Construction method and application of fingerprint of tangerine seeds and their traditional Chinese medicine preparations

Abstract

The present invention discloses a method for constructing a fingerprint of a tangerine seed and a traditional Chinese medicine preparation thereof and its application, and relates to the technical field of traditional Chinese medicine identification. The method for constructing a fingerprint of a tangerine seed and a traditional Chinese medicine preparation thereof comprises: extracting the tangerine seed or the traditional Chinese medicine preparation thereof with a first extraction solvent to obtain a test solution; dissolving or extracting a reference substance and / or a tangerine seed reference medicinal material with a second extraction solvent to obtain a reference substance solution and / or a reference medicinal material solution; measuring the test solution, the reference substance solution and / or the reference medicinal material solution with a liquid chromatograph to obtain a fingerprint of the tangerine seed or the traditional Chinese medicine preparation thereof; wherein the chromatographic column of the liquid chromatograph uses octadecylsilane bonded silica gel as the stationary phase, acetonitrile as the mobile phase A, and a phosphoric acid aqueous solution with a concentration of 0.1 vol% to 0.5 vol% as the mobile phase B for gradient elution, and the reference substances include a vecinin-2 reference substance, a hesperidin reference substance, a limonin reference substance, a nomilin reference substance, and a flavonoid reference substance; the first extraction solvent and the second extraction solvent are methanol aqueous solutions with a concentration of 30 vol% to 100 vol%. The fingerprints constructed by the present invention contain rich characteristic peak information, enabling identification from common counterfeits. Furthermore, the construction method is simple, stable, and highly specific, making it applicable to various types of tangerine seed slices and their standard decoctions and granules.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine identification, and in particular to a method for constructing a fingerprint of tangerine seeds and a traditional Chinese medicine preparation thereof and applications thereof. Background Art

[0002] Tangerine seeds are the dried, mature seeds of Citrus reticulata Blanco (Rutaceae) and its cultivated varieties. They have the properties of regulating qi, dispersing stagnation, and relieving pain, and are commonly used for ailments such as hernia pain, testicular swelling and pain, and mastitis. The tangerine, from which the tangerine seed originates, is a dual-purpose medicinal and edible plant, with multiple parts used as medicine. The 2020 edition of the Chinese Pharmacopoeia stipulates that the origin of tangerine seeds is the dried mature seeds of the Rutaceae plant Citrus reticulata Blanco and its cultivated varieties, mainly Citrus reticulata 'Dahongpao' and Citrus reticulata 'Tangerina'. To meet market demand, new varieties with seedless seeds or relatively few seeds have gradually emerged. The emergence of these new varieties has gradually eliminated the traditional medicinal variety Dahongpao from the market. In addition, these new varieties are grown by grafting on traditional varieties, and citrus species such as orange, mandarin, and pomelo can hybridize with each other, resulting in confusion in germplasm resources, potential changes in active ingredients, and uncertainty in quality, leading to uncertainty in clinical efficacy. Contaminants of tangerine seeds mainly come from varieties of citron, orange, and pomelo from the subgenus Eucitrus, as well as their hybrids, such as orange, pomelo, citron, and lemon. There is a certain correlation between plant genetic diversity and differences in the accumulation of their secondary metabolites. It is necessary to establish a method to distinguish genuine orange seeds from their counterfeits.

[0003] In addition, historical herbal texts record the following methods for processing tangerine seeds: clean, stir-fry, salt, wine, and salt-wine. The stir-frying method, which has been used throughout history, has become less common since the Qing Dynasty, while the salt-roasting method, which emerged in the Qing Dynasty, has been consistently used to this day. With the exception of the 2018 edition of the Hubei Province Traditional Chinese Medicine Preparation Specifications, which only includes stir-fried tangerine seeds (the stir-frying method), the processed tangerine seeds listed in the 1963-2020 editions of the Chinese Pharmacopoeia and other provincial and municipal processing specifications nationwide are all "salted tangerine seeds." Raw tangerine seeds have a strong effect in regulating qi and dispersing nodules, and can be used to treat mastitis. Salting them guides the drug downward, traveling to the kidney meridian, enhancing their efficacy in treating hernias and relieving pain. Currently, little research has been conducted on the processing of tangerine seeds, and in-depth research is needed on the chemical composition characteristics of their processed products. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for constructing a fingerprint of tangerine seeds and their traditional Chinese medicine preparations. The resulting fingerprint contains rich characteristic peak information, enabling identification from common counterfeits. Furthermore, the construction method is simple, stable, and highly specific, and is applicable to a variety of tangerine seed slices, as well as their standard decoctions and granules.

[0005] The technical problem that the present invention also aims to solve is to provide a method for constructing the fingerprint of the above-mentioned tangerine seeds and their traditional Chinese medicine preparations and to apply it in the identification of tangerine seeds and their counterfeits.

[0006] The technical problem that the present invention also aims to solve is to provide a method for identifying orange seeds, which can distinguish orange seeds from common counterfeits.

[0007] The technical problem that the present invention also aims to solve is to provide a method for detecting the chemical components of tangerine seeds and their traditional Chinese medicine preparations, which can realize the determination of the chemical components of a variety of tangerine seed pieces and provide a basis for the study of their effective substances and processing mechanisms before and after processing.

[0008] In order to solve the above technical problems, the present invention provides a method for constructing a fingerprint of tangerine seeds and their traditional Chinese medicine preparations, which comprises:

[0009] Extracting the orange seeds or the Chinese medicine preparation thereof with a first extraction solvent to obtain a test solution;

[0010] Dissolving or extracting the reference substance and / or the orange seed reference medicinal material with a second extraction solvent to obtain a reference substance solution and / or a reference medicinal material solution;

[0011] The test solution, reference solution and / or reference medicinal material solution are measured by liquid chromatography to obtain a fingerprint of the orange seed or its Chinese medicine preparation;

[0012] The chromatographic column of the liquid chromatograph is composed of octadecylsilane bonded silica gel as the stationary phase, acetonitrile as the mobile phase A, and a 0.1 vol% to 0.5 vol% phosphoric acid aqueous solution as the mobile phase B for gradient elution. The gradient elution curve is:

[0013] 0 min to 5 min, mobile phase A is 9%, mobile phase B is 91%;

[0014] 5-6 min, mobile phase A from 9% to 12%, mobile phase B from 91% to 88%;

[0015] 6 min to 20 min, mobile phase A from 12% to 20%, mobile phase B from 88% to 80%;

[0016] 20-30 min, mobile phase A from 20% to 23%, mobile phase B from 80% to 77%;

[0017] 30-40 min, mobile phase A from 23% to 44%, mobile phase B from 77% to 56%;

[0018] 40-50 min, mobile phase A from 44% to 45%, mobile phase B from 56% to 55%;

[0019] Among them, the reference substances include, but are not limited to, a reference substance of vincain-2, a reference substance of hesperidin, a reference substance of limonin, a reference substance of nomilin and a reference substance of chloroquine.

[0020] The first extraction solvent and the second extraction solvent are methanol aqueous solutions with a concentration of 30 vol% to 100 vol%. Exemplarily, the concentration of the methanol aqueous solution is 38 vol%, 42 vol%, 50 vol%, 58 vol%, 66 vol%, 74 vol%, 82 vol%, or 93 vol%, but is not limited thereto. Preferably, the concentration is 50 vol% to 100 vol%, and more preferably, 65 vol% to 100 vol%.

[0021] Exemplarily, the concentration of the aqueous phosphoric acid solution is 0.14 vol%, 0.18 vol%, 0.22 vol%, 0.26 vol%, 0.3 vol%, 0.34 vol%, 0.38 vol%, 0.42 vol% or 0.48 vol%, but is not limited thereto; preferably, it is 0.15 vol% to 0.3 vol%, and more preferably, it is 0.15 vol% to 0.2 vol%.

[0022] It should be noted that, in the present invention, only the reference solution and the test solution may be used for determination, or the reference medicinal material solution, the reference solution and the test solution may be used simultaneously for determination.

