Method for identifying aging time of fructus aurantii crude product and processed product based on HPLC fingerprint spectrum in combination with chemometrics and application

Abstract

The invention discloses a method for identifying the aging time of a raw fructus aurantii product and a processed fructus aurantii product based on HPLC fingerprint spectrum combined with chemometrics, which comprises the following steps: performing HPLC fingerprint spectrum analysis on the raw fructus aurantii product and the processed fructus aurantii product with different aging time to respectively obtain retention time and peak area; the method comprises the following steps of: performing PCA (Principal Component Analysis) by taking the peak areas of 18 common peaks of a fructus aurantii crude product as variables, determining two main components, taking a sample with the comprehensive score of 0.64-0.95 of the fructus aurantii crude product as a short-term sample, taking a sample with the comprehensive score of 0.01-0.61 of the fructus aurantii crude product as a medium-term sample, and taking a sample with the comprehensive score of-1.41--1.26 of the fructus aurantii crude product as a long-term sample; taking the peak area of 22 common peaks of the fructus aurantii processed product as a variable, performing PCA analysis, determining 3 principal components, determining a sample with the comprehensive score of more than 0 of the fructus aurantii processed product as a short-term sample, and determining a sample with the comprehensive score of less than 0 of the fructus aurantii crude product as a long-term sample; the invention discloses an application of identifying the aging time of a fructus aurantii crude product and a processed product based on an HPLC (High Performance Liquid Chromatography) fingerprint spectrum in combination with chemometrics.

Description

Technical Field

[0001] The present invention relates to the field of traditional Chinese medicine identification, and in particular to a method and application for identifying the aging time of raw and processed Fructus Aurantii Immaturus based on HPLC fingerprint combined with chemometrics. Background Art

[0002] Fructus Aurantii Immaturus (AF) is the dried immature fruit of Citrus aurantium L. and its cultivated varieties of Rutaceae, and is mainly produced in Jiangxi, Sichuan, Hunan, Zhejiang and other places. Fructus Aurantii Immaturus is bitter, pungent, sour, and slightly cold in nature, and belongs to the spleen and stomach meridians. It has the effects of regulating qi and relieving distension, promoting qi movement and eliminating stagnation, etc. Clinically, it is mostly used to treat diseases such as chest and hypochondrium qi stagnation, fullness and pain, indigestion, phlegm retention, and visceral ptosis, and mainly contains components such as flavonoids, volatile oils, coumarins, and alkaloids. Research shows that the raw Fructus Aurantii Immaturus has a strong drying property, and long-term use is likely to damage body fluids, so it is mostly used after processing clinically. The processed medicinal materials can not only relieve the drastic property, but also enhance the effect of regulating qi and strengthening the spleen. There are many processing methods for Fructus Aurantii Immaturus, and different methods produce different effects. "Leigong's Treatise on Processing of Medicinal Herbs" records that for Fructus Aurantii Immaturus, "when using, first remove the core, stir-fry with bran until the bran turns dark brown, then take out, wipe off the black on the upper part with a cloth, and then pound it into powder alone for use". This is also the first time to propose the processing methods of removing the core and stir-frying with bran, and these two methods have been used as the mainstream processing methods until now. In addition, "Taiping Holy Prescriptions" proposed stir-frying Fructus Aurantii Immaturus with vinegar, "stir-fry three liang until slightly yellow with bran, remove the core, pound and sift into powder, and use two liters of rice vinegar to simmer slowly until it becomes like maltose", believing that Fructus Aurantii Immaturus can guide the medicine to enter the liver after being stir-fried with vinegar. "Compendium of Materia Medica Justice" in the Qing Dynasty also recorded the method of stir-frying without any adjuvant, believing that Fructus Aurantii Immaturus is "bitter, cool, slightly sour, and flat in nature after being stir-fried", and the property of the medicine can be relieved after stir-frying without any adjuvant. There are many other processing methods of Fructus Aurantii Immaturus recorded in ancient books. Mr. Wang Xiaotao summarized 23 processing methods of Fructus Aurantii Immaturus from the Han Dynasty to the Qing Dynasty in "Compendium of Processing Methods of Traditional Chinese Medicines in Past Dynasties", including purification, cutting, stir-frying, stir-frying with bran, wine processing, vinegar processing, sophora flower processing, etc. At present, the most commonly used processing method for Fructus Aurantii Immaturus is stir-frying with bran.

[0003] Traditional Chinese medicine fingerprint is a fingerprint of chemical components of traditional Chinese medicine obtained by modern technology analysis based on the understanding of the overall effect of a group of substances, mainly including mass spectrometry, high performance liquid chromatography, gas chromatography, etc. This technology can completely present all the information of the types and quantities of internal chemical components of medicinal materials, and then reflect the quality of traditional Chinese medicinal materials. The traditional Chinese medicine fingerprint technology can detect multiple chemical components simultaneously in a short time, which can not only improve the analysis efficiency, but also avoid the deficiencies of single component analysis. Therefore, it has the characteristics of comprehensiveness, high efficiency and stability, and has been widely used in evaluating the quality, authenticity identification and species differentiation of medicinal materials in recent years. Chemometrics is a multivariate analysis technology. Based on chemical experimental data, it combines computer algorithms with disciplines such as statistics and mathematics to realize the visual analysis of complex data, and then reveals the laws behind the data. Its analysis modes mainly include unsupervised pattern recognition and supervised pattern recognition, and it is now mostly used for parsing the data of fingerprint spectra. This technology can quickly synthesize, simplify and classify the complex information in the fingerprint spectrum in a short time, greatly improving the separation and analysis performance of the complex system of traditional Chinese medicine, thus making up for the limitations of analytical instruments.

