A method for determining the content of oral liquid for stimulating appetite and promoting digestion

Abstract

The present invention discloses a method for determining the content of an oral liquid for stimulating appetite and promoting digestion, and belongs to the technical field of detection of components of traditional Chinese medicine. The method of the present invention comprises the following technical steps: (1) preparation of a reference solution; (2) preparation of a test solution; (3) liquid chromatography conditions; and (4) determination. The method of the present invention adds the determination of nobiletin on the basis of the original quality standard that only determines the single component of hesperidin in tangerine peel, that is, the content of tangerine peel is calculated as the total amount of hesperidin and nobiletin; and adds the determination of the content of forsythia suspensa, and the content of forsythia suspensa is calculated as the total amount of forsythiaside A and forsythin. The method makes the control of drug quality more comprehensive and effective, so that the drug quality reaches the purpose of stable, controllable, efficient and safe standards. The method is simple, has high accuracy, good stability and good reproducibility, and is conducive to effective control of the quality of the oral liquid and improves the safety and stability of the oral liquid.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine component detection, and particularly relates to a method for determining the content of an appetizer-stimulating and digestion-promoting oral liquid. Background Art

[0002] The oral liquid for stimulating appetite and digestion is an oral liquid made from seven drugs, namely, Cistanche deserticola, hawthorn (fried), malt (fried), Liushenqu (fried), betel nut, tangerine peel, and forsythia, through water boiling and alcohol extraction and concentration. Functions and indications: digestion and relieving stagnation, spleen and appetite. It is used for children with anorexia, with symptoms of not being greedy for food for a long time, loss of appetite, even refusal to eat, pale complexion, pale lips and tongue, thin body, loose stools or stools mixed with undigested food, sour and smelly. The drug is prepared from a traditional prescription, and the drugs in the prescription have good synergistic effects. In the long-term clinical use process, it has been proved to have significant clinical efficacy, and the drug is deeply trusted by patients, especially parents of pediatric patients. Tangerine peel and forsythia in the prescription are important active ingredients of this product. Tangerine peel is a well-known raw material for medicine and food. It was recorded in the Compendium of Materia Medica more than 400 years ago. It contains active substances such as flavonoids, volatile oils, alkaloids, polysaccharides, etc., and has the effects of strengthening the spleen and appetite, regulating qi and digestion, and removing dampness and phlegm. Forsythia suspensa is one of the commonly used Chinese medicinal materials in my country. It contains active substances such as phenylethanol glycosides, lignans, and terpenes. The multi-component and multi-target characteristics of Forsythia suspensa make it have multiple pharmacological activities, such as antibacterial, antiviral, and anti-inflammatory.

[0003] There are many ingredients in the oral liquid for aphrodisiac and digestion, and the system is complex. The instability and uncontrollability of the quality can easily lead to the instability and uncontrollability of the therapeutic effect of the oral liquid. The content determination of the current quality standard of the oral liquid for aphrodisiac and digestion is relatively simple, and only the single component of tangerine peel is determined, which cannot comprehensively monitor the quality of the drug, which is extremely unfavorable for market supervision and clinical medication guidance. The current quality standard of the oral liquid for aphrodisiac and digestion is included in the National Drug Standard Promulgation-New Chinese Medicine Drug Regularization-2019 (National Bureau Single-page Standard), standard number: WS-398 (Z-054)-2019Z. For the content determination of tangerine peel, high performance liquid chromatography is used to determine only a single component, hesperidin. The test ingredients and components are relatively simple, and other components in the prescription are not involved, making it difficult to comprehensively evaluate the quality of the drug. In addition, the test operation is cumbersome and the reproducibility is poor. The extraction method uses the extraction method, which has a large deviation, and the extraction solvent used is n-butanol, which will produce an unpleasant irritating odor.

[0004] In order to improve the controllability of product quality and provide a scientific basis for scientific utilization and drug supervision by medical and other related departments, the present invention conducts innovative research on the content determination of dried tangerine peel and forsythia suspensa in the appetizer and digestion oral liquid. 28 H 34 O 15) was added with nobiletin (C 21 H 22 O8) detection, that is, the content of dried orange peel is measured by the total amount of hesperidin and nobiletin; the content of Forsythia suspensa was also determined, and the content was measured by forsythiaside A (C 27 H 34 O 11 ) and forsythiaside (C 29 H 36 O 15 ) to make the control of drug quality more comprehensive and effective, so that the drug quality can reach the goal of stable, controllable, efficient and safe standards. Summary of the invention

[0005] In view of the above problems, the present invention provides a method for determining the content of an appetizer and digestion-promoting oral liquid, which determines the contents of dried tangerine peel and forsythia in the oral liquid, thereby achieving the purpose of controlling the quality of the drug more comprehensively and effectively.

