Method for constructing HPLC fingerprint of pericarpium citri sarcodactylis exocarp and endocarp and method for determining content of index components

Abstract

The present invention discloses a method for constructing the HPLC fingerprint of the outer pericarp and inner pericarp of fingered citron and a method for determining the content of index components, including preparing a mixed reference substance stock solution, preparing a test sample, preparing a negative control solution, and determining the HPLC fingerprint of the outer pericarp and inner pericarp of fingered citron. The present invention comparatively analyzes the chemical components and contents of the outer pericarp and inner pericarp of fingered citron to find out the differences in the chemical components and contents of the outer pericarp and inner pericarp. At the same time, the contents of 6 components in the outer pericarp and 2 components in the inner pericarp of fingered citron are determined, providing technical support for the full development and utilization of fingered citron resources and providing a scientific basis for improving the quality evaluation of fingered citron.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a method for constructing an HPLC fingerprint of bergamot exocarp and endocarp and a method for determining the content of index components thereof. Background Art

[0002] Buddha's hand is the dried fruit of the Rutaceae plant Buddha's hand (Citrus medica L.var.sarco-dactylis Swingle). It has the effects of soothing the liver and regulating qi, harmonizing the stomach and relieving pain. It is mainly used for symptoms such as fullness in the stomach, distension and pain in the chest and ribs, loss of appetite and vomiting, cough and sputum. The 2020 edition of the "Chinese Pharmacopoeia" stipulates that Buddha's hand should be harvested in autumn when the fruit has not yet turned yellow or has turned yellow. According to literature reports, Sichuan and Guangdong have a large planting area of ​​​​Buddha's hand and high yield. Sichuan Buddha's hand is popular in domestic and foreign markets because of its large yield and good quality, forming the authentic Sichuan Buddha's hand brand. As a Sichuan authentic Chinese medicinal material and a traditional famous medicinal material with both medicinal and edible properties, it has great economic value.

[0003] Fingerprints are widely used in the quality evaluation of traditional Chinese medicines and can fully reflect the distribution of various components in traditional Chinese medicines. Currently, most existing technologies use fingerprints to evaluate the components and content of bergamot, but there are no reports on the differences in the chemical composition and content between the exocarp and endocarp of bergamot. Summary of the invention

[0004] In order to solve the defects existing in the prior art, the present invention provides a method for constructing an HPLC fingerprint of the exocarp and endocarp of bergamot and a method for determining the content of its index components, compares and analyzes the chemical components and contents of the exocarp and endocarp of bergamot to find out the differences in the chemical components and contents of the exocarp and endocarp, and simultaneously determines the contents of 6 components of the exocarp and 2 components of the endocarp of bergamot, thereby providing technical support for the full development and utilization of bergamot resources and providing a scientific basis for improving the quality evaluation of bergamot.

[0005] In order to achieve the above purpose, the following technical solutions are adopted:

[0006] In a first aspect, the present invention provides a method for constructing an HPLC fingerprint of bergamot exocarp and endocarp, comprising the following steps:

[0007] Prepare mixed reference stock solution:

[0008] Weigh the reference substances of scopoletin, scopolamine, hesperidin, diosmin, 5,7-dimethoxycoumarin, and bergamotolide respectively, add methanol to dissolve, and obtain reference substance solutions of scopoletin, scopolamine, hesperidin, diosmin, 5,7-dimethoxycoumarin, and bergamotolide; mix the reference substance solutions and dilute with methanol to obtain a mixed reference substance stock solution;

[0009] Preparation of test sample:

[0010] Take appropriate amounts of the dried outer and inner pericarp of fingered citron, add methanol and reflux, cool, make up the reduced weight with methanol, shake well, filter, and obtain the sample solution;

[0011] Preparation of negative control solution:

[0012] Prepare a negative control solution without the outer and inner pericarp of fingered citron according to the method for preparing the test sample;

[0013] Determination of HPLC fingerprint of the outer and inner pericarp of fingered citron:

[0014] Precisely pipette the mixed reference substance stock solution, test sample, and negative control solution respectively, and detect them by HPLC to obtain the HPLC fingerprints of the outer and inner pericarp of fingered citron. Import the obtained fingerprints into the similarity evaluation system software for similarity evaluation; among them, the HPLC conditions are: C18 chromatographic column; the mobile phase is methanol - ammonium acetate; gradient elution; detection wavelength: 320 nm.

