Pericarpium citri reticulatae fingerprint spectrum, construction method and application thereof, multi-index component detection method of pericarpium citri reticulatae, identification standard of pericarpium citri reticulatae variety and construction method thereof

Abstract

The invention provides a pericarpium citri reticulatae fingerprint spectrum, a construction method and application thereof, a multi-index component detection method of pericarpium citri reticulatae, an identification standard of pericarpium citri reticulatae varieties and a construction method of the identification standard, and relates to the technical field of analysis and detection. The pericarpium citri reticulatae fingerprint spectrum provided by the invention has 17 characteristic peaks, wherein the 17 characteristic peaks comprise the characteristic peaks of narirutin, hesperidin, neohesperidin, sweet orange flavone, nobiletin, 5-demethylnobiletin, hesperetin and heptamethoxyflavone. According to the pericarpium citri reticulatae fingerprint spectrum provided by the invention, a plurality of substances are used as quality indexes of the pericarpium citri reticulatae, so that accurate and comprehensive quality control on the quality of the pericarpium citri reticulatae can be realized, and an excessively simple currently executed quality standard of the pericarpium citri reticulatae is perfected. According to the method, the mass ratio of narirutin to hesperidin to neohesperidin to sweet orange flavone to nobiletin to 5-demethylnobiletin is taken as an identification standard of the dried orange peel variety, and accurate identification of six different dried orange peels including Citrus chachiensis, Dahongpao tea, Satsuma orange, Fujian orange, local early orange and Nanfeng orange can be realized.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, and in particular to a tangerine peel fingerprint spectrum and a construction method and application thereof, a tangerine peel multi-index component detection method, and a tangerine peel variety identification standard and a construction method thereof. Background Art

[0002] The 2020 edition of the Pharmacopoeia of the People's Republic of China stipulates that the main cultivated varieties of dried tangerine peel are C. reticulata 'Chachi' (Guangdong tangerine peel), C. reticulata 'Dahongpao', C. reticulata 'Unshiu' and C. reticulata 'langerina'. However, based on literature and research, it is found that in addition to these four types of dried tangerine peel on the market, there are other varieties such as Nanfeng tangerine and local early, which brings instability and uncertainty to the quality safety and clinical efficacy of dried tangerine peel. Many literatures have studied the differences between different cultivated varieties of dried tangerine peel, but most of them focus on the differences in the ingredients contained in dried tangerine peel, especially the differences in volatile oils and flavonoids. There are few reports on how to distinguish different varieties of dried tangerine peel. The 2020 edition of the Pharmacopoeia of the People's Republic of China uses the content of hesperidin as an indicator for the quality evaluation of "dried tangerine peel" medicinal materials, but it is difficult to identify different cultivars and objectively evaluate the intrinsic quality of medicinal materials by only measuring the content of this component. Therefore, it is particularly important to establish an accurate and comprehensive quality control method. Summary of the invention

[0003] In view of this, the purpose of the present invention is to provide a tangerine peel fingerprint spectrum and its construction method and application, a multi-index component detection method for tangerine peel, and an identification standard for tangerine peel varieties and its construction method. The tangerine peel fingerprint spectrum provided by the present invention uses multiple substances as the quality indicators of tangerine peel, and can achieve accurate and comprehensive quality control of tangerine peel quality.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The invention provides a tangerine peel fingerprint spectrum, comprising 17 characteristic peaks, wherein the characteristic peaks include characteristic peaks of naringin, hesperidin, neohesperidin, sweet orange flavonoids, nobiletin, 5-demethylnobiletin, tangeretin and heptamethoxyflavone.

[0006] The present invention also provides a method for constructing the tangerine peel fingerprint spectrum described in the above technical solution, comprising the following steps:

[0007] Mixing tangerine peel with an extractant to obtain a test solution; the extractant includes methanol; the tangerine peel includes one or more of tea-branch tangerine, Dahongpao, mandarin orange, Fuju, Bendi Zao and Nanfeng mandarin orange;

[0008] The test solution and the mixed reference solution are subjected to high performance liquid chromatography to obtain a test liquid phase spectrum and a reference liquid phase spectrum, respectively;

[0009] The liquid phase spectrum of the test sample is introduced into the similarity evaluation system of the characteristic spectrum of traditional Chinese medicine chromatography to generate a common mode of the characteristic spectrum, and a fingerprint spectrum of dried orange peel with 17 characteristic peaks is obtained; by comparing the retention time of the fingerprint spectrum with that of the liquid phase spectrum of the reference substance, characteristic peaks of naringin, hesperidin, neohesperidin, sweet orange flavonoids, nobiletin, 5-demethylnobiletin, tangeretin and heptamethoxyflavonoids in the fingerprint spectrum of dried orange peel are obtained;

[0010] The reference substances in the mixed reference substance solution include naringin, hesperidin, neohesperidin, sweet orange flavonoids, nobiletin, 5-demethylnobiletin, tangeretin and heptamethoxyflavone;

[0011] The conditions for the high performance liquid chromatography detection include: the mobile phase system includes mobile phase A and mobile phase B, the mobile phase A is acetonitrile, and the mobile phase B is 0.1 v / v% formic acid aqueous solution; the elution method is gradient elution, and the procedure of the gradient elution includes:

[0012] 0-0.2min, the volume fraction of the mobile phase A is 5%;

[0013] 0.2-2.5min, the volume fraction of the mobile phase A is increased from 5% to 6%;

[0014] 5-15 min, the volume fraction of the mobile phase A is increased from 6% to 25%;

[0015] 15-18 min, the volume fraction of the mobile phase A is increased from 25% to 45%;

[0016] 18-27 min, the volume fraction of the mobile phase A is increased from 45% to 60%;

[0017] 27-31 min, the volume fraction of the mobile phase A increases from 60% to 95%;

[0018] 31-34.9 min, the volume fraction of the mobile phase A is 95%;

[0019] From 34.9 to 35 min, the volume fraction of the mobile phase A is reduced from 95% to 5%.

[0020] Preferably, the solid-liquid ratio of the tangerine peel to the extractant is 0.95-1.05 g: 49.5-50.5 mL;

[0021] The volume fraction of methanol in the extractant is 50 to 100%;

[0022] The extraction protection ultrasonic extraction; the power of the ultrasonic extraction is 240-260W, the frequency is 75-85kHz, and the time is 29-31min.

[0023] Preferably, in the mixed reference solution, the concentration of nobiletin is 0.149-0.151 mg / mL, the concentration of tangeretin is 0.019-0.021 mg / mL, the concentration of hesperidin is 0.319-0.321 mg / mL, the concentration of naringin is 0.009-0.011 mg / mL, the concentration of neohesperidin is 0.0019-0.0021 mg / mL, the concentration of sweet orange flavonoids is 0.201-0.203 mg / mL, the concentration of 5-demethylnobiletin is 0.200-0.202 mg / mL, and the concentration of heptamethoxyflavone is 0.185-0.195 mg / mL; the solvent in the mixed reference solution includes methanol.