[0023] Preferably, in some embodiments of the present invention, the mobile phase B is 0.2 vol% phosphoric acid, the first extraction solvent is 70 vol% methanol aqueous solution, and the second extraction solvent is methanol.

[0024] Specifically, in some embodiments of the present invention, the column length of the chromatographic column is 100 mm to 300 mm, exemplarily 100 mm, 150 mm, 200 mm, or 250 mm, but not limited thereto. The column diameter of the chromatographic column is 2.1 mm to 5 mm, exemplarily 3.0 mm, 3.9 mm, or 4.6 mm, but not limited thereto. The particle size of the stationary phase is 1.7 μm to 3 μm, exemplarily 2.5 μm, 2.7 μm, or 3 μm, but not limited thereto.

[0025] The column temperature of the chromatographic column is 20° C. to 40° C., exemplified by 22° C., 25° C., 28° C., 31° C., 34° C., or 37° C., but not limited thereto.

[0026] The flow rate of the liquid chromatograph is 0.5 mL / min to 1.5 mL / min, exemplified by 0.55 mL / min, 0.7 mL / min, 0.85 mL / min, 1.05 mL / min, 1.2 mL / min, or 1.35 mL / min, but not limited thereto.

[0027] The detection wavelength of the liquid chromatograph is 200 nm to 250 nm, exemplified by, but not limited to, 205 nm, 215 nm, 225 nm, 235 nm or 245 nm.

[0028] The injection volume of the test solution is 0.8 μL to 5 μL, exemplified by 1.1 μL, 1.7 μL, 2.3 μL, 2.9 μL, 3.5 μL, 4.1 μL or 4.7 μL, but not limited thereto.

[0029] The injection volume of the reference solution is 0.8 μL to 5 μL, exemplified by 1.1 μL, 1.7 μL, 2.3 μL, 2.9 μL, 3.5 μL, 4.1 μL or 4.7 μL, but not limited thereto.

[0030] The injection volume of the control medicinal material solution is 0.8 μL to 5 μL, exemplified by 1.1 μL, 1.7 μL, 2.3 μL, 2.9 μL, 3.5 μL, 4.1 μL or 4.7 μL, but not limited thereto.

[0031] Preferably, in some embodiments of the present invention, the column length of the chromatographic column is 150 mm, the column diameter is 4.6 mm, and the particle size of the stationary phase is 2.7 μm;

[0032] The column temperature of the chromatographic column is 30°C;

[0033] The flow rate of the liquid chromatograph was 0.9 mL / min, the detection wavelength was 220 nm, the injection volume of the test solution was 2 μL, the injection volume of the reference solution was 2 μL, and the injection volume of the reference medicinal material solution was 2 μL.

[0034] Specifically, in some embodiments of the present invention, in the step of extracting the orange seed or the Chinese medicine preparation thereof with a first extraction solvent to obtain a test solution, the orange seed or the Chinese medicine preparation thereof is mixed with a methanol aqueous solution having a concentration of 30 vol% to 100 vol%, and ultrasonically treated or heated under reflux for 10 min to 60 min;

[0035] The ratio of orange seeds or their Chinese medicine preparation to methanol aqueous solution is 0.1g-2g:10mL-50mL. Exemplary ratios are 0.15g:14mL, 0.46g:13mL, 0.7g:13mL, 0.9g:19mL, 1.2g:21mL, 1.4g:25mL, 1.6g:37mL, or 1.8g:47mL, but are not limited thereto. Preferably, the ratio is 0.8g-1.5g:22mL-40mL, and more preferably, the ratio is 0.8g-1.2g:24mL-28mL.

[0036] The extraction time is exemplified by, but not limited to, 12 min, 24 min, 35 min, 40 min, or 50 min, preferably 30 min to 50 min, and more preferably 40 min to 50 min.

[0037] Preferably, in one embodiment of the present invention, when the sample is tangerine seed medicinal material or decoction piece, the ratio of the sample to the methanol aqueous solution is 1 g:25 mL, and heating reflux extraction is adopted, and the extraction time is 45 min.

[0038] In another embodiment of the present invention, when the sample is a standard decoction or formula granules, the ratio of the sample to the methanol aqueous solution is 0.2 g:15 mL, and ultrasonic extraction is performed, wherein the ultrasonic power is 200 W to 400 W, the ultrasonic frequency is 30 kHz to 50 kHz, and the ultrasonic treatment time is 45 min.

[0039] Specifically, in some embodiments of the present invention, in the step of dissolving or extracting the reference substance and / or orange seed control medicinal material with a second extraction solvent to obtain a reference substance solution and / or a control medicinal material solution, the orange seed control medicinal material is mixed with a methanol aqueous solution with a concentration of 30 vol% to 100 vol%, and ultrasonically treated or heated under reflux for 10 min to 60 min; wherein the ratio of orange seed control medicinal material to methanol aqueous solution is 0.5 g to 2 g: 10 mL to 50 mL.

[0040] Specifically, in some embodiments of the present invention, in the reference solution, the concentration of each reference substance is 5 μg / mL to 20 μg / mL, exemplified by 8 μg / mL, 11 μg / mL, 13 μg / mL, 15 μg / mL or 17 μg / mL, but not limited thereto.

[0041] Specifically, in some embodiments of the present invention, the fingerprint includes 25 common peaks, wherein peak 3 is vetacnin-2, peak 7 is kaempferol-3-O-rutinoside, peak 8 is naringin, peak 9 is hesperidin, peak 17 is hesperetin, peak 22 is limonin, peak 24 is nomilin, and peak 25 is phloxetine;

[0042] Taking Peak 9 as the S peak, calculate the relative retention times of Peaks 10 and 11. The relative retention times of each peak are within ±10% of the specified values, where the specified values ​​for Peaks 10 and 11 are 1.02 and 1.05, respectively.

[0043] The relative peak areas of Peak 10 and Peak 11 are calculated, and the ratio of the relative peak areas of Peak 10 and Peak 11 is less than or equal to 1.0.

[0044] Specifically, in some embodiments of the present invention, the Chinese medicine preparation of tangerine peel includes tangerine peel slices and tangerine peel standard decoction and tangerine peel formula granules processed from the tangerine peel slices; the tangerine peel slices refer to the slices obtained after purification of the tangerine peel medicinal material, and the specific preparation method can be found in the 2020 edition of the "Chinese Pharmacopoeia". Correspondingly, the tangerine peel standard decoction refers to the preparation obtained by decocting, concentrating and freeze-drying the above-mentioned tangerine peel slices with reference to the provisions of the "Technical Requirements for Quality Control and Standardization of Traditional Chinese Medicine Formula Granules". The tangerine peel formula granules refer to the preparation obtained by extracting, separating, concentrating, drying and granulating the above-mentioned slices with reference to the provisions of the "Technical Requirements for Quality Control and Standardization of Traditional Chinese Medicine Formula Granules", but are not limited thereto.

[0045] Specifically, in some embodiments of the present invention, the Chinese medicine preparation of tangerine seed includes salt tangerine seed slices and salt tangerine seed standard decoction and salt tangerine seed formula granules processed from the salt tangerine seed slices; the salt tangerine seed slices refer to the slices obtained after the tangerine seed medicinal material is salted, and its specific preparation method can be referred to the 2020 edition of the "Chinese Pharmacopoeia".

[0046] Specifically, in some embodiments, the Chinese medicine preparation of tangerine peel includes stir-fried tangerine peel slices and stir-fried tangerine peel standard decoction and stir-fried tangerine peel formula granules obtained from the stir-fried tangerine peel slices. Stir-fried tangerine peel slices refer to slices obtained by stir-frying tangerine peel medicinal materials. The specific preparation method can be found in the 2018 edition of the "Hubei Province Traditional Chinese Medicine Piece Preparation Specifications."

[0047] It should be noted that the fingerprint construction method of the present invention can also be applied to other types of tangerine seed medicinal pieces and their standard decoctions and formula granules, such as tangerine seed medicinal pieces obtained by stir-frying with wine, salt and wine, etc.

