A quality detection method of wolfberry leaf bagged tea

Abstract

The application discloses a quality detection method of a box tea of Chinese wolfberry leaves, adopts a thin layer chromatography method to qualitatively identify pericarpium citri reticulatae and hawthorn, adopts HPLC-ELSD to detect the content of betaine, adopts HPLC-UV to detect the content of hesperidin and hyperoside, and establishes a quality evaluation standard by three index components and thin layer chromatography identification results. The method established by the application has the advantages of good speciality, high accuracy, good repeatability and the like, and can be used for quality control, product inspection and quality evaluation of the box tea of Chinese wolfberry leaves.

Description

Technical Field

[0001] This invention relates to a method for establishing quality standards for wolfberry leaf tea bags, specifically involving the application of thin-layer chromatography identification and high-performance liquid chromatography content determination techniques to establish a quality testing method for wolfberry leaf tea bags. Background Technology

[0002] Lycium barbarum leaves are from the plant Lycium barbarum (Gynostemma pentaphyllum) of the Solanaceae family. Lycium barbarum The dried leaves of wolfberry (L.) have high medicinal and edible value. They can be used as a traditional Chinese medicine and as a raw material for developing health products such as tea. Modern research shows that wolfberry leaves are rich in flavonoids, phenolic acids, polysaccharides, alkaloids, and various vitamins and trace elements. They have various biological activities such as anti-oxidation, hypoglycemia, and blood lipid regulation, and have broad prospects for development and application in the field of functional foods and health products.

[0003] Currently, wolfberry leaves are mainly processed into tea products, among which tea bags have good market prospects due to their convenience in drinking, storage, and portability. With the increasing demand from consumers for natural and healthy products, the industrial development of wolfberry leaf tea bags is receiving more and more attention.

[0004] However, existing research on wolfberry leaves mainly focuses on chemical composition analysis, pharmacological activity evaluation, and processing technology optimization, with relatively little research on the quality control of wolfberry leaf tea bags. In particular, regarding product quality standards, there is currently a lack of a unified, scientific, and operable quality evaluation system. Comprehensive detection methods have not yet been established for the identification of specific components and the determination of indicative component content, making it difficult to achieve consistent product quality control and hindering standardized production, quality supervision, and industrial promotion.

[0005] Therefore, it is necessary to establish a quality standard applicable to wolfberry leaf tea bags, apply thin-layer chromatography for qualitative identification of the product, and combine it with high-performance liquid chromatography for quantitative determination of index components, so as to provide a reliable technical basis for the production quality control, product inspection and quality evaluation of wolfberry leaf tea bags. Summary of the Invention

[0006] Purpose of the Invention: The purpose of this invention is to overcome the shortcomings of existing technologies for wolfberry leaf tea bags, which lack unified and scientific quality standards and make it difficult to effectively evaluate and control the authenticity, stability, and quality consistency of the product. This invention establishes a quality testing method suitable for wolfberry leaf tea bags. The method employs thin-layer chromatography (TLC) for qualitative identification of tangerine peel and hawthorn in the formula, high-performance liquid chromatography-evaporative light scattering (HPLC-ELSD) for determining betaine content, and high-performance liquid chromatography-ultraviolet (HPLC-UV) for determining hesperidin and hyperoside content. By controlling these three representative components in the tea bags, the quality of wolfberry leaf tea bags can be evaluated, providing technical support for the establishment of quality standards, production quality control, and product quality evaluation for wolfberry leaf tea bags.

[0007] Technical solution: To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing wolfberry leaf tea bags includes the following steps: (1) Take 10-30 parts of wolfberry leaves and grind them to 60-100 mesh, 6-18 parts of hawthorn and grind them to 60-100 mesh, and 4-12 parts of dried tangerine peel and grind them to 100-150 mesh respectively; (2) Dry the obtained powder and then mix it evenly; (3) The obtained mixed powder is put into a food-grade filter bag to make wolfberry leaf tea bags.

[0008] As a preferred option, in step (1), 10 parts of wolfberry leaves are crushed to 60-100 mesh, 6 parts of hawthorn are crushed to 60-100 mesh, and 4 parts of dried tangerine peel are crushed to 100-150 mesh.

[0009] A quality testing method for wolfberry leaf tea bags includes the following steps: (1) Thin-layer chromatography was used to qualitatively identify tangerine peel and hawthorn in wolfberry leaf tea bags; (2) The content of betaine in wolfberry leaf tea bags was determined by high performance liquid chromatography-evaporative light scattering detection method; (3) The contents of hesperidin and hyperoside in wolfberry leaf tea bags were determined by high performance liquid chromatography-ultraviolet detection method; (4) Using the thin-layer chromatography specific identification results of tangerine peel and hawthorn obtained in step (1), the betaine content obtained in step (2), and the hesperidin and hyperoside content obtained in step (3) as quality evaluation indicators, a quality standard for wolfberry leaf tea bags was established and used for product quality evaluation and production quality control.

[0010] The wolfberry leaf tea bag is composed of wolfberry leaves, hawthorn and dried tangerine peel. The mass ratio of each raw material is 1.0g of wolfberry leaves, 0.6g of hawthorn and 0.4g of dried tangerine peel. The preparation method is to grind the wolfberry leaves into a fine powder of 60-100 mesh; take 0.6g of hawthorn and grind it into 60-100 mesh and 0.4g of dried tangerine peel and grind it into 150 mesh, mix them with the above wolfberry leaf powder, and dry them to obtain the tea bag.

