A colorimetric card for rapid detection of hesperidin concentration, and its preparation method and application

Abstract

A colorimetric card for rapidly detecting hesperidin concentration, as well as its preparation method and application, relates to the technical field of hesperidin concentration detection. A colorimetric card for rapidly detecting hesperidin concentration is provided. The colorimetric card is a collection of color development degrees obtained by developing hesperidin solutions of a certain gradient concentration in a fluorescent support medium under ultraviolet light. The colorimetric card can be compared with the color development degree obtained by developing a hesperidin solution extracted from tangerine peel to be tested in a fluorescent support medium under ultraviolet light, thereby rapidly determining the concentration range of the hesperidin solution and thereby determining the quality of the tangerine peel.

Description

Technical Field

[0001] The present invention relates to the technical field of hesperidin concentration detection, in particular to a colorimetric card for rapidly detecting hesperidin concentration, and a preparation method and application thereof. Background Art

[0002] Tangerine peel (Citrus tangerine) is the dried, mature peel of the Rutaceae plant (Citrus sinensis) and its cultivated varieties. It's slightly hard and brittle, fragrant, and pungent with a slightly bitter taste. It regulates qi and dampness, calms the stomach, stimulates appetite, and eliminates phlegm. It's primarily used to treat spleen and stomach qi stagnation and dampness, chest fullness and stuffiness, abdominal distension and pain, loss of appetite, vomiting, constipation, lung qi stagnation, cough with excessive sputum, and early-stage mastitis. Tangerine peel is primarily produced in Guangdong, Fujian, Sichuan, Chongqing, Zhejiang, Jiangxi, and Hunan. The highest quality comes from Xinhui and Sihui in Guangdong and the suburbs of Guangzhou, while Sichuan and Chongqing boast significant production.

[0003] Dried tangerine peel is prized for its age; the older it is, the more distinct its flavor. From source to store, the price of dried tangerine peel passes through four stages: dealers, wholesalers, agents, and retailers, with prices being repeatedly inflated. To maximize profits, counterfeit and inferior medicinal materials, such as sulfur-fumigated peel and industrial-grade peel, have emerged. To ensure the quality of dried tangerine peel in the medicinal market, a method for quickly identifying genuine and inferior dried tangerine peel is urgently needed, enabling both businesses and the public to better utilize high-quality dried tangerine peel.

[0004] Methods for authenticating the quality of traditional Chinese medicines (TCMs), such as chemometrics, instrumental analysis, and mass spectrometry, often require expensive, high-precision instruments that are difficult for most TCM vendors and the general public to purchase and use. Common methods for identifying tangerine peel include characterization, physical and chemical analysis, and content determination. Spectrophotometry, fluorescence spectrophotometry, nuclear magnetic resonance, radioimmunoassay, capillary electrophoresis, and HPLC are commonly used to identify its main active ingredient, hesperidin. Each of these methods has its own advantages and disadvantages, but they are often cumbersome, require sophisticated laboratory equipment, and require a strong biological background, hindering widespread adoption.

[0005] In the process of identifying the quality of dried tangerine peel, the quality control of dried tangerine peel currently focuses on the qualitative and quantitative analysis of a single substance, namely hesperidin. For example, the 2015 edition of the Pharmacopoeia of the People's Republic of China (ChP2015) uses the hesperidin content as an important indicator for measuring the quality of dried tangerine peel. That is, when the hesperidin content in dried tangerine peel is high, it can be considered that the quality of the dried tangerine peel is good, and when the hesperidin content in dried tangerine peel is low, it can be considered that the quality of the dried tangerine peel is poor. Currently, there is a lack of an article and method for quickly detecting the quality of dried tangerine peel on the market. Therefore, based on this, the present application proposes a colorimetric card for quickly detecting the concentration of hesperidin, as well as a preparation method and application thereof. Summary of the Invention

[0006] The object of the present invention is to provide a colorimetric card for rapidly detecting the concentration of hesperidin, so as to solve the problem in the above-mentioned background art of the lack of an article for rapidly detecting the quality of tangerine peel.