[0048] Correspondingly, the present invention also discloses the application of the method for constructing the characteristic spectrum of the tangerine seeds and their traditional Chinese medicine preparations in distinguishing tangerine seeds from their counterfeits.

[0049] Preferably, in some embodiments of the present invention, the counterfeit products include orange seeds, tribute orange seeds, pomelo seeds, small green orange seeds, wolfberry seeds and lemon seeds, but are not limited thereto.

[0050] Accordingly, the present invention also discloses a method for identifying orange seeds, which is used to identify orange seeds and their counterfeits, and comprises:

[0051] Provide the substance to be identified;

[0052] Constructing the fingerprint of the substance to be identified using the above-mentioned method for constructing the fingerprint of the orange seed and its traditional Chinese medicine preparation;

[0053] If the fingerprint spectrum shows peaks 1 to 25 at the same time, and the ratio of the relative peak areas of peak 10 to peak 11 is less than or equal to 1.0, then the substance to be identified includes or is orange seeds; otherwise, it is a counterfeit.

[0054] The counterfeit products include orange seeds, tribute orange seeds, pomelo seeds, small green orange seeds, goji tangerine seeds and lemon seeds, but are not limited thereto. For example, the identification method of the present invention can also identify pomelo seeds.

[0055] Correspondingly, the present invention also discloses a method for detecting the chemical components of tangerine seeds and their traditional Chinese medicine preparations, which comprises:

[0056] Extracting the orange seeds or the Chinese medicine preparation thereof with a first extraction solvent to obtain a test solution;

[0057] dissolving or extracting the reference substance with a second extraction solvent to obtain a reference substance solution;

[0058] The test solution and the reference solution are detected by ultra-high performance liquid spectrometry to obtain;

[0059] The chromatographic conditions of the ultra-high performance liquid chromatography-mass spectrometry instrument include: a chromatographic column with octadecylsilane bonded silica gel as the stationary phase, acetonitrile as the mobile phase A, and a 0.1 vol% to 0.5 vol% formic acid aqueous solution as the mobile phase B for gradient elution, and the gradient elution curve is:

[0060] 0 min to 5 min, mobile phase A is 9%, mobile phase B is 91%;

[0061] 5-6 min, mobile phase A from 9% to 12%, mobile phase B from 91% to 88%;

[0062] 6 min to 20 min, mobile phase A from 12% to 20%, mobile phase B from 88% to 80%;

[0063] 20-30 min, mobile phase A from 20% to 23%, mobile phase B from 80% to 77%;

[0064] 30-40 min, mobile phase A from 23% to 44%, mobile phase B from 77% to 56%;

[0065] 40-50 min, mobile phase A from 44% to 45%, mobile phase B from 56% to 55%;

[0066] The mass spectrometry conditions of the ultra-high performance liquid chromatography-mass spectrometry instrument include: a mass scanning range of m / z 120 to 1000;

[0067] Among them, the reference substances include vinacetin-2 reference substance, kaempferol-3-O-rutinoside reference substance, naringin reference substance, astragalin reference substance, hesperidin reference substance, naringenin reference substance, hesperetin reference substance, limonin reference substance, nobiletin reference substance, nomilin reference substance, tangeretin reference substance, and phloxetine reference substance;

[0068] The first extraction solvent and the second extraction solvent are methanol aqueous solutions with a concentration of 30 vol% to 100 vol%. Exemplarily, the concentration of the methanol aqueous solution is 38 vol%, 42 vol%, 50 vol%, 58 vol%, 66 vol%, 74 vol%, 82 vol%, or 93 vol%, but is not limited thereto. Preferably, the concentration is 50 vol% to 100 vol%, and more preferably, 65 vol% to 100 vol%.

[0069] Exemplarily, the concentration of the formic acid aqueous solution is 0.14 vol%, 0.18 vol%, 0.22 vol%, 0.26 vol%, 0.3 vol%, 0.34 vol%, 0.38 vol%, 0.42 vol% or 0.48 vol%, but is not limited thereto; preferably, it is 0.15 vol% to 0.3 vol%, and more preferably, it is 0.15 vol% to 0.2 vol%.

[0070] Preferably, in some embodiments of the present invention, the mobile phase B is 0.2 vol% formic acid, the first extraction solvent is a methanol aqueous solution with a concentration of 70 vol%, and the second extraction solvent is methanol.

[0071] Specifically, in some embodiments of the present invention, the chromatographic conditions of the ultra-high performance liquid spectrometer further include:

[0072] The column length of the chromatographic column is 100 mm to 300 mm, exemplarily 100 mm, 150 mm, 200 mm, or 250 mm, but not limited thereto. The column diameter of the chromatographic column is 2.1 mm to 5 mm, exemplarily 3.0 mm, 3.9 mm, or 4.6 mm, but not limited thereto. The particle size of the stationary phase is 1.7 μm to 3 μm, exemplarily 2.5 μm, 2.7 μm, or 3 μm, but not limited thereto.

[0073] The column temperature of the chromatographic column is 20° C. to 40° C., exemplified by 22° C., 25° C., 28° C., 31° C., 34° C., or 37° C., but not limited thereto.

[0074] The flow rate of the liquid chromatograph is 0.5 mL / min to 1.5 mL / min, exemplified by 0.55 mL / min, 0.7 mL / min, 0.85 mL / min, 1.05 mL / min, 1.2 mL / min, or 1.35 mL / min, but not limited thereto.

[0075] The detection wavelength of the liquid chromatograph is 200 nm to 250 nm, exemplified by 205 nm, 215 nm, 225 nm, 235 nm or 245 nm, but not limited thereto.

[0076] The injection volume of the test solution is 0.8 μL to 5 μL, exemplified by 1.1 μL, 1.7 μL, 2.3 μL, 2.9 μL, 3.5 μL, 4.1 μL or 4.7 μL, but not limited thereto.

[0077] The injection volume of the reference solution is 0.8 μL to 5 μL, exemplified by 1.1 μL, 1.7 μL, 2.3 μL, 2.9 μL, 3.5 μL, 4.1 μL or 4.7 μL, but not limited thereto.

[0078] Preferably, in some embodiments of the present invention, the chromatographic conditions of the ultra-high performance liquid spectrometer further include:

[0079] The column length of the chromatographic column is 150 mm, the column diameter is 4.6 mm, and the particle size of the stationary phase is 2.7 μm;

[0080] The column temperature of the chromatographic column is 30°C;

[0081] The flow rate of the liquid chromatograph was 0.9 mL / min, the detection wavelength was 220 nm, the injection volume of the test solution was 2 μL, and the injection volume of the reference solution was 2 μL.

[0082] Specifically, in some embodiments of the present invention, the mass spectrometry conditions of the ultra-high performance liquid chromatography-mass spectrometry instrument further include:

[0083] Electrospray ionization source, positive or negative ion mode, sheath gas flow rate of 30 to 50 arb, exemplified by, but not limited to, 32 arb, 35 arb, 39 arb, 42 arb, 47 arb, or 49 arb. Auxiliary gas flow rate of 5 to 20 arb, exemplified by, but not limited to, 7 arb, 11 arb, 13 arb, 17 arb, or 19 arb. Spray voltage of 3 kV to 5 kV, exemplified by, but not limited to, 3.4 kV, 3.7 kV, 4 kV, 4.3 kV, 4.6 kV, or 4.9 kV. S-lens voltage of 40 V to 60 V, exemplified by, but not limited to, 43 V, 45 V, 48 V, 51 V, 53 V, 55 V, or 58 V. The auxiliary heating temperature is 350°C to 450°C, and is exemplified by, but not limited to, 360°C, 380°C, or 420°C. The capillary temperature is 350°C to 450°C, and is exemplified by, but not limited to, 350°C, 370°C, or 390°C. The normalized collision energy is 20eV to 40eV, and is exemplified by, but not limited to, 22eV, 25eV, 27eV, 33eV, 37eV, or 39eV.