[0004] The aging of traditional Chinese medicine is a special treatment method summarized by the ancient Chinese through continuous experiments. It was first recorded in "Mencius * Lilou" during the Warring States period, "It's like treating a seven-year illness with mugwort that has been stored for three years". After the traditional Chinese medicine is stored, its components and efficacy will change, so as to better meet the clinical needs. During the Southern and Northern Dynasties, the famous medical scientist Tao Hongjing first clearly stated in "Compendium of Materia Medica" that six kinds of aged medicines, namely Stellera chamaejasme, Fructus Aurantii Immaturus, Pericarpium Citri Reticulatae, Pinellia ternata, Ephedra sinica, and Evodia rutaecarpa, all need to be "better when aged". Since the appearance changes of new medicines and aged medicines are not obvious and can only be subjectively judged by the naked eye, it is necessary to explore a simple and rapid method to identify the different aging years of medicinal materials. Summary of the Invention

[0005] The present invention designs and develops a method for identifying the aging time of raw and processed Fructus Aurantii based on HPLC fingerprint combined with chemometrics. The object of the present invention is to solve the problems of quickly processing and analyzing a large number of sample data and accurately identifying the raw and processed Fructus Aurantii with different aging years.

[0006] The present invention designs and develops an application of a method for identifying the aging time of raw and processed Fructus Aurantii based on HPLC fingerprint combined with chemometrics. The object of the present invention is to provide an application for identifying the aging time of raw and processed Fructus Aurantii.

[0007] The technical solution provided by the present invention is as follows:

[0008] A method for identifying the aging time of raw and processed Fructus Aurantii based on HPLC fingerprint combined with chemometrics, comprising the following steps:

[0009] Perform HPLC fingerprint analysis on raw and processed Fructus Aurantii Immaturus with different aging times, and obtain the retention time and peak area respectively;

[0010] Taking the peak areas of 18 common peaks of the raw Fructus Aurantii Immaturus as variables, perform PCA analysis. Taking the eigenvalue greater than 1 as the standard, determine 2 principal components, and calculate the comprehensive score of the raw Fructus Aurantii Immaturus according to the first formula. Samples with a comprehensive score of 0.64 - 0.95 for the raw Fructus Aurantii Immaturus are short-term samples, samples with a comprehensive score of 0.01 - 0.61 are medium-term samples, and samples with a comprehensive score of -1.41 - -1.26 are long-term samples; and

[0011] Taking the peak areas of 22 common peaks of the processed Fructus Aurantii Immaturus as variables, perform PCA analysis. Taking the eigenvalue greater than 1 as the standard, determine 3 principal components, and calculate the comprehensive score of the processed Fructus Aurantii Immaturus according to the second formula. Samples with a comprehensive score greater than 0 for the processed Fructus Aurantii Immaturus are short-term samples, and samples with a comprehensive score less than 0 for the raw Fructus Aurantii Immaturus are long-term samples;

[0012] Among them, the first formula is Y1 = 0.858×PC 1A + 0.061×PC 2B , and the second formula is Y2 = 0.591×PC 1a + 0.204×PC 2b + 0.072×PC 3c ; In the formula, PC 1A and PC 2B are the scores of the 2 principal components of the raw Fructus Aurantii Immaturus respectively, and PC 1a , PC 2b and PC 3c are the scores of the 3 principal components of the processed Fructus Aurantii Immaturus respectively.

[0013] Preferably, the relative retention times of the 18 common peaks of the raw Fructus Aurantii Immaturus include: Peak 1: 0.16; Peak 2: 0.76; Peak 3: 0.82; Peak 4: 0.89; Peak 5: 0.94; Peak 6 as the reference peak: 1.00; Peak 7: 1.05; Peak 8: 1.13; Peak 9: 1.30; Peak 10: 1.92; Peak 11: 2.01; Peak 12: 2.06; Peak 13: 2.11; Peak 14: 2.24; Peak 15: 2.31; Peak 16: 2.50; Peak 17: 3.25; Peak 18: 3.28.

[0014] Preferably, the relative retention times of the 22 common peaks of the processed Fructus Aurantii Immaturus are as follows: Peak 1: 0.16; Peak 2: 0.19; Peak 3: 0.23; Peak 4: 0.48; Peak 5: 0.76; Peak 6: 0.82; Peak 7: 0.94; Peak 8 is the reference peak: 1.00; Peak 9: 1.03; Peak 10: 1.05; Peak 11: 1.13; Peak 12: 1.30; Peak 13: 1.92; Peak 14: 2.01; Peak 15: 2.06; Peak 16: 2.11; Peak 17: 2.19; Peak 18: 2.24; Peak 19: 2.31; Peak 20: 2.50; Peak 21: 3.25; Peak 22: 3.28.

[0015] Preferably, the short-term samples of the raw Fructus Aurantii Immaturus are from 0 to 2 months, the medium-term samples are from 4 to 8 months, and the long-term samples are from 10 to 12 months.

[0016] Preferably, the short-term samples of the processed Fructus Aurantii Immaturus are from 0 to 2 months, and the long-term samples are from 4 to 12 months.

[0017] Preferably, the process of determining the main components includes: after standardizing the common peak area data according to the formula then calculating the scores of the eigenvalues according to the formula PC i =Z i1 ×D i1 +Z i2 ×D i2 +……+Z ij ×D ij ;

[0018] In the formula, x ij is the measured data of each input quantity, μ j is the average value of each input quantity, σ j is the standard deviation of each input quantity, Z ij is the standardized data, and D ij is the component score coefficient of each input quantity on the main component.

[0019] Preferably, the aging processes of the raw Fructus Aurantii Immaturus and the processed Fructus Aurantii Immaturus include: storing the raw Fructus Aurantii Immaturus and the processed Fructus Aurantii Immaturus in a simulated natural environment with a temperature of 25 °C and a humidity of 40% for 12 months, sampling every two months for HPLC analysis, and storing the Fructus Aurantii Immaturus sampled at each time point in a freezer at -18 °C.

[0020] Preferably, the HPLC chromatographic conditions are as follows: an octadecylsilane-bonded silica gel is used as the stationary phase in the chromatographic column, the column temperature is 35 °C, the detection wavelength is 283 nm, the flow rate is 1 mL / min, the analysis time is 40 min, and a sample injection volume of 10 μL is used to inject into the HPLC chromatograph for gradient elution.

