Raman determination method of total flavonoids content in propolis extract

Abstract

The application discloses a Raman determination method for the total flavone content in propolis extract, and comprises the following steps: gold nanospheres, metal-phenolic aldehyde network, epoxy resin and polymethyl methacrylate-microneedle composite detection materials for detecting flavone in propolis are prepared, and a rapid detection method for propolis flavone based on surface-enhanced Raman technology is established in sequence, so that rapid qualitative judgment and quantitative detection of propolis flavone are realized.

Description

Technical Field

[0001] This invention relates to the field of rapid food testing equipment, specifically a Raman spectroscopy method for determining the total flavonoid content in propolis extract. Background Technology

[0002] Propolis has a long history of medicinal use and contains abundant and unique bioactive substances, mainly flavonoids, phenolic acids, and terpenes. Due to its wide range of biological activities, it has become a hot topic of research and development both domestically and internationally. Flavonoid content is an important indicator for evaluating propolis quality and plays a crucial role in the quality control and industrialization of propolis health products. However, the quality of propolis products varies greatly, and it is difficult to distinguish between good and counterfeit products based on appearance, color, and odor alone. Therefore, achieving rapid quantitative detection of flavonoids in propolis is of great value in judging propolis quality.

[0003] Existing techniques for detecting flavonoids in propolis are mostly chromatographic, but their procedures are complex and cannot achieve portable, rapid detection. Raman spectroscopy has been used to detect flavonoids in vinegar mud, wolfberry, and citrus fruits; through pretreatment and mathematical methods, the interference of matrix components on the Raman signal can be greatly reduced. However, in the propolis detection system, the Raman spectral signals of flavonoids and phenolic acids show significant overlap, and their signal response intensity is easily affected by terpenes and fatty acids. Therefore, there is a need to develop a Raman method for determining the total flavonoid content in propolis extract to provide a basis for propolis quality control and authenticity identification. Summary of the Invention

[0004] The purpose of this invention is to provide a Raman spectroscopy method for determining the total flavonoid content in propolis extract.

[0005] The objective of this invention can be achieved through the following technical solutions:

[0006] A Raman spectroscopy method for determining the total flavonoid content in propolis extract includes: preparing gold nanospheres, a metal-phenolic network, an epoxy resin, and a polymethyl methacrylate-microneedle composite detection material for the detection of flavonoids in propolis; and establishing a rapid detection method for propolis flavonoids based on surface-enhanced Raman spectroscopy to achieve rapid qualitative and quantitative determination of propolis flavonoids.

[0007] As a further aspect of the present invention, the specific steps are as follows:

[0008] 1. Draw the standard curve of total flavonoids in propolis;

[0009] First, rutin standard solutions of different concentrations, gold nanospheres, metal-phenolic networks, epoxy resin, and polymethyl methacrylate-microneedle composite detection materials were prepared. Then, rutin standard solutions of different concentrations were taken in equal volumes, denoted as V1. The gold nanospheres, metal-phenolic networks, epoxy resin, and polymethyl methacrylate-microneedle composite detection materials were immersed in V1 volumes of standard solutions of different concentrations and incubated at room temperature. After incubation, microneedle conjugates in standard solutions of different concentrations were obtained.

[0010] After drying, the Raman spectrum of the microneedle conjugate was obtained by scanning it with a Raman spectrometer. The characteristic Raman peak was used as the quantitative reference peak, and the Raman peak intensity value I at the shift of the quantitative reference peak was measured. A standard curve was plotted with the concentration of rutin standard solution and its corresponding Raman peak intensity value I.

[0011] As a further aspect of the present invention: in step one, the concentration range of the rutin standard solution is 0-5 mg / mL; the concentration of the rutin standard solution is 0.1-5 mg / mL; the ratio of the amount of the gold nanospheres, metal-phenolic network, epoxy resin and polymethyl methacrylate-microneedle composite detection material to V1 is 1 piece: 10-20 μL.

[0012] As a further aspect of the present invention: in step one, the incubation time at room temperature is 10-20 s; the Raman characteristic peak is 1580-1630 cm⁻¹. -1 .