[0006] The present invention is achieved through the following technical solutions:

[0007] A method for determining the content of an oral liquid for stimulating appetite and promoting digestion, comprising the following steps:

[0008] (1) Preparation of reference solution

[0009] ① Tangerine peel: Accurately weigh appropriate amounts of hesperidin reference substance and nobiletin reference substance, add methanol to make a mixed solution containing 80 μg of hesperidin and 25 μg of nobiletin per 1 mL, and obtain reference substance solution I;

[0010] ②Forsythia: Accurately weigh appropriate amounts of forsythiaside reference substance and forsythiaside A reference substance, add 50% ethanol to prepare a mixed solution containing 150 μg forsythiaside and 20 μg forsythiaside A per 1 mL, and obtain reference substance solution II;

[0011] (2) Preparation of test solution

[0012] ① Tangerine peel: accurately measure 10 mL of the test sample of the appetizer and digestion oral solution, add 10 mL of methanol to dissolve, ultrasonicate for 10 min, fix the volume, shake well, filter through a 0.45 μm filter membrane, and take the filtrate to obtain the test sample solution I;

[0013] ②Forsythia suspensa: Accurately measure 5 mL of the test sample of the appetizer and digestion oral solution, add 50% ethanol to dilute, make up to volume, and shake well to obtain the test sample solution II;

[0014] (3) Liquid chromatography conditions

[0015] ① Tangerine peel: octadecylsilane bonded silica gel was used as filler, acetonitrile was used as mobile phase A, 0.1% phosphoric acid aqueous solution was used as mobile phase B, gradient elution was performed, the detection wavelength was 209 nm, the flow rate was 1.0 mL / min, the column temperature was 30°C, and the theoretical plate number was calculated based on the hesperidin base peak and was not less than 5000;

[0016] ② Forsythia: octadecylsilane bonded silica gel was used as filler, methanol was used as mobile phase A, 0.1% formic acid aqueous solution was used as mobile phase B, gradient elution was performed, the detection wavelength was 228nm, the flow rate was 0.7mL / min, the column temperature was 30℃, and the theoretical plate number was calculated based on the forsythiaside A base peak and was not less than 5000;

[0017] (4) Determination

[0018] ① Tangerine peel: Accurately pipette 10 μL of reference solution Ⅰ and test solution Ⅰ, respectively, inject into liquid chromatograph, and measure to obtain the content of hesperidin and nobiletin in test solution Ⅰ;

[0019] ② Forsythia: Accurately pipette 10 μL of reference solution II and test solution II respectively, inject into liquid chromatograph and measure to obtain the contents of forsythiaside and forsythiaside A in test solution II.

[0020] Furthermore, in step (3), the specific procedure of the gradient elution in step ① is: 0-12 min, 20% A; 12-18 min, 20→45% A; 18-30 min, 45% A; 30-35 min, 45→60% A.

[0021] Furthermore, in step (3), the specific procedure of the gradient elution in step ② is: 0-30 min, 25→48% A; 30-40 min, 48→60% A; 40-41 min, 60→80% A; 41-50 min, 80% A; 50-51 min, 80→25% A; 51-58 min, 25% A.

[0022] Furthermore, in step (2), the power of the ultrasonic treatment is 200 W and the frequency is 37 kHz.

[0023] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0024] 1. The content determination method of the oral liquid for stimulating appetite and digestion of the present invention is that the original quality standard only determines the hesperidin (C 28 H 34 O 15 ) was added to the single component. 21 H 22O8), that is, the content of dried orange peel is measured by the total amount of hesperidin and nobiletin; and the content of Forsythia suspensa is measured by forsythiaside A (C 27 H 34 O 11 ) and forsythiaside (C 29 H 36 O 15 ) is measured. This determination method makes the control of drug quality more comprehensive and effective, so that the drug quality reaches the standard of stability, controllability, efficiency and safety.

[0025] 2. Compared with the extraction method using n-butanol in the existing standard, the present invention uses methanol and 50% ethanol to ultrasonically treat the test solution. The extraction method is simple, easy to operate, has better reproducibility, small error, and less unpleasant irritating odor.