[0024] Preferably, the HPLC detection conditions include: the chromatographic column includes a Waters Cortecs UPLC T3 chromatographic column, the mobile phase flow rate is 0.24-0.26 mL / min, the detection wavelength is 283 nm, the column temperature is 39-41° C., and the injection volume is 1.99-2.01 μL.

[0025] The present invention also provides the application of the tangerine peel fingerprint spectrum described in the above technical solution or the tangerine peel fingerprint spectrum obtained by the construction method described in the above technical solution in the detection of components in tangerine peel or the identification of tangerine peel varieties.

[0026] The present invention also provides a method for detecting multiple index components of tangerine peel, comprising the following steps:

[0027] The tangerine peel to be tested is mixed with an extractant to obtain a test solution; the extractant includes methanol; the tangerine peel includes one or more of the following: tea-branch tangerine, dahongpao, mandarin orange, fu ju, bendi zao and nanfeng tangerine;

[0028] The sample solution to be tested is subjected to high performance liquid chromatography to obtain a multi-index component test result of tangerine peel; the multi-index components include at least two of naringin, hesperidin, neohesperidin, sweet orange flavonoids, nobiletin, 5-demethylnobiletin, tangeretin and heptamethoxyflavone; the test result includes a quantitative test result and / or a qualitative test result; the qualitative test result is obtained by comparing the liquid phase spectrum of the test solution with the tangerine peel fingerprint spectrum described in the above technical solution or the tangerine peel fingerprint spectrum obtained by the construction method described in the above technical solution;

[0029] The conditions for the high performance liquid chromatography detection include: the mobile phase system includes mobile phase A and mobile phase B, the mobile phase A is acetonitrile, and the mobile phase B is 0.1 v / v% formic acid aqueous solution; the elution method is gradient elution, and the procedure of the gradient elution includes:

[0030] 0-0.2min, the volume fraction of the mobile phase A is 5%;

[0031] 0.2-2.5min, the volume fraction of the mobile phase A is increased from 5% to 6%;

[0032] 5-15 min, the volume fraction of the mobile phase A is increased from 6% to 25%;

[0033] 15-18 min, the volume fraction of the mobile phase A is increased from 25% to 45%;

[0034] 18-27 min, the volume fraction of the mobile phase A is increased from 45% to 60%;

[0035] 27-31 min, the volume fraction of the mobile phase A increases from 60% to 95%;

[0036] 31-34.9 min, the volume fraction of the mobile phase A is 95%;

[0037] From 34.9 to 35 min, the volume fraction of the mobile phase A is reduced from 95% to 5%.

[0038] Preferably, the solid-liquid ratio of the tangerine peel to the extractant is 0.95-1.05 g: 49.5-50.5 mL;

[0039] The volume fraction of methanol in the extractant is 50 to 100%;

[0040] The extraction protection ultrasonic extraction; the power of the ultrasonic extraction is 240-260W, the frequency is 75-85kHz, and the time is 29-31min;

[0041] The high performance liquid chromatography detection conditions include: the chromatographic column includes a Waters Cortecs UPLC T3 chromatographic column, the mobile phase flow rate is 0.24-0.26 mL / min, the detection wavelength is 283 nm, the column temperature is 39-41° C., and the injection volume is 1.99-2.01 μL.

[0042] The present invention also provides a standard for identifying tangerine peel varieties, wherein the tangerine peel varieties include one or more of the following: tea-branch tangerine, dahongpao, mandarin orange, fu ju, local early tangerine and nanfeng mandarin orange;

[0043] When the ratio between the components is 0.87-2.35:23.0-37.62:0.79-1.00:1.25-2.00:11.90-17.00:0.85-1.50, the tangerine peel variety is tea-branch tangerine peel;

[0044] When the ratio between the components is 0.77-1.62: 28.33-41.50: 1.49-13.10: 1.85-5.10: 24.00-29.90: 0.40-0.98, the tangerine peel variety is Da Hong Pao;

[0045] When the ratio between the components is 23.40-23.50: 36.30-36.36: 0.20-0.23: 0.10-0.12: 0.78-0.80: 0.38-0.39, the orange peel variety is mandarin orange;

[0046] When the ratio between the components is 0.7-0.75: 21.96-21.97: 2.25-2.26: 1.99-2.00: 9.50-9.55: 5.25-5.26, the orange peel variety is Fuju;

[0047] When the ratio between the components is 0.96-5.96:29.75-43.74:0.22-0.23:1.08-1.33:7.13-7.41:1.11-1.95, the tangerine peel variety is local early;

[0048] When the ratio between the components is 1.74-1.76: 18.55-18.56: 5.14-5.15: 6.85-6.86: 38.43-38.44: 1.23-1.25, the tangerine peel variety is Nanfeng mandarin orange;

[0049] The ratio between the components is the mass ratio of naringin, hesperidin, neohesperidin, citrus aurantium, nobiletin and 5-demethylnobiletin.

[0050] The present invention also provides a method for constructing the identification standard of tangerine peel varieties described in the above technical solution, comprising the following steps:

[0051] The quantitative detection results of the multi-index components of tangerine peel are obtained according to the multi-index component detection method described in the above technical scheme; the multi-index components include naringin, hesperidin, neohesperidin, sweet orange flavonoids, nobiletin and 5-demethylnobiletin; the tangerine peel includes one or more of tea-branch tangerine, dahongpao, mandarin orange, fu ju, local early and Nanfeng mandarin orange;

[0052] The mass ratios of naringin, hesperidin, neohesperidin, citrus aurantium, nobiletin and 5-demethylnobiletin are calculated according to the quantitative detection results of the multi-index components to obtain the identification standard of the tangerine peel variety.

[0053] The tangerine peel fingerprint provided by the present invention has 17 characteristic peaks, including characteristic peaks of naringin, hesperidin, neohesperidin, citrus flavonoids, nobiletin, 5-demethylnobiletin, tangeretin and heptamethoxyflavone. The tangerine peel fingerprint provided by the present invention uses multiple substances as quality indicators of tangerine peel, can achieve accurate and comprehensive quality control of tangerine peel quality, and improves the overly simple existing quality standards for tangerine peel.

[0054] The method for constructing the tangerine peel fingerprint provided by the present invention has high precision, good stability and good reproducibility, and can comprehensively control the quality of tangerine peel, thereby better ensuring the quality stability, consistency and controllability of tangerine peel. The method provided by the present invention overcomes the problem that the existing tangerine peel quality standard is single and cannot accurately reflect the quality of tangerine peel.

[0055] The present invention establishes a multi-index component synchronous detection method for tangerine peel, which has the advantages of strong specificity, simple operation, etc., and has good stability, precision, reproducibility and sample recovery rate. The methodological investigation test results are good. The content of 8 main effective ingredients in tangerine peel, including naringin, hesperidin, neohesperidin, sweet orange flavonoids, nobiletin, 5-demethylnobiletin, tangeretin and heptamethoxyflavone, can be comprehensively detected, and the method can be used as a marker component for evaluating the quality of tangerine peel medicinal materials.