[0013] As a further aspect of the present invention: In step one, the specific preparation method of the gold nanospheres, metal-phenolic network, epoxy resin, and polymethyl methacrylate-microneedle composite detection material is as follows:

[0014] a. Preparation of metal-phenolic network solution: Polyvinylpyrrolidone and ferric chloride hexahydrate were added to a flask containing deionized water, and then tannic acid was added to the mixture and stirred at room temperature for 20 min; the lower precipitate was collected by centrifugation at 3000 rpm / min for 10 min; and then resuspended in deionized water to obtain metal-phenolic network solution.

[0015] As a further embodiment of the present invention: in step a, the concentration of polyvinylpyrrolidone is 1~4 mg / mL, the concentration of ferric chloride hexahydrate is 0.1~0.3 mg / mL, the concentration of tannic acid is 0.1~1 mg / mL, the volume of deionized water is 10~20 mL, the stirring time at room temperature is 10~20 min, and the centrifugation conditions are 2000~5000 rpm / min for 5~10 min.

[0016] b. Preparation of epoxy resin solution: Dissolve epoxy resin in chloroform to obtain mixture A; then dissolve epoxy curing agent in chloroform to obtain mixture B; sonicate mixtures A and B separately, then mix them, seal and continue sonicating to obtain epoxy resin solution.

[0017] As a further aspect of the present invention: in step b, the ratio of epoxy resin to epoxy curing agent is 1g:(1~5)g; the ratio of chloroform in mixtures A and B is 25mL:(20~50)mL; the ultrasonic conditions are ultrasonication at room temperature for 10~30 min.

[0018] c. Synthesis of epoxy resin and polymethyl methacrylate microneedles: Wash polymethyl methacrylate microneedles with anhydrous ethanol and deionized water, then immerse the polymethyl methacrylate microneedles in an epoxy resin solution, remove them and dry them in an oven to obtain epoxy resin and polymethyl methacrylate microneedles.

[0019] As a further aspect of the present invention: in step c, the washing conditions are as follows: ultrasonic cleaning at room temperature for 10-15 minutes, and the amount of epoxy resin solution used is 100-300 μL; the immersion time in EPR solution is 12-24 h; and the drying conditions in the oven are 40°C for 12-24 h.

[0020] d. Synthesis of gold nanospheres, metal-phenolic network, epoxy resin, and polymethyl methacrylate-microneedle composite detection materials: Epoxy resin and polymethyl methacrylate-microneedles were immersed in a metal-phenolic network solution and washed with deionized water to obtain the metal-phenolic network, epoxy resin, and polymethyl methacrylate-microneedles. 100 mL of 0.01% chloroauric acid was added to a round-bottom flask and boiled, followed by the addition of 1 mL of 1% sodium citrate solution and reaction for 30 min. After cooling to room temperature, a gold seed solution was obtained. Then, the metal-phenolic network, epoxy resin, and polymethyl methacrylate-microneedles were immersed in the gold seed solution to obtain gold seed-modified metal-phenolic network, epoxy resin, and polymethyl methacrylate-microneedles. Next, a gold growth solution was prepared by adding 57 μL of 0.01M silver nitrate, 1.5 mL of 0.2M hexadecyltrimethylammonium bromide, 96 μL of 40mM chloroauric acid, 2.75 mL of deionized water, and 240... μL of ascorbic acid and 0.1M are mixed thoroughly in sequence;

[0021] Then, the gold seed-modified metal-phenolic network, epoxy resin, and polymethyl methacrylate-microneedles were immersed in the prepared gold growth solution. Subsequently, the mixture was washed with anhydrous ethanol and deionized water to obtain a composite detection material consisting of gold nanospheres, metal-phenolic network, epoxy resin, and polymethyl methacrylate-microneedles.

[0022] As a further aspect of the present invention: in step d, the amount of the metal-phenolic network solution used is 100~300 μL; the immersion time in the metal-phenolic network solution is 12~24 h; and the washing condition with deionized water is rinsing with running water for 10~30 s.