[0015] Preferably, in the process of preparing the mixed reference substance stock solution, the concentrations of each reference substance solution are as follows: scopoletin reference substance 0.4649 mg / mL, scoparone reference substance 0.4190 mg / mL, hesperidin reference substance 0.5218 mg / mL, diosmin reference substance 0.5057 mg / mL, 5,7 - dimethoxycoumarin reference substance 0.4677 mg / mL, bergapten reference substance 0.1706 mg / mL;

[0016] In the obtained mixed reference substance stock solution, scopoletin is 14.8768 μg / mL, scoparone reference substance is 13.4096 μg / mL, hesperidin reference substance is 166.976 μg / mL, diosmin reference substance is 161.8176 μg / mL, 5,7 - dimethoxycoumarin reference substance is 149.6496 μg / mL, bergapten reference substance is 6.8241 μg / mL.

[0017] Specifically, the preparation process of the mixed reference substance stock solution is as follows: precisely pipette 0.8 mL, 0.8 mL, 8.0 mL, 8.0 mL, 8.0 mL, and 1.0 mL of the reference substance solution in sequence, place them in the same 25 mL volumetric flask, dissolve with methanol and dilute to the scale to obtain the mixed reference substance stock solution.

[0018] Preferably, the specific process for preparing the test sample is as follows: Take fresh fingered citron, separate its outer peel and inner peel, dry them in an oven at 60°C until the moisture content is less than 10.0%, take 0.5 g each of the outer peel and inner peel, pulverize them to 50 mesh, accurately weigh, place them in a 100 mL conical flask, accurately add 25 mL of methanol, reflux for 1 hour, cool, make up the reduced weight with methanol, shake well, filter, and obtain. Prepare a negative control solution without the test sample in the same way.

[0019] Preferably, the HPLC conditions are as follows:

[0020] The chromatographic column is Kromasil 100-5-C18, 4.6×250 mm, 5 μm; mobile phase A is methanol, and mobile phase B is 0.1 mol / L ammonium acetate; gradient elution is as follows: 0 - 60 min, 5% A → 90% A; 60 - 62 min, 90% A → 5% A; 62 - 68 min, 5% A; detection wavelengths: 284 nm, 320 nm, 350 nm; flow rate: 1 mL·min -1 ; column temperature: 30°C - 40°C; injection volume: 10 μL.

[0021] In the second aspect, the present invention provides a fingerprint obtained by the method for constructing the HPLC fingerprint of the outer peel and inner peel of fingered citron as described above: The fingerprint of the outer peel of fingered citron calibrates 18 common peaks, and the fingerprint of the inner peel of fingered citron calibrates 8 common peaks; 6 peaks are simultaneously identified in the outer peel of fingered citron, and peaks 8, 11, 13, 14, 15, and 16 are scopoletin, scoparone, hesperidin, diosmin, 5,7-dimethoxycoumarin, and bergapten respectively; 8 common peaks are simultaneously identified in the inner peel of fingered citron, and peaks 6 and 7 are hesperidin and diosmin respectively; peaks 1, 5, 13, and 14 in the chromatogram of the outer peel are common peaks of the outer peel and the inner peel.

[0022] Specifically, the content of 5,7-dimethoxycoumarin is the highest in the outer peel of fingered citron, and the content of hesperidin is the highest in the inner peel of fingered citron; the similarity between the outer peel and the inner peel is 0.10 - 0.35.

[0023] To further compare the changes in the chemical component contents of the outer peel and inner peel of fingered citron, the present invention determines the contents of the index components in the outer peel and inner peel of fingered citron based on the established fingerprint:

[0024] In the third aspect, the present invention provides a method for determining the content of the index components in the outer peel and inner peel of fingered citron, including the following steps:

[0025] According to the HPLC fingerprint construction method of the pericarp and endocarp of Citrus medica L. var. sarcodactylis (Noot.) Swingle, six index components including scopoletin, scoparone, hesperidin, diosmin, 5,7-dimethoxycoumarin, and bergapten in the mixed reference stock solution, test sample, and negative control solution were determined by HPLC, and the contents of the six index components in the pericarp and endocarp of Citrus medica L. var. sarcodactylis (Noot.) Swingle were calculated by the external standard method.

[0026] Specifically, each reference solution obtained during the preparation of the mixed reference stock solution was diluted with methanol to different concentrations, injected and determined under the above chromatographic conditions. With the solution mass concentration as the abscissa and the peak area as the ordinate, regression was performed to establish the standard curves of the six index components, namely scopoletin, scoparone, hesperidin, diosmin, 5,7-dimethoxycoumarin, and bergapten, and the linear regression equations were obtained. According to the peak areas of the six index components in the HPLC chromatogram of the test sample and the linear regression equations, the contents of the six index components were calculated.

[0027] Preferably, in the content determination, 284 nm was selected as the detection wavelength for hesperidin; 320 nm was selected as the detection wavelength for 5,7-dimethoxycoumarin and bergapten; 350 nm was selected as the detection wavelength for scopoletin, scoparone, and diosmin.