[0026] 3. The determination method of the present invention is simple, highly accurate, stable and reproducible, and can more comprehensively and accurately evaluate the quality of the oral liquid for stimulating appetite and digestion, which is beneficial to effectively control the quality of the oral liquid, improve the safety and stability of the oral liquid, and provide a reliable method for scientifically evaluating and effectively controlling the quality of the oral liquid. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The linear chromatograms of hesperidin and nobiletin in the oral liquid for stimulating appetite and digestion in the present invention are shown in the figure above, wherein the upper figure is the spectrum of curve 1 and the lower figure is the spectrum of curve 6.

[0028] Figure 2 The above figure is the precision chromatogram of hesperidin and nobiletin in the oral liquid for stimulating appetite and digestion in the present invention. The upper figure is the second injection spectrum, and the lower figure is the sixth injection spectrum.

[0029] Figure 3 The above diagram is the repetitive chromatogram of hesperidin and nobiletin in the oral liquid for stimulating appetite and digestion in the present invention. The upper diagram is the second sample spectrum, and the lower diagram is the sixth sample spectrum.

[0030] Figure 4 The stability chromatograms of hesperidin and nobiletin in the oral liquid for stimulating appetite and digestion in the present invention are shown in the figure above for the 0-hour spectrum and below for the 51-hour spectrum.

[0031] Figure 5 The chromatograms are the recovery rates of hesperidin and nobiletin in the oral liquid for stimulating appetite and promoting digestion in the present invention. The upper figure is the first recovery rate solution spectrum, and the lower figure is the sixth recovery rate solution spectrum.

[0032] Figure 6 The chromatogram is of the solvent methanol in the oral liquid for stimulating appetite and promoting digestion.

[0033] Figure 7 This is a specificity test of dried tangerine peel in the oral liquid for stimulating appetite and digestion in the present invention, and the chromatogram of the negative solution of dried tangerine peel is missing.

[0034] Figure 8 The linear chromatograms of forsythiaside A and forsythiaside in the oral liquid for stimulating appetite and digestion in the present invention are shown in the upper figure, which is the curve 1 spectrum and the lower figure is the curve 5 spectrum.

[0035] Fig. 9 The above figure is the precision chromatogram of forsythiaside A and forsythiaside in the oral liquid for stimulating appetite and digestion in the present invention. The upper figure is the second injection spectrum, and the lower figure is the sixth injection spectrum.

[0036] Fig.10 It is the repetitive chromatogram of forsythiaside A and forsythiaside in the oral liquid for stimulating appetite and digestion in the present invention. The upper figure is the chromatogram of the second sample, and the lower figure is the chromatogram of the sixth sample.

[0037] Fig.11 The stability chromatograms of forsythiaside A and forsythiaside in the oral liquid for stimulating appetite and digestion in the present invention are shown in the figure above for 0 hours and the figure below for 24 hours.

[0038] Fig.12 The chromatograms are the recovery rates of forsythiaside A and forsythiaside in the oral liquid for stimulating appetite and digestion in the present invention. The upper figure is the first recovery rate solution spectrum, and the lower figure is the sixth recovery rate solution spectrum.

[0039] Fig.13 It is a chromatogram of the solvent 50% ethanol in the oral liquid for stimulating appetite and promoting digestion.

[0040] Fig.14 This is a specificity test for the Forsythia suspensa in the oral liquid for stimulating appetite and digestion in the present invention, and the chromatogram of the Forsythia suspensa negative solution is missing. DETAILED DESCRIPTION

[0041] The present invention is further described in detail below by way of examples. These examples are only used to illustrate the present invention and do not limit the protection scope of the present invention.

[0042] Example 1

[0043] The oral liquid for stimulating appetite and promoting digestion comprises 7 traditional Chinese medicines, including Cistanche deserticola, hawthorn (fried), malt (fried), Liushenqu (fried), betel nut, tangerine peel and forsythia.

[0044] The present invention uses the oral liquid for stimulating appetite and digestion produced by Guangxi Huibaoyuan Pharmaceutical Technology Co., Ltd. to conduct experiments, and uses liquid chromatography to determine the total content of hesperidin and nobiletin in tangerine peel; and determines the total content of forsythiaside A and forsythin in forsythia, comprising the following steps:

[0045] 1. Tangerine peel

[0046] Step 1: Liquid chromatography conditions

[0047] Octadecylsilane bonded silica gel is used as filler, and acetonitrile-0.1% phosphoric acid solution, methanol-0.1% phosphoric acid solution, and acetonitrile-0.2% phosphoric acid solution in a volume ratio of 20-60:80-40, and 20-60:80-40, respectively, are used as mobile phases; an ultraviolet detector is used, and the detection wavelength is 200-400 nm; the flow rate is 1.0 mL / min; the column temperature is 30° C.; and the theoretical plate number calculated based on the hesperidin base peak should be no less than 5000.