[0056] The present invention also provides an identification standard for tangerine peel varieties. The present invention uses the mass ratio of naringin, hesperidin, neohesperidin, sweet orange flavonoids, nobiletin and 5-demethylnobiletin as the identification standard for tangerine peel varieties, and can achieve accurate identification of six different kinds of tangerine peels, namely, tea-branch tangerine, dahongpao, mandarin orange, lucky orange, local early tangerine and Nanfeng mandarin orange, providing a more scientific basis for ensuring the quality of medicinal materials.

[0057] The method for constructing the identification standard of tangerine peel varieties provided by the invention is simple to operate and provides a more scientific basis for judging the quality of different tangerine peel medicinal materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 is the UPLC chromatogram of sample S1;

[0059] Figure 2 is the UPLC chromatogram of sample S2;

[0060] Figure 3 is the UPLC chromatogram of sample S3;

[0061] Figure 4 is the UPLC chromatogram of sample S4;

[0062] Figure 5 is the UPLC chromatogram of sample S5;

[0063] Figure 6 is the UPLC chromatogram of sample S6;

[0064] Figure 7 is the UPLC chromatogram of sample S7;

[0065] Figure 8 is the UPLC chromatogram of sample S8;

[0066] Fig. 9 is the UPLC chromatogram of sample S9;

[0067] Fig.10 is the UPLC chromatogram of sample S10;

[0068] Fig.11 is the UPLC chromatogram of sample S11;

[0069] Fig.12 is the UPLC chromatogram of sample S12;

[0070] Fig.13 Overlay UPLC spectra for 12 batches of sample fingerprints;

[0071] Fig.14 This is the common pattern diagram of UPLC fingerprint of dried tangerine peel;

[0072] Figure 1 to Figure 14 Among them, peak 10 is naringin, peak 11 is hesperidin, peak 12 is neohesperidin, peak 13 is sweet orange flavonoids, peak 14 is nobiletin, peak 15 is 5-demethylnobiletin, peak 16 is hesperidin, and peak 17 is heptamethoxyflavone;

[0073] Fig.15 is the UPLC chromatogram of the test solution using 50 v / v% methanol aqueous solution as the extractant;

[0074] Fig.16 is the UPLC chromatogram of the test solution using 70 v / v% methanol aqueous solution as the extractant;

[0075] Fig.17 It is the UPLC chromatogram of the test solution using anhydrous methanol water as the extractant;

[0076] Fig.18 It is the UPLC chromatogram of the test solution when gradient elution program 1 is used;

[0077] Fig.19 The UPLC chromatogram of the test solution using gradient elution program 2. DETAILED DESCRIPTION

[0078] The invention provides a tangerine peel fingerprint spectrum, comprising 17 characteristic peaks, wherein the characteristic peaks include characteristic peaks of naringin, hesperidin, neohesperidin, sweet orange flavonoids, nobiletin, 5-demethylnobiletin, tangeretin and heptamethoxyflavone.

[0079] The present invention provides a method for constructing a tangerine peel fingerprint spectrum described in the above technical solution, comprising the following steps:

[0080] Mixing tangerine peel with an extractant to obtain a test solution; the extractant includes methanol; the tangerine peel includes one or more of tea-branch tangerine, Dahongpao, mandarin orange, Fuju, Bendi Zao and Nanfeng mandarin orange;

[0081] The test solution and the mixed reference solution are subjected to high performance liquid chromatography to obtain a test liquid phase spectrum and a reference liquid phase spectrum, respectively;

[0082] The liquid phase spectrum of the test sample is introduced into the similarity evaluation system of the characteristic spectrum of traditional Chinese medicine chromatography to generate a common mode of the characteristic spectrum, and a fingerprint spectrum of dried orange peel with 17 characteristic peaks is obtained; by comparing the retention time of the fingerprint spectrum with that of the liquid phase spectrum of the reference substance, characteristic peaks of naringin, hesperidin, neohesperidin, sweet orange flavonoids, nobiletin, 5-demethylnobiletin, tangeretin and heptamethoxyflavonoids in the fingerprint spectrum of dried orange peel are obtained;

[0083] The reference substances in the mixed reference substance solution include naringin, hesperidin, neohesperidin, sweet orange flavonoids, nobiletin, 5-demethylnobiletin, tangeretin and heptamethoxyflavone;

[0084] The conditions for the high performance liquid chromatography detection include: the mobile phase system includes mobile phase A and mobile phase B, the mobile phase A is acetonitrile, and the mobile phase B is 0.1 v / v% formic acid aqueous solution; the elution method is gradient elution, and the procedure of the gradient elution includes:

[0085] 0-0.2min, the volume fraction of the mobile phase A is 5%;

[0086] 0.2-5min, the volume fraction of the mobile phase A increases from 5% to 6%;

[0087] 5-15 min, the volume fraction of the mobile phase A is increased from 6% to 25%;

[0088] 15-18 min, the volume fraction of the mobile phase A is increased from 25% to 45%;

[0089] 18-27 min, the volume fraction of the mobile phase A is increased from 45% to 60%;

[0090] 27-31 min, the volume fraction of the mobile phase A increases from 60% to 95%;

[0091] 31-34.9 min, the volume fraction of the mobile phase A is 95%;

[0092] From 34.9 to 35 min, the volume fraction of the mobile phase A is reduced from 95% to 5%.

[0093] Unless otherwise specified, the materials and equipment used in the present invention are all commercially available products in the art.

[0094] The present invention mixes tangerine peel with an extractant to obtain a test solution; the extractant comprises methanol; the tangerine peel comprises one or more of the following: tangerine peel of tea-branch citrus, dahongpao, mandarin orange, fu ju, benbenzao and nanfeng mandarin orange, and more preferably tangerine peel of tea-branch citrus, dahongpao, mandarin orange, fu ju, benbenzao and nanfeng mandarin orange.

[0095] In the present invention, the extractant includes methanol or a methanol aqueous solution, more preferably a methanol aqueous solution; the volume fraction of methanol in the extractant is preferably 50-100%, and in specific embodiments it can be 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

[0096] In the present invention, the solid-liquid ratio of the tangerine peel to the extractant is preferably 0.95-1.05 g:49.5-50.5 mL, more preferably 1 g:50 mL. In the present invention, the tangerine peel is preferably crushed before use to obtain a tangerine peel product.

[0097] In the present invention, the extraction preferably includes ultrasonic extraction, the temperature of the ultrasonic extraction is preferably room temperature, the power is preferably 240-260W, more preferably 250W, the frequency is preferably 75-85kHz, more preferably 80kHz, and the time is preferably 29-31min, more preferably 30min.

[0098] After the extraction, the present invention preferably further comprises passing the obtained extraction system through a 0.22 μm microporous filter membrane to obtain a test solution.