[0028] The beneficial effects of the present invention are as follows:

[0029] Firstly, the HPLC fingerprints of the pericarp and endocarp of Citrus medica L. var. sarcodactylis (Noot.) Swingle were established respectively, and the chromatographic peak data were imported into the Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines (version 2012.130723), and the similarity calculation results were obtained. The results showed that the similarities of the pericarp and endocarp of Citrus medica L. var. sarcodactylis (Noot.) Swingle in Leshan City to their respective reference fingerprints were both greater than 0.90, indicating good similarity among different batches in this area of Leshan. For the pericarp reference fingerprint of Citrus medica L. var. sarcodactylis (Noot.) Swingle from other provinces, except for the relatively low similarity in the Yunnan area, those from Fujian and Zhaoqing were both above 0.90; for the endocarp reference fingerprint of Citrus medica L. var. sarcodactylis (Noot.) Swingle from other provinces, except for the relatively low similarity in the Zhaoqing area, those from Fujian and Yunnan were both above 0.90. Since the sample size of Citrus medica L. var. sarcodactylis (Noot.) Swingle from other provinces selected in this study was low, it could not well reflect the quality of local Citrus medica L. var. sarcodactylis (Noot.) Swingle. From the reference fingerprints of the pericarp and endocarp of Citrus medica L. var. sarcodactylis (Noot.) Swingle, it can be seen that the chromatogram of the pericarp of Citrus medica L. var. sarcodactylis (Noot.) Swingle has more calibrated chromatographic peaks, including 4 peaks calibrated in the endocarp, more intuitively indicating the inconsistency in the types and contents of chemical components between the pericarp and endocarp.

[0030] The present invention found in the research process that under the condition of wavelength 320nm, there are more chromatographic peaks and the absorption value of each peak is good, so 320nm is selected as the detection wavelength of the fingerprint. The multi-component content determination requires the detection wavelength to be the maximum absorption wavelength. When the wavelength is 284nm, hesperidin has the maximum absorption; when the wavelength is 320nm, 5,7-dimethoxycoumarin and bergamot lactone absorb the maximum; when the wavelength is 350nm, scopoletin, scopoletin and diosmin absorb the maximum; so 284nm is selected as the detection wavelength of hesperidin in the content determination; 320nm is used as the detection wavelength of 5,7-dimethoxycoumarin and bergamot lactone; 350nm is used as the detection wavelength of scopoletin, scopoletin and diosmin.

[0031] In order to further compare the changes in the contents of chemical components in the exocarp and endocarp of bergamot, the present invention determined the contents of 6 index components in the exocarp and endocarp of 16 batches of bergamot based on the established fingerprint. The results showed that there were significant differences in the contents of hesperidin and 5,7-dimethoxycoumarin in the exocarp and endocarp of bergamot; the hesperidin content was higher in the endocarp, while the 5,7-dimethoxycoumarin content was higher in the exocarp; the measurement results also found that the content of 5,7-dimethoxycoumarin in the exocarp of yellow fruit from the same origin was significantly higher than that of green fruit (about 3 to 5 times); and the content of hesperidin in the endocarp of green fruit was significantly higher than that of yellow fruit (about 0.5 to 3 times).

[0032] Hesperidin is a dihydroflavonoid glycoside compound, which has been proven to have a variety of pharmacological activities such as anti-inflammatory analgesia, antioxidant, immunomodulatory, and anti-tumor. In order to improve the therapeutic effect of bergamot in this regard, the endocarp of bergamot can be selected, and the green fruit is preferably selected. 5,7-Dimethoxycoumarin belongs to the coumarin class of compounds, which has antiviral, anti-infective, anti-tumor, anti-thrombotic, antioxidant, and antibacterial pharmacological activities. The anti-tumor effects of coumarin compounds have become a research hotspot and have high medicinal value. In addition, coumarin compounds have aromatic odors and can be used as flavoring agents and spices. They are also used in food, plastic products and other products, and the prospects are very promising. In order to make bergamot play a good role in these fields, the exocarp of bergamot can be selected, and the yellow fruit is preferably selected.