[0084] Specifically, in some embodiments of the present invention, the mass spectrometry conditions of the ultra-high performance liquid chromatography-mass spectrometry instrument further include:

[0085] Electrospray ion source, positive and negative ion mode, sheath gas flow rate of 35arb, auxiliary gas flow rate of 10arb, spray voltage of 3.8kV, S-lens voltage of 50V, auxiliary heating temperature of 350℃, capillary temperature of 350℃, normalized collision energy of 20eV~40eV.

[0086] Specifically, in some embodiments of the present invention, in the step of extracting the orange seed or the Chinese medicine preparation thereof with a first extraction solvent to obtain a test solution, the orange seed or the Chinese medicine preparation thereof is mixed with a methanol aqueous solution having a concentration of 30 vol% to 100 vol%, and ultrasonically treated or heated under reflux for 10 min to 60 min;

[0087] The ratio of orange seeds or their Chinese medicine preparation to methanol aqueous solution is 0.1g-2g:10mL-50mL. Exemplary ratios are 0.15g:14mL, 0.46g:13mL, 0.7g:13mL, 0.9g:19mL, 1.2g:21mL, 1.4g:25mL, 1.6g:37mL, or 1.8g:47mL, but are not limited thereto. Preferably, the ratio is 0.8g-1.5g:22mL-40mL, and more preferably, the ratio is 0.8g-1.2g:24mL-28mL.

[0088] The extraction time is exemplified by, but not limited to, 12 min, 24 min, 35 min, 40 min, or 50 min, preferably 30 min to 50 min, and more preferably 40 min to 50 min.

[0089] Preferably, in one embodiment of the present invention, when the sample is tangerine seed medicinal material or decoction piece, the ratio of the sample to the methanol aqueous solution is 1 g:25 mL, and heating reflux extraction is adopted, and the extraction time is 45 min.

[0090] In another embodiment of the present invention, when the sample is a standard decoction or formula granules, the ratio of the sample to the methanol aqueous solution is 0.2 g:15 mL, and ultrasonic extraction is performed, wherein the ultrasonic power is 200 W to 400 W, the ultrasonic frequency is 30 kHz to 50 kHz, and the ultrasonic treatment time is 45 min.

[0091] Specifically, in some embodiments of the present invention, in the reference solution, the concentration of each reference substance is 5 μg / mL to 20 μg / mL, exemplified by 8 μg / mL, 11 μg / mL, 13 μg / mL, 15 μg / mL or 17 μg / mL, but not limited thereto.

[0092] Specifically, in some embodiments of the present invention, the Chinese medicine preparation of tangerine peel includes tangerine peel slices and tangerine peel standard decoction and tangerine peel formula granules obtained by processing the tangerine peel slices; the tangerine peel slices refer to the slices obtained after purification of the tangerine peel medicinal materials, and the specific preparation method thereof can be found in the 2020 edition of the "Chinese Pharmacopoeia". Correspondingly, the tangerine peel standard decoction refers to the preparation obtained by decocting, concentrating and freeze-drying the above-mentioned tangerine peel slices with reference to the provisions of the "Technical Requirements for Quality Control and Standardization of Traditional Chinese Medicine Formula Granules". The tangerine peel formula granules refer to the preparation obtained by extracting, separating, concentrating, drying and granulating the above-mentioned tangerine peel slices with reference to the provisions of the "Technical Requirements for Quality Control and Standardization of Traditional Chinese Medicine Formula Granules", but are not limited thereto.

[0093] Specifically, in some embodiments of the present invention, the Chinese medicine preparation of tangerine seed includes salt tangerine seed slices and salt tangerine seed standard decoction and salt tangerine seed formula granules processed from the salt tangerine seed slices; the salt tangerine seed slices refer to the slices obtained after the tangerine seed medicinal material is salted, and its specific preparation method can be referred to the 2020 edition of the "Chinese Pharmacopoeia".

[0094] Specifically, in some embodiments, the Chinese medicine preparation of tangerine peel includes stir-fried tangerine peel slices and stir-fried tangerine peel standard decoction and stir-fried tangerine peel formula granules obtained from the stir-fried tangerine peel slices. Stir-fried tangerine peel slices refer to slices obtained by stir-frying tangerine peel medicinal materials. The specific preparation method can be found in the 2018 edition of the "Hubei Province Traditional Chinese Medicine Piece Preparation Specifications."

[0095] It should be noted that the detection method of the present invention can also be applied to other types of tangerine seed medicinal pieces and their standard decoctions and formula granules, such as medicinal pieces obtained by frying tangerine seed medicinal materials with wine, salt and wine, etc.

[0096] The implementation of the present invention has the following beneficial effects:

[0097] The present invention establishes a fingerprint of tangerine seeds and their traditional Chinese medicine preparations, which contains 25 common peaks, identifies 8 characteristic peaks, and calculates the relative peak areas of peaks 10 and 11. Based on this, a method for identifying tangerine seeds and their counterfeits is established. The fingerprint establishment method of the present invention is applicable to raw products (tangerine seeds medicinal materials), various processed products and their processed products (standard decoctions, formula granules, etc.), and has a wide range of applications.

[0098] The present invention also identified the chemical components of tangerine seeds and their traditional Chinese medicine preparations by liquid chromatography-mass spectrometry, and a total of 46 chemical components were identified, and 12 of them were confirmed using reference substances. Based on this, the medicinal substances and processing mechanisms of tangerine seeds before and after salt roasting can be further explored. BRIEF DESCRIPTION OF THE DRAWINGS

[0099] Figure 1 This is a graph showing the specificity investigation results of the orange seed fingerprint in Example 1;

[0100] Figure 2 This is an overlay of the fingerprints of 13 batches of tangerine seed medicinal materials in Example 1;

[0101] Figure 3 is the control fingerprint of the tangerine seed medicinal material in Example 1;

[0102] Figure 4 is the fingerprint of the reference medicinal material of tangerine seed in Example 1;

[0103] Figure 5 It is an overlay of the fingerprints of 13 batches of salt orange core decoction pieces in Example 1;

[0104] Figure 6 is the control fingerprint of the salt orange core decoction piece in Example 1;

[0105] Figure 7 This is a comparison of the fingerprints of the tangerine seed medicinal material and the salt tangerine seed decoction piece in Example 1;

[0106] Figure 8 This is the fingerprint identification result of the reference substance of the orange seed in Example 1;

[0107] Figure 9 This is a comparison of the fingerprints of the tangerine seed medicinal material, the salted tangerine seed medicinal piece, and the stir-fried tangerine seed medicinal piece in Example 2;

[0108] Figure 10is an overlay of the fingerprints of 10 batches of standard decoction of tangerine seeds in Example 3;

[0109] Figure 11 is an overlay of the fingerprints of 10 batches of salt orange seed standard decoction in Example 3;

[0110] Figure 12 This is a comparison of the fingerprints of the standard decoction of tangerine seed and the standard decoction of salt tangerine seed in Example 3;

[0111] Figure 13 This is an overlay of the fingerprints of three batches of orange seed formula granules in Example 3;

[0112] Figure 14 This is an overlay of the fingerprints of three batches of salt orange seed granule standard decoction in Example 3;

[0113] Figure 15 This is a comparison of the fingerprints of the orange seed formula granules and the salt orange seed formula granules in Example 3;

[0114] Figure 16 This is a comparison of the fingerprints of the orange pit and its counterfeit in Example 4;

[0115] Figure 17 This is an overlay of fingerprints of six batches of orange cores (fake orange cores) in Example 4;

[0116] Figure 18 It is the positive and negative total ion current (TIC) of the tangerine seed medicinal material and the salt tangerine seed decoction piece in Example 5;

[0117] Figure 19 This is a comparison of the mass spectra of the reference substance and the test substance of orange seeds in Example 5;

[0118] Figure 20 : These are the OPLS-DA analysis results of the raw tangerine seeds and the salted tangerine seeds fried for different times in Example 6, wherein (A) and (B) are the OPLS-DA score graphs and S-plot graphs of the raw tangerine seeds and the samples fried for 3 min, respectively; (C) and (D) are the OPLS-DA score graphs and S-plot graphs of the raw tangerine seeds and the samples fried for 5 min, respectively; (E) and (F) are the OPLS-DA score graphs and S-plot graphs of the raw tangerine seeds and the samples fried for 8 min, respectively; (G) and (H) are the OPLS-DA score graphs and S-plot graphs of the samples fried for 3 min and the samples fried for 8 min, respectively;

[0119] Figure 21 This is the chromatogram of tangerine and orange core obtained using the chromatographic conditions of comparative document 1 in comparative example 1;

[0120] Figure 22 This is the chromatogram of tangerine and orange core obtained using the chromatographic conditions of comparative document 2 in comparative example 1;

[0121] In the figure, peak 3 is vetacnin-2, peak 7 is kaempferol-3-O-rutinoside, peak 8 is naringin, peak 9 is hesperidin, peak 17 is hesperetin, peak 22 is limonin, peak 24 is nomilin, and peak 25 is flavonoid. DETAILED DESCRIPTION

[0122] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings and specific implementation methods.