[0048] (1) Determine wavelength and select the optimal wavelength

[0049] Weigh an appropriate amount of hesperidin and nobiletin reference substances, dissolve them in methanol, and perform spectral scanning in the range of 200-400nm. The results show that hesperidin and nobiletin have maximum absorption at wavelengths of 209nm and 284nm. Although 209nm is an end absorption, the sample baseline is not much different from 284nm, and the response of the substance to be tested is higher than 284nm, so the detection wavelength is determined to be 209nm.

[0050] (2) Chromatographic conditions and system suitability test: Acetonitrile-0.2% phosphoric acid solution

[0051] Different mobile phase systems including acetonitrile-0.1% phosphoric acid solution, methanol-0.1% phosphoric acid solution and acetonitrile-0.2% phosphoric acid solution were investigated. The chromatographic peak of the analyte in methanol-0.1% phosphoric acid solution was delayed, the detection time was prolonged, and the baseline fluctuated greatly at 209nm. The peak shape and baseline of the analyte in acetonitrile-0.1% phosphoric acid solution and acetonitrile-0.2% phosphoric acid solution were not much different, and both could be completely separated from the impurity peaks. Therefore, acetonitrile-0.1% phosphoric acid solution was selected as the mobile phase in this experiment, and gradient elution was performed according to the provisions in Table 1.

[0052] Table 1 Gradient elution program of tangerine peel mobile phase

[0053]

[0054] (3) Durability test

[0055] The effects of two different brands of liquid chromatography and three different brands of chromatographic columns on the separation of hesperidin, nobiletin and other impurity peaks in the sample were investigated. All of them can effectively separate the components to be tested and impurities. Therefore, the theoretical plate number is proposed to be calculated based on the hesperidin peak and shall not be less than 5000. The results are shown in Table 2.

[0056] Table 2 System suitability test

[0057]

[0058] Step 2: Preparation of reference solution

[0059] Accurately weigh appropriate amounts of hesperidin reference substance and nobiletin reference substance, accurately weigh, and add methanol to prepare a mixed solution containing 80 μg of hesperidin and 25 μg of nobiletin per 1 mL.

[0060] Step 3: Preparation of test solution

[0061] Accurately measure 1-10 mL of the test sample, place it in a 25-100 mL volumetric flask, add appropriate amount of methanol, 70% methanol, 50% methanol, or ethanol to dissolve it, shake and ultrasonically treat it for 10-60 minutes, then add the corresponding solvent to the scale, shake well, filter through a 0.45 μm filter membrane, and take the filtrate to obtain the product.

[0062] (1) Investigation of extraction methods

[0063] Ultrasonication for 10 min and shaking at 60°C for 10 min were investigated. The results showed that the total amount of hesperidin and nobiletin was the highest during ultrasonic extraction, so ultrasonic extraction was selected as the sample sampling method. The results are shown in Table 3.

[0064] Table 3 Calculation of hesperidin and nobiletin contents by different extraction methods

[0065]

[0066]

[0067] (2) Investigation of sampling quantity

[0068] The sampling volumes of 1 mL, 3 mL, 5 mL, and 10 mL were investigated respectively. The results showed that when the sampling volume was 10 mL, the total amount of hesperidin and nobiletin was the highest, so 10 mL was selected as the sample sampling volume. The results are shown in Table 4.

[0069] Table 4 Calculation of hesperidin and nobiletin contents in different sampling amounts

[0070]

[0071] (3) Investigation of solvents

[0072] Methanol and ethanol were investigated respectively, and the results showed that the total amount of hesperidin and nobiletin was the highest when extracted with methanol, so methanol was selected as the sample extraction solvent. The results are shown in Table 5.

[0073] Table 5 Calculation of the content of hesperidin and nobiletin in different solvents

[0074]

[0075] (4) Investigation of solvent concentration

[0076] Methanol, 70% methanol and 50% methanol were investigated respectively. The results showed that the total amount of hesperidin and nobiletin was the highest when methanol was used for extraction. Therefore, methanol was selected as the sample extraction solvent. The results are shown in Table 6.