[0021] Preferably, the elution program for the gradient elution is as follows:

[0022] At 0 minute, mobile phase A is a 90% formic acid solution and mobile phase B is a 10% acetonitrile solution;

[0023] At 10 minutes, mobile phase A is a 79% formic acid solution and mobile phase B is a 21% acetonitrile solution;

[0024] At 20 minutes, mobile phase A is a 78% formic acid solution and mobile phase B is a 22% acetonitrile solution;

[0025] At 25 minutes, mobile phase A is a 54% formic acid solution and mobile phase B is a 46% acetonitrile solution;

[0026] At 35 minutes, mobile phase A is a 53% formic acid solution and mobile phase B is a 47% acetonitrile solution;

[0027] At 40 minutes, mobile phase A is a 0% methanol solution and mobile phase B is a 100% acetonitrile solution;

[0028] Among them, the formic acid solution is an aqueous solution of formic acid with a mass fraction of 0.3%.

[0029] An application of a method for identifying the aging time of raw and processed Fructus Aurantii Immaturus by combining HPLC fingerprint and chemometrics, using the said method to identify the aging time of raw and processed Fructus Aurantii Immaturus.

[0030] The beneficial effects of the present invention are as follows:

[0031] 1. The present invention constructs the fingerprint of raw and processed Fructus Aurantii Immaturus, and combines chemometric analysis methods to achieve accurate identification of the aging time of raw and processed Fructus Aurantii Immaturus;

[0032] 2. The present invention obtains the characteristic fingerprint of the sample by high performance liquid chromatography (HPLC), and combines principal component analysis (PCA) to obtain the comprehensive score of the sample, which can quickly and effectively distinguish the aging time of raw and processed Fructus Aurantii Immaturus. This method has the advantages of simple operation, reliable results, good reproducibility, etc., provides a new technical means for the scientific evaluation of the storage time of traditional Chinese medicine, and helps to ensure the quality of medicinal materials and the stability of clinical efficacy;

[0033] 3. The fingerprint method for the crude and processed Fructus Aurantii provided by the present invention, combined with partial least squares discriminant analysis (PLS-DA), can quickly distinguish the aging time of the crude and processed Fructus Aurantii. Among them, variables 17, 14 (limonin), and 4 are the key chemical markers for distinguishing different aging times of the crude Fructus Aurantii, and variables 21, 4, 18 (limonin), 2, 20 (tangeretin), 5 (eriocitrin), and 1 are the key chemical markers for distinguishing different aging times of the processed Fructus Aurantii. Description of the Drawings

[0034] Figure 1a Chromatogram of the crude Fructus Aurantii described in the present invention. (2 Eriocitrin; 5 Narirutin; 6 Naringin; 7 Hesperidin; 8 Neohesperidin; 9 Hydroxycitronellal; 10 Poncitrin; 11 Naringenin; 12 Hesperetin; 14 Limonin; 15 Nobiletin; 16 Tangeretin)

[0035] Figure 1b Chromatogram of the processed Fructus Aurantii described in the present invention. (2 Eriocitrin; 5 Narirutin; 6 Naringin; 7 Hesperidin; 8 Neohesperidin; 9 Hydroxycitronellal; 10 Poncitrin; 11 Naringenin; 12 Hesperetin; 14 Limonin; 15 Nobiletin; 16 Tangeretin)

[0036] Figure 1c Chromatogram of the reference substance described in the present invention. (2 Eriocitrin; 5 Narirutin; 6 Naringin; 7 Hesperidin; 8 Neohesperidin; 9 Hydroxycitronellal; 10 Poncitrin; 11 Naringenin; 12 Hesperetin; 14 Limonin; 15 Nobiletin; 16 Tangeretin)

[0037] Figure 2a HPLC fingerprint of the crude Fructus Aurantii with different aging times described in the present invention.

[0038] Figure 2b HPLC fingerprint of the processed Fructus Aurantii with different aging times described in the present invention.

[0039] Figure 3a HCA analysis chart of the crude Fructus Aurantii with different aging times described in the present invention.

[0040] Figure 3b HCA analysis chart of the processed Fructus Aurantii with different aging times described in the present invention.

[0041] Figure 4a PCA analysis chart of the crude Fructus Aurantii with different aging times described in the present invention.

[0042] Figure 4b PCA analysis chart of the processed Fructus Aurantii with different aging times described in the present invention.

[0043] Figure 5a VIP analysis chart of raw Fructus Aurantii Immaturus with different aging times described in the present invention.

[0044] Figure 5b VIP analysis chart of processed Fructus Aurantii Immaturus with different aging times described in the present invention. Detailed implementation manners

[0045] The present invention will be further described in detail below with reference to the accompanying drawings, so that those skilled in the art can implement it according to the description in the specification.

[0046] The present invention provides a method for identifying the aging time of raw and processed Fructus Aurantii Immaturus based on HPLC fingerprint combined with chemometrics, including the following steps:

[0047] Perform HPLC fingerprint analysis on raw Fructus Aurantii Immaturus and processed Fructus Aurantii Immaturus with different aging times, and obtain the retention time and peak area respectively;

[0048] Taking the peak areas of 18 common peaks of raw Fructus Aurantii Immaturus as variables, perform PCA analysis. Taking the eigenvalue greater than 1 as the standard, determine 2 principal components, and calculate the comprehensive score of raw Fructus Aurantii Immaturus according to the first formula. The comprehensive score of raw Fructus Aurantii Immaturus is 0.64 - 0.95 for short-term samples, 0.01 - 0.61 for medium-term samples, and -1.41 - -1.26 for long-term samples;

[0049] Taking the peak areas of 22 common peaks of processed Fructus Aurantii Immaturus as variables, perform PCA analysis. Taking the eigenvalue greater than 1 as the standard, determine 3 principal components, and calculate the comprehensive score of processed Fructus Aurantii Immaturus according to the second formula. The comprehensive score of processed Fructus Aurantii Immaturus greater than 0 is for short-term samples, and the comprehensive score of raw Fructus Aurantii Immaturus less than 0 is for long-term samples;

[0050] Among them, the first formula is Y1 = 0.858×PC 1A +0.061×PC 2B , and the second formula is Y2 = 0.591×PC 1a +0.204×PC 2b +0.072×PC 3c ; in the formula, PC 1A and PC 2B are the scores of the 2 principal components of raw Fructus Aurantii Immaturus respectively, and PC 1a , PC 2b and PC 3c are the scores of the 3 principal components of processed Fructus Aurantii Immaturus respectively.