[0123] The instruments, reagents and reagent information involved in each embodiment and comparative example of the present invention are as follows:

[0124] Instruments: Waters ultra-high-performance liquid chromatograph (H-Class, Waters Corporation, USA), Waters Cortecs C18 column (4.6 mm × 150 mm, 2.7 μm), microbalance (ME204E, Mettler-Toledo), microbalance (XP26, Mettler-Toledo), electric thermostatic water bath (HWS-28, Shanghai Yiheng Technology Co., Ltd.), ultrapure water machine (Milli-Q Direct 8 / 16 system, Merck & Co., Ltd.). UHPLC-Q-Exactive Orbitrap MS quadrupole-electrostatic field orbitrap high-resolution mass spectrometer, Thermo Vanquish ultra-high-performance liquid chromatograph with electrospray ionization (HESI) source, Xcalibur 4.1 workstation, and Compound Discoverer 3.3 software (Thermo Fisher Scientific, USA).

[0125] Reagents: ethanol (Xilong Scientific Co., Ltd.) and methanol (Xilong Scientific Co., Ltd.) were of analytical grade; phosphoric acid (Tianjin Komiou Chemical Reagent Co., Ltd., chromatographic grade), acetonitrile (Merck Co., Ltd., chromatographic grade), and water was ultrapure water (prepared in the laboratory).

[0126] Test drugs: Weicaining-2 (batch number: 112096-202201, content 88%, China Food and Drug Inspection Institute); hesperidin (batch number: 110721-202019, content 95.3%, China Food and Drug Inspection Institute); limonin (batch number: 110800-201707, content 97.9%, China Food and Drug Inspection Institute); nomilin (batch number: CFS202101, content ≥98%, Wuhan Tianzhi Biological Co., Ltd.); flavonoids (batch number: 111923-202105, content 99.7%, China Food and Drug Inspection Institute); kaempferol-3-O-rutinoside (batch number: 112007-202103, content 94.0%, China Food and Drug Inspection Institute); naringin (batch number: ST 05590120, content ≥98%, Shanghai Shidande Standard Technology Service Co., Ltd.); hesperetin (batch number: 150816; content ≥98%, Chengdu Pufeide Biotechnology Co., Ltd.); tangerine seed control medicinal material (batch number: 125028-202006A, tested by Guangdong Provincial Institute for Drug Control); astragalin (batch number: DSTDZ000101, content calculated as 99.19%, Chengdu Lemeitian Pharmaceutical Technology Co., Ltd.); naringenin (batch number: wkq20030401, content ≥98%, Sichuan Weikeqi Biotechnology Co., Ltd.); nobiletin (batch number: wkq20031701, content ≥98%, Sichuan Weikeqi Biotechnology Co., Ltd.); tangeretin (batch number: wkq16011401, content ≥98%, Sichuan Weikeqi Biotechnology Co., Ltd.). The sources of the medicinal materials and their traditional Chinese medicine preparations used in the study are shown in Table 1.

[0127] Table 1 Sample information table

[0128]

[0129]

[0130] Example 1

[0131] This embodiment provides a method for constructing a fingerprint of orange seeds, which is as follows:

[0132] 1 Preparation of test solution

[0133] Medicinal materials and decoction pieces: Take about 1g of the powder of this product (passed through No. 2 sieve), accurately weigh, accurately add 25mL of 70% methanol, heat and reflux for 45 minutes, take out, cool, filter, and take the filtrate as the test solution.

[0134] Standard decoction / formulated granules: Take about 0.2 g of the powder of this product (passed through a No. 2 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 15 mL of 70% methanol, weigh it, and ultrasonically treat it (power 300 W, frequency 40 kHz) for 45 minutes. Remove it, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the product.

[0135] 2 Preparation of reference solution

[0136] Reference substance solution: Take appropriate amounts of vincin-2 reference substance, hesperidin reference substance, limonin reference substance, nomilin reference substance, and flavonoid reference substance, add methanol to dissolve them and prepare a mixed reference substance solution containing 10 μg each of vincin-2, hesperidin, limonin, nomilin, and flavonoid per 1 mL, as the reference substance solution.

[0137] Reference solution of control medicinal material: take 1 g of tangerine seed control medicinal material, accurately add 25 mL of 70% methanol, heat and reflux for 45 minutes, take out, cool, filter, and take the filtrate as the reference solution of tangerine seed control medicinal material.

[0138] 3 Chromatographic conditions

[0139] Chromatographic column: Octadecyl bonded silica gel as the filler (Waters Cortecs C18 column, column length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm), acetonitrile as mobile phase A, and 0.2% phosphoric acid solution as mobile phase B; gradient elution as specified in Table 2; flow rate 0.9 mL / min, column temperature 30°C, detection wavelength 220 nm, and the number of theoretical plates calculated based on the hesperidin peak must not be less than 5000.

[0140] Table 2 Gradient elution program conditions

[0141]

[0142]

[0143] 4 Determination

[0144] Accurately pipette 2 μL of reference solution and test solution respectively, inject into liquid chromatograph, and measure to obtain the result.

[0145] 5 Methodological Investigation

[0146] 5.1 Specificity Investigation

[0147] Prepare the test sample solution and blank solvent solution using the preparation method in item "1" for the tangerine seed medicinal material, the salt tangerine seed decoction piece, and 70% methanol blank solvent. Take 2 μL of the reference solution in item "2" and inject 2 μL into the liquid chromatograph. Analyze according to the chromatographic conditions in item "3."

[0148] The analysis results are as follows Figure 1 As shown in the figure, it can be seen that the test sample chromatogram has the same chromatographic peak within the retention time corresponding to the reference sample chromatogram, and there is no interference from the blank solvent, indicating that the method has good specificity.

[0149] 5.2 Precision experiment

[0150] Prepare the test solution from tangerine seed according to the preparation method in item "1". Repeat the analysis six times using the chromatographic conditions in item "3". Using hesperidin as the reference peak (S), the relative retention time RSD of the common peaks was <0.06%, and the relative peak area RSD was <2.22%, indicating good instrument precision.

[0151] 5.3 Repeatability Experiment

[0152] Prepare a test solution from tangerine seed according to the preparation method in item "1" (parameter 1). Repeat the test solution in six replicates and analyze the solution using the chromatographic conditions in item "3" (parameter 3). Using hesperidin as the reference peak (S), the relative retention time RSD of the common peaks was <0.53%, and the relative peak area RSD was <2.97%, indicating good reproducibility.

[0153] 5.4 Stability test

[0154] Prepare a test solution from tangerine seed according to the preparation method in item "1". Inject the solution at 0, 2, 4, 6, 14, and 24 hours. Determine the concentration according to the chromatographic conditions in item "3". Using hesperidin as the reference peak (S), the relative retention time RSD of the common peak was <1.10%, and the relative peak area RSD was <4.70%, indicating that the test solution was stable within 24 hours.