[0033] The HPLC fingerprint and multi-component simultaneous quantitative analysis method established in this study can be used for the quality control of bergamot and provide data support for the rational and full utilization of the medicinal value of bergamot. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 HPLC fingerprints of 16 batches of bergamot peel;

[0035] Figure 2 HPLC fingerprints of 16 batches of bergamot endocarp;

[0036] Figure 3 It is the HPLC chromatogram of the outer pericarp of fingered citron with number (1);

[0037] Figure 4 It is the HPLC chromatogram of the inner pericarp of fingered citron with number (1);

[0038] Figure 5 It is the HPLC chromatogram of each component in the mixed reference stock solution;

[0039] Figure 6 It is the HPLC chromatogram of each component in the test solution of the outer pericarp of fingered citron from Jiajiang area;

[0040] Figure 7 It is the HPLC chromatogram of each component in the test solution of the inner pericarp of fingered citron from Jiajiang area;

[0041] Figure 8 It is the HPLC chromatogram of each component in the negative control solution. Specific embodiments

[0042] In order to enable those skilled in the art to better understand the technical solution of the invention, the present invention will be further described in detail below in conjunction with specific embodiments.

[0043] In the embodiment of the present invention, HPLC was used to establish the fingerprint and multi-component quantitative analysis methods for 10 batches of Sichuan fingered citron and 6 batches of fingered citron from other provinces. At the same time, the chemical components and contents of the outer pericarp and inner pericarp of fingered citron were compared and analyzed to find out the differences in the chemical components and contents of the outer pericarp and inner pericarp. At the same time, the contents of 6 components in the outer pericarp and 2 components in the inner pericarp of fingered citron were determined, providing technical support for the full development and utilization of fingered citron resources and scientific basis for improving the quality evaluation of fingered citron.

[0044] Embodiment 1 Construction method of HPLC fingerprint of the outer pericarp and inner pericarp of fingered citron and its fingerprint

[0045] 1. Materials and instruments

[0046] Fresh fruit test samples of Sichuan fingered citron were purchased from Jiajiang County, Wutongqiao District, Qianwei County, Muchuan County, and Shawan District in Leshan City, Sichuan Province, 2 batches each, a total of 10 batches (numbered S1 - S10); 2 batches each of fingered citron from Zhaoqing, Yunnan, and Fujian in other provinces were purchased, a total of 6 batches (numbered S11 - S16).

[0047] Methanol, ethanol (analytical pure, Chengdu Kelong Chemical Reagent Factory); distilled water: prepared in the laboratory. Altus10 liquid chromatograph (PerkinElmer Enterprise Management (Shanghai) Co., Ltd.); liquid chromatography column: Kromasil 100-5-C18 (4.6×250 mm, 5 μm) (Akzo Nobel, Sweden); XS205DU electronic analytical balance (Mettler Toledo Instruments Shanghai Co., Ltd.); ULUP-11-10 ultrapure water machine (Chengdu Ultra-pure Water Technology Co., Ltd.).

[0048] Scopoletin, scoparone, hesperidin, diosmin, 5,7-dimethoxycoumarin, bergapten reference substances (Chengdu Pusi Biotechnology Co., Ltd., content greater than 98%).

[0049] 2. Experimental methods

[0050] 2.1 Liquid chromatography conditions

[0051] Chromatography column: Kromasil 100-5-C18 (4.6×250 mm, 5 μm); mobile phase: methanol (A) - 0.1 mol / L ammonium acetate (B), gradient elution (0 - 60 min, 5% A → 90% A; 60 - 62 min, 90% A → 5% A; 62 - 68 min, 5% A); flow rate: 1 mL·min -1 ; column temperature 30 °C; injection volume: 10 μL; detection wavelength for fingerprint: 320 nm; detection wavelengths for determination of the contents of target components: 284 nm (hesperidin), 320 nm (5,7-dimethoxycoumarin, bergapten), 350 nm (scopoletin, scoparone);.

[0052] 2.2 Preparation of the mixed reference substance stock solution

[0053] Accurately weigh each reference substance and dissolve them separately in methanol to obtain solutions containing scopoletin 0.4649 mg / mL, scoparone reference substance 0.4190 mg / mL, hesperidin reference substance 0.5218 mg / mL, diosmin reference substance 0.5057 mg / mL, 5,7-dimethoxycoumarin reference substance 0.4677 mg / mL, bergapten reference substance 0.1706 mg / mL;

[0054] Accurately pipette 0.8mL, 0.8mL, 8.0mL, 8.0mL, 8.0mL, and 1.0mL of each of the above-mentioned reference substance solutions in turn, place them in the same 25mL volumetric flask, dissolve and dilute to the scale with methanol to obtain a mixed reference substance stock solution (containing scopoletin 14.8768μg / mL, artemisinin reference substance 13.4096μg / mL, hesperidin reference substance 166.976μg / mL, diosmin reference substance 161.8176μg / mL, 5,7-dimethoxycoumarin reference substance 149.6496μg / mL, and bergamotolide reference substance 6.8241μg / mL).