[0051] In another embodiment, the process of determining 2 principal components for raw Fructus Aurantii Immaturus includes: performing standardization processing on the peak area data of 18 common peaks of raw Fructus Aurantii Immaturus, according to the formula Wherein, x ij refers to the measured data of each input quantity, and μ j refers to the average value of each input quantity, and σ j is the standard deviation of each input quantity; combined with the eigenvalues, variance contribution rates, cumulative variance contribution rates, and component score coefficient values obtained by PCA analysis, according to the formula PC i = Z i1 × D i1 + Z i2 × D i2 + …… + Z ij × D ij calculate the scores of the eigenvalues. In the formula, Z ij is the standardized data, and D ij is the component score coefficient of each input quantity on 2 principal components;

[0052] The process of determining 3 principal components for the processed products of Fructus Aurantii includes: standardizing the peak area data of 22 common peaks of the processed products of Fructus Aurantii, according to the formula Wherein, x ij refers to the measured data of each input quantity, and μ j refers to the average value of each input quantity, and σ j is the standard deviation of each input quantity; combined with the eigenvalues, variance contribution rates, cumulative variance contribution rates, and component score coefficient values obtained by PCA analysis, according to the formula PC i = Z i1 × D1 + Z i2 × D2 + …… + Z ij × D j calculate the scores of the eigenvalues. In the formula, Z ij is the standardized data, and D ij is the component score coefficient of each input quantity on 3 principal components.

[0053] In another embodiment, the relative retention times of 18 common peaks of the raw products of Fructus Aurantii are as follows: Peak 1: 0.16; Peak 2: 0.76; Peak 3: 0.82; Peak 4: 0.89; Peak 5: 0.94; Peak 6 is the reference peak: 1.00; Peak 7: 1.05; Peak 8: 1.13; Peak 9: 1.30; Peak 10: 1.92; Peak 11: 2.01; Peak 12: 2.06; Peak 13: 2.11; Peak 14: 2.24; Peak 15: 2.31; Peak 16: 2.50; Peak 17: 3.25; Peak 18: 3.28;

[0054] The relative retention times of 22 common peaks in the processed product of Fructus Aurantii Immaturus include: Peak 1: 0.16; Peak 2: 0.19; Peak 3: 0.23; Peak 4: 0.48; Peak 5: 0.76; Peak 6: 0.82; Peak 7: 0.94; Peak 8 is the reference peak: 1.00; Peak 9: 1.03; Peak 10: 1.05; Peak 11: 1.13; Peak 12: 1.30; Peak 13: 1.92; Peak 14: 2.01; Peak 15: 2.06; Peak 16: 2.11; Peak 17: 2.19; Peak 18: 2.24; Peak 19: 2.31; Peak 20: 2.50; Peak 21: 3.25; Peak 22: 3.28.

[0055] In another embodiment, the short-term samples of the raw product of Fructus Aurantii Immaturus are from 0 to 2 months, the medium-term samples are from 4 to 8 months, and the long-term samples are from 10 to 12 months; the short-term samples of the processed product of Fructus Aurantii Immaturus are from 0 to 2 months, and the long-term samples are from 4 to 12 months.

[0056] In another embodiment, the processed product of Fructus Aurantii Immaturus is the stir-fried Fructus Aurantii Immaturus with bran, and its preparation process includes: preheating the frying container, then evenly sprinkling wheat bran, when a large amount of thick white smoke emerges, immediately putting in the slices of the raw product of Fructus Aurantii Immaturus, quickly stirring, frying until the surface of Fructus Aurantii Immaturus turns dark yellow, taking out, sifting off the bran, and cooling. 10 kg of wheat bran is used for every 100 kg of Fructus Aurantii Immaturus.

[0057] In another embodiment, the aging processes of the raw product and the processed product of Fructus Aurantii Immaturus include: storing the raw product and the processed product of Fructus Aurantii Immaturus in a simulated natural environment with a temperature of 25°C and a humidity of 40% for a total of 12 months, sampling once every two months for HPLC analysis, and storing the Fructus Aurantii Immaturus sampled at each time point in a freezer at -18°C for preservation.

[0058] In another embodiment, the HPLC chromatographic conditions are as follows: using a chromatographic column with octadecylsilane-bonded silica gel as the stationary phase, the column temperature is 35°C, the detection wavelength is 283 nm, the flow rate is 1 mL / min, the analysis time is 40 min, injecting 10 μL of the sample volume into the HPLC chromatograph for gradient elution, and the elution program for gradient elution is as follows:

[0059] At 0 minute, mobile phase A is a 90% formic acid solution, and mobile phase B is a 10% acetonitrile solution;

[0060] At 10 minutes, mobile phase A is a 79% formic acid solution, and mobile phase B is a 21% acetonitrile solution;

[0061] At 20 minutes, mobile phase A is a 78% formic acid solution, and mobile phase B is a 22% acetonitrile solution;

[0062] At 25 minutes, mobile phase A is a 54% formic acid solution, and mobile phase B is a 46% acetonitrile solution;

[0063] At 35 min, mobile phase A was 53% formic acid solution and mobile phase B was 47% acetonitrile solution;

[0064] At 40 min, mobile phase A was 0% methanol solution, and mobile phase B was 100% acetonitrile solution;

[0065] The formic acid solution is a formic acid aqueous solution with a mass fraction of 0.3%.

[0066] The present invention also provides an application of a method for identifying the aging time of raw and processed Fructus Aurantii Immaturus products based on HPLC fingerprint in combination with chemometrics, for identifying the aging time of raw and processed Fructus Aurantii Immaturus products.

[0067] Example

[0068] 1. Instruments, reagents and medicinal materials:

[0069] The instruments required for this example are shown in Table 1.