[0099] After obtaining the test solution, the present invention preferably performs high performance liquid chromatography on the test solution and the mixed reference solution to obtain a test liquid phase spectrum and a reference liquid phase spectrum, respectively;

[0100] In the present invention, the reference substances in the mixed reference solution include naringin, hesperidin, neohesperidin, sweet orange flavonoids, nobiletin, 5-demethylnobiletin, tangeretin and heptamethoxyflavone. In the present invention, in the mixed reference solution, the concentration of nobiletin is preferably 0.149-0.151 mg / mL, more preferably 0.150 mg / mL; the concentration of tangeretin is preferably 0.019-0.021 mg / mL, more preferably 0.020 mg / mL; the concentration of hesperidin is preferably 0.319-0.321 mg / mL, more preferably 0.320 mg / mL; the concentration of naringin is preferably 0.009-0.011 mg / mL, more preferably 0.010 mg / mL; the concentration of neohesperidin is preferably 0. The concentration of sweet orange flavonoids is preferably 0.0019-0.0021 mg / mL, more preferably 0.002 mg / mL; the concentration of 5-demethylnobiletin is preferably 0.201-0.203 mg / mL, more preferably 0.202 mg / mL; the concentration of heptamethoxyflavone is preferably 0.185-0.195 mg / mL, more preferably 0.19 mg / mL; the solvent in the mixed reference solution preferably includes methanol.

[0101] In the present invention, the HPLC detection conditions include: the chromatographic column is preferably Waters The specification of the chromatographic column is preferably 150mm×2.1mm, 1.6μm; the mobile phase system includes mobile phase A and mobile phase B, the mobile phase A is acetonitrile, the mobile phase B is formic acid aqueous solution, the volume fraction of formic acid in the formic acid aqueous solution is 0.095-0.15%, preferably 0.098-0.12%, more preferably 0.1%; the elution mode is gradient elution, and the procedure of the gradient elution includes: 0-0.2min, the volume fraction of the mobile phase A is 5%; 0.2-5min, the volume fraction of the mobile phase A increases from 5% to 6%; 5-15min, the volume fraction of the mobile phase A increases from 6% to 25%; 15-18min, the volume fraction of the mobile phase A increases from 6% to 25%; The volume fraction of mobile phase A increases from 25% to 45%; from 18 to 27 min, the volume fraction of mobile phase A increases from 45% to 60%; from 27 to 31 min, the volume fraction of mobile phase A increases from 60% to 95%; from 31 to 34.9 min, the volume fraction of mobile phase A is 95%; from 34.9 to 35 min, the volume fraction of mobile phase A decreases from 95% to 5%; the mobile phase flow rate is preferably 0.24 to 0.26 mL / min, more preferably 0.25 mL / min, the detection wavelength is preferably 283 nm, the column temperature is preferably 39 to 41° C., more preferably 40° C., and the injection volume is preferably 1.99 to 2.01 μL, more preferably 2 μL.

[0102] The liquid phase spectrum of the test sample is introduced into the similarity evaluation system of the Chinese medicine chromatographic characteristic spectrum, and a common mode of the characteristic spectrum is generated to obtain a tangerine peel fingerprint spectrum with 17 characteristic peaks; by comparing the retention time of the fingerprint spectrum with the reference liquid phase spectrum, characteristic peaks of rutin, hesperidin, neohesperidin, sweet orange flavonoids, nobiletin, 5-demethylnobiletin, tangeretin and heptamethoxyflavonoids are obtained in the tangerine peel fingerprint spectrum. In the present invention, the liquid phase spectrum of the test sample is preferably calculated by the software of "Chinese medicine chromatographic characteristic spectrum similarity evaluation system", and the similarity of each tangerine peel sample is between 0.756 and 0.995. In the present invention, the software of "Chinese medicine chromatographic characteristic spectrum similarity evaluation system" is preferably "Chinese medicine chromatographic characteristic spectrum similarity evaluation system software (2012 edition)" of the State Pharmacopoeia Committee. In the present invention, by comparing and analyzing the retention time of the reference substance in the tangerine peel fingerprint spectrum and the reference substance liquid phase spectrum, it is obtained that peak 10 in the tangerine peel fingerprint spectrum is naringin, peak 11 is hesperidin, peak 12 is neohesperidin, peak 13 is sweet orange flavonoids, peak 14 is nobiletin, peak 15 is 5-demethylnobiletin, peak 16 is hesperidin, and peak 17 is heptamethoxyflavone.

[0103] The present invention also provides the application of the tangerine peel fingerprint spectrum described in the above technical solution or the tangerine peel fingerprint spectrum obtained by the construction method described in the above technical solution in the detection of components in tangerine peel or the identification of tangerine peel varieties.

[0104] The present invention provides a method for detecting multiple index components of dried tangerine peel, comprising the following steps:

[0105] The tangerine peel to be tested is mixed with an extractant to obtain a test solution; the extractant includes methanol; the tangerine peel includes one or more of the following: tea-branch tangerine, dahongpao, mandarin orange, fu ju, bendi zao and nanfeng tangerine;

[0106] The sample solution to be tested is subjected to high performance liquid chromatography to obtain a multi-index component test result of tangerine peel; the multi-index components include at least two of naringin, hesperidin, neohesperidin, sweet orange flavonoids, nobiletin, 5-demethylnobiletin, tangeretin and heptamethoxyflavone; the test result includes a quantitative test result and / or a qualitative test result; the qualitative test result is obtained by comparing the liquid phase spectrum of the test solution with the tangerine peel fingerprint spectrum described in the above technical solution or the tangerine peel fingerprint spectrum obtained by the construction method described in the above technical solution;

[0107] The conditions for the high performance liquid chromatography detection include: the mobile phase system includes mobile phase A and mobile phase B, the mobile phase A is acetonitrile, and the mobile phase B is 0.1 v / v% formic acid aqueous solution; the elution method is gradient elution, and the procedure of the gradient elution includes:

[0108] 0-0.2min, the volume fraction of the mobile phase A is 5%;

[0109] 0.2-2.5min, the volume fraction of the mobile phase A is increased from 5% to 6%;

[0110] 5-15 min, the volume fraction of the mobile phase A is increased from 6% to 25%;

[0111] 15-18 min, the volume fraction of the mobile phase A is increased from 25% to 45%;

[0112] 18-27 min, the volume fraction of the mobile phase A is increased from 45% to 60%;

[0113] 27-31 min, the volume fraction of the mobile phase A increases from 60% to 95%;

[0114] 31-34.9 min, the volume fraction of the mobile phase A is 95%;

[0115] From 34.9 to 35 min, the volume fraction of the mobile phase A is reduced from 95% to 5%.

[0116] In the present invention, the preparation method of the sample solution to be tested is preferably the same as the preparation method of the test solution; the conditions of the HPLC detection are the same as the conditions of the HPLC detection in the process of constructing the labeled tangerine peel spectrum, and will not be repeated here.

[0117] In the present invention, the quantitative detection result is preferably obtained by quantitatively using the area normalization method.