[0023] The amounts of gold seed solution and gold growth solution used are 100~300μL: 200~300μL; the immersion time in the gold seed solution is 12~24 h, and the immersion time in the prepared gold growth solution is 10~30 min.

[0024] II. Preparation of the sample solution to be tested:

[0025] Using propolis as raw material, after removing impurities, the propolis was weighed and recorded as w. Ethanol solution was added, and the mixture was extracted by shaking at room temperature to obtain a crude extract. The crude extract was centrifuged and the supernatant was collected. Metal salt was added, and the mixture was washed twice with n-hexane to obtain the test solution. The volume V0 was recorded and the solution was refrigerated for later use.

[0026] The weighed propolis mass w ranges from 10 to 25 g, the ethanol solution concentration is 70%, and the volume ranges from 50 to 100 mL; the centrifugation conditions are 3000 to 5000 r / min for 3 to 10 min; the shaking extraction time is 10 to 20 min; the metal salt is one or a combination of manganese, zinc, or copper ion salts; the ratio of the metal salt to the supernatant is 1 g: 300 to 1000 mL; the hexane washing operation involves shaking 10 to 30 times and then separating and collecting the ethanol layer.

[0027] As a further aspect of the present invention: III. Raman detection of the test liquid:

[0028] The gold nanospheres, metal-phenolic network, epoxy resin, and polymethyl methacrylate-microneedle composite detection material were immersed in the test solution prepared in step two and incubated at room temperature to obtain the microneedle conjugate. After drying, the Raman spectrum was obtained by spectral scanning using a Raman spectrometer. The incubation time at room temperature was 10-20 s. The instrument parameters were as follows: the handheld Raman spectrometer emitted a laser wavelength of 785 nm, the fixed laser power was 50-200 mW, the scanning range was 200-3500 cm⁻¹, the integration time was 3-10 s, and each sample was scanned 10 times and the average value was taken.

[0029] IV. Qualitative Judgment:

[0030] After preprocessing the Raman spectra obtained in step three, when the spectrum is in the range of 900~1000 cm⁻¹ -1 1130~1190cm -1 1230~1300 cm-1 1340~1390 cm -1 1560~1630 cm -1 If a Raman characteristic peak is present, then the test solution contains propolis flavonoids;

[0031] In step four, the preprocessing is a data processing method that uses optimized baselines to improve the spectral data;

[0032] V. Quantitative determination:

[0033] Based on the Raman spectrum of the test solution obtained in step three, the concentrations were measured at 1580–1630 cm⁻¹. -1 The characteristic Raman peak at the position is used as the quantitative reference peak. The Raman peak intensity value at the displacement of the quantitative reference peak is measured. The concentration of total flavonoids in the test solution obtained according to the rutin standard curve obtained in step one is recorded as m. The content of total flavonoids X in the propolis sample is further calculated according to the formula.

[0034]

[0035] X: Total flavonoid content in the propolis sample, μg / kg;

[0036] m: The concentration of total flavonoids in the test solution obtained from the standard curve, in μg / L;

[0037] V0: Volume of liquid to be measured, L;

[0038] w: Mass of propolis sample, kg.

[0039] The beneficial effects of this invention are:

[0040] (1) In the process of preparing the extract, the metal salt is introduced and washed with n-hexane, which can eliminate the interference of overlapping spectra of most phenolic acid substances; in the Raman substrate, the epoxy resin coating can selectively affinity adsorb flavonoids in propolis, which can effectively reduce the interference of terpenoids, aromatic substances and fatty acids in propolis on the Raman signal of flavonoids, and the epoxy resin layer attracts propolis flavonoids to bind with gold nanospheres, promoting the enhancement of Raman signal response;

[0041] (2) The present invention prepares a composite Raman substrate material of gold nanospheres, metal-phenolic network, epoxy resin, and polymethyl methacrylate-microneedles. This material greatly enhances the original Raman spectral signal intensity of propolis flavonoids. The Raman detection method provided by the present invention can obtain the fingerprint spectrum of propolis flavonoids and accurately quantify them. The operation is simple, the detection cost is low, it is portable, and it is suitable for on-site detection. It can provide technical reference for the quality judgment of propolis. Attached Figure Description

[0042] To facilitate understanding by those skilled in the art, the present invention will be further described below with reference to the accompanying drawings.