[0155] 6 Establishment of fingerprint of medicinal material of tangerine seed

[0156] Take 13 batches of orange seed medicinal materials respectively, prepare the test solution according to the method of item "1", inject the sample for determination, and use the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software" to identify the common peaks of the fingerprints of the 13 batches of orange seed medicinal materials. The overlay diagram is shown in Figure 2 , and generate a comparison map see Figure 3 ; Orange seed reference medicinal material atlas see Figure 4The results of calculating the relative peak area ratio of Peak 10 to Peak 11 are shown in Table 3. The measured range of the relative peak area of ​​Peak 10 to Peak 11 in the fingerprint of the tangerine seed medicinal material is 0.17 to 0.61. The requirements for determining the fingerprint of the tangerine seed medicinal material are as follows: 25 common peaks should be present in the test sample, and the retention times should correspond to the 25 characteristic peaks in the chromatographic peaks of the reference medicinal material reference; Peaks 3, 9, 22, 24, and 25 should correspond to the retention times of the corresponding reference peaks. The peak corresponding to the hesperidin reference peak is the S peak. The relative retention times of Peaks 10, 11, and S peak are calculated. The relative retention times should be within ±10% of the specified values, which are: 1.02 (Peak 10) and 1.05 (Peak 11). The relative peak areas of Peak 10 and Peak 11 are calculated. The relative peak area ratio should be within the specified range, which is: not greater than 1.0.

[0157] 7 Establishment of fingerprint of salt orange seed slices

[0158] Take 13 batches of salt orange core slices respectively, prepare the test solution according to the method under "1", inject the sample for determination, and use the "Chinese medicine chromatographic fingerprint similarity evaluation software" to identify the common peaks of the 13 batches of orange core medicinal material fingerprints. The overlay diagram is shown in Figure 5 , and generate a comparison map see Figure 6 ; Comparison of fingerprints of orange seed and salt orange seed is shown in Figure 7 The results of calculating the relative peak area ratio of Peak 10 to Peak 11 are shown in Table 3. The measured relative peak area range of Peak 10 to Peak 11 in the fingerprint of Salt Orange Hexagonal Pieces is 0.12 to 0.51, both less than 1.0. This confirms that the fingerprint of Salt Orange Hexagonal Pieces is consistent with that of Orange Hexagonal Pieces, namely: 25 common peaks should be present in the test sample, and the retention times should correspond to the 25 characteristic peaks in the chromatographic peaks of the reference medicinal material; among them, Peak 3, Peak 9, Peak 22, Peak 24, and Peak 25 should correspond to the retention times of the corresponding reference material peaks, and the peak corresponding to the hesperidin reference peak is the S peak. The relative retention times of Peak 10, Peak 11, and S peak are calculated, and their relative retention times should be within the range of ±10% of the specified value, which is: 1.02 (Peak 10) and 1.05 (Peak 11); the relative peak areas of Peak 10 and Peak 11 are calculated, and their relative peak area ratios should be within the specified range, which is: not greater than 1.0.

[0159] Table 3 Calculation results of the relative peak area ratio of peak 10 and peak 11 of tangerine seed medicinal materials and salt tangerine seed decoction pieces

[0160]

[0161]

[0162] 8 Reference Material Identification

[0163] Take the orange seed medicinal material and prepare the test solution according to the preparation method under "1". Take the mixed standard solution (each single standard concentration is 50μg / mL) or single standard solution (concentration is 50μg / mL) containing the reference substance of vecinin-2, kaempferol-3-O-rutinoside, rutin reference substance, hesperidin reference substance, hesperetin reference substance, champrodone reference substance, nomilin reference substance, and limonin reference substance respectively, and inject and analyze according to the chromatographic conditions under "3". The results are shown in Figure 8 .from Figure 8 Peak 3 was confirmed to be vetacnin-2, peak 7 was confirmed to be kaempferol-3-O-rutinoside, peak 8 was confirmed to be naringin, peak 9 was confirmed to be hesperidin, peak 17 was confirmed to be hesperetin, peak 22 was confirmed to be limonoid, peak 24 was confirmed to be nomilin, and peak 25 was confirmed to be flavonoid.

[0164] Example 2

[0165] This example uses the construction method of Example 1 to establish the fingerprints of orange core medicinal materials, salt orange core medicinal pieces and stir-fried orange core medicinal pieces, and compares them. Specifically, 3 batches of orange cores (origin: Zhejiang) were taken, and according to the processing method of "salt orange core" included in the orange core item of the 2020 edition of the "Chinese Pharmacopoeia" and the processing method of "stir-fried orange core" recorded in the 2018 edition of the "Hubei Province Traditional Chinese Medicine Preparation Specifications", stir-fried orange core and salt orange core medicinal pieces were prepared respectively, and the test solution was prepared and the fingerprint was measured according to the method determined in item "1" of Example 1. The results are as follows: Figure 9 As shown in the figure, the chromatograms of the test samples of tangerine seeds, fried tangerine seeds and salted tangerine seeds can all show 25 chromatographic peaks, that is, the established fingerprint method can be applied to the raw tangerine seeds and the two processed products of fried tangerine seeds and salted tangerine seeds.

[0166] Example 3

[0167] This example uses the construction method of Example 1 to establish fingerprints of the standard decoction of tangerine seed, the standard decoction of salt tangerine seed, the formula granules of tangerine seed, and the formula granules of salt tangerine seed. The details are as follows:

[0168] 1 Fingerprint of standard decoction of tangerine seeds and salt tangerine seeds

[0169] Prepare the standard decoction of tangerine core and salt tangerine core according to the following method: take tangerine core slices (clean preparation, refer to the tangerine core slices under the tangerine core item of the 2020 edition of the Chinese Pharmacopoeia) and salt tangerine core slices, crush them, add 8 times the amount of water and soak for 30 minutes, boil over high heat (500W), and keep it slightly boiling for 30 minutes over low heat (200W), filter it while hot with a 200-mesh sieve, and cool the filtrate quickly with cold water. Add 6 times the amount of water for the second decoction, boil over high heat (500W), and keep it slightly boiling for 25 minutes over low heat (200W), filter it while hot with a 200-mesh sieve, and cool the filtrate quickly with cold water, and combine the two filtrates. The decoction was transferred to a 2000mL round-bottom flask and concentrated at low temperature under reduced pressure using a rotary evaporator (temperature: 65°C, vacuum degree: -0.08MPa~-0.1MPa) to 100mL of fluid extract, and freeze-dried to obtain tangerine core / salt tangerine core standard decoction freeze-dried powder.

[0170] Preparation of test solution: Take the freeze-dried powder of standard decoction of orange seed and salt orange seed respectively (sample number see Table 1), grind it into powder, take about 0.2g, accurately weigh it, place it in a stoppered conical flask, accurately add 15mL of 70% methanol, weigh it, and ultrasonically treat it (power 300W, frequency 40kHz) for 45 minutes. Take it out, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0171] The fingerprints were determined according to the chromatographic conditions specified under item "3" in Example 1. The common peaks of the fingerprints of the freeze-dried powder of the standard decoction of orange seed and salt orange seed were identified using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software" and a control spectrum was generated. The overlay diagram is shown in FIG. Figures 10-11 As shown in Table 4, all of them can present 25 common peaks. The results of calculating the relative peak area ratio of peak 10 to peak 11 are shown in Table 4. The comparison of the fingerprints of the standard decoction of orange seed and salt orange seed is shown in Figure 12 .

[0172] 2 Fingerprint of orange seed and salt orange seed formula granules

[0173] Formula granules are prepared according to the following method: (1) Tangerine seed formula granules: take 5000g of tangerine seed slices (pure preparation, refer to the tangerine seed slices under the tangerine seed item in the 2020 edition of the "Chinese Pharmacopoeia"), add water to decoct, filter, concentrate the filtrate into a clear paste, add appropriate amount of auxiliary materials, dry (or dry and crush), add appropriate amount of auxiliary materials, mix, granulate, and make 1000g; (2) Salt tangerine seed formula granules: take 4500g of salt tangerine seed slices, add water to decoct, filter, concentrate the filtrate into a clear paste, add appropriate amount of auxiliary materials, dry (or dry and crush), add appropriate amount of auxiliary materials, mix, granulate, and make 1000g.