[0007] To achieve the above objectives, the present invention provides a colorimetric card for rapidly detecting the concentration of hesperidin. The colorimetric card is a collection of color development degrees obtained when a hesperidin solution with a certain gradient concentration is developed in a fluorescent support medium under ultraviolet light.

[0008] The colorimetric card can be used to compare the color of the hesperidin solution extracted from the tangerine peel to be tested in a fluorescent support medium under ultraviolet light, so as to quickly obtain the concentration range of the hesperidin solution and then judge the quality of the tangerine peel.

[0009] In order to obtain the colorimetric card for rapid detection of hesperidin concentration, the present invention also provides a method for preparing the colorimetric card for rapid detection of hesperidin concentration, the preparation method specifically comprising the following steps:

[0010] S1: Selection of fluorescence support medium;

[0011] S2: Determination of the relationship between hesperidin concentration and fluorescence intensity;

[0012] A 2000 μg / mL hesperidin solution was used as a hesperidin standard solution, and diluted to prepare a hesperidin solution with a concentration gradient of 30-400 μg / mL. The solution was placed on the fluorescent support medium selected in step S1, and then developed under ultraviolet light to obtain the fluorescence intensity. Based on the fluorescence intensity, a fluorescence development method was determined, and a preliminary relationship between fluorescence intensity and hesperidin concentration was obtained.

[0013] S2: Verification of fluorescence intensity maintenance;

[0014] A 2000 μg / mL hesperidin solution is used as a hesperidin standard solution, and diluted to prepare a hesperidin solution with a concentration gradient of 30-400 μg / mL. The solution is placed on the fluorescent support medium selected in step S1, and then developed under ultraviolet light to obtain fluorescence intensity. Based on the fluorescence intensity, the relationship between fluorescence intensity and hesperidin concentration is derived.

[0015] S4: Based on the results obtained in S1, S2, and S3, dilute the 2000 μg / mL hesperidin standard solution to prepare a hesperidin solution with a concentration gradient of 10-2000 μg / mL, place the solution in a selected fluorescent support medium, and develop the color under ultraviolet light to obtain a hesperidin color card.

[0016] As a further solution of the present invention: in the step S1, when selecting the fluorescent support medium, three materials, namely a black circular hole plate, a PVDF membrane, and an absorbent paper, are first determined as candidates for the fluorescent support medium. Then, a standard hesperidin solution of the same concentration and volume is added dropwise to the black circular hole plate, the PVDF membrane, and the absorbent paper, respectively. Then, a magnesium acetate methanol solution is added to each of the two materials. The images are immediately developed at an excitation light wavelength of 365 nm, photographed and compared, and the black circular hole plate with the brightest fluorescence intensity and no background color is selected as the fluorescent support medium. However, the fluorescence image produced by the black circular hole plate has a relatively obvious coffee ring effect. To make the fluorescence more representative, the square area in the center of the fluorescence image is captured as the representative fluorescence image.

[0017] As a further solution of the present invention: the hesperidin standard solution in step S2 is a solution obtained by placing hesperidin in methanol with a concentration of 75%.

[0018] In order to realize the practical use significance of the colorimetric card for rapidly detecting the concentration of hesperidin, the present invention also provides an application of the colorimetric card for rapidly detecting the concentration of hesperidin, which is used for detecting the quality of tangerine peel.

[0019] As a further solution of the present invention: when testing the quality of tangerine peel, the steps of extracting hesperidin from tangerine peel are as follows:

[0020] Z1: preprocessing;

[0021] The dust on the surface of the tangerine peel is removed, and then the tangerine peel is dried. After drying, the tangerine peel is crushed by a traditional Chinese medicine grinder, sieved to obtain a powder of 1-2 mm, and then packaged and stored at 4° C.