[0043] Figure 1 Raman spectra of rutin standards.

[0044] Figure 2 This is a standard curve graph of rutin.

[0045] Figure 3 This is an example of the Raman spectrum of total flavonoids in the test solution. Detailed Implementation

[0046] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0047] like Figure 1-3 The image shows a Raman spectroscopy method for determining the total flavonoid content in propolis extract, including experimental samples, main reagents, and instruments.

[0048] Samples: Six different sources of propolis were used as the test subjects;

[0049] Reagents: Rutin standard was purchased from Chengdu Medtronic Technology Co., Ltd., with a purity greater than 98%; all water used in the experiment was deionized water, and all reagents were of analytical grade; polymethyl methacrylate microneedles were purchased from Taizhou Microchip Pharmaceutical Technology Co., Ltd.

[0050] Instrument: Portable Raman spectrometer, model SR-510 Pro, Shanghai Weihai Optical Instrument Co., Ltd.;

[0051] I. Plotting the standard curve of total flavonoids in propolis

[0052] First, prepare rutin standard solutions of different concentrations: accurately weigh 5 mg of rutin standard, dissolve it in 1 mL of ethanol to prepare a standard stock solution, dilute the standard stock solution with ethanol to prepare a series of rutin solutions of different concentrations of 0.10, 0.50, 1.50, 2.50 and 5.00 mg / mL, and store them at 4℃ in the dark.

[0053] The specific preparation process of gold nanospheres, metal-phenolic network, epoxy resin, and polymethyl methacrylate-microneedle signal detection microneedles is as follows:

[0054] a. Preparation of metal-phenolic network solution: 1.5 mg / mL polyvinylpyrrolidone and 0.12 mg / mL ferric chloride hexahydrate were added to a flask containing 10 mL of deionized water; then 0.4 mg / mL tannic acid was added to the mixture and stirred at room temperature for 20 min; the lower precipitate was collected by centrifugation at 3000 rpm / min for 10 min and resuspended in deionized water to obtain the metal-phenolic network solution;

[0055] b. Preparation of epoxy resin solution: Dissolve 1 g of epoxy resin in 25 mL of chloroform to obtain mixture A, then dissolve 1 g of epoxy curing agent in 20 mL of chloroform to obtain mixture B; sonicate mixtures A and B separately for 10 min; then mix the two, seal and continue sonicating for 10 min to obtain epoxy resin solution.

[0056] c. Synthesis of epoxy resin and polymethyl methacrylate microneedles: Wash polymethyl methacrylate microneedles with anhydrous ethanol and deionized water, then immerse polymethyl methacrylate microneedles in 200 μL epoxy resin solution for 24 h, remove them and dry them in an oven at 40℃ for 12~24 h to obtain epoxy resin and polymethyl methacrylate microneedles.

[0057] d. Synthesis of gold nanospheres, metal-phenolic network, epoxy resin, and polymethyl methacrylate-microneedle composite detection materials: Epoxy resin and polymethyl methacrylate-microneedles were immersed in 200 μL of metal-phenolic network solution for 24 h, and washed with deionized water to obtain metal-phenolic network, epoxy resin, and polymethyl methacrylate-microneedles; 100 mL of 0.01% chloroauric acid was added to a round-bottom flask and boiled, followed by the addition of 1 mL of 1% sodium citrate solution and reaction for 30 min. After cooling to room temperature, gold seed solution was obtained; then, metal-phenolic network, epoxy resin, and polymethyl methacrylate-microneedles were immersed in 200 μL of gold seed solution for 12 h to obtain gold seed-modified metal-phenolic network, epoxy resin, and polymethyl methacrylate-microneedles; then, gold growth solution was prepared by adding 57 μL of 0.01M silver nitrate, 1.5 mL of 0.2M hexadecyltrimethylammonium bromide, and 96 μL of 40% chloroauric acid. The gold nanospheres, metal-phenolic network, epoxy resin, and polymethyl methacrylate-microneedles were then thoroughly mixed sequentially with 240 μL of ascorbic acid (0.1 M), 2.75 mL of deionized water, and 240 μL of ascorbic acid (0.1 M). The gold-seed-modified metal-phenolic network, epoxy resin, and polymethyl methacrylate-microneedles were then immersed in 200 μL of the prepared gold growth solution for 30 min. Subsequently, the mixture was rinsed with anhydrous ethanol and deionized water to obtain a composite detection material consisting of gold nanospheres, metal-phenolic network, epoxy resin, and polymethyl methacrylate-microneedles.

