Application of 6-demethylnobiletin in identification of aging years of pericarpium citri reticulatae

Abstract

The invention discloses application of 6-demethylnobiletin in identification of aging years of dried orange peel. The invention also discloses a method for predicting the aging year of the dried orange peel, and the method comprises the following steps: measuring the content of 6-demethylnobiletin in a dried orange peel sample to be detected, and calculating the predicted aging year of the dried orange peel sample to be detected according to a pre-established calculation model of the content of 6-demethylnobiletin in dried orange peel years. According to the present invention, the significant linear positive correlation between the 6-demethylnobiletin content and the dried orange peel aging year is found for the first time, the quantitative prediction is performed by establishing the linear model, the traditional sensory dependence identification method is converted into the objective chemical data index, and the result is accurate and reliable.

Description

Technical Field

[0001] This invention relates to the field of quality testing and identification technology of Chinese medicinal materials, specifically to the application of 6-demethylhesperidin in the identification of the aging years of dried tangerine peel. Background Technology

[0002] Dried tangerine peel (Chenpi) is the dried, mature peel of the citrus fruit (Citrus reticulata) and its cultivated varieties, belonging to the Rutaceae family. It is believed to have the effects of regulating qi, strengthening the spleen, and resolving dampness and phlegm. In traditional Chinese medicine theory and practice, it is generally accepted that the medicinal value of dried tangerine peel is positively correlated with its aging period, hence the saying "the older the better," and the market consensus that "century-old dried tangerine peel is better than gold." The principle behind this is that as aging progresses, the chemical components within the tangerine peel undergo a series of dynamic transformations and accumulations, which not only directly affects its medicinal efficacy but also leads to significant differences in its market value.

[0003] Currently, the industry still mainly relies on traditional experience-based methods such as "observing color, smelling aroma, tasting flavor, and feeling texture" to identify the age of dried tangerine peel. This method is highly dependent on the personal experience of the appraiser, is subjective, has low repeatability, and lacks objective and unified quantitative standards. Therefore, it is most prone to misjudgment in practice and cannot meet the standardized requirements of modern Chinese medicinal materials markets for circulation efficiency and quality control.

[0004] To overcome the limitations of traditional methods, some studies have attempted to use modern analytical techniques such as chromatography and spectroscopy to objectively analyze dried tangerine peel. However, most of these studies focus on a few known main components such as hesperidin and nonotrimonin. Unfortunately, there is no clear and strong correlation between the content of these components and the aging years, resulting in low accuracy and insufficient stability in the age prediction models built based on this, making them difficult to apply in practical identification scenarios.

[0005] It is evident that existing technologies have failed to reveal the key chemical markers closely related to the aging process of dried tangerine peel. Therefore, there is an urgent need in this field to identify a characteristic chemical indicator that is highly correlated with the aging years and has a clear pattern of change, and on this basis, to establish an objective, accurate, and quantifiable method for detecting the age of dried tangerine peel, in order to fill the gap in existing technologies. Summary of the Invention

[0006] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, this invention proposes an application of 6-demethylhesperidin in the identification of the aging age of dried tangerine peel. For the first time, 6-demethylhesperidin is used as an identification marker. By accurately detecting its content, the aging age of dried tangerine peel can be accurately identified.

[0007] The present invention also proposes a method for detecting 6-demethylhesperidin.

[0008] According to one aspect of the present invention, the application of 6-demethylhesperidin in the identification of the aging age of dried tangerine peel is proposed.

[0009] According to some embodiments of the present invention, the structural formula of 6-demethylhesperidin is as follows: .

[0010] It can also be called 6-hydroxyhesperidin.

[0011] According to another aspect of the present invention, a method for predicting the aging years of dried tangerine peel is proposed, comprising the following steps: The content of 6-demethylhesperidin in the tangerine peel sample was determined, and the predicted aging year of the tangerine peel sample was calculated based on the pre-established calculation model of "tangerine peel year-6-demethylhesperidin content".

[0012] According to some embodiments of the present invention, the content of 6-demethylhesperidin in the tangerine peel sample is determined by high performance liquid chromatography.

[0013] According to some embodiments of the present invention, the content of 6-demethylhesperidin in the tangerine peel sample is determined by high performance liquid chromatography-mass spectrometry (LCMS).

[0014] According to some embodiments of the present invention, the computational model is obtained by linear regression analysis using the least squares method.

[0015] According to some embodiments of the present invention, the calculation model uses the known aging years of the standard tangerine peel sample as the dependent variable and the corresponding 6-demethylhesperidin content as the independent variable, and performs linear regression analysis to obtain the model.