[0022] Z2: extraction of hesperidin;

[0023] The tangerine peel powder in Z1 was taken out and placed in a room until the tangerine peel powder reached room temperature. A hesperidin extract was prepared in a 50 mL centrifuge tube. The extract was placed in an ultrasonic cleaning machine with an ultrasonic extraction power set to 200 W to extract hesperidin and obtain an extract.

[0024] As a further solution of the present invention: in the ultrasonic extraction in step Z2, the extraction conditions adopted are a solid-liquid ratio of 1:50, 1g of tangerine peel powder is added to 50mL of 75% methanol, the extraction is carried out for 15min, and the temperature is maintained at 70°C.

[0025] As a further solution of the present invention: the extract obtained in Z2 is subjected to a fluorescence intensity test to obtain the fluorescence intensity, which is then compared with a colorimetric card.

[0026] As a further solution of the present invention: the fluorescence intensity experiment is to place a portion of the extract in Z2 in a fluorescent support medium, and then develop the color under ultraviolet light to obtain the fluorescence intensity status.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The invention obtains the main index for detecting the quality of tangerine peel by using hesperidin, thereby preparing a fluorescent colorimetric card, and can judge the quality of tangerine peel very quickly. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The fluorescence results of the same hesperidin reference substance under different supporting media.

[0030] Figure 2 This is the fluorescence intensity diagram under gradient hesperidin concentration at 0 h, where the unit is μg / mL.

[0031] Figure 3 This is the fluorescence intensity graph under gradient hesperidin concentration for 12 h, where the unit is μg / mL.

[0032] Figure 4 This is the hesperidin fluorescence colorimetric chart, where the unit is μg / mL.

[0033] Figure 5 HPLC fingerprint and hesperidin standard curve.

[0034] Figure 6 This is the fluorescence intensity diagram of the reaction results of standard hesperidin D, tangerine peel extracts E and F with magnesium acetate, where the unit is mg / mL. DETAILED DESCRIPTION

[0035] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0036] In addition, when an element in the present invention is referred to as being "fixed to" or "disposed on" another element, it may be directly on the other element or there may be an intermediate element. When an element is referred to as being "connected to" another element, it may be directly connected to the other element or there may be an intermediate element. The terms "vertical," "horizontal," "left," "right," and similar expressions used herein are for illustrative purposes only and do not represent the only implementation method.

[0037] In order to verify the relationship between the quality of dried tangerine peel and hesperidin, the HPLC chromatogram analysis and standard curve drawing method were used, such as Figure 5 As shown, A is the fingerprint of the hesperidin standard, B is the fingerprint of the tangerine peel extract, and C is the standard curve of hesperidin. Specifically, through the results of high-performance liquid chromatography gradient elution, the reference substance and the tangerine peel extract have a single peak with the same peak time, and the peak area accounts for a large proportion, which can be determined that the extract contains hesperidin. In addition, with concentration as the horizontal axis (x) and peak area as the vertical axis (y), linear regression is performed to draw a standard curve, and the obtained value is y=10.791x-134.98, R 2 =0.9935 approximate functional relationship.

[0038] In order to determine the optimal extraction conditions for hesperidin from tangerine peel, an orthogonal experiment was designed as follows:

[0039] The material-liquid ratio (X), methanol concentration (Y), extraction time (G) and extraction temperature (H) were used as the investigation factors. Each factor was set at 3 levels, with a total of 9 samples. The L9 (3 4 ) Orthogonal array was used to arrange the experiment. The factor levels are shown in Table 1.