[0058] Then, 10 μL of rutin standard solutions of different concentrations were taken. Five gold nanospheres, metal-phenolic network, epoxy resin, and polymethyl methacrylate-microneedle composite detection material were immersed in 10 μL of standard solutions of different concentrations and incubated at room temperature for 10 s. After being removed, microneedle conjugates in standard solutions of different concentrations were obtained.

[0059] After drying, use a Raman spectrometer at 200–3500 cm⁻¹ -1 Raman spectra of the microneedle conjugates were obtained by scanning within a certain range, and the spectra were smoothed and baseline optimized. Figure 1 This is the standard curve Raman spectrum of rutin at an excitation wavelength of 785 nm. The horizontal axis represents the Raman shift, and the vertical axis represents the Raman signal. (The last line appears to be a separate, unrelated statement: "with a wavelength of 1630 cm⁻¹...") -1 The characteristic Raman peak at the specified location is used as the quantitative reference peak. The Raman peak intensity value I at the shift of the quantitative reference peak is measured. A standard curve is plotted using the concentration of rutin standard solution and its corresponding Raman peak intensity value I. Figure 2 As shown, the functional relationship of the standard curve in the linear range of 0.1~5 mg / mL is y=153.81x+31.9, and the correlation coefficient R is . 2 =0.9971, showing good linearity, and can be used for quantitative analysis of flavonoids in propolis; the working conditions of the Raman spectrometer are: laser power: 5 mW; integration time: 5 s; averaging times: 10; smoothing parameter: 2;

[0060] II. Preparation of the sample solution to be tested:

[0061] Using propolis as raw material, after removing impurities, 20 g of propolis was weighed and added to 75 mL of 80% ethanol solution. The crude extract was extracted by shaking at room temperature for 18 min to obtain crude extract. The crude extract was centrifuged at 3000 r / min for 5 min, and the supernatant was collected. Metal salt was added, and the mixture was washed twice with n-hexane. After shaking 10-30 times each time, the ethanol layer was separated and collected to obtain the test solution. The volume V0 was recorded and stored for later use.

[0062] III. Raman Detection of the Test Solution:

[0063] The gold nanospheres, metal-phenolic network, epoxy resin, and polymethyl methacrylate-microneedle composite detection material were immersed in the test solution prepared in step two and incubated at room temperature for 10 s to obtain the microneedle conjugate. After drying, the conjugate was analyzed using a Raman spectrometer at 200–3500 cm⁻¹. -1 Raman spectra were obtained by performing spectral scanning within the range, and the spectra were smoothed and baseline optimized. Figure 3 The image shows the Raman spectrum of the test liquid at an excitation wavelength of 785 nm. The horizontal axis represents the Raman shift, and the vertical axis represents the Raman intensity.

[0064] IV. Qualitative Judgment:

[0065] After preprocessing the Raman spectra obtained in step three, such as smoothing and baseline optimization, when the spectrum is in the range of 900~1000 cm⁻¹ -1 1130~1190 cm -1 1230~1300 cm -1 1340~1390 cm -1 1560~1630 cm -1 If a Raman characteristic peak is present, then the test solution contains propolis flavonoids;

[0066] (5) Quantitative determination:

[0067] Based on the Raman spectrum of the test solution obtained in step three, at 1630 cm⁻¹... -1 The characteristic Raman peak at the position is used as the quantitative reference peak. The Raman peak intensity value at the displacement of the quantitative reference peak is measured. The concentration of total flavonoids in the test solution obtained according to the rutin standard curve obtained in step one is recorded as m. The content of total flavonoids X in the propolis sample is further calculated according to the formula.