[0077] Table 6 Calculation of the content of hesperidin and nobiletin at different methanol concentrations

[0078]

[0079] (5) Investigation of ultrasonic treatment time

[0080] The ultrasonic treatment was conducted for 10 min, 20 min, 30 min and 60 min respectively. The results showed that the total amount of hesperidin and nobiletin was the highest when the ultrasonic treatment was conducted for 10 min. Therefore, the ultrasonic treatment for 10 min was selected as the sample extraction time. The results are shown in Table 7.

[0081] Table 7 Calculation of hesperidin and nobiletin contents at different ultrasound times

[0082]

[0083] (6) Selection of dilution multiple

[0084] The results of dilutions of 2.5 times, 5 times, and 10 times showed that when the volume was fixed to 25 mL (diluted 2.5 times), the total amount of hesperidin and nobiletin was the highest, so a dilution of 2.5 times (fixed to 25 mL) was selected as the sample dilution multiple. The results are shown in Table 8.

[0085] Table 8 Calculation of hesperidin and nobiletin content at different dilution multiples

[0086]

[0087] The above test results show that the preparation of the preferred test solution is as follows: accurately measure 10 mL of the test sample, place it in a 25 mL volumetric flask, add 10 mL of methanol to dissolve it, ultrasonically treat it (power: 200 W, frequency: 37 KHZ) for 10 minutes, add methanol to the scale, shake well, filter through a 0.45 μm filter membrane, and take the filtrate to obtain the solution.

[0088] Step 4: Determination

[0089] Accurately pipette 10 μL of the reference solution and the test solution respectively, inject into the liquid chromatograph, and measure to obtain the measurement results.

[0090] Methodological study on the detection method in the present invention:

[0091] (1) Linearity: Accurately pipette the mixed reference solution of hesperidin and nobiletin, and dilute it step by step with methanol to form a series of mixed reference solutions of different concentrations. According to the above chromatographic conditions, 10 μL of each solution was injected and measured (n=6). The injection concentration of the reference solution (μg / mL) was used as the abscissa (X) and the peak area was used as the ordinate (Y). The regression equations were calculated to examine the linear relationship between hesperidin and nobiletin. The results are shown in Table 1. Figure 1 and Tables 9 and 10.

[0092] Table 9 Investigation of linear relationship of hesperidin

[0093]

[0094] Table 10 Investigation of linear relationship of nobiletin

[0095]

[0096] The results showed that the linear relationship was good in the range of hesperidin from 6.34 to 317.2 μg / mL and that of nobiletin from 1.94 to 97.21 μg / mL.

[0097] (2) Precision: Under the above chromatographic conditions, take the same sample solution and inject it into the liquid chromatograph 6 times continuously. The 6 measurement results are used for evaluation. The acceptable range is RSD of peak area <3.0%. The results are shown in Figure 2 and Table 11.

[0098] Table 11 Precision test

[0099]

[0100] The results in the above table show that this method has good precision.

[0101] (3) Repeatability: Take an appropriate amount of the test solution and mix it evenly. Take 6 samples in parallel and prepare repeatable sample solutions according to the test solution preparation method. Determine them separately under the above chromatographic conditions. The acceptable range is RSD (relative standard deviation) of the content <3.0%. The results are shown in Figure 3 and Tables 12 and 13.

[0102] Table 12 Hesperidin repeatability test

[0103]

[0104] Table 13 Nobiletin repeatability test

[0105]

[0106] The results in the above table show that this method has good repeatability.

[0107] (4) Stability: Under the above chromatographic conditions, take the same sample solution and measure it at 0, 1, 3, 12, 24, 35, and 51 hours respectively. The 7 measurement results are used for evaluation. The acceptable range is the RSD (relative standard deviation) of the peak area < 3.0%. The results are shown in Figure 4 and Table 14.

[0108] Table 14 Stability test

[0109]

[0110] The test results in the above table show that this product has good stability within 51 hours.

[0111] (5) Recovery rate: Take 6 portions of the test sample with known content, accurately add a certain amount of mixed reference solution to each portion, prepare recovery solution in the same way as the test sample solution, measure under the above-mentioned chromatographic conditions, and calculate the recovery rate.

[0112] Recovery rate % = (CA) / B × 100%

[0113] Where: A is the amount of the component to be measured contained in the test sample; B is the amount of reference substance added; C is the actual measured value.

[0114] Standard requirements: recovery rate 95% to 105%; RSD < 3.0%, see the results Figure 5 and Table 15.

[0115] Table 15 Recovery test

[0116]

[0117] The results in the above table show that the recovery rate is good.