[0174] Take orange seed and salt orange seed formula granules (sample numbers are shown in Table 1) respectively. The preparation method of the test sample is the same as that of the standard decoction freeze-dried powder. The fingerprints thereof are determined according to the chromatographic conditions specified under item "3" in Example 1. The "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software" is used to identify the common peaks of the fingerprints of the orange seed and salt orange seed formula granules, and a control spectrum is generated. The overlay diagram is as follows: Figures 13-14 As shown in Table 4, all of them can present 25 common peaks. The results of calculating the relative peak area ratio of peak 10 to peak 11 are shown in Table 4. The comparison of the fingerprints of the orange seed and salt orange seed formula granules is shown in Figure 15 .

[0175] Table 4 Calculation results of the relative peak area ratios of peak 10 and peak 11 for standard decoction and formula granules

[0176]

[0177]

[0178] Example 4

[0179] This embodiment provides a method for identifying orange seeds and their counterfeits, as follows:

[0180] Take the counterfeit orange seeds, tangerine seeds, pomelo seeds, small green orange seeds, goji seeds, lemon seeds samples, and determine their fingerprints according to the method specified in Example 1. The fingerprint comparison diagram of orange seeds and counterfeit products is shown in Figure 1. Figure 16 , 6 batches of orange core fingerprint superposition map see Figure 17 The results of calculating the relative peak area ratio of Peak 10 to Peak 11 are shown in Table 5. Except for the tangerine seeds and lemon seeds that lack Peak 10 and cannot calculate the relative peak area, the relative peak area ratios of the other counterfeit products are measured in the range of 1.53 to 11.24, all greater than 1.0, and can be distinguished from the genuine tangerine seeds.

[0181] Table 5 Calculation results of the relative peak area ratio of peak 10 and peak 11 of the counterfeit orange seed

[0182]

[0183] Example 5

[0184] This example provides a method for rapid identification of the chemical components of tangerine seeds and their traditional Chinese medicine preparations based on UHPLC-QE-Orbitrap-MS technology, as follows:

[0185] 1 Chromatographic conditions and mass spectrometry conditions

[0186] Chromatographic conditions: Octadecyl bonded silica gel as the filler (Waters Cortecs C18 column, column length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm), acetonitrile as mobile phase A, 0.2% formic acid solution as mobile phase B, gradient elution according to the specifications in Table 6; flow rate 0.9 mL / min, column temperature 30°C; injection volume 1 μL.

[0187] Table 6 Gradient elution program conditions

[0188]

[0189]

[0190] Mass spectrometry conditions: an electrospray ionization source (HESI) was used, with a sheath gas flow rate of 35 arb; an auxiliary gas flow rate of 10 arb; a spray voltage of 3.80 kV; an S-lens voltage of 50 V; an auxiliary heating temperature of 350°C; a capillary temperature of 350°C; a scan mode of Full MS / dd-MS2 Discovery (positive and negative ion modes); a mass scan range of m / z 120 to 1000; and a normalized collision energy (NCE) of 20 to 40 eV.

[0191] 2. Preparation of reference solution

[0192] Reference solution: Take appropriate amount of vinacetin-2 reference substance, kaempferol-3-O-rutinoside reference substance, naringin reference substance, astragalin reference substance, hesperidin reference substance, naringenin reference substance, hesperetin reference substance, limonin reference substance, nobiletin reference substance, nomilin reference substance, tangeretin reference substance, and phloxetine reference substance, dissolve them in methanol and prepare a reference solution containing 10 μg of each reference substance per 1 mL.

[0193] 2.3 Preparation of test solution

[0194] Take about 1 g of orange seed medicinal material and salt orange seed decoction piece powder (pass through No. 2 sieve), accurately add 25 mL of 70% methanol, heat and reflux for 45 minutes, take out, cool, filter, and take the filtrate as the test solution.

[0195] 3. Identification results of components of tangerine seeds and salt tangerine seeds

[0196] The total ion chromatograms of the chemical components of tangerine seeds and salt tangerine seeds in positive and negative ion modes were obtained by UHPLC-QE-Orbitrap-MS technology. Figure 18Compound Discoverer 3.3 and Thermo mzVault software were used to analyze the mass spectrometry data, and the mzCloud mass spectrometry library and the OTCML local mass spectrometry database were searched. Based on the principle that the deviation between the actual mass-to-charge ratio and the theoretical accurate value is less than 5ppm, the molecular formula of the compound corresponding to each chromatographic peak was determined. Based on the database matching results and relevant literature reports, the preliminary identification of the compound was completed, and further confirmation was achieved by comparison with reference substances. A total of 46 compounds were identified in orange seeds and salt orange seeds, including 19 flavonoids and their glycosides, 19 limonoids and their glycosides, 6 phenolic components, and 2 other components; the structures of 12 of them were confirmed by comparison with reference substances (see Table 7, Figure 19 ), which are 2-vinylpyrrolidone (10), kaempferol-3-O-rutinoside (15), rutin (16), astragalin (18), hesperidin (21), naringenin (29), hesperetin (32), limonin (39), nobiletin (42), nomilin (44), tangeretin (45), and phlorothalonol (46).

[0197] Table 7 Identification results of chemical components of tangerine seed medicinal materials and salt tangerine seed

[0198]

[0199]

[0200]

[0201] In the table, “*” indicates the results confirmed by comparison with reference substances.

[0202] Example 6

[0203] This example provides a method for analyzing the composition changes of orange seeds after frying for different times based on UHPLC-QE-Orbitrap-MS technology, as follows:

[0204] According to the "Salt Orange Core" decoction piece included in the 2020 edition of the Chinese Pharmacopoeia, the following provisions apply: Clean the orange core and stir-fry it dry using the salt water roasting method (General Rule 0213). The "Salt Roasting Method" section of the General Rules for Processing (General Rule 0213 of the 2020 edition of the Chinese Pharmacopoeia) stipulates: Take the product to be processed, add salt water, mix well, and simmer thoroughly. Transfer the product to the stir-frying container and heat over low heat. When stir-frying to the specified degree, remove the product and let it cool. When salt roasting, use table salt. First, add an appropriate amount of water to dissolve it, filter it, and set aside. Unless otherwise specified, use 2 kg of table salt for every 100 kg of the product to be processed.

[0205] Preparation of salted tangerine core samples: Take 20 g of clean tangerine core, add 8 mL of 5% salt water, mix well, and simmer for 1.5 h. Place in a wok preheated to 120°C and fry for 3 min, 5 min, and 8 min respectively to obtain salted tangerine core samples with different processing times. Prepare 4 samples for each process.

[0206] The orange core samples and the salted orange core samples with different frying times were collected and mass spectrometry data were collected in positive ion mode according to the method in Example 4. The mass spectrometry data were analyzed using Compound Discoverer 3.3 and Thermo mzVault software. The results showed that the mass error was 10 ppm, the retention time error was 0.2 min, and the response intensity was greater than 1×10 5 Through ion peak extraction, matching and normalization analysis, the peak information was screened as shown in Table 8. The multivariate data matrix containing compound numbers and peak intensities was imported into SIMCA-P14.1 software for orthogonal partial least squares-discriminant analysis (OPLS-DA). The inter-group difference components were screened according to the variable projection importance (VIP) value. The OPLS-DA analysis score graph and S-plot graph are shown in Figure 8. Figure 20 One-way analysis of variance was used to examine the significance of differences among the groups, and components with P < 0.1 were screened as differential components. The results are shown in Table 9.

[0207] The method for rapid identification of the chemical components of tangerine seeds and their traditional Chinese medicine preparations based on UHPLC-QE-Orbitrap-MS technology is as follows:

[0208] Table 8 Mass spectrometry analysis results of raw orange seeds and salted orange seeds fried for different times

[0209]

[0210] In the table, “*” indicates the results confirmed by comparison with reference substances.