[0068]

[0069] X: Total flavonoid content in the propolis sample, mg / g;

[0070] m: The concentration of total flavonoids in the test solution obtained from the standard curve, in mg / mL;

[0071] V0: Volume of the liquid to be tested, mL;

[0072] w: Mass of propolis sample, g;

[0073] VI. Verification Experiment

[0074] The total flavonoid content in six test solutions was determined and calculated using HPLC to verify the accuracy of the method. For HPLC-MS detection, rutin standard was used as the standard sample. Rutin was dissolved in ethanol to prepare a series of standard solutions of different concentrations for detection. A standard curve was plotted with rutin concentration on the x-axis and peak area on the y-axis. 200 μL of each test solution was diluted to 2 mL with ethanol, and 1.5 mL was filtered through a 0.22 μm filter before HPLC detection. The results were analyzed using a retention time of 32-34 seconds. The peak at min was taken as the target peak. Based on the sum of the calculated peak areas and the linear regression equation corresponding to the standard curve, the total flavonoid concentration in the test solution was calculated. Then, the total flavonoid content in the propolis sample was converted according to the formula described in step five. The total flavonoid content determined by Raman detection method and HPLC is shown in Table 1. The relative error range between the HPLC determination value and the Raman spectroscopy method for different propolis samples was 3.61%-7.92%, all less than 8%. The results show that Raman spectroscopy can well obtain the total flavonoid content in propolis samples, and its results are highly similar to those of HPLC.

[0075] Table 1 Comparison of results from HPLC and Raman spectroscopy

[0076]

[0077] VII. Rutin Recovery Rate and Precision Spiking Recovery Results

[0078] Propolis samples No. 1, 3, and 6 were selected. Following step three of the implementation method, different amounts of rutin standard were added during the ultrasonic extraction process to prepare unspecified and rutin-standardized test solutions. Then, following steps three, four, and five of the implementation method, the rutin concentration in the unspecified and spiked test solutions was detected and calculated. The results are shown in Table 2.

[0079] Table 2 Results of Spiked Recovery Experiment

[0080]

[0081] As shown in Table 2, the recovery rate ranged from 91.98% to 106.15%, indicating that the detection method established in this invention is reliable.

[0082] In summary, by utilizing an optimized flavonoid extraction method to remove phenolic acids from the matrix and by employing gold nanospheres, metal-phenolic networks, epoxy resins, and polymethyl methacrylate-microneedles with characteristic affinity for propolis flavonoids, interference from matrix components was achieved. The established surface-enhanced Raman spectroscopy method enabled the quantitative detection of flavonoids in propolis.