[0118] (6) Specificity: Under the above chromatographic conditions, accurately pipette 10 μL of the mixed reference solution, test solution, solvent methanol solution and negative sample solution lacking tangerine peel into the liquid chromatograph and record the chromatogram. Requirement: There should be no impurity peak interference at the position corresponding to the chromatographic peak of hesperidin and nobiletin reference substances in the solvent methanol solution and the negative sample lacking tangerine peel.

[0119] The results showed that the solvent methanol solution and the negative sample lacking tangerine peel were at the corresponding position of the chromatographic peaks of hesperidin and nobiletin reference substances, without any interference from impurity peaks. This indicated that the negative sample had no interference and had strong specificity. Figure 6 , 7 .

[0120] 2. Forsythia

[0121] Step 1: Liquid chromatography conditions

[0122] Octadecylsilane bonded silica gel is used as filler, and mobile phases include methanol-water with a volume ratio of 25-80:75-20, methanol-0.1% phosphoric acid with a volume ratio of 25-80:75-20, methanol-0.1% acetic acid with a volume ratio of 25-80:75-20, acetonitrile-0.1% formic acid with a volume ratio of 25-80:75-20, methanol-0.2% formic acid with a volume ratio of 25-80:75-20, and methanol-0.1% formic acid solution with a volume ratio of 25-80:75-20; ultraviolet detector, detection wavelength 200-400nm; flow rate 0.6-1.0mL / min; column temperature 30℃; theoretical plate number calculated based on forsythiaside A base peak should be no less than 5000.

[0123] (1) Determine wavelength and select the optimal wavelength

[0124] Weigh appropriate amount of forsythiaside A and forsythiaside reference substance, dissolve them in 50% ethanol, and perform spectral scanning in the range of 200-400nm. The results show that forsythiaside has maximum absorption at wavelengths of 228nm and 277nm, and forsythiaside A has maximum absorption at wavelengths of 329nm. The content of forsythiaside A in the oral liquid for stimulating appetite and digestion is relatively high, which is 5-8 times that of forsythiaside, so its detection wavelength is considered to be set at 228nm or 277nm, which is conducive to dual content determination. However, at a wavelength of 277nm, there are many impurity peaks adjacent to the peak of forsythiaside A, which interferes, so 228nm is used as the determination wavelength after comprehensive consideration.

[0125] (2) Chromatographic conditions and system suitability test

[0126] Different mobile phase systems such as methanol-water, methanol-0.1% phosphoric acid, methanol-0.1% acetic acid, acetonitrile-0.1% formic acid, methanol-0.2% formic acid, and methanol-0.1% formic acid were investigated. Results When methanol-0.1% formic acid, methanol-0.1% phosphoric acid, and methanol-0.1% acetic acid were used as mobile phases, the separation and peak shape of the reference and sample were good. When methanol-0.1% formic acid was used as the mobile phase, the peak height of the sample was good, the peak area was large, and the content was high. The results are shown in Table 16. Therefore, the mobile phase was determined to be methanol-0.1% formic acid, and gradient elution was performed according to the provisions in Table 17.

[0127] Table 16 Investigation of mobile phase

[0128]

[0129] Table 17 Forsythia suspensa mobile phase gradient elution program

[0130]

[0131] (3) Durability test

[0132] The effects of two different brands of liquid chromatography and three different brands of chromatographic columns on the separation of forsythiaside A, forsythiaside and other impurity peaks in the sample were investigated. All of them can effectively separate the components to be tested and impurities. Therefore, the theoretical plate number is proposed to be calculated based on the forsythiaside A peak and shall not be less than 5000. The results are shown in Table 18.

[0133] Table 18 System suitability test

[0134]

[0135] Step 2: Preparation of reference solution

[0136] Take appropriate amount of forsythiaside reference substance and forsythiaside A reference substance, weigh accurately, add 50% ethanol to prepare a mixed solution containing 150 μg forsythiaside and 20 μg forsythiaside A per 1 mL.

[0137] Step 3: Preparation of test solution

[0138] Accurately measure 5 mL of the test sample, place it in a 10 mL volumetric flask, and add 50% ethanol to dilute to the mark.

[0139] (1) Investigation of extraction methods

[0140] Direct volume fixation, ultrasonic treatment for 10 min and ultrasonic treatment for 30 min were investigated. The results showed that the total amount of forsythiaside A and forsythiaside was the highest during ultrasonic extraction, so ultrasonic extraction was selected as the sample sampling method. The results are shown in Table 19.