[0016] According to some embodiments of the present invention, the prediction method includes the following steps: S1. Sample pretreatment: Take the tangerine peel sample to be tested and extract it with an organic solvent to obtain the test solution; S2. Content determination: The content of 6-demethylhesperidin in the test solution was determined by high performance liquid chromatography-mass spectrometry. S3. Age determination: Substitute the 6-demethylhesperidin content measured in step S2 into the pre-established linear regression model of "tangerine peel year-6-demethylhesperidin content" to calculate the predicted aging year of the tangerine peel sample to be tested. The expression for the linear regression model is as follows: y = a × x + b In the formula, x Indicates the predicted year of dried tangerine peel.y This indicates the content of 6-demethylhesperidin as determined by LCMS. a The slope of the model, b The intercept of the model; the model parameters a and b The determination was made by measuring and regression analysis on a standard tangerine peel sample set with known aging years.

[0017] According to some embodiments of the present invention, in step S1, the tangerine peel sample to be tested is also subjected to pulverization.

[0018] According to some embodiments of the present invention, in step S1, the organic solvent is methanol, ethanol or acetonitrile.

[0019] According to some embodiments of the present invention, in step S2, the chromatographic conditions of the high performance liquid chromatography-mass spectrometry (HPLC-MS) are as follows: the chromatographic column is packed with octadecylsilane-bonded silica gel; the mobile phase includes phase A and phase B, wherein phase A is an aqueous phase and phase B is an acetonitrile phase, and gradient elution is performed; a mass spectrometer is used for detection, and the mass spectrometry detection adopts selected ion monitoring or positive and negative ion full scan mode, with a mass range of 100-1500 m / z.

[0020] According to some embodiments of the present invention, formic acid is added to the aqueous phase.

[0021] According to some embodiments of the present invention, the amount of formic acid added to the aqueous phase is 0.05~0.15%, such as 0.1%.

[0022] According to some embodiments of the present invention, the gradient elution program is as follows: 0-50 min, the volume percentage of phase B changes linearly from 5% to 95%.

[0023] According to some embodiments of the present invention, the chromatographic column is a Hypersil GOLD C18 with specifications including 100 mm × 2.1 mm and 1.9 µm.

[0024] According to some embodiments of the present invention, the method for establishing the linear regression model of "aged tangerine peel - 6-demethylhesperidin content" includes: M1. Collect a series of standard dried tangerine peel samples with known exact aging years to form a training sample set; M2. For each standard tangerine peel sample, process it according to steps S1 and S2 and determine its 6-demethylhesperidin content; M3. Using the known aging years of the standard tangerine peel sample as the dependent variable and the corresponding 6-demethylhesperidin content as the independent variable, a linear regression analysis was performed to obtain the linear regression model. y = a ×x + b .

[0025] According to some embodiments of the present invention, the expression of the linear regression model is: y =11.97 x +0.52.

[0026] According to another aspect of the present invention, the above-mentioned prediction method is also provided for application in the quality grading, authenticity identification, or market circulation supervision of dried tangerine peel.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Objective and accurate, eliminating subjective errors: This invention is the first to discover a significant linear positive correlation between the content of 6-demethylhesperidin and the aging years of dried tangerine peel. A linear model is established to achieve quantitative prediction, transforming traditional sensory-based identification methods into objective chemical data indicators, resulting in accurate and reliable results. This method requires only a single indicator to accurately predict the aging years of dried tangerine peel, effectively overcoming the shortcomings of traditional empirical identification methods' strong subjectivity and the low accuracy of existing instrumental analysis methods. It provides a reliable technical means for achieving objective, accurate, and quantifiable identification of the age of dried tangerine peel.

[0028] 2. Strong scientific basis: The present invention is based on LCMS, a modern analytical technique with high precision and good reproducibility, and combined with a rigorous mathematical modeling and verification process, which gives the detection method a solid scientific basis.

[0029] 3. High practicality: The method has a standardized operation process, is simple and easy to use, and is highly repeatable. It is easy to standardize and promote, and can be used by Chinese medicinal material testing institutions, pharmaceutical factories, market supervision departments and collectors. It provides a scientific basis for the quality grading, authenticity identification and market pricing of dried tangerine peel.

[0030] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0031] Figure 1 This is a chromatogram of the 6-demethylhesperidin sample obtained in Example 1 of the present invention.

[0032] Figure 2 This is the mass spectrum of the 6-demethylhesperidin sample obtained in Example 1 of the present invention.

[0033] Figure 3 The content of 6-demethylhesperidin in different years was measured in Example 1 of the present invention.