[0070] Table 1 Experimental instruments

[0071]

[0072] The reagents required in this example include: naringenin (batch number: YJ0603HA13, purity: ≥98%), all purchased from Shanghai Yuanye Biotechnology Co., Ltd.; eriocitrin (batch number: WP23090508, purity: ≥98%); naringin (batch number: WP22122302, purity: ≥98%); naringin (batch number: WP23011805, purity: ≥98%); hydrated hesperidin (batch number: WP 23030705, purity: ≥98%); nobiletin (batch number: WP23050904, purity: ≥98%); neohesperidin (batch number: WP23061802, purity: ≥98%); hesperidin (batch number: WP22122302, purity: ≥98%); hesperidin (batch number: WP23110701, purity: ≥98%); citrus glycosides (batch number: WP23020702, purity: ≥98%); tangerine (batch number: WP23031905, purity: ≥98%); limonin (batch number: WP23031701, purity: ≥98%) were purchased from Sichuan Weikeqi Biotechnology Co., Ltd. Methanol, analytical grade, was purchased from Tianjin Kangkede Technology Co., Ltd.; methanol, chromatographic grade, was purchased from Sigma, USA; formic acid, chromatographic grade, was purchased from Tianjin Komiou Chemical Reagent Co., Ltd.; distilled water was purchased from Guangzhou Watsons Food and Beverage Co., Ltd.;

[0073] The medicinal materials required in this example include: 4 kinds of processed Fructus Aurantii products prepared according to the general rules of processing in the Pharmacopoeia of the People's Republic of China (2020 Edition, Part IV) and the Jiangxi Zhangbang processing method. The raw Fructus Aurantii products and the processed Fructus Aurantii products (Fructus Aurantii stir-fried with bran) are numbered as shown in Table 2. The specific processing methods are as follows:

[0074] The processed Fructus Aurantii product in this example is Fructus Aurantii stir-fried with bran. The process of stir-frying with bran includes: preheating the frying container, then evenly sprinkling wheat bran. When a large amount of thick white smoke emerges, immediately put in the raw Fructus Aurantii slices, quickly stir, and fry until the surface of the Fructus Aurantii turns dark yellow, then take out, sift off the bran, and let it cool. 10 kg of wheat bran is used for every 100 kg of Fructus Aurantii.

[0075] Table 2 Sample numbers

[0076]

[0077] 2. Establishment method

[0078] (1) Aging experiment method: Store the raw Fructus Aurantii products and the processed Fructus Aurantii products in an indoor medicinal material cabinet (temperature 25 °C, humidity 40%) to simulate aging in a natural environment. Store for a total of 12 months, take samples every two months for HPLC analysis, and store the Fructus Aurantii samples taken at each time point in a freezer at -18 °C.

[0079] (2) HPLC chromatographic conditions: Chromatographic column: Symmetry C18 (4.6×150 mm, 5 μm); Mobile phase: acetonitrile - 0.3% formic acid in water; Detection wavelength: 283 nm; Injection volume: 10 μL; Flow rate: 1 mL / min; Column temperature: 35 °C; The gradient elution program is shown in Table 3.

[0080] Table 3 Gradient elution program table

[0081]

[0082] (3) Preparation of sample solution: Accurately weigh 50 mg of the raw Fructus Aurantii products and the processed Fructus Aurantii products at different aging stages, add 5 mL of 100% methanol, extract ultrasonically for 30 min (power 400 W, frequency 40 kHz), centrifuge at 8000 r / min for 5 min with a high-speed centrifuge to obtain the supernatant, filter through a 0.22 μm nylon filter membrane, and take the subsequent filtrate for HPLC analysis. Each sample is processed in parallel 3 times.

[0083] (4) Preparation of reference substance solution: Accurately weigh appropriate amounts of each reference substance, eriocitrin, narirutin, naringin, hesperidin, neohesperidin, hydrated hesperetin, poncirin, naringenin, hesperetin, limonin, neohesperidin dihydrochalcone, and tangeritin, and prepare a reference substance solution with a concentration of 5 mg / mL with pure water. The chromatograms of the raw Fructus Aurantii products, the processed Fructus Aurantii products, and each reference substance are as Figures 1a to 1c shown.

[0084] 3. Methodological investigation

[0085] (1) Precision test: Take the same samples of crude and processed Fructus Aurantii Immaturus respectively, prepare the sample solutions according to the method under "Preparation of Sample Solution", and inject samples continuously 6 times according to the chromatographic conditions under "HPLC Chromatographic Conditions". Using naringin (peak No. 6) as the reference peak, calculate the relative standard deviation (RSD) values of relative retention time and relative peak area. The RSDs of relative retention time and relative peak area of each common peak are less than 1.0% and 5.0% respectively, indicating that the instrument has good precision. The results are shown in Tables 4 and 5 below.

[0086] Table 4 Results of precision test for relative retention time

[0087]

[0088] Table 5 Results of precision test for relative peak area

[0089]

[0090]

[0091] (2) Repeatability test: Take the same samples of crude and processed Fructus Aurantii Immaturus respectively, prepare the sample solutions according to the method under "Preparation of Sample Solution", inject samples for detection according to the chromatographic conditions under "HPLC Chromatographic Conditions", and use naringin (peak No. 6) as the reference peak to calculate the relative standard deviation (RSD) values of relative retention time and relative peak area. The RSDs of relative retention time and relative peak area of each common peak are less than 1.0% and 5.0% respectively, indicating good repeatability of the method. The results are shown in Tables 6 and 7 below.

[0092] Table 6 Results of repeatability test for relative retention time

[0093]

[0094]

[0095] Table 7 Results of repeatability test for relative peak area

[0096]

[0097] (3) Stability test: Samples of raw and processed Fructus Aurantii Immaturus were taken respectively and prepared into sample solutions according to the method under "Preparation of Sample Solution". According to the chromatographic conditions under "HPLC Chromatographic Conditions", the samples were injected for detection at 0, 2, 4, 8, 12, and 24 h. Using naringin (peak No. 6) as the reference peak, the relative standard deviation (RSD) values of relative retention time and relative peak area were calculated. The RSDs of relative retention time and relative peak area of each common peak were less than 1.0% and 5.0% respectively, indicating that the sample solution was stable within 24 h. The results are shown in Tables 8 and 9.