[0040] Table 1 Experimental factor levels

[0041]

[0042] The results of the orthogonal experiment to optimize the optimal extraction conditions are shown in Table 2 below:

[0043] Table 2 Orthogonal experimental design and results of optimal extraction process

[0044]

[0045]

[0046] Table 3 Analysis of variance

[0047]

[0048]

[0049] Visual analysis reveals that the order of influence on hesperidin extraction concentration is: X > H > Y > G, meaning the solid-liquid ratio is the most important factor, followed by temperature, methanol concentration, and extraction time. The optimal extraction process for this medicinal material was determined to be X1-H3-Y3-G3, defined as a solid-liquid ratio of 1:50, a methanol concentration of 75%, an extraction time of 60 minutes, and a temperature of 70°C. As shown in Table 3, analysis of variance results indicate that there was no significant difference in the effect of ultrasonication time on the dissolution rate of hesperidin. Therefore, after comprehensive considerations, the optimal extraction process for hesperidin from tangerine peel was determined to be A1D3B3C1, defined as a solid-liquid ratio of 1:50, a methanol concentration of 75%, a temperature of 70°C, and an extraction time of 15 minutes. This method achieves excellent extraction results with a shorter extraction time.

[0050] After confirming that the fluorescent support medium is a black circular well plate, after the reaction of hesperidin with magnesium acetate, the change in fluorescence intensity is proportional to the change in hesperidin concentration, that is, the greater the hesperidin concentration measured by HPLC method, the stronger the fluorescence intensity of the reaction of hesperidin with magnesium acetate. Figure 6 shown.

[0051] In order to detect the quality of tangerine peel, the present invention selects a strategy of using the content of hesperidin in tangerine peel to judge the quality of tangerine peel. The main basis is that the ketone group in hesperidin can react with the magnesium ions in the magnesium acetate methanol solution to produce a complex reaction, and under ultraviolet light, it can emit a specific sky blue fluorescence.

[0052] Specifically, the present invention includes a method for preparing a colorimetric card for rapidly detecting hesperidin concentration, which method specifically comprises the following steps:

[0053] S1: Selection of fluorescence support medium;

[0054] When selecting the fluorescent support medium, the first choice is to determine three substances: black circular hole plate, PVDF membrane and absorbent paper as alternative fluorescent support media. Standard hesperidin solution of the same concentration and volume is added to them. When preparing the standard hesperidin solution, hesperidin is added to methanol with a concentration of 75%; then magnesium acetate methanol solution is added at the same time, and the solution is immediately developed at an excitation light wavelength of 365nm. After taking pictures for comparison, the appropriate fluorescent support is selected for subsequent experiments.

[0055] like Figure 1 The following are the fluorescence effects of the same hesperidin reference substance under different supporting media. Figure 1 It can be found that under the irradiation of 365nm ultraviolet light, the fluorescence intensity in the black circular hole is the brightest, the background is colorless, and the shape is regular; while the fluorescence intensity of the PVDF membrane and absorbent paper is weak and the shape is irregular. Therefore, it is determined to use the black circular hole plate as the fluorescent support medium for the light spot in the hesperidin fluorescence color development reaction.

[0056] S2: Determination of the relationship between hesperidin concentration and fluorescence intensity;

[0057] A hesperidin solution with a concentration of 2000 μg / mL was used as the hesperidin standard solution, and a hesperidin solution with a concentration gradient of 30-400 μg / mL was diluted and prepared. The solution was placed on the fluorescent support medium selected in step S1, and then developed under ultraviolet light to obtain the fluorescence intensity status. The fluorescence color development method was preliminarily determined, and the relationship between the fluorescence intensity and the hesperidin concentration was obtained based on the fluorescence intensity status.

[0058] Depend on Figure 2 As shown, it can be clearly seen that within a certain concentration range, the fluorescence intensity has an obvious linear correlation with the hesperidin concentration.

[0059] S3: Verification of fluorescence intensity maintenance;

[0060] Add the hesperidin standard solution in S2 to the fluorescent support medium confirmed in S1, then add magnesium acetate methanol solution, immediately observe the fluorescence intensity under ultraviolet light, record the fluorescence intensity at this time, and then observe the fluorescence intensity continuously at intervals of 12 hours. Each time observation is made, add colorimetric solution to replenish the original volume to prevent changes in fluorescence intensity caused by changes in solution volume. Then, place it under ultraviolet light to observe the fluorescence intensity and compare it with the fluorescence intensity at 0 hour to observe whether fluorescence quenching occurs.