[0034] Figure 4This is a fitted curve of "aged tangerine peel - 6-demethylhesperidin content" measured in Example 1 of the present invention.

[0035] Figure 5 This is the curve of "aged tangerine peel - content of hesperidin" measured in Comparative Example 1 of this invention.

[0036] Figure 6 This is the curve of "aged tangerine peel - tangeretin content" measured in Comparative Example 1 of this invention.

[0037] Figure 7 The curve showing the content of "aged tangerine peel - 5-demethylhesperidin (5-hydroxyhesperidin)" measured in Comparative Example 1 of this invention is shown. Detailed Implementation

[0038] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available. Unless otherwise specified, the same parameter value is the same in all embodiments. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0039] In the description of this invention, references to terms such as "some embodiments" indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0040] In the following examples, the 6-demethylnobiletin sample was obtained by fermentation and conversion of nobiletin. It was prepared according to existing techniques, and the reference information is as follows: Su S, Zhao D, Yuan B, et al. Biosynthesis of 6- and 7-Mono-Demethylated Nobiletins by a Newly Isolated Strain of Yeast[J]. Journal of agricultural and food chemistry, 2022, 70(49):15439-15448. DOI:10.1021 / acs.jafc.2c03822. Example 1 This example provides a method for predicting the aging years of dried tangerine peel. The specific steps are as follows: (a) Standard sample collection: Collect 10 standard samples of dried tangerine peel with known exact aging years (0.5 years, 2.5 years, 3.5 years, 6.5 years, 8.5 years, 10.5 years) confirmed by the place of origin and authoritative experts, for a total of 60 samples, to form a training sample set.

[0041] (II) Sample pretreatment: Crush each standard sample and pass it through a No. 3 sieve. Accurately weigh about 0.1 g of each powder and place it in a stoppered volumetric flask. Accurately add 100 mL of methanol, make up to volume, and sonicate for 1 hour. After cooling, shake well and filter through a 0.45 μm microporous membrane. Take the filtrate as the test solution.

[0042] (III) Determination of content by LCMS: Chromatographic conditions: Hypersil GOLD C18 (100 mm × 2.1 mm, 1.9 µm); mobile phase A was water containing 0.1 vol% formic acid, mobile phase B was acetonitrile, gradient elution program was: 0-50 min, acetonitrile linearly changed from 5% to 95%; flow rate was 0.4 mL / min; column temperature was 40℃; ion scan mode (Polarity): positive ion full scan mode; molecular weight range (Scan Range): 100-1500 m / z; resolution (Resolution): 70000; injection volume was 2 μL.

[0043] (iv) Year determination: Record the chromatographic peak area of ​​6-demethylhesperidin in each standard sample (pre-determine the standard curve of chromatographic peak content and area of ​​6-demethylhesperidin sample; the chromatogram and mass spectrum of 6-demethylhesperidin sample are as follows). Figures 1-2 (As shown) The exact content (μg / g) of 6-demethylhesperidin in dried tangerine peel from different years was calculated based on a pre-plotted standard curve. The relationship between the content of 6-demethylhesperidin in dried tangerine peel from different years and the year is shown below. Figure 3 As shown. From Figure 3 It can be seen that the content of 6-demethylhesperidin increases with the increase of aging years.

[0044] Model establishment: Using the known year of the standard sample as the x-value and the corresponding 6-demethylhesperidin content as the y-value, a curve showing the relationship between the year of dried tangerine peel and the 6-demethylhesperidin content is plotted as follows: Figure 4 As shown. Linear regression analysis using the least squares method yields the linear equation y = 11.97x + 0.52. The coefficient of determination R0 of the linear equation is... 2 =0.998, indicating good linearity, which meets the requirements for precise analysis.

[0045] A sample of dried tangerine peel, labeled "5 years old," was collected from the market. Sample pretreatment and high-performance liquid chromatography (HPLC) analysis were performed using the same methods as the standard sample, and the 6-demethylhesperidin content was determined to be 57.14 μg / g. This content value was then substituted into the established linear model: y =11.97 x +0.52. The predicted year for this sample is calculated. x ≈4.73 years. This shows that the predicted year for the sample is approximately 4.7 years, which is basically consistent with its labeled 5-year period, confirming the accuracy of the year label. Similar predictions using samples from other years also yield accurate year data. The prediction method of this invention has a high accuracy rate.