[0098] Table 8 Results of Stability Experiment of Relative Retention Time

[0099]

[0100]

[0101] Table 9 Results of Stability Experiment of Relative Peak Area

[0102]

[0103]

[0104] 4. Results of Examples

[0105] (1) HPLC analysis: The aged samples of raw and processed Fructus Aurantii Immaturus were subjected to HPLC analysis, and the generated fingerprint chromatograms are as Figures 2a to 2b shown. Similarity evaluation was performed on the obtained chromatographic data. The similarities of the raw and processed Fructus Aurantii Immaturus samples stored for 12 months were both greater than 0.950. This indicates that different samples at different storage times cannot be distinguished by traditional fingerprint chromatograms, and further analysis using chemometrics is required.

[0106] (2) Chemometric analysis:

[0107] Using the data management function of the liquid chromatography workstation, relevant information such as retention time and peak area of Fructus Aurantii Immaturus samples was obtained. The obtained data matrix was imported into SIMCA 14.1, and hierarchical cluster analysis (HCA) was performed on the samples using Ward's method. When the distance scale was 40, the raw Fructus Aurantii Immaturus could be divided into 3 categories according to short-term, medium-term, and long-term storage; when the distance scale was 250, the stir-fried Fructus Aurantii Immaturus with bran could be divided into 2 categories according to short-term and long-term storage. This indicates that during the storage process, the internal chemical components of both raw and stir-fried Fructus Aurantii Immaturus gradually changed.

[0108] Taking the peak areas of 18 common peaks in the crude product of Fructus Aurantii Immaturus as variables, import them into SPSS 26 software for PCA analysis; with the eigenvalue > 1 as the standard, 2 principal components are determined for the crude product of Fructus Aurantii Immaturus, and the cumulative variance contribution rate is 91.97%, as shown in Table 10, indicating that the first 2 principal component factors have good representativeness and can be used to evaluate the quality of the crude product of Fructus Aurantii Immaturus.

[0109] In this embodiment, the process of determining 2 principal components for the crude product of Fructus Aurantii Immaturus includes: standardizing the peak area data of 18 common peaks of the crude product of Fructus Aurantii Immaturus according to the formula ; where x ij is the test data of each input quantity, μ j is the average value of each input quantity, and σ j is the standard deviation of each input quantity; then, combined with the scores of eigenvalues, variance contribution rates, cumulative variance contribution rates, and component score coefficient values obtained from PCA analysis, calculate the scores of eigenvalues according to the formula PC i = Z i1 × D i1 + Z i2 × D i2 +……+ Z ij × D ij ; where Z ij is the standardized data, and D ij is the component score coefficient of each input quantity on 2 principal components; in this embodiment, i is 1A or 2B, j is 1 - 18, and the specific values of D ij are shown in Table 11, and the specific calculation formula is:

[0110] PC 1A = 0.068Z 1A1 + 0.065Z 1A2 + 0.071Z 1A3 + 0.056Z 1A4 + 0.068Z 1A5 + 0.069Z 1A6 + 0.068Z 1A7 + 0.07Z 1A8 + 0.059Z 1A9 + 0.068Z 1A10 + 0.052Z 1A11 + 0.066Z 1A12 + 0.071Z 1A13 + 0.051Z 1A14 + 0.074Z 1A15 + 0.031Z 1A16 - 0.08Z 1A17 + 0.041Z 1A18 ;

[0111] PC2B = -0.046Z 2B1 -0.007Z 2B2 -0.067Z 2B3 +0.014Z 2B4 -0.035Z 2B5 -0.041Z 2B6 -0.032Z 2B7 -0.057Z 2B8 +0.035Z 2B9 -0.038Z 2B10 +0.108Z 2B11 -0.057Z 2B12 -0.075Z 2B13 +0.048Z 2B14 -0.103Z 2B15 +0.19Z 2B16 +0.877Z 2B17 +0.203Z 2B18 ;

[0112] Table 10 PCA eigenvalue and variance contribution rate of raw Fructus Aurantii samples

[0113]

[0114] Table 11 PCA component score coefficient D of raw Fructus Aurantii samples ij Matrix

[0115]

[0116]

[0117] Two principal component factors extracted from 18 common peaks in the raw Fructus Aurantii samples were comprehensively evaluated. Using the variance contribution rate corresponding to each principal component as the weight, a linear weighting was performed on the principal component scores and the corresponding weights. The original data was projected into the principal component space using the eigenvector to obtain the scores of each sample on each principal component, and then the comprehensive score of the principal component was calculated. The formula is Y1 = 0.858 × PC 1A + 0.061 × PC 2B , where Y1 is the comprehensive score of the raw Fructus Aurantii, and PC 1A and PC 2B are the scores of the two principal components. The results are shown in Table 12. According to the factor load and comprehensive score ranking of the principal component analysis of the raw Fructus Aurantii samples, the short-term samples from 0 to 2 months can be classified into the first category, with a comprehensive score of 0.64 - 0.95; the medium-term samples from 4 to 8 months are classified into the second category, with a comprehensive score of 0.01 - 0.61; the long-term samples from 10 to 12 months are classified into the third category, with a comprehensive score of -1.41 - -1.26.

[0118] Table 12 Principal Component Scores, Comprehensive Scores, and Excellent Rankings of Raw Fructus Aurantii Samples

[0119]

[0120]

[0121] Taking the peak areas of 22 common peaks in the stir-fried Fructus Aurantii samples as variables, import them into SPSS 26 software for PCA analysis; with the eigenvalue > 1 as the standard, 3 principal components are determined for the stir-fried Fructus Aurantii samples, and the cumulative variance contribution rate is 86.66%, as shown in Table 13, indicating that the first 3 principal component factors have good representativeness and can be used to evaluate the quality of stir-fried Fructus Aurantii.