[0061] Depend on Figure 3 It can be seen that the experimental results show that there is no obvious quenching phenomenon of hesperidin fluorescence intensity within 12 hours.

[0062] S4: Based on the results obtained in S1, S2, and S3, the 2000 μg / mL hesperidin standard solution was diluted to prepare a hesperidin solution with a concentration gradient of 10-2000 μg / mL, placed in a selected fluorescent support medium, and developed under ultraviolet light to obtain a hesperidin colorimetric card. The final colorimetric card can be obtained as follows: Figure 4 shown.

[0063] When testing the quality of tangerine peel, the extraction method of hesperidin, the raw material in tangerine peel, is also involved. The following steps can be used:

[0064] Z1: preprocessing;

[0065] The dust on the surface of the dried tangerine peel was removed by using absorbent paper. After the dust removal was completed, 40 g of dried tangerine peel was weighed and dried. After the drying was completed, the dried tangerine peel was crushed using a traditional Chinese medicine grinder, sieved to obtain a powder of 1-2 mm, and then packaged and stored at 4 ° C.

[0066] Z2: extraction of hesperidin;

[0067] Remove the tangerine peel powder from Z1 and place it indoors until it reaches room temperature. Prepare the hesperidin extract in a 50mL centrifuge tube and place the extract in an ultrasonic cleaner according to the experimental protocol number. Since 200W is the optimal power for hesperidin extraction, the ultrasonic extraction power for this experiment is set to 200W. Accurately weigh the corresponding mass of tangerine peel powder for each experimental protocol and place it into the centrifuge tube already in the ultrasonic cleaner to extract hesperidin.

[0068] During the ultrasonic extraction, the extraction conditions adopted were a solid-liquid ratio of 1:50, that is, 1 g of tangerine peel powder was added to 50 mL of 75% methanol, extracted for 15 minutes, and the temperature was maintained at 70°C.

[0069] The results showed that among the three fluorescent support media (black circular plate, PVDF membrane, and absorbent paper), the black circular plate had a brighter fluorescence intensity and a clearer background. The hesperidin concentration was linearly correlated with its fluorescence intensity, and no significant fluorescence quenching occurred within 12 hours. A preliminary hesperidin colorimetric chart was developed, providing a basis for the tangerine peel quality detection kit.

[0070] The invention determines that the most suitable extraction process for extracting hesperidin from tangerine peel is as follows: a material-liquid ratio of 1:50, a methanol concentration of 75%, an extraction time of 15 minutes, and a temperature of 70°C, which has short extraction time and good extraction effect.

[0071] The fluorescence intensity of the dried tangerine peel extract after reaction with magnesium acetate is similar to that of the standard hesperidin solution, indicating that the fluorescence in the dried tangerine peel extract mainly comes from hesperidin. The approximate hesperidin concentration can be obtained by reacting the extract with magnesium acetate methanol solution, and then the quality of the dried tangerine peel can be inferred.

[0072] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims, not the foregoing description, and all variations within the meaning and range of equivalents of the claims are intended to be included therein. Any reference sign in a claim should not be construed as limiting the claim to which it relates.