[0055] 2.3 Preparation of test samples

[0056] Take fresh bergamot and separate its outer peel and inner peel, bake in an oven at 60°C until the moisture content is less than 10.0%, take 0.5g of the outer peel and inner peel respectively, crush them into 50-mesh particles, accurately weigh them, place them in a 100mL conical flask, accurately add 25mL of methanol, reflux for 1 hour, cool, make up the reduced weight with methanol, shake well, filter, and obtain.

[0057] 2.4 Negative control solution: Prepare a negative control solution without the test sample according to the method under "2.3".

[0058] 2.5 System suitability and specificity test

[0059] Accurately pipette 10 μl of each mixed reference solution, test solution and negative control solution, and inject and measure according to the chromatographic conditions under "2.1". The results are detailed in Figures 1 to 4 The results showed that under the chromatographic conditions, each component achieved baseline separation with a resolution of >1.5, and there was no interference from other components.

[0060] 2.6 Research Methodology of Fingerprint Spectroscopy

[0061] 2.6.1 Reference peak selection

[0062] The 2020 edition of the "Chinese Pharmacopoeia" uses hesperidin as a single indicator component to evaluate the quality of bergamot. Hesperidin has good separation in the chromatogram, high peak area and good stability. Therefore, it is used as the chromatographic reference peak (S) to calculate the relative retention time and relative peak area of ​​each chromatographic peak.

[0063] 2.6.2 Precision test

[0064] Prepare a solution from the test sample (sample number S1). Repeat the injection 6 times under the chromatographic conditions described in "2.1", record the chromatograms for the 6 times respectively, and calculate the RSDs of the relative peak areas and relative retention times of each common peak. The results show that the RSDs of the relative peak areas of each common peak in the test sample are 0.52% - 1.75%; the RSDs of the relative retention times are 0.35% - 1.88%, both less than 2.0%, indicating good instrument precision.

[0065] 2.6.3 Repeatability Test

[0066] Take 6 portions of the powder of the test sample (sample number S1), prepare the test sample solution according to the method described in "2.3", inject and determine under the chromatographic conditions described in "2.1", and calculate the RSDs of the relative peak areas and relative retention times of each common peak. The results show that the RSDs of the relative peak areas of each common peak in the test sample are 1.36% - 3.75%; the RSDs of the relative retention times are 0.78% - 3.12%, both less than 4.0%, indicating good repeatability of this experimental method.

[0067] 2.6.4 Stability Test

[0068] Take the solution of the test sample (sample number S1), inject and determine under the chromatographic conditions described in "2.1" at 0, 2, 4, 8, 12, and 24 h at room temperature respectively. Calculate the RSDs of the relative peak areas and relative retention times of each common peak. The results show that the RSDs of the relative peak areas of each common peak in the test sample are 0.41% - 2.68%; the RSDs of the relative retention times are 0.053% - 0.47%, both less than 3.0%, indicating good stability of the test sample solution within 24 h at room temperature.

[0069] 2.7 Construction of Fingerprint

[0070] 2.7.1 Construction of Fingerprints of Exocarp and Endocarp of 16 Batches of Fructus Citri Sarcodactylis

[0071] Take the exocarp and endocarp of 16 batches of Fructus Citri Sarcodactylis, prepare the test sample solutions respectively according to the method described in "2.3", inject and determine under the chromatographic conditions described in "2.1", record the chromatograms of the test samples, export the HPLC data chromatograms, and import them into the software "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (Version 2012.130723)". Select the exocarp and endocarp of the test sample (sample number S1) of Fructus Citri Sarcodactylis as the reference chromatograms respectively, with a time window width of 0.3, select multiple characteristic peaks for multi-point calibration and automatic peak matching. The fingerprint of the exocarp of Fructus Citri Sarcodactylis calibrated 18 common peaks, and the fingerprint of the endocarp of Fructus Citri Sarcodactylis calibrated 8 common peaks. Generate the reference fingerprints of the pericarp and pulp of Fructus Citri Sarcodactylis respectively, as shown in Figures 1 to 4Six peaks were simultaneously identified in the outer pericarp of Citrus medica L. var. sarcodactylis (Noot.) Swingle. Among them, peaks 8, 11, 13, 14, 15, and 16 were scopoletin, scoparone, hesperidin, diosmin, 5,7-dimethoxycoumarin, and bergapten, respectively. Eight common peaks were simultaneously identified in the inner pericarp of Citrus medica L. var. sarcodactylis (Noot.) Swingle. Among them, peaks 6 and 7 were hesperidin and diosmin, respectively. When the fingerprint similarity evaluation was carried out on the outer and inner pericarps of the same batch of Citrus medica L. var. sarcodactylis (Noot.) Swingle, it was found that there were 4 common peaks, namely peaks 1, 5, 13, and 14 in the chromatogram of the outer pericarp. Due to the large difference in the content values of each component between the outer and inner pericarps, the similarity between the two was very low. By visually comparing the control chromatogram, it can be seen that hesperidin and 5,7-dimethoxycoumarin are the differential substances that distinguish the outer and inner pericarps of Citrus medica L. var. sarcodactylis (Noot.) Swingle.