[0122] In this embodiment, the process of determining 3 principal components for the stir-fried Fructus Aurantii samples includes: standardizing the peak area data of 22 common peaks of stir-fried Fructus Aurantii according to the formula where x ij is the measured data of each input quantity, μ j is the average value of each input quantity, and σ j is the standard deviation of each input quantity; then, combined with the eigenvalue, variance contribution rate, cumulative variance contribution rate, and component score coefficient values obtained from PCA analysis, calculate the eigenvalue according to the formula PC i = Z i1 × D1 + Z i2 × D2 + …… + Z ij × D j where Z ij is the standardized data, and D ij is the component score coefficient of each input quantity on 3 principal components; in this embodiment, i is 1a, 2b, or 3c, j is 1 to 22, and the specific values of D ij are shown in Table 14, and the specific calculation formula is:

[0123] PC 1a = -0.001Z 1a1 - 0.145Z 1a2 - 0.136Z 1a3 - 0.069Z 1a4 - 0.09Z 1a5 - 0.026Z 1a6 - 0.053Z 1a7 + 0.014Z 1a8 + 0.12Z 1a9 + 0.07z 1a10 + 0.042Z 1a11 + 0.013Z 1a12 + 0.027Z 1a13 + 0.068Z 1a14 + 0.066Z1a15 +0.108Z 1a16 -0.112Z 1a17 -0.017Z 1a18 +0.101Z 1a19 +0.086Z 1a20 +0.014Z 1a21 -0.121Z 1a22 ;

[0124] PC 2b =0.104Z 2b1 +0.149Z 2b2 +0.056Z 2b3 +0.11Z 2b4 +0.154Z 2b5 +0.127Z 2b6 +0.149Z 2b7 +0.098Z 2b8 -0.034Z 2b9 +0.035Z 2b10 +0.071Z 2b11 +0.071Z 2b12 +0.084Z 2b13 +0.034Z 2b14 +0.024Z 2b15 -0.024Z 2b16 +0.065Z 2b17 -0.026Z 2b18 -0.01Z 2b19 -0.06Z 2b20 -0.08Z 2b21 +0.032Z 2b22 ;

[0125] PC 3c =-0.094Z 3c1 +0.005Z 3c2 +0.05Z 3c3 +0.2Z 3c4 +0.003Z 3c5 -0.032Z 3c6 -0.097Z 3c7 -0.084Z 3c8 +0.03Z 3c9 -0.021Z 3c10 -0.068Z 3c11 +0.129Z 3c12 -0.054Z 3c13 +0.035Z 3c14 +0.077Z 3c15 +0.082Z 3c16 +0.206Z3c17 +0.549Z 3c18 +0.056z 3c19 +0.412Z 3c20 +0.002Z 3c21 +0.086Z 3c22 ;

[0126] Table 13 PCA eigenvalue and variance contribution rate of stir-fried Fructus Aurantii samples

[0127]

[0128] Table 14 PCA component score coefficient D of stir-fried Fructus Aurantii samples ij matrix

[0129]

[0130]

[0131] A comprehensive evaluation was carried out on the three principal component factors extracted from 22 common peaks in the stir-fried Fructus Aurantii samples. Using the variance contribution rate corresponding to each principal component as the weight, a linear weighting was performed on the principal component scores and the corresponding weights. The original data was projected into the principal component space using the eigenvector to obtain the scores of each sample on each principal component, and then the comprehensive score of the principal component was calculated. The formula is Y2 = 0.591×PC 1a +0.204×PC 2b +0.072×PC 3c , where Y2 is the comprehensive score of the processed Fructus Aurantii, and PC 1a , PC 2b and PC 3c are the scores of the three principal components. The results are shown in Table 15. According to the factor loadings and comprehensive score rankings of the principal component analysis of the stir-fried Fructus Aurantii samples, the short-term samples from 0 to 2 months can be classified into the first category, and the comprehensive scores are all greater than 0; the long-term samples from 4 to 12 months are classified into the second category, and the comprehensive scores are all less than 0.

[0132] Table 15 Principal component scores, comprehensive scores and excellent rankings of stir-fried Fructus Aurantii samples

[0133]

[0134] In summary, according to the factor loadings and comprehensive score rankings of the principal component analysis of the samples, the aging time of the Fructus Aurantii samples can be distinguished. The results show that the raw Fructus Aurantii samples can be divided into 3 categories according to the classification method of short-term, medium-term or long-term storage, and the stir-fried Fructus Aurantii can be divided into 2 categories. The PCA classification results are consistent with the HCA classification results, further proving the rationality of the established model. The specific staging is shown in Table 16. This indicates that the types and quantities of internal chemical components of Fructus Aurantii with different aging times are significantly different.

[0135] Table 16 Aging Stages of Immature Bitter Orange

[0136]

[0137] In order to further explore the quality difference markers among immature bitter oranges with different aging times, a supervised PLS-DA model was established for the samples based on the HCA and PCA models. The variable importance in projection (VIP value) of each type of sample variable is shown in Figures 3 to 5, and variables with a VIP value greater than 1 were selected as chemical markers. The chemical markers screened out from the raw immature bitter orange were mainly peak 17; 14 (limonin) and 4; the chemical markers of the processed immature bitter orange were mainly peak 21; 4; 18 (limonin); 2; 20 (citroxanthin); 5 (eriocitrin) and 1. This indicates that these variables play a major role in the classification of raw and processed immature bitter oranges with different aging times, and also shows that the content changes of these components are more obvious during the aging process of immature bitter orange.

[0138] Although the embodiments of the present invention have been disclosed as above, it is not limited to the applications listed in the specification and embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the field, additional modifications can be easily made. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to the specific details and the illustrated and described examples here.

Claims

1. A method for identifying the aging time of raw and processed Fructus Aurantii Immaturus based on HPLC fingerprint combined with chemometrics, which is characterized in that, It includes the following steps: Perform HPLC fingerprint analysis on crude Fructus Aurantii Immaturus and processed Fructus Aurantii Immaturus with different aging times, and obtain the retention time and peak area respectively; Taking the peak areas of 18 common peaks of the crude Fructus Aurantii Immaturus as variables, perform PCA analysis. Taking the eigenvalue greater than 1 as the standard, determine 2 principal components, and calculate the comprehensive score of the crude Fructus Aurantii Immaturus according to the first formula. The crude Fructus Aurantii Immaturus with a comprehensive score of 0.64 - 0.95 is a short-term sample, the crude Fructus Aurantii Immaturus with a comprehensive score of 0.01 - 0.61 is a medium-term sample, and the crude Fructus Aurantii Immaturus with a comprehensive score of -1.41 - -1.26 is a long-term sample; and Taking the peak areas of 22 common peaks of the processed Fructus Aurantii Immaturus as variables, perform PCA analysis. Taking the eigenvalue greater than 1 as the standard, determine 3 principal components, and calculate the comprehensive score of the processed Fructus Aurantii Immaturus according to the second formula. The processed Fructus Aurantii Immaturus with a comprehensive score greater than 0 is a short-term sample, and the crude Fructus Aurantii Immaturus with a comprehensive score less than 0 is a long-term sample; Among them, the first formula is Y1 = 0.858×PC 1A + 0.061×PC 2B , and the second formula is Y2 = 0.591×PC 1a + 0.204×PC 2b + 0.072×PC 3c ; in the formula, PC 1A and PC 2B are the scores of two main components of raw Fructus Aurantii Immaturus respectively, and PC 1a , PC 2b and PC 3c are the scores of three main components of processed Fructus Aurantii Immaturus respectively.