[0011] (1) The thin-layer chromatography identification includes the following steps: (1) Using hesperidin as a reference standard, the tangerine peel was identified. A 0.5% sodium hydroxide silica gel G thin-layer plate was used for double development with the upper layer solutions of ethyl acetate-methanol-water (100:17:13) and toluene-ethyl acetate-formic acid-water (20:10:1:1). After color development with aluminum trichloride reagent, the sample was examined under a 365 nm ultraviolet lamp. The chromatogram of the test sample showed fluorescent spots of the same color as those of the hesperidin reference standard and the tangerine peel reference material at the corresponding positions. There was no interference from the negative control.

[0012] (2) Using ursolic acid as a reference standard, hawthorn was identified; silica gel G thin layer plate was used, toluene-ethyl acetate-formic acid (20:4:0.5) was used as the developing solvent, 30% sulfuric acid ethanol solution was sprayed and heated to 80℃ for color development, and examined under 365 nm ultraviolet light. In the chromatogram of the test sample, the same characteristic spots appeared at the corresponding positions as the ursolic acid reference standard and hawthorn reference material. There was no interference from the negative control.

[0013] (2) The HPLC-ELSD determination steps include: (1) Preparation of the test solution Accurately weigh 2g of wolfberry leaf tea bag sample, add ultrapure water for ultrasonic extraction, centrifuge, take the supernatant, filter through a 0.22 μm microporous membrane, and take the filtrate as the test solution. (2) Preparation of reference solution Accurately weigh an appropriate amount of the reference standard, add ultrapure water to prepare a betaine solution with a concentration of 1.00 mg / mL, and dilute different volumes of the above stock solution to prepare a series of reference standard solutions of different concentrations for linearity investigation. (3) Determination of betaine content The test solution prepared in step (1) and the reference solution prepared in step (2) were injected into a high-performance liquid chromatograph to obtain chromatograms. A standard curve was plotted with the logarithm of the concentration of the reference solution as the abscissa and the logarithm of the peak area as the ordinate. The content of betaine in the test sample was calculated based on the standard curve. The HPLC-ELSD determination conditions described above are as follows: Column: Ecosil Carbohydrate ES (250 mm × 4.6 mm, 5 μm); Mobile phase: Deionized water as phase A, acetonitrile as phase B, gradient elution; Flow rate: 1.0 mL / min; Gradient elution conditions: 0–4 min, 15%A → 30%A; 4–12 min, 30%A → 45%A; Drift tube temperature: 50 ℃; Nitrogen flow rate: 2.7 L / min.

[0014] (3) The HPLC-UV determination steps include: (1) Preparation of the test solution Accurately weigh the sample of wolfberry leaf tea bags, add 70% methanol for ultrasonic extraction, centrifuge, filter through a 0.22 μm microporous membrane, and take the filtrate as the test solution. (2) Preparation of reference solution Weigh appropriate amounts of hesperidin and hyperoside, add 70% methanol to prepare a reference solution with a concentration of 1 mg / g, and dilute different volumes of the above stock solution to prepare a series of reference solutions with different concentrations for linearity investigation. (3) Determination of hesperidin and hyperoside content The test solution prepared in step (1) and the reference solution prepared in step (2) were injected into a high performance liquid chromatograph to obtain chromatograms. A standard curve was plotted with the concentration of the reference solution as the abscissa and the peak area as the ordinate. The content of betaine in the test sample was calculated based on the standard curve. The HPLC-UV determination conditions described above are as follows: Chromatographic column: 4.6 mm × 250 mm, 5 μm C18 column; Mobile phase: 0.1% formic acid aqueous solution as phase A, acetonitrile as phase B, gradient elution; Flow rate: 1 mL / min; Gradient elution conditions: 0–7 min, 76% A → 70% A; 7–9 min, 70% A → 70% A; Detection wavelengths: hesperidin is detected at 280 nm; hyperoside is detected at 360 nm.

[0015] As a preferred option, the quality testing method uses betaine, hesperidin and hyperoside as evaluation indicators, respectively. Alternatively, the comprehensive evaluation value of the content of the three indicators can be used as the quality evaluation indicator.

[0016] As a preferred option, the specificity, linearity, detection limit, quantitation limit, precision, repeatability, stability, and recovery rate of the method were investigated during its establishment, and the results all met the requirements of the analytical methodology.

[0017] This invention establishes a specific identification method for tangerine peel and hawthorn in wolfberry leaf tea bags using thin-layer chromatography. Hesperidin is used as a reference for qualitative identification of tangerine peel, and ursolic acid is used as a reference for qualitative identification of hawthorn. Through verification with reference standards, reference medicinal materials, and negative controls, the test samples all showed corresponding characteristic spots, and the negative controls did not interfere, achieving accurate identification of tangerine peel and hawthorn in tea bags. Based on this, betaine, hesperidin, and hyperoside, derived from wolfberry leaves, tangerine peel, and hawthorn, were selected as quantitative evaluation indicators. Betaine reflects the quality characteristics of wolfberry leaves, hesperidin reflects the quality characteristics of tangerine peel, and hyperoside reflects the quality characteristics of hawthorn. These three indicator components together constitute a quantitative quality evaluation system for wolfberry leaf tea bags, achieving quality control combining qualitative identification and quantitative analysis.

[0018] Beneficial effects: The method for quality