[0211] Table 9 OPLS-DA analysis results of raw orange seeds and salted orange seeds fried for different times

[0212]

[0213] The results showed that after frying for 3 minutes, rosmarinic acid-4-O-glucoside showed a downward trend compared with the raw product; after frying for 5 minutes, astragalin, ychang neritin, marinic acid-4-O-glucoside, and deacetylnomironin methyl ester 17-glucoside showed a downward trend; after frying for 8 minutes, nomilin showed a downward trend, and flavonoids showed an upward trend.

[0214] Comparative Example 1

[0215] This embodiment provides an application comparison between comparative documents 1 and 2, as follows:

[0216] Referring to the methods disclosed in comparative document 1 (CN202310203875.0) and comparative document 2 (Luo Jing, HPLC fingerprint study and cluster analysis of tangerine seeds), the chromatographic column Dikma C18 (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm) was selected, and the fingerprints of tangerine seeds and orange seeds were determined according to the following two methods, respectively.

[0217] Comparative Document 1 Method: Octadecyl bonded silica gel was used as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm), acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, gradient elution (0-3 min, 20% A; 3-8 min, 20%-23% A; 8-19 min, 23%-23.5% A; 19-25 min, 23.5%-24% A; 25-28 min, 24% A; 28-34 min, 24%-36% A; 34-49 min, 36%-41% A; 49-55 min, 41%-43% A; 55-65 min, 43%-47% A; 65-87 min, 47%-100% A; 87-97 min, 100% A); the flow rate was 1.2 mL per minute, the column temperature was 30°C, and the detection wavelength was 220 nm.

[0218] Comparative document 2 method: octadecyl bonded silica gel is used as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm), acetonitrile is used as mobile phase A, 0.3% phosphoric acid solution is used as mobile phase B, gradient elution (0-6 min, 5%-9% A; 6-15 min, 9%-20% A; 15-45 min, 20%-35% A; 45-70 min, 35%-60% A); the flow rate is 1.0 mL per minute, the column temperature is 30°C, and the detection wavelength is 210 nm.

[0219] The results showed that there was a corresponding higher chromatographic peak near the hesperidin peak in the chromatogram of the orange core sample, and the separation effect of the chromatographic peak and the hesperidin peak was poor under the above two chromatographic conditions ( Figure 21 、 Figure 22 ), which shows that the methods of comparative documents 1 and 2 are difficult to effectively distinguish tangerine pits from orange pits.

[0220] The above is a preferred embodiment of the invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the invention. These improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A method for constructing a fingerprint of a tangerine seed medicinal material and a traditional Chinese medicine preparation thereof, characterized in that: include: Extracting the tangerine seed medicinal material or its traditional Chinese medicine preparation with a first extraction solvent to obtain a test solution; wherein the traditional Chinese medicine preparation of the tangerine seed medicinal material includes tangerine seed decoction pieces and tangerine seed standard decoction and tangerine seed formula granules processed from the tangerine seed decoction pieces; or the traditional Chinese medicine preparation of the tangerine seed medicinal material includes salted tangerine seed decoction pieces and salted tangerine seed standard decoction and salted tangerine seed formula granules processed from the salted tangerine seed decoction pieces; or the traditional Chinese medicine preparation of the tangerine seed medicinal material includes stir-fried tangerine seed decoction pieces and stir-fried tangerine seed standard decoction and stir-fried tangerine seed formula granules processed from the stir-fried tangerine seed decoction pieces; Dissolving or extracting the reference substance and the tangerine seed reference medicinal material with a second extraction solvent to obtain a reference substance solution and a reference medicinal material solution; wherein the reference substances include a vecinin-2 reference substance, a hesperidin reference substance, a limonin reference substance, a nomilin reference substance, and a flavonoid reference substance; The test solution, reference solution and reference medicinal material solution are measured by liquid chromatography to obtain a fingerprint of the orange seed or its Chinese medicine preparation; The chromatographic column of the liquid chromatograph is a Waters Cortecs C18 chromatographic column with a column length of 150 mm, a column diameter of 4.6 mm, and a particle size of the stationary phase of 2.7 μm. The liquid chromatograph uses acetonitrile as mobile phase A and a phosphoric acid aqueous solution with a concentration of 0.1 vol% to 0.5 vol% as mobile phase B for gradient elution. The gradient elution curve is: 0 min to 5 min, mobile phase A was 9%, mobile phase B was 91%; 5min~6min, mobile phase A from 9% to 12%, mobile phase B from 91% to 88%; 6 min to 20 min, mobile phase A from 12% to 20%, mobile phase B from 88% to 80%; 20 min to 30 min, mobile phase A from 20% to 23%, mobile phase B from 80% to 77%; From 30 to 40 minutes, mobile phase A increased from 23% to 44%, and mobile phase B increased from 77% to 56%. 40-50 min, mobile phase A from 44% to 45%, mobile phase B from 56% to 55%; The detection wavelength of the liquid chromatograph is 220 nm; Wherein, the first extraction solvent and the second extraction solvent are methanol aqueous solutions with a concentration of 30 vol% to 100 vol%; The fingerprint spectrum includes 25 common peaks, among which peak 3 is vetacnin-2, peak 7 is kaempferol-3-O-rutinoside, peak 8 is naringin, peak 9 is hesperidin, peak 17 is hesperetin, peak 22 is limonin, peak 24 is nomilin, and peak 25 is flavonoid; Taking Peak 9 as the S peak, calculate the relative retention times of Peaks 10 and 11. The relative retention times of each peak are within ±10% of the specified values. The specified values ​​for Peaks 10 and 11 are 1.02 and 1.05, respectively. The relative peak areas of Peak 10 and Peak 11 are calculated, and the ratio of the relative peak areas of Peak 10 and Peak 11 is less than or equal to 1.

0.

2. The method for constructing the fingerprint of the tangerine seed medicinal material and its traditional Chinese medicine preparation according to claim 1, characterized in that: The mobile phase B is 0.2 vol% phosphoric acid, the first extraction solvent is a methanol aqueous solution with a concentration of 70 vol%, and the second extraction solvent is methanol.

3. The method for constructing the fingerprint of the tangerine seed medicinal material and its traditional Chinese medicine preparation according to claim 1, characterized in that: The injection volume of the test solution is 0.8μL~5μL, the injection volume of the reference solution is 0.8μL~5μL, and the injection volume of the control medicinal material solution is 0.8μL~5μL.

4. The method for constructing the fingerprint of the tangerine seed medicinal material and its traditional Chinese medicine preparation according to claim 3, characterized in that: The column temperature of the chromatographic column is 30°C; The flow rate of the liquid chromatograph was 0.9 mL / min, the injection volume of the test solution was 2 μL, the injection volume of the reference solution was 2 μL, and the injection volume of the reference medicinal material solution was 2 μL.

5. The method for constructing the fingerprint of the tangerine seed medicinal material and its traditional Chinese medicine preparation according to claim 1, characterized in that: In the step of extracting the tangerine seed medicinal material or the Chinese medicine preparation thereof with a first extraction solvent to obtain a test solution, the tangerine seed medicinal material or the Chinese medicine preparation thereof is mixed with a methanol aqueous solution with a concentration of 30 vol% to 100 vol%, and ultrasonically treated or heated under reflux for 10 min to 60 min; The ratio of the orange seed medicinal material or its Chinese medicine preparation to the methanol aqueous solution is 0.1g~2g:10mL~50mL.

6. The method for constructing the fingerprint of the tangerine seed medicinal material and its traditional Chinese medicine preparation according to claim 1, characterized in that: In the step of dissolving or extracting the reference substance and the tangerine seed control medicinal material with a second extraction solvent to obtain the reference substance solution and the control medicinal material solution, the tangerine seed control medicinal material is mixed with a methanol aqueous solution having a concentration of 30 vol% to 100 vol%, and ultrasonically treated or heated under reflux for 10 min to 60 min; wherein the ratio of the tangerine seed control medicinal material to the methanol aqueous solution is 0.5 g to 2 g: 10 mL to 50 mL; In the reference substance solution, the concentration of each reference substance is 5 μg / mL to 20 μg / mL.

Images