[0118] The present invention also provides a standard for identifying tangerine peel varieties, wherein the tangerine peel varieties include one or more of the following: tea-branch tangerine, dahongpao, mandarin orange, fu ju, local early tangerine and nanfeng mandarin orange;

[0119] When the ratio between the components is 0.87-2.35:23.0-37.62:0.79-1.00:1.25-2.00:11.90-17.00:0.85-1.50, the tangerine peel variety is tea-branch tangerine peel;

[0120] When the ratio between the components is 0.77-1.62: 28.33-41.50: 1.49-13.10: 1.85-5.10: 24.00-29.90: 0.40-0.98, the tangerine peel variety is Da Hong Pao;

[0121] When the ratio between the components is 23.40-23.50: 36.30-36.36: 0.20-0.23: 0.10-0.12: 0.78-0.80: 0.38-0.39, the orange peel variety is mandarin orange;

[0122] When the ratio between the components is 0.7-0.75: 21.96-21.97: 2.25-2.26: 1.99-2.00: 9.50-9.55: 5.25-5.26, the orange peel variety is Fuju;

[0123] When the ratio between the components is 0.96-5.96:29.75-43.74:0.22-0.23:1.08-1.33:7.13-7.41:1.11-1.95, the tangerine peel variety is local early;

[0124] When the ratio between the components is 1.74-1.76: 18.55-18.56: 5.14-5.15: 6.85-6.86: 38.43-38.44: 1.23-1.25, the tangerine peel variety is Nanfeng mandarin orange;

[0125] The ratio between the components is the mass ratio of naringin, hesperidin, neohesperidin, citrus aurantium, nobiletin and 5-demethylnobiletin.

[0126] The present invention also provides a method for constructing the identification standard of tangerine peel varieties described in the above technical solution, comprising the following steps:

[0127] The quantitative detection results of the multi-index components of tangerine peel are obtained according to the multi-index component detection method described in the above technical scheme; the multi-index components include naringin, hesperidin, neohesperidin, sweet orange flavonoids, nobiletin and 5-demethylnobiletin; the tangerine peel includes one or more of tea-branch tangerine, dahongpao, mandarin orange, fu ju, local early and Nanfeng mandarin orange;

[0128] The mass ratios of naringin, hesperidin, neohesperidin, citrus aurantium, nobiletin and 5-demethylnobiletin are calculated according to the quantitative detection results of the multi-index components to obtain the identification standard of the tangerine peel variety.

[0129] To further illustrate the present invention, the tangerine peel fingerprint provided by the present invention, its construction method and application, the multi-index component detection method of tangerine peel, the identification standard of tangerine peel varieties and its construction method are described in detail below in conjunction with the embodiments, but they should not be understood as limiting the scope of protection of the present invention.

[0130] In the following examples, instruments are shown in Table 1, chemical reagents are shown in Table 2, reference substances are shown in Table 3, and tangerine peel samples are shown in Table 4.

[0131] Table 1 Experimental instruments

[0132] Instrument Name model Manufacturer Ultra-high performance liquid chromatography Ultimate3000RSLC Thermo-Fisher, USA Ultrasonic Cleaner KQ-500CDE Thermo-Fisher, USA Electronic balance SartoriusBP121s Beijing Sartorius Scientific Instruments Co., Ltd. Circulation vacuum pump SHZ-DⅢ Gongyi Yuhua Instrument Co., Ltd.

[0133] Table 2 Chemical reagents

[0134] Reagents level Manufacturer Methanol Chromatographic pure Thermo-Fisher, USA Formic acid Chromatographic pure Thermo-Fisher, USA Methanol Analytical grade Chengdu Kelong Chemicals Co., Ltd. Ultrapure water Chromatographic pure Merck Mili-Q water purification system

[0135] Table 3 Reference substances

[0136]

[0137]

[0138] Table 4 Tangerine peel sample information

[0139] Tangerine peel sample number Cultivar Origin Harvest time Weight / g S1 Da Hong Pao Tailong Town, Chongqing 2023.11 1000 S2 Da Hong Pao Jintang County, Sichuan Province 2023.12 1000 S3 Da Hong Pao Xingan County, Jiangxi Province 2023.12 1000 S4 Tea Branch Tangerine Xinhui District, Guangdong Province 2023.11 1000 S5 Tea Branch Tangerine Meijiang Town, Guangdong Province 2023.11 1000 S6 Tea Branch Tangerine Sanjiang Town, Guangdong Province 2023.11 1000 S7 Tea Branch Tangerine Pubei County, Guangxi Zhuang Autonomous Region 2023.11 1000 S8 Local breakfast Taizhou City, Zhejiang Province 2023.11 1000 S9 Local breakfast Wenzhou City, Zhejiang Province 2023.11 1000 S10 Nanfeng Mandarin Orange Nanfeng County, Jiangxi Province 2023.12 1000 S11 Fukuju Fu'an City, Fujian Province 2023.12 1000 S12 Satsuma mandarin orange Taizhou City, Zhejiang Province 2023.11 1000

[0140] Preparation of mixed reference solution: dissolve nobiletin, tangeretin, hesperidin, naringin and neohesperidin reference substances in methanol, shake well, and filter through a 0.22 μm microporous membrane to prepare mixed reference solutions containing nobiletin, tangeretin, hesperidin, naringin, neohesperidin, citrus flavonoids, 5-demethylnobiletin and heptamethoxyflavone reference substances with concentrations of 0.150 mg / mL, 0.020 mg / mL, 0.320 mg / mL, 0.010 mg / mL, 0.002 mg / mL, 0.202 mg / mL, 0.201 mg / mL and 0.19 mg / mL, respectively.

[0141] Preparation of test solution: Accurately weigh 0.5 g of tangerine peel sample powder from each batch, place it in a stoppered conical flask, accurately add 25 mL of 50% methanol (50 v / v% methanol aqueous solution), weigh the initial weight, ultrasonically extract at room temperature, 250 W, 80 kHz for 30 min, cool to room temperature, make up the lost weight with 50% methanol, shake well, and filter through a 0.22 μm microporous filter membrane to obtain the test solution.

[0142] UPLC chromatographic conditions: chromatographic column is Waters (150mm×2.1mm, 1.6μm), mobile phase flow rate of 0.25mL / min, column temperature of 40℃, detection wavelength of 283nm, injection volume of 2μL, mobile phase A of acetonitrile, mobile phase B of 0.1v / v% formic acid aqueous solution. Gradient elution, elution program see Table 5.

[0143] Table 5 Gradient elution program

[0144]

[0145]

[0146] Example 1

[0147] Fingerprint Methodology Investigation-Precision Test

[0148] The prepared test solution of sample S1 was injected 6 times continuously, and peak 11 was used as the reference peak to calculate the relative peak area of ​​each peak. The results are shown in Table 6, and the relative retention time is shown in Table 7. The results show that the RSD of the relative retention time of each common peak is 0.10-0.63%, and the RSD of the relative peak area is 0.33-2.73%, which meets the requirements of the fingerprint spectrum and has good precision.