[0072] 2.7.2 Fingerprint Similarity Analysis

[0073] Using the software "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines (Version 2012.130723)", the similarity results between the outer and inner pericarps of Citrus medica L. var. sarcodactylis (Noot.) Swingle and their respective control fingerprint chromatograms, as well as the similarity results between the outer and inner pericarps of the same batch of Citrus medica L. var. sarcodactylis (Noot.) Swingle, are shown in the overlapping fingerprint chromatograms of the outer and inner pericarps of Citrus medica L. var. sarcodactylis (Noot.) Swingle, respectively Figures 1 to 4 , and the similarity results of the fingerprint chromatograms are shown in Table 1.

[0074] Table 1 Similarity Calculation Results of the Outer and Inner Pericarps of 16 Batches of Citrus medica L. var. sarcodactylis (Noot.) Swingle

[0075]

[0076]

[0077] The results show that there are many components and rich contents in the outer pericarp of Citrus medica L. var. sarcodactylis (Noot.) Swingle, and the content of 5,7-dimethoxycoumarin is the highest; the similarity results of the control fingerprint chromatograms of the outer pericarps of 10 batches of Citrus medica L. var. sarcodactylis (Noot.) Swingle in Leshan City are above 0.90, the similarity of the control fingerprint chromatograms of the outer pericarps of Citrus medica L. var. sarcodactylis (Noot.) Swingle from Yunnan region among 6 batches of Citrus medica L. var. sarcodactylis (Noot.) Swingle from other provinces are relatively low, and the rest are above 0.90; while there are fewer components in the inner pericarp of Citrus medica L. var. sarcodactylis (Noot.) Swingle, scopoletin, scoparone, 5,7-dimethoxycoumarin and bergapten are missing in the inner pericarp, the contents of each component in the inner pericarp are relatively flat, and the content of hesperidin is the highest; the similarity of the control fingerprint chromatograms of the inner pericarps of 10 batches of Citrus medica L. var. sarcodactylis (Noot.) Swingle in Leshan City is above 0.90, the similarity of the control fingerprint chromatograms of the inner pericarps of Citrus medica L. var. sarcodactylis (Noot.) Swingle from Zhaoqing region among 6 batches of Citrus medica L. var. sarcodactylis (Noot.) Swingle from other provinces are relatively low, and the rest are above 0.9; while the similarity calculation results between the outer and inner pericarps of the same batch of Citrus medica L. var. sarcodactylis (Noot.) Swingle are very low, only 0.10 - 0.35. The possible reason is that there are significant differences in the components and contents between the outer and inner pericarps of Citrus medica L. var. sarcodactylis (Noot.) Swingle, resulting in a low similarity calculation result.

[0078] Example 2 Determination of the Content of Index Components in the Outer and Inner Pericarps of Citrus medica L. var. sarcodactylis (Noot.) Swingle

[0079] The fingerprint spectra of the outer pericarp and inner pericarp of Citrus medica L. var. sarcodactylis Swingle were established by HPLC. Six marker components were identified in the outer pericarp and two marker components were identified in the inner pericarp. In order to further clarify the contents and differences of the marker components in the outer pericarp and inner pericarp of Citrus medica L. var. sarcodactylis Swingle, a method for simultaneous determination of the contents of the marker components in the outer pericarp and inner pericarp was established in this study. Scoparone, scopoletin, hesperidin, diosmin, 5,7-dimethoxycoumarin, and bergapten in the outer pericarp and inner pericarp were simultaneously determined, as shown in Figures 5 to 8 .

[0080] 2.8.1 Methodology investigation of content determination

[0081] 2.8.1.1 Investigation of linear relationship

[0082] Precisely measure the reference substance stock solution under item "2.2", dilute it with methanol to form 5 concentrations, precisely pipette 10 μl of each, and inject and determine under the chromatographic conditions of item "2.1" in Example 1. Using the solution mass concentration as the abscissa (X) and the peak area as the ordinate (Y) for regression, the results are shown in Table 2, indicating that the linear relationships of each component are good within their respective ranges.

[0083] Table 2 Results of investigation of linear relationship of each component

[0084]

[0085] 2.8.1.2 Precision test

[0086] Precisely pipette 10 μl of the mixed reference substance solution under item "2.2" in Example 1, and repeat the injection 6 times under the chromatographic conditions of item "2.1" in Example 1. As a result, the RSDs of the peak areas of scoparone, scopoletin, hesperidin, diosmin, 5,7-dimethoxycoumarin, and bergapten are 0.85%, 1.02%, 0.46%, 0.58%, 0.52%, and 1.36% (n = 6) respectively, indicating good instrument precision.