[0141] Table 19 Calculation of the content of hessyphilitic acid A glycoside and forsythiaside by different extraction methods

[0142]

[0143] (2) Investigation of solvents

[0144] Methanol, ethanol, 50% methanol and 50% ethanol were investigated respectively. The results showed that the four extraction solvents had similar extraction effects on forsythiaside A and forsythin, so 50% ethanol was selected as the extraction solvent because it is less harmful to the human body and has a lower economic cost. The results are shown in Table 20.

[0145] Table 20 Calculation of the content of forsythiaside A and forsythiaside in different solvents

[0146]

[0147] (3) Selection of dilution multiple

[0148] The dilutions of 1.25 times, 2 times, and 5 times were investigated respectively. The results showed that when the sample volume was 5 mL and the volume was fixed to 10 mL (diluted 2 times), the total amount of forsythiaside A and forsythiaside was the highest. Therefore, a dilution of 2 times (fixed to 10 mL) was selected as the sample dilution multiple. The results are shown in Table 21.

[0149] Table 21 Calculation of forsythiaside A and forsythiaside content at different dilution multiples

[0150]

[0151] (4) Investigation of different flow rates

[0152] Dilutions of 0.6, 0.7, and 1.0 mL / min were investigated respectively. The results showed that when the flow rate was 0.7 mL / min, the retention times of forsythiaside A and forsythiaside in the sample were moderate and well separated from other impurity peaks, so the flow rate was selected as 0.7 mL / min.

[0153] The above test results in the preparation of the preferred test solution: take the product under the item of filling amount difference, mix well, accurately measure 5mL, put it in a 10mL volumetric flask, add 50% ethanol to dilute to the scale, shake well, filter through a 0.45μm filter membrane, and take the filtrate to obtain.

[0154] Step 4: Determination

[0155] Accurately pipette 10 μL of the reference solution and the test solution respectively, inject into the liquid chromatograph, and measure to obtain the measurement results.

[0156] Methodological study on the detection method in the present invention:

[0157] (1) Linearity: Precisely pipette forsythiaside A and forsythiaside mixed reference solution, dilute with 50% ethanol to form a series of mixed reference solutions of different concentrations, and inject 10 μL of each solution according to the above chromatographic conditions for determination (n=5). With the injection concentration of the reference solution (μg / mL) as the abscissa (X) and the peak area as the ordinate (Y), calculate the regression equations to investigate the linear relationship between forsythiaside A and forsythiaside. The results are shown in Table 1. Figure 8 and Tables 22 and 23.

[0158] Table 22 Investigation of the linear relationship of Forsythiaside A

[0159]

[0160] Table 23 Investigation of the linear relationship of forsythiaside

[0161]

[0162] The results showed that the linear relationship was good in the range of Forsythiaside A from 15.08 to 603.4 μg / mL and in the range of Forsythiaside from 2.063 to 82.52 μg / mL.

[0163] (2) Precision: Under the above chromatographic conditions, take the same sample solution and inject it into the liquid chromatograph 6 times continuously. The 6 measurement results are used for evaluation. The acceptable range is RSD of peak area <3.0%. The results are shown in Fig. 9 and Table 24:

[0164] Table 24 Precision test

[0165]

[0166] The results in the above table show that this method has good precision.

[0167] (3) Repeatability: Take an appropriate amount of the test solution and mix it evenly. Take 6 samples in parallel and prepare repeatable sample solutions according to the test solution preparation method. Determine them separately under the above chromatographic conditions. The acceptable range is RSD (relative standard deviation) of the content <3.0%. For details, see Fig.10 and Tables 25, 25.

[0168] Table 25 Repeatability test of forsythiaside A

[0169]

[0170] Table 26 Forsythiaside repeatability test

[0171]

[0172]

[0173] The results in the above table show that this method has good repeatability.

[0174] (4) Stability: Under the above chromatographic conditions, take the same sample solution and measure it at 0, 2, 4, 8, 16, and 24 hours respectively. The 6 measurement results are used for evaluation. The acceptable range is the RSD (relative standard deviation) of the peak area <3.0%. For details of the results, see Fig.11 and Table 27.

[0175] Table 27 Stability test

[0176]

[0177] The test results in the above table show that this product has good stability within 24 hours.

[0178] (5) Recovery rate: Take 6 portions of the test sample with known content, accurately add a certain amount of mixed reference solution to each portion, prepare recovery solution in the same way as the test sample solution, measure under the above-mentioned chromatographic conditions, and calculate the recovery rate.