[0046] Comparative Example 1 The LCMS method described in Example 1 was used to detect the contents of hesperidin, tangeretin, and 5-demethylhesperidin (also known as 5-hydroxyhesperidin) in the standard samples obtained during the aging process (0.5 years, 2.5 years, 3.5 years, 6.5 years, 8.5 years, and 10.5 years), and to investigate whether the contents of other flavonoids were related to the aging years. The results are as follows: Figure 5-7 As shown, with the increase of aging years, the contents of norihesperidin, tangeretin, and 5-demethylnorihesperidin fluctuated, with no obvious trend, and were unrelated to the year.

[0047] Comparative Example 2 Three senior pharmacists conducted a blind evaluation of the test samples in Example 1 using a traditional empirical method. One pharmacist estimated the time to be approximately 4-5 years, another approximately 5-6 years, and the third approximately 3-4 years. The results showed that the traditional method had certain subjective differences, while the method of the present invention provided a precise quantitative result (4.7 years), avoiding the fluctuations caused by human judgment.

[0048] In summary, this invention is the first to discover a significant linear relationship between the content of 6-demethylhesperidin and the aging years of dried tangerine peel. Only a single indicator is needed to accurately predict the aging years of dried tangerine peel, thus overcoming the shortcomings of traditional empirical identification methods, which are highly subjective, and existing instrumental analysis methods, which have low accuracy. This provides a reliable technical means for achieving objective, accurate, and quantifiable identification of the age of dried tangerine peel.

[0049] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. Application of 6-Demethylhesperidin in the identification of the aging years of dried tangerine peel.

2. A method for predicting the aging years of dried tangerine peel, characterized in that: Includes the following steps: The content of 6-demethylhesperidin in the tangerine peel sample was determined, and the aging year of the tangerine peel sample was calculated based on the pre-established calculation model of "tangerine peel year - 6-demethylhesperidin content".

3. The method for predicting the aging years of dried tangerine peel according to claim 2, characterized in that: The procedure includes the following steps: determining the content of 6-demethylhesperidin in the tangerine peel sample by high performance liquid chromatography.

4. The method for predicting the aging years of dried tangerine peel according to any one of claims 2 to 3, characterized in that: Includes the following steps: S1. Sample pretreatment: Take the tangerine peel sample to be tested and extract it with an organic solvent to obtain the test solution; S2. Content determination: The content of 6-demethylhesperidin in the test solution was determined by high performance liquid chromatography-mass spectrometry. S3. Age determination: Substitute the 6-demethylhesperidin content measured in step S2 into the pre-established linear regression model of "tangerine peel year-6-demethylhesperidin content" to calculate the predicted aging year of the tangerine peel sample to be tested. The expression for the linear regression model is as follows: y = a × x + b In the formula, x Indicates the predicted year for dried tangerine peel. y This indicates the content of 6-demethylhesperidin as determined by LCMS. a The slope of the model, b The intercept of the model; the model parameters a and b The determination was made by measuring and regression analysis on a standard tangerine peel sample set with known aging years.

5. The method for predicting the aging years of dried tangerine peel according to claim 4, characterized in that: In step S1, the organic solvent is methanol, ethanol, or acetonitrile.

6. The method for predicting the aging year of dried tangerine peel according to claim 4, characterized in that: In step S2, the chromatographic conditions for the high performance liquid chromatography-mass spectrometry (HPLC-MS) are as follows: the chromatographic column is packed with octadecylsilane-bonded silica gel; the mobile phase includes phase A and phase B, wherein phase A is an aqueous phase and phase B is an acetonitrile phase, and gradient elution is performed; a mass spectrometer is used for detection, and the mass spectrometry detection adopts selected ion monitoring or positive and negative ion full scan mode, with a mass range of 100-1500 m / z.

7. The method for predicting the aging year of dried tangerine peel according to claim 6, characterized in that: The gradient elution program was as follows: 0-50 min, with the volume percentage of phase B changing linearly from 5% to 95%.

8. The method for predicting the aging year of dried tangerine peel according to claim 4, characterized in that: The method for establishing the linear regression model of "aged tangerine peel - 6-demethylhesperidin content" includes: M1. Collect a series of standard dried tangerine peel samples with known exact aging years to form a training sample set; M2. For each standard tangerine peel sample, process it according to steps S1 and S2 and determine its 6-demethylhesperidin content; M3. Using the known aging years of the standard tangerine peel sample as the dependent variable and the corresponding 6-demethylhesperidin content as the independent variable, a linear regression analysis was performed to obtain the linear regression model. y = a × x + b .

9. The method for predicting the aging years of dried tangerine peel according to claim 4, characterized in that: The expression for the linear regression model is: y =11.97 x +0.

52.

10. The application of the prediction method according to any one of claims 2 to 9 in the quality grading, authenticity identification, or market circulation supervision of dried tangerine peel.

Images