[0087] 2.8.1.3 Stability test

[0088] Take the solution of the same batch of test samples (serial number S1), and inject and determine under the chromatographic conditions of item "2.1" in Example 1 at 0, 2, 4, 8, 12, and 24 h at room temperature respectively. As a result, the RSDs of the peak areas of scoparone, scopoletin, hesperidin, diosmin, 5,7-dimethoxycoumarin, and bergapten are 1.04%, 0.97%, 0.48%, 0.64%, 0.42%, and 1.25% (n = 6) respectively, indicating good stability of the test sample solution within 24 h at room temperature.

[0089] 2.8.1.4 Repeatability test

[0090] Take an appropriate amount of the test samples of the same batch (No. S1), a total of 6 portions, prepare the test sample solution according to the method under item "2.3" of Example 1, inject and determine under the chromatographic conditions under item "2.1" of Example 1. The RSDs of the contents of scopoletin, scoparone, hesperidin, diosmin, 5,7-dimethoxycoumarin, and bergapten are 1.58%, 1.41%, 0.95%, 0.99%, 1.08%, and 1.43% respectively, indicating that this method has good repeatability.

[0091] 2.8.1.5 Test for recovery of added samples

[0092] Take 0.5 g of the test samples of the same batch (No. S1) with known content, accurately weigh, a total of 6 portions, and add a mixed reference substance stock solution (containing 14.8768 μg / mL of scopoletin, 13.4096 μg / mL of scoparone reference substance, 166.976 μg / mL of hesperidin reference substance, 161.8176 μg / mL of diosmin reference substance, 149.6496 μg / mL of 5,7-dimethoxycoumarin reference substance, 6.8241 μg / mL of bergapten reference substance), prepare the test sample solution according to the method under item "2.3" of Example 1, and then inject and determine under the chromatographic conditions under item "2.1" of Example 1, calculate the recovery of added samples. The average recoveries of scopoletin, scoparone, hesperidin, diosmin, 5,7-dimethoxycoumarin, and bergapten are 100.45%, 94.36%, 92.25%, 99.74%, 93.25%, and 93.05% respectively, and the RSDs are 1.02%, 1.14%, 1.74%, 1.26%, 1.35%, and 1.41% respectively.

[0093] 2.8.2 Determination of the content of bergamot in 16 batches

[0094] Take the test samples of bergamot in 16 batches respectively, prepare the test sample solution according to the method under item "2.3" of Example 1, inject and determine under the chromatographic conditions under item "2.1" of Example 1, and calculate the content of each component by the external standard method. The results are shown in Table 3.

[0095] Table 3 Determination results of the contents of each component in the outer and inner peels of bergamot from different regions (%, n = 3)

[0096]

[0097]

[0098] As can be seen from Examples 1 to 2, under the condition of a wavelength of 320 nm, there are more chromatographic peaks and the absorption values of each peak are good. Therefore, 320 nm is selected as the detection wavelength for the fingerprint. For the determination of the contents of multiple components, the detection wavelength is required to be the maximum absorption wavelength. When the wavelength is 284 nm, hesperidin has the maximum absorption; when the wavelength is 320 nm, 5,7-dimethoxycoumarin and bergapten have the maximum absorption; when the wavelength is 350 nm, scopoletin, scoparone, and diosmin have the maximum absorption. Therefore, 284 nm is selected as the detection wavelength for hesperidin; 320 nm is selected as the detection wavelength for 5,7-dimethoxycoumarin and bergapten; 350 nm is selected as the detection wavelength for scopoletin, scoparone, and diosmin in the content determination.

[0099] Based on the established fingerprint, the contents of 6 index components in the outer and inner peels of 16 batches of fingered citron were determined. As can be seen from Table 3, there are significant differences in the contents of hesperidin and 5,7-dimethoxycoumarin between the outer and inner peels of fingered citron; the content of hesperidin is higher in the inner peel, while the content of 5,7-dimethoxycoumarin is higher in the outer peel; the determination results also found that the content of 5,7-dimethoxycoumarin in the outer peel of yellow fruits from the same origin is significantly higher than that of green fruits (about 3 to 5 times); while the content of hesperidin in the inner peel of green fruits is significantly higher than that of yellow fruits (about 0.5 to 3 times).