[0149] Table 6 Relative peak areas of precision experiments

[0150]

[0151] Table 7 Relative retention time of precision experiment

[0152]

[0153] Example 2

[0154] Fingerprint Methodology Investigation-Repeatability Test

[0155] The sample solution of sample S1 was injected and measured at 0, 4, 8, 12, 18, and 24 hours. Peak 11 was used as the reference peak to calculate the relative peak area and relative retention time of each peak. The results are shown in Tables 8 and 9. The results showed that the relative peak area RSD of each common peak was 2.05-3.43%, and the relative retention time RSD was 0.07-0.58%, which met the requirements of the fingerprint spectrum and had good repeatability.

[0156] Table 8 Relative peak areas of repeatability experiments

[0157]

[0158] Table 9 Relative retention time of repeatability experiment

[0159]

[0160] Example 3

[0161] Fingerprint Methodology Investigation-Stability Test

[0162] Six test solutions (1 to 6) were prepared in parallel with sample S1 and injected for determination. Peak 11 was used as the reference peak to calculate the relative peak area and relative retention time of each peak, and the results are shown in Tables 10 and 11. The results showed that the RSD of the relative peak area of ​​each common peak was 0.43 to 3.13%, and the RSD of the relative retention time was 0.07 to 2.00%, which met the requirements of the fingerprint spectrum and had good stability.

[0163] Table 10 Relative peak areas of stability test

[0164]

[0165] Table 11 Relative retention time of stability experiment

[0166]

[0167]

[0168] Example 4

[0169] 1.1 Establishment and analysis of UPLC fingerprint of dried tangerine peel

[0170] The 12 test solutions were injected and tested respectively to obtain 12 chromatograms of dried orange peel. The chromatograms of samples S1 to S12 are shown in Figures 1 to 12 The fingerprints of each sample were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software to obtain the superimposed fingerprints of 12 dried tangerine peel samples, see Fig.13 . Figures 1 to 13 In the middle, peak 10 is naringin, peak 11 is hesperidin, peak 12 is neohesperidin, peak 13 is sweet orange flavonoids, peak 14 is nobiletin, peak 15 is 5-demethylnobiletin, peak 16 is hesperidin, and peak 17 is heptamethoxyflavone; Fig.13 In the figure (17), 17 common peaks were identified in the fingerprint, and R represents the common pattern peak of the UPLC fingerprint.

[0171] Set sample S1 as the reference spectrum and generate the common pattern map of dried tangerine peel by “median”. The results are shown in Fig.14 , 17 common peaks were identified. By comparing with the UPLC spectrum of the mixed reference substance, the common peaks of the common pattern were identified, and 8 chromatographic peaks were identified, namely peak 10 was naringin, peak 11 was hesperidin, peak 12 was neohesperidin, peak 13 was sweet orange flavonoids, peak 14 was nobiletin, peak 15 was 5-demethylnobiletin, peak 16 was hesperidin, and peak 17 was heptamethoxyflavone.

[0172] 1.2 Fingerprint similarity evaluation

[0173] The common pattern of fingerprints was used as the control (R) to calculate the similarity of 12 dried tangerine peel samples, and the results are shown in Table 12. It can be seen that the similarity is between 0.756 and 0.995.

[0174] Table 1 Similarity of 1212 dried tangerine peel samples

[0175] S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 R S1 1.000 0.930 0.579 0.912 0.832 0.959 0.910 0.982 0.936 0.925 0.784 0.911 0.950 S2 0.930 1.000 0.749 0.980 0.957 0.969 0796 0.968 0.998 0.996 0.930 0.992 0.995 S3 0.579 0.749 1.000 0.722 0.809 0.679 0.335 0.647 0.729 0.747 0.885 0.764 0.756 S4 0.912 0.980 0.722 1.000 0.944 0.957 0.763 0.947 0.975 0.979 0.914 0.975 0.976 S5 0.832 0.957 0.809 0.944 1.000 0.916 0.631 0.898 0.939 0.965 0.984 0.974 0.954 S6 0.959 0.969 0.679 0.957 0.916 1.000 0.916 0.962 0.967 0.968 0.882 0.964 0.979 S7 0.910 0.796 0.335 0.763 0.631 0.832 1.000 0.861 0.827 0.776 0.546 0.738 0.809 S8 0.982 0.968 0.647 0.947 0.989 0.962 0.861 1.000 0.968 0.970 0.860 0.961 0.979 S9 0.936 0.998 0.729 0.975 0.939 0.967 0.827 0.968 1.000 0.990 0.908 0.982 0.992 S10 0.925 0.996 0.747 0.979 0.965 0.968 0.771 0.970 0.990 1.000 0.942 0.998 0993 S11 0.784 0.930 0.885 0.914 0.984 0.882 0.546 0.860 0.908 0.942 1.000 0.956 0.929 S12 0.911 0.992 0.764 0.975 0.974 0.964 0.738 0.961 0.982 0.998 0.956 1.000 0.989

[0176] Depend on Figure 13-14 As shown in Table 12, among the 12 samples, S3 and S7 have lower similarities, which are 0.756 and 0.809 respectively. S3 is mandarin orange and S7 is Nanfeng mandarin orange, which indicates that the chemical composition of mandarin orange and Nanfeng mandarin orange is quite different from that of the other four kinds of tangerine peel.

[0177] Example 5

[0178] Screening of extraction solvents

[0179] 50v / v% methanol aqueous solution, 70v / v% methanol aqueous solution, anhydrous methanol,

[0180] Preparation of test solution: Accurately weigh 0.5 g of tea-branch orange peel sample, place it in a stoppered conical flask, accurately add 25 mL of extraction solvent (50 v / v% methanol aqueous solution, 70 v / v% methanol aqueous solution, anhydrous methanol, respectively), weigh the initial weight, ultrasonically extract at room temperature, 250 W, 80 kHz for 30 min, cool to room temperature, make up the lost weight with 50% methanol, shake well, pass through 0.22 μm microporous filter membrane, and obtain test solutions with different extraction solvents.

[0181] Each test sample was tested. Figure 15 to Figure 17 The fingerprints of the test solution obtained with 50v / v% methanol aqueous solution, 70v / v% methanol aqueous solution and anhydrous methanol as extraction solvents are shown in sequence. It can be seen that the fingerprints obtained with 50v / v% methanol aqueous solution, 70v / v% methanol aqueous solution and anhydrous methanol as extraction solvents have good peak shapes and high separation. Among them, more peaks appear when 50v / v% methanol aqueous solution is used as the extraction solvent, so 50v / v% methanol aqueous solution is used as the extraction solvent.

[0182] Example 6

[0183] Gradient elution program investigation: The test solution was tested by HPLC using the gradient elution programs shown in Table 13 and Table 14, respectively. The results are shown in Fig.18 and Fig.19 .

[0184] Table 13 Gradient elution program 1

[0185] Time / min Mobile phase A volume fraction / % Mobile phase B volume fraction / % 0 10 90 5 20 80 7 20 80 13 22 78 16 35 65 25 55 45 30 95 5 34 95 5 34.5 10 90 39 10 90

[0186] Table 14 Gradient elution program 2

[0187]

[0188]

[0189] Fig.18 The UPLC chromatogram of the test solution when the gradient elution program 1 shown in Table 13 is used. It can be seen that it takes 39 minutes, but the peak times of some peaks are relatively early, making it inconvenient to judge whether the peaks are complete.