2. The method for identifying the aging time of raw and processed Fructus Aurantii Immaturus based on HPLC fingerprint combined with chemometrics according to claim 1, characterized in that, The relative retention times of the 18 common peaks of the crude Fructus Aurantii Immaturus are as follows: Peak 1: 0.16; Peak 2: 0.76; Peak 3: 0.82; Peak 4: 0.89; Peak 5: 0.94; Peak 6 is the reference peak: 1.00; Peak 7: 1.05; Peak 8: 1.13; Peak 9: 1.30; Peak 10: 1.92; Peak 11: 2.01; Peak 12: 2.06; Peak 13: 2.11; Peak 14: 2.24; Peak 15: 2.31; Peak 16: 2.50; Peak 17: 3.25; Peak 18: 3.

28.

3. The method for identifying the aging time of raw and processed Fructus Aurantii Immaturus based on HPLC fingerprint combined with chemometrics according to claim 1 or 2, characterized in that, The relative retention times of the 22 common peaks of the processed Fructus Aurantii Immaturus are as follows: Peak 1: 0.16; Peak 2: 0.19; Peak 3: 0.23; Peak 4: 0.48; Peak 5: 0.76; Peak 6: 0.82; Peak 7: 0.94; Peak 8 is the reference peak: 1.00; Peak 9: 1.03; Peak 10: 1.05; Peak 11: 1.13; Peak 12: 1.30; Peak 13: 1.92; Peak 14: 2.01; Peak 15: 2.06; Peak 16: 2.11; Peak 17: 2.19; Peak 18: 2.24; Peak 19: 2.31; Peak 20: 2.50; Peak 21: 3.25; Peak 22: 3.

28.

4. The method for identifying the aging time of raw and processed Fructus Aurantii Immaturus based on HPLC fingerprint combined with chemometrics according to claim 3, characterized in that, The short-term samples of the crude Fructus Aurantii Immaturus are 0 - 2 months, the medium-term samples are 4 - 8 months, and the long-term samples are 10 - 12 months.

5. The method for identifying the aging time of raw and processed Fructus Aurantii Immaturus based on HPLC fingerprint combined with chemometrics according to claim 3, characterized in that, The short-term samples of the processed Fructus Aurantii Immaturus are 0 - 2 months, and the long-term samples are 4 - 12 months.

6. The method for identifying the aging time of raw and processed Fructus Aurantii Immaturus based on HPLC fingerprint combined with chemometrics according to claim 1, 2, 4 or 5, characterized in that, The process of determining the principal components includes: After normalizing the common peak area data according to the formula , then calculate the score of the eigenvalue according to the formula PC i = Z i1 × D i1 + Z i2 × D i2 + …… + Z ij × D ij ; where x ij is the test data of each input quantity, μ j is the average value of each input quantity, σ j is the standard deviation of each input quantity, Z ij is the standardized data, D ij is the component score coefficient of each input quantity on the principal component.

7. The method for identifying the aging time of raw and processed Fructus Aurantii Immaturus based on HPLC fingerprint combined with chemometrics according to claim 6, characterized in that, The aging process of the crude Fructus Aurantii Immaturus and the processed Fructus Aurantii Immaturus includes: storing the crude Fructus Aurantii Immaturus and the processed Fructus Aurantii Immaturus in a simulated natural environment with a temperature of 25°C and a humidity of 40% for a total of 12 months. Sampling is taken every two months for HPLC analysis, and the Fructus Aurantii Immaturus sampled at each time point is stored in a freezer at -18°C for preservation.

8. The method for identifying the aging time of raw and processed Fructus Aurantii Immaturus based on HPLC fingerprint combined with chemometrics according to claim 7, characterized in that, The HPLC chromatographic conditions are as follows: an octadecylsilane-bonded silica gel column is used as the stationary phase, the column temperature is 35 °C, the detection wavelength is 283 nm, the flow rate is 1 mL / min, the analysis time is 40 min, and a sample injection volume of 10 μL is injected into the HPLC chromatograph for gradient elution.

9. The method for identifying the aging time of raw and processed Fructus Aurantii Immaturus based on HPLC fingerprint combined with chemometrics according to claim 8, characterized in that, The elution program for the gradient elution is as follows: At 0 min, mobile phase A is a 90% formic acid solution and mobile phase B is a 10% acetonitrile solution; At 10 min, mobile phase A is a 79% formic acid solution and mobile phase B is a 21% acetonitrile solution; At 20 min, mobile phase A is a 78% formic acid solution and mobile phase B is a 22% acetonitrile solution; At 25 min, mobile phase A is a 54% formic acid solution and mobile phase B is a 46% acetonitrile solution; At 35 min, mobile phase A is a 53% formic acid solution and mobile phase B is a 47% acetonitrile solution; At 40 min, mobile phase A is a 0% methanol solution and mobile phase B is a 100% acetonitrile solution; Among them, the formic acid solution is an aqueous solution of formic acid with a mass fraction of 0.3%.

10. Application of a method for identifying the aging time of raw and processed Fructus Aurantii Immaturus based on HPLC fingerprint combined with chemometrics, characterized in that, Use the method described in any one of claims 1-9 to identify the aging time of raw and processed Fructus Aurantii Immaturus.

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