[0100] The above is only the preferred embodiment of the present invention. It should be noted that the above preferred embodiment should not be regarded as a limitation of the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. For those of ordinary skill in the art of this technology, without departing from the spirit and scope of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for constructing the HPLC fingerprint of the outer and inner pericarp of fingered citron, characterized in that, It includes the following steps: Prepare a mixed reference stock solution: Weigh the reference substances of scopoletin, scoparone, hesperidin, diosmin, 5,7-dimethoxycoumarin, and bergapten respectively, dissolve them in methanol to obtain the reference substance solutions of scopoletin, scoparone, hesperidin, diosmin, 5,7-dimethoxycoumarin, and bergapten; mix the above reference substance solutions and dilute them with methanol to obtain a mixed reference stock solution; Prepare the test sample: Take an appropriate amount of the dried outer and inner pericarp of fingered citron, add methanol and reflux, cool, make up the reduced weight with methanol, shake well, and filter to obtain it; Prepare the negative control solution: Prepare a negative control solution without the outer and inner pericarp of fingered citron according to the method for preparing the test sample; Determination of the HPLC fingerprint of the outer and inner pericarp of fingered citron: Precisely pipette the mixed reference stock solution, the test sample, and the negative control solution respectively, and perform detection by HPLC to obtain the HPLC fingerprint of the outer and inner pericarp of fingered citron, and import the obtained fingerprint into the similarity evaluation system software for similarity evaluation; among them, the HPLC conditions are: C18 chromatographic column; the mobile phase is methanol - 0.1mol / L ammonium acetate; the gradient elution is: 0 - 60min, 5% A → 90% A; 60 - 62min, 90% A → 5% A; 62 - 68min, 5% A; the detection wavelengths are: 284nm, 320nm, 350nm.

2. The method for constructing the HPLC fingerprint of the outer and inner pericarp of fingered citron according to claim 1, characterized in that, During the process of preparing the mixed reference stock solution, in the obtained mixed reference stock solution, the content of scopoletin is 14.8768μg / mL, the content of scoparone reference substance is 13.4096μg / mL, the content of hesperidin reference substance is 166.976μg / mL, the content of diosmin reference substance is 161.8176μg / mL, the content of 5,7-dimethoxycoumarin reference substance is 149.6496μg / mL, and the content of bergapten reference substance is 6.8241μg / mL.

3. The method for constructing the HPLC fingerprint of the outer and inner pericarp of fingered citron according to claim 1, characterized in that, The specific process for preparing the test sample is: take fresh fingered citron, separate its outer and inner pericarp, dry it until the moisture content is lower than 10.0%, take 0.5g of the outer and inner pericarp respectively, pulverize, precisely weigh, place in a 100mL conical flask, precisely add 25mL of methanol, reflux for 1 hour, cool, make up the reduced weight with methanol, shake well, and filter to obtain it.

4. The method for constructing the HPLC fingerprint of the outer and inner pericarp of fingered citron according to claim 1, characterized in that, The HPLC conditions are as follows: the chromatographic column is Kromasil 100-5-C18, 4.6×250 mm, 5 μm; mobile phase A is methanol, and mobile phase B is 0.1 mol / L ammonium acetate; flow rate: 1 mL·min -1 ; column temperature is 30°C to 40°C; sample injection volume: 10 μL.

5. A method for determining the content of index components in the outer and inner pericarp of fingered citron, characterized in that, It includes the following steps: According to the HPLC fingerprint construction method of the outer and inner pericarp of fingered citron described in any one of claims 1 - 4, perform HPLC determination on 6 index components of scopoletin, scoparone, hesperidin, diosmin, 5,7-dimethoxycoumarin, and bergapten in the mixed reference stock solution, the test sample, and the negative control solution, and calculate the contents of the 6 index components in the outer and inner pericarp of fingered citron by the external standard method.

6. The method for determining the content of index components in the outer and inner pericarp of fingered citron according to claim 5, characterized in that, The reference substance solutions obtained during the preparation of the mixed reference substance stock solution were diluted with methanol to different concentrations, and then injected for determination. Using the solution mass concentration as the abscissa and the peak area as the ordinate for regression, standard curves of 6 index components, scopoletin, scoparone, hesperidin, diosmin, 5,7-dimethoxycoumarin, and bergapten, were established respectively to obtain the linear regression equations; according to the peak areas of the 6 index components in the HPLC chromatogram of the test sample and the linear regression equations, the contents of the 6 index components were calculated.

7. The method for determining the content of index components in the outer and inner pericarp of fingered citron according to claim 5, characterized in that, In the content determination, 284 nm was selected as the detection wavelength for hesperidin; 320 nm was selected as the detection wavelength for 5,7-dimethoxycoumarin and bergapten; 350 nm was selected as the detection wavelength for scopoletin, scoparone, and diosmin.

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