[0190] Fig.19 This is the UPLC chromatogram of the test solution when the gradient elution program 2 shown in Table 14 is used. It can be seen that the time consumption is 60 minutes, which is relatively long.

[0191] Finally, the optimal elution gradient was determined to be the gradient elution program shown in Table 5. Using the optimal gradient elution program, more chromatographic peaks can be obtained and the separation effect is better.

[0192] Example 7

[0193] Weigh 0.5g of each medicinal material powder accurately, place it in a stoppered conical bottle, accurately add 25mL of 50v / v% methanol aqueous solution, weigh the initial weight, and ultrasonically extract for 30min at room temperature (power: 250W; frequency: 80kHz). After cooling, add 50% methanol to make up the lost weight, shake well, filter through a 0.22μm microporous filter membrane, and put into a sample injection vial to obtain the sample solution. Inject under the above chromatographic conditions. The control peak area of ​​each sample is obtained, see Table 15.

[0194] Table 1 Control peak area of ​​1512 samples

[0195] serial number Naringin Hesperidin Neohesperidin Sweet orange flavonoids Nobiletin 5-Demethylnobiletin Hesperidin Heptamethoxyflavone S4 2.327 27.726 0.947 1.242 13.157 1.083 12.917 1.133 S7 2.485 22.984 0.797 1.248 11.892 0.842 12.048 1.161 S5 1.504 37.613 0.789 2.002 16.978 1.471 13.981 1.596 S6 0.877 31.497 0.988 1.636 12.679 0.934 10.050 1.103 S1 1.613 28.328 6.904 4.278 29.587 0.972 13.021 2.841 S2 0.762 37.539 1.480 5.041 29.806 0.935 14.025 1.523 S3 1.055 41.461 13.077 1.831 23.999 0.398 17.720 3.039 S12 23.497 36.354 0.221 0.111 0.793 0.386 0.506 0.108 S11 0.743 21.961 2.251 1.993 9.512 5.257 8.886 0.495 S8 0.966 29.750 0.230 1.085 7.137 1.118 7.144 0.964 S9 5.962 43.734 0.220 1.322 7.403 1.194 6.310 0.711 S10 1.750 18.559 5.142 6.855 38.438 1.240 31.092 4.137

[0196] The average peak areas of 6 varieties (Chazhigan, Dahongpao, Mitangerine, Fuju, Bendi Zao, and Nanfeng Mitangerine) among the 12 tangerine peel samples are shown in Table 16.

[0197] Table 1 Average peak area of ​​66 reference substances

[0198]

[0199]

[0200] It can be seen from Tables 15 and 16 that according to the HPLC fingerprints of 12 batches of different varieties of dried tangerine peel medicinal materials, the peak areas of the 8 identified common peaks were analyzed, and it was found that the six components of naringin, hesperidin, neohesperidin, sweet orange flavonoids, nobiletin and 5-demethylnobiletin had great differences and fluctuations among different varieties.

[0201] According to the analysis of the relevant experimental data in Tables 15 and 16 above, different varieties of tangerine peel were roughly distinguished by the proportion between the components (the mass ratio of naringin, hesperidin, neohesperidin, flavonoids, nobiletin and 5-demethylnobiletin). The mass ratios of naringin, hesperidin, neohesperidin, flavonoids, nobiletin and 5-demethylnobiletin in Citrus aurantium were 0.87-2.35: 23.0-37.62: 0.79-1.00: 1.25-2.00: 11.90-17.00: 0.85-1.50. The mass ratio of naringin, hesperidin, neohesperidin, flavonoids, nobiletin and 5-demethylnobiletin in Dahongpao is 0.77-1.62: 28.33-41.50: 1.49-13.10: 1.85-5.10: 24.00-29.90: 0.40-0.98. The mass ratio of naringin, hesperidin, neohesperidin, flavonoids, nobiletin and 5-demethylnobiletin in mandarin orange is 23.40-23.50: 36.30-36.36: 0.20-0.23: 0.10-0.12: 0.78-0.80: 0.38-0.39. The mass ratio of naringin, hesperidin, neohesperidin, sweet orange flavonoids, nobiletin and 5-demethylnobiletin in Fuju is 0.7-0.75: 21.96-21.97: 2.25-2.26: 1.99-2.00: 9.50-9.55: 5.25-5.26. The mass ratio of naringin, hesperidin, neohesperidin, sweet orange flavonoids, nobiletin and 5-demethylnobiletin in local Zao is 0.96-5.96: 29.75-43.74: 0.22-0.23: 1.08-1.33: 7.13-7.41: 1.11-1.95. The mass ratios of naringin, hesperidin, neohesperidin, flavonoids, nobiletin and 5-demethylnobiletin in Nanfeng mandarin oranges are 1.74-1.76:18.55-18.56:5.14-5.15:6.85-6.86:38.43-38.44:1.23-1.25.

[0202] The above is only a preferred embodiment of the present 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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A tangerine peel fingerprint, characterized in that: The invention comprises 17 characteristic peaks, which include characteristic peaks of naringin, hesperidin, neohesperidin, sweet orange flavonoids, nobiletin, 5-demethylnobiletin, tangeretin and heptamethoxyflavone.

2. The method for constructing the tangerine peel fingerprint spectrum according to claim 1, characterized in that: The following steps are involved: Mixing tangerine peel with an extractant to obtain a test solution; the extractant includes methanol; the tangerine peel includes one or more of tea-branch tangerine, Dahongpao, mandarin orange, Fuju, Bendi Zao and Nanfeng mandarin orange; The test solution and the mixed reference solution are subjected to high performance liquid chromatography to obtain a test liquid phase spectrum and a reference liquid phase spectrum, respectively; The liquid phase spectrum of the test sample is introduced into the similarity evaluation system of the characteristic spectrum of traditional Chinese medicine chromatography to generate a common mode of the characteristic spectrum, and a fingerprint spectrum of dried orange peel with 17 characteristic peaks is obtained; by comparing the retention time of the fingerprint spectrum with that of the liquid phase spectrum of the reference substance, characteristic peaks of naringin, hesperidin, neohesperidin, sweet orange flavonoids, nobiletin, 5-demethylnobiletin, tangeretin and heptamethoxyflavonoids in the fingerprint spectrum of dried orange peel are obtained; The reference substances in the mixed reference substance solution include naringin, hesperidin, neohesperidin, sweet orange flavonoids, nobiletin, 5-demethylnobiletin, tangeretin and heptamethoxyflavone; The conditions for the high performance liquid chromatography detection include: the mobile phase system includes mobile phase A and mobile phase B, the mobile phase A is acetonitrile, the mobile phase B is a formic acid aqueous solution, and the volume fraction of formic acid in the formic acid aqueous solution is 0.095-0.15%; the elution mode is gradient elution, and the procedure of the gradient elution includes: 0-0.2min, the volume fraction of the mobile phase A is 5%; 0.2-2.5min, the volume fraction of the mobile phase A is increased from 5% to 6%; 5-15 min, the volume fraction of the mobile phase A is increased from 6% to 25%; 15-18 min, the volume fraction of the mobile phase A is increased from 25% to 45%; 18-27 min, the volume fraction of the mobile phase A is increased from 45% to 60%; 27-31 min, the volume fraction of the mobile phase A increases from 60% to 95%; 31-34.9 min, the volume fraction of the mobile phase A is 95%; From 34.9 to 35 min, the volume fraction of the mobile phase A is reduced from 95% to 5%.