[0179] Recovery rate % = (CA) / B × 100%

[0180] Where: A is the amount of the component to be measured contained in the test sample; B is the amount of reference substance added; C is the actual measured value.

[0181] Standard requirements: recovery rate 95% to 105%; RSD < 3.0%, see the results for details Fig.12 and Table 28.

[0182] Table 28 Recovery test

[0183]

[0184]

[0185] The results in the above table show that the recovery rate is good.

[0186] (6) Specificity: Under the above chromatographic conditions, accurately pipette 10 μL of the mixed reference solution, test solution, 50% ethanol solution of solvent and negative sample solution lacking Forsythia suspensa into the liquid chromatograph and record the chromatogram. Requirement: There should be no impurity peak interference at the corresponding position of the chromatographic peak of Forsythia suspensa A and Forsythia suspensa reference.

[0187] The results showed that the solvent 50% ethanol solution and the negative sample solution lacking forsythia suspensa were at the corresponding positions of the chromatographic peaks of forsythiaside A and forsythiaside reference substances, and there was no interference from impurity peaks. This indicated that the negative sample had no interference and had strong specificity. Fig.13 , 14 .

[0188] The method of the present invention establishes a method for determining the content of dried tangerine peel and forsythia in the oral liquid for stimulating appetite and digestion, improves the safety and effectiveness of the product, makes up for the deficiencies of the existing standards, and further improves the accuracy of the detection, which can better control the stability of the overall quality of the product.

Claims

1. A method for determining the content of an oral liquid for stimulating appetite and promoting digestion, characterized in that: The following steps are involved: (1) Preparation of reference solution ① Tangerine peel: Accurately weigh appropriate amounts of hesperidin reference substance and nobiletin reference substance, add methanol to make a mixed solution containing 80 μg of hesperidin and 25 μg of nobiletin per 1 mL, and obtain reference substance solution I; ② Forsythia: Accurately weigh appropriate amounts of forsythiaside reference substance and forsythiaside A reference substance, add 50% ethanol to prepare a mixed solution containing 150 μg forsythiaside and 20 μg forsythiaside A per 1 mL, and obtain reference substance solution II; (2) Preparation of test solution ① Tangerine peel: accurately measure 10 mL of the test sample of the appetizer and digestion oral solution, add 10 mL of methanol to dissolve, ultrasonicate for 10 min, fix the volume, shake well, filter through a 0.45 μm filter membrane, and take the filtrate to obtain the test sample solution I; ②Forsythia suspensa: accurately measure 5 mL of the test sample of the appetizer and digestion oral solution, add 50% ethanol to dilute, fix the volume, and shake well to obtain the test sample solution II; (3) Liquid chromatography conditions ① Tangerine peel: octadecylsilane bonded silica gel was used as filler, acetonitrile was used as mobile phase A, 0.1% phosphoric acid aqueous solution was used as mobile phase B, gradient elution was performed, the detection wavelength was 209 nm, the flow rate was 1.0 mL / min, the column temperature was 30°C, and the theoretical plate number was calculated based on the hesperidin base peak and was not less than 5000; the specific procedure of the gradient elution was: 0-12 min, 20%A; 12-18 min, 20→45%A; 18-30 min, 45%A; 30-35 min, 45→60%A; ② Forsythia: octadecylsilane bonded silica gel was used as filler, methanol was used as mobile phase A, 0.1% formic acid aqueous solution was used as mobile phase B, gradient elution was performed, detection wavelength was 228 nm, flow rate was 0.7 mL / min, column temperature was 30°C, the theoretical plate number was calculated based on the forsythiaside A base peak and was not less than 5000; the specific procedure of the gradient elution was: 0-30 min, 25→48%A; 30-40 min, 48→60%A; 40-41 min, 60→80%A; 41-50 min, 80%A; 50-51 min, 80→25%A; 51-58 min, 25%A; (4) Determination ① Tangerine peel: Accurately pipette 10 μL of reference solution Ⅰ and test solution Ⅰ, respectively, inject into liquid chromatography, and measure to obtain the content of hesperidin and nobiletin in test solution Ⅰ; ② Forsythia: Accurately pipette 10 μL of reference solution II and test solution II respectively, inject into liquid chromatograph and measure to obtain the contents of forsythiaside and forsythiaside A in test solution II.

2. The method for determining the content of the oral liquid for stimulating appetite and promoting digestion according to claim 1, characterized in that: In step (2), the power of the ultrasonic treatment is 200 W and the frequency is 37 kHz.

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