[0073] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. An application of a colorimetric card for rapid detection of hesperidin concentration, characterized in that: Used to detect the quality of dried tangerine peel; The color chart is a collection of color development degrees obtained when a hesperidin solution with a certain gradient concentration is developed under ultraviolet light in a fluorescent support medium; Also included is a method for preparing a colorimetric card for rapidly detecting hesperidin concentration, the method specifically comprising the following steps: S1: Selection of fluorescence support medium; S2: Determination of the relationship between hesperidin concentration and fluorescence intensity; A 2000 μg / mL hesperidin solution was used as a hesperidin standard solution, and diluted to prepare a hesperidin solution with a concentration gradient of 30-400 μg / mL. The solution was placed on the fluorescent support medium selected in step S1, and then developed under ultraviolet light to obtain the fluorescence intensity. Based on the fluorescence intensity, a fluorescence development method was determined, and a preliminary relationship between fluorescence intensity and hesperidin concentration was obtained. S3: Verification of fluorescence intensity maintenance; A 2000 μg / mL hesperidin solution was used as the hesperidin standard solution, and diluted to prepare a hesperidin solution with a concentration gradient of 30-400 μg / mL. The solution was placed on the fluorescent support medium selected in step S1, and then developed under ultraviolet light to obtain the fluorescence intensity status, which was recorded as the fluorescence intensity graph under the gradient hesperidin concentration at 0 h. As the methanol evaporates, the fluorescence disappears. Therefore, after 12 h, the color developing solution was added to the well again to obtain the fluorescence intensity status, which was recorded as the fluorescence intensity graph under the gradient hesperidin concentration at 12 h. The relationship between fluorescence intensity and time was obtained based on the fluorescence intensity status, and the fluorescence quenching was observed. S4: Based on the results obtained in S1, S2, and S3, a 2000 μg / mL hesperidin standard solution was diluted to prepare a hesperidin solution with a concentration gradient of 10-2000 μg / mL, placed in a selected fluorescent support medium, and developed under ultraviolet light to obtain a hesperidin colorimetric card; In step S1, when selecting the fluorescent support medium, three materials, namely, a black circular hole plate, a PVDF membrane, and an absorbent paper, are first determined as candidates for the fluorescent support medium. Then, a standard hesperidin solution of the same concentration and volume is added dropwise to the black circular hole plate, the PVDF membrane, and the absorbent paper, respectively. Then, a magnesium acetate methanol solution is added to each of the two materials. The two materials are immediately developed at an excitation light wavelength of 365 nm, photographed and compared, and the one with the brightest fluorescence intensity, no background color, and a regular shape is selected as the fluorescent support medium; The hesperidin standard solution in step S2 is a solution obtained by placing hesperidin in methanol with a concentration of 75%.

2. The use of the colorimetric card for rapid detection of hesperidin concentration according to claim 1, characterized in that: When testing the quality of tangerine peel, the steps for extracting hesperidin from tangerine peel are as follows: Z1: preprocessing; The dust on the surface of the tangerine peel is removed, and then the tangerine peel is dried. After drying, the tangerine peel is crushed by a traditional Chinese medicine grinder, sieved to obtain a powder of 1-2 mm, and then packaged and stored at 4° C. Z2: extraction of hesperidin; The tangerine peel powder in Z1 was taken out and placed in a room until the tangerine peel powder reached room temperature. A hesperidin extract was prepared in a 50 mL centrifuge tube. The extract was placed in an ultrasonic cleaning machine with an ultrasonic extraction power set to 200 W to extract hesperidin and obtain an extract.

3. The use of the colorimetric card for rapid detection of hesperidin concentration according to claim 2, characterized in that: In the ultrasonic extraction in step Z2, the extraction conditions adopted are a solid-liquid ratio of 1:50, 1 g of tangerine peel powder is added to 50 mL of 75% methanol, the extraction is carried out for 15 minutes, and the temperature is maintained at 70°C.

4. The use of the colorimetric card for rapid detection of hesperidin concentration according to claim 2, characterized in that: The extract obtained in Z2 was subjected to a fluorescence intensity experiment and the fluorescence intensity was obtained and then compared with a colorimetric card.

5. The use of the colorimetric card for rapid detection of hesperidin concentration according to claim 4, characterized in that: The fluorescence intensity experiment is to place a portion of the extract in Z2 in a fluorescent support medium, and then develop the color under ultraviolet light to obtain the fluorescence intensity status.

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