3. The construction method according to claim 2, characterized in that: The solid-liquid ratio of the tangerine peel to the extractant is 0.95-1.05 g: 49.5-50.5 mL; The volume fraction of methanol in the extractant is 50 to 100%; The extraction protection ultrasonic extraction; the power of the ultrasonic extraction is 240-260W, the frequency is 75-85kHz, and the time is 29-31min.

4. The construction method according to claim 2, characterized in that: In the mixed reference solution, the concentration of nobiletin is 0.145-0.155 mg / mL, the concentration of tangeretin is 0.015-0.025 mg / mL, the concentration of hesperidin is 0.315-0.325 mg / mL, the concentration of naringin is 0.005-0.015 mg / mL, the concentration of neohesperidin is 0.0015-0.0025 mg / mL, the concentration of sweet orange flavonoids is 0.197-0.207 mg / mL, the concentration of 5-demethylnobiletin is 0.196-0.206 mg / mL, and the concentration of heptamethoxyflavone is 0.185-0.195 mg / mL; the solvent in the mixed reference solution includes methanol.

5. The construction method according to claim 2, characterized in that: The high performance liquid chromatography detection conditions include: the chromatographic column includes a Waters Cortecs UPLC T3 chromatographic column, the mobile phase flow rate is 0.24-0.26 mL / min, the detection wavelength is 283 nm, the column temperature is 39-41° C., and the injection volume is 1.99-2.01 μL.

6. Application of the tangerine peel fingerprint described in claim 1 or the tangerine peel fingerprint obtained by the construction method described in any one of claims 2 to 5 in the detection of components in tangerine peel or the identification of tangerine peel varieties.

7. A multi-index component detection method for dried tangerine peel, characterized in that: The following steps are involved: The tangerine peel to be tested is mixed with an extractant to obtain a test solution; the extractant includes methanol; the tangerine peel includes one or more of the following: tea-branch tangerine, dahongpao, mandarin orange, fu ju, bendi zao and nanfeng tangerine; The sample solution to be tested is subjected to high performance liquid chromatography to obtain a multi-index component test result of tangerine peel; the multi-index components include at least two of naringin, hesperidin, neohesperidin, sweet orange flavonoids, nobiletin, 5-demethylnobiletin, tangeretin and heptamethoxyflavone; the test result includes a quantitative test result and / or a qualitative test result; the qualitative test result is obtained by comparing the liquid phase spectrum of the test solution with the tangerine peel fingerprint spectrum described in claim 1 or the tangerine peel fingerprint spectrum obtained by the construction method according to any one of claims 2 to 5; The conditions for the high performance liquid chromatography detection include: the mobile phase system includes mobile phase A and mobile phase B, the mobile phase A is acetonitrile, and the mobile phase B is 0.1 v / v% formic acid aqueous solution; the elution method is gradient elution, and the procedure of the gradient elution includes: 0-0.2min, the volume fraction of the mobile phase A is 5%; 0.2-2.5min, the volume fraction of the mobile phase A is increased from 5% to 6%; 5-15 min, the volume fraction of the mobile phase A is increased from 6% to 25%; 15-18 min, the volume fraction of the mobile phase A is increased from 25% to 45%; 18-27 min, the volume fraction of the mobile phase A is increased from 45% to 60%; 27-31 min, the volume fraction of the mobile phase A increases from 60% to 95%; 31-34.9 min, the volume fraction of the mobile phase A is 95%; From 34.9 to 35 min, the volume fraction of the mobile phase A is reduced from 95% to 5%.

8. The multi-index component detection method according to claim 7, characterized in that: The solid-liquid ratio of the tangerine peel to the extractant is 0.95-1.05 g: 49.5-50.5 mL; The volume fraction of methanol in the extractant is 50 to 100%; The extraction protection ultrasonic extraction; the power of the ultrasonic extraction is 240-260W, the frequency is 75-85kHz, and the time is 29-31min; The high performance liquid chromatography detection conditions include: the chromatographic column includes a Waters Cortecs UPLC T3 chromatographic column, the mobile phase flow rate is 0.24-0.26 mL / min, the detection wavelength is 283 nm, the column temperature is 39-41° C., and the injection volume is 1.99-2.01 μL.

9. An identification standard for a variety of tangerine peel, the tangerine peel varieties including one or more of the following: tea-branch tangerine, dahongpao, mandarin orange, fu ju, local early tangerine and nanfeng mandarin orange; When the ratio between the components is 0.87-2.35:23.0-37.62:0.79-1.00:1.25-2.00:11.90-17.00:0.85-1.50, the tangerine peel variety is tea-branch tangerine peel; When the ratio between the components is 0.77-1.62: 28.33-41.50: 1.49-13.10: 1.85-5.10: 24.00-29.90: 0.40-0.98, the tangerine peel variety is Da Hong Pao; When the ratio between the components is 23.40-23.50: 36.30-36.36: 0.20-0.23: 0.10-0.12: 0.78-0.80: 0.38-0.39, the orange peel variety is mandarin orange; When the ratio between the components is 0.7-0.75: 21.96-21.97: 2.25-2.26: 1.99-2.00: 9.50-9.55: 5.25-5.26, the orange peel variety is Fuju; When the ratio between the components is 0.96-5.96:29.75-43.74:0.22-0.23:1.08-1.33:7.13-7.41:1.11-1.95, the tangerine peel variety is local early; When the ratio between the components is 1.74-1.76: 18.55-18.56: 5.14-5.15: 6.85-6.86: 38.43-38.44: 1.23-1.25, the tangerine peel variety is Nanfeng mandarin orange; The ratio between the components is the mass ratio of naringin, hesperidin, neohesperidin, citrus aurantium, nobiletin and 5-demethylnobiletin.

10. The method for constructing the identification standard of dried tangerine peel varieties according to claim 9 comprises the following steps: The quantitative detection result of the multi-index components of tangerine peel is obtained according to the multi-index component detection method of claim 7 or 8; the multi-index components include naringin, hesperidin, neohesperidin, sweet orange flavonoids, nobiletin and 5-demethylnobiletin; the tangerine peel includes one or more of tea-branch tangerine, dahongpao, mandarin orange, fu ju, local early and Nanfeng mandarin orange; The mass ratios of naringin, hesperidin, neohesperidin, citrus aurantium, nobiletin and 5-demethylnobiletin are calculated according to the quantitative detection results of the multi-index components to obtain the identification standard of the tangerine peel variety.

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