[0083] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A Raman spectroscopy method for determining the total flavonoid content in propolis extract, characterized in that, include: Gold nanospheres, metal-phenolic networks, epoxy resins, and polymethyl methacrylate-microneedle composite detection materials for the detection of flavonoids in propolis were prepared, and a rapid detection method for propolis flavonoids based on surface-enhanced Raman spectroscopy was established. The specific steps include: Gold nanospheres, metal-phenolic network, epoxy resin, and polymethyl methacrylate-microneedle composite detection material were immersed in the prepared test solution and incubated at room temperature to obtain the test microneedle conjugate. After drying, Raman spectra were obtained by spectral scanning using a Raman spectrometer. The specific preparation methods of the gold nanospheres, metal-phenolic network, epoxy resin, and polymethyl methacrylate-microneedle composite detection material are as follows: a. Preparation of metal-phenolic network solution: Polyvinylpyrrolidone and ferric chloride hexahydrate were added to a flask containing deionized water, and then tannic acid was added to the mixture and stirred at room temperature for 20 min; the lower precipitate was collected by centrifugation at 3000 rpm / min for 10 min; and then resuspended in deionized water to obtain metal-phenolic network solution. b. Preparation of epoxy resin solution: Dissolve epoxy resin in chloroform to obtain mixture A; then dissolve epoxy curing agent in chloroform to obtain mixture B; sonicate mixtures A and B separately, then mix them, seal and continue sonicating to obtain epoxy resin solution; c. Synthesis of epoxy resin and polymethyl methacrylate microneedles: Wash polymethyl methacrylate microneedles with anhydrous ethanol and deionized water, then immerse the polymethyl methacrylate microneedles in epoxy resin solution, remove them and dry them in an oven to obtain epoxy resin and polymethyl methacrylate microneedles. d. Synthesis of gold nanospheres, metal-phenolic network, epoxy resin, and polymethyl methacrylate-microneedle composite detection materials: Epoxy resin and polymethyl methacrylate-microneedles were immersed in a metal-phenolic network solution and washed with deionized water to obtain the metal-phenolic network, epoxy resin, and polymethyl methacrylate-microneedles. 100 mL of 0.01% chloroauric acid was added to a round-bottom flask and boiled, followed by the addition of 1 mL of 1% sodium citrate solution and reaction for 30 min. After cooling to room temperature, a gold seed solution was obtained. Then, the metal-phenolic network, epoxy resin, and polymethyl methacrylate-microneedles were immersed in the gold seed solution to obtain gold seed-modified metal-phenolic network, epoxy resin, and polymethyl methacrylate-microneedles. Next, a gold growth solution was prepared by adding 57 μL of 0.01 M silver nitrate, 1.5 mL of 0.2 M hexadecyltrimethylammonium bromide, 96 μL of 40 mM chloroauric acid, and 2.75... mL of deionized water and 240 μL of 0.1 M ascorbic acid were mixed thoroughly in sequence; Then, the gold seed-modified metal-phenolic network, epoxy resin, and polymethyl methacrylate-microneedles were immersed in the prepared gold growth solution, and subsequently rinsed with anhydrous ethanol and deionized water to obtain a composite detection material of gold nanospheres, metal-phenolic network, epoxy resin, and polymethyl methacrylate-microneedles.

