 → 22%; 46-60 minutes, mobile phase acetonitrile (A) 22 → 23%; 60-61 minutes, mobile phase acetonitrile (A) 23 → 32%; 61-80 minutes, mobile phase acetonitrile (A) 32 → 40%.

2. The application of the method described in claim 1 in the quality testing of food and medicine homologous beverages.

3. The application of the method described in claim 1 in the optimization of the process for extracting active ingredients in the production of food and medicine homologous beverages.

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