2. The Raman spectroscopy method for determining the total flavonoid content in propolis extract according to claim 1, characterized in that, 1. Draw the standard curve of total flavonoids in propolis; First, rutin standard solutions of different concentrations, gold nanospheres, metal-phenolic networks, epoxy resin, and polymethyl methacrylate-microneedle composite detection materials were prepared. Then, rutin standard solutions of different concentrations were taken in equal volumes, denoted as V1. The gold nanospheres, metal-phenolic networks, epoxy resin, and polymethyl methacrylate-microneedle composite detection materials were immersed in V1 volumes of standard solutions of different concentrations and incubated at room temperature. After incubation, microneedle conjugates in standard solutions of different concentrations were obtained. After drying, the microneedle conjugate was scanned using a Raman spectrometer to obtain the Raman spectrum. Using the characteristic Raman peak as the quantitative reference peak, the Raman peak intensity value I at the displacement of the quantitative reference peak was measured, and a standard curve was plotted with the concentration of rutin standard solution and its corresponding Raman peak intensity value I. In step one, the concentration range of the rutin standard solution is 0–5 mg / mL; the concentration of the rutin standard solution is 0.1–5 mg / mL; the ratio of the amount of gold nanospheres, metal-phenolic network, epoxy resin and polymethyl methacrylate-microneedle composite detection material to V1 is 1 piece: 10–20 μL; II. Preparation of the sample solution to be tested: Using propolis as raw material, after removing impurities, the propolis was weighed and recorded as w. Ethanol solution was added, and the mixture was extracted by shaking at room temperature to obtain a crude extract. The crude extract was centrifuged and the supernatant was collected. Metal salt was added, and the mixture was washed twice with n-hexane to obtain the test solution. The volume V0 was recorded and the solution was refrigerated for later use. The weighed propolis mass w ranges from 10 to 25 g; the concentration of the ethanol solution is 70%, and the volume ranges from 50 to 100 mL; the centrifugation conditions are 3000 to 5000 r / min for 3 to 10 min; the shaking extraction time is 10 to 20 min; the metal salt is one or a combination of manganese, zinc, or copper ion salts; the ratio of the metal salt to the supernatant is 1 g: 300 to 1000 mL; the hexane washing operation involves shaking 10 to 30 times, followed by separation and collection of the ethanol layer; III. Raman Detection of the Test Solution: In step three, the incubation time at room temperature is 10-20 s; the instrument parameters are as follows: handheld Raman spectrometer with an emitted laser wavelength of 785 nm, a fixed laser power of 50-200 mW, and a scanning range of 200-3500 cm⁻¹. -1 The integration time is 3~10 s, and each sample is scanned 10 times and the average value is taken; IV. Qualitative Judgment: After preprocessing the Raman spectra obtained in step three, when the spectrum is in the range of 900~1000 cm⁻¹ -1 1130~1190 cm -1 1230~1300 cm -1 1340~1390 cm -1 1560~1630 cm -1 If a Raman characteristic peak is present, then the test solution contains propolis flavonoids; In step four, the preprocessing is a data processing method that uses optimized baselines to improve the spectral data; V. Quantitative determination: Based on the Raman spectrum of the test solution obtained in step three, the concentrations were measured at 1580–1630 cm⁻¹. -1 The characteristic Raman peak at the position is used as the quantitative reference peak. The Raman peak intensity value at the displacement of the quantitative reference peak is measured. The concentration of total flavonoids in the test solution obtained according to the rutin standard curve obtained in step one is recorded as m. The content of total flavonoids X in the propolis sample is further calculated according to the formula. ， X: Total flavonoid content in the propolis sample, μg / kg; m: The concentration of total flavonoids in the test solution obtained from the standard curve, in μg / L; V0: Volume of liquid to be measured, L; w: Mass of propolis sample, kg.

3. The Raman spectroscopy method for determining the total flavonoid content in propolis extract according to claim 2, characterized in that, In step one, the incubation time at room temperature is 10-20 s; the Raman characteristic peak is 1580-1630 cm⁻¹. -1 .

4. The Raman spectroscopy method for determining the total flavonoid content in propolis extract according to claim 2, characterized in that, In step a, the concentration of polyvinylpyrrolidone is 1-4 mg / mL, the concentration of ferric chloride hexahydrate is 0.1-0.3 mg / mL, the concentration of tannic acid is 0.1-1 mg / mL, the volume of deionized water is 10-20 mL, the stirring time at room temperature is 10-20 min, and the centrifugation conditions are 2000-5000 rpm / min for 5-10 min.

5. The Raman spectroscopy method for determining the total flavonoid content in propolis extract according to claim 2, characterized in that, In step b, the ratio of epoxy resin to epoxy curing agent is 1g:(1~5)g; the ratio of chloroform in mixtures A and B is 25mL:(20~50)mL; and the ultrasonic conditions are ultrasonication at room temperature for 10~30 min.

6. The Raman spectroscopy method for determining the total flavonoid content in propolis extract according to claim 2, characterized in that, In step c, the washing conditions are as follows: ultrasonic cleaning at room temperature for 10-15 min, and the amount of epoxy resin solution used is 100-300 μL; the immersion time in epoxy resin solution is 12-24 h; and the drying conditions in the oven are 40℃ for 12-24 h.

7. The Raman spectroscopy method for determining the total flavonoid content in propolis extract according to claim 2, characterized in that, In step d, the amount of the metal-phenolic network solution used is 100~300 μL; the immersion time in the metal-phenolic network solution is 12~24 h; and the washing condition with deionized water is rinsing with running water for 10~30 s. The amounts of gold seed solution and gold growth solution used are 100~300μL: 200~300μL; the immersion time in the gold seed solution is 12~24 h, and the immersion time in the prepared gold growth solution is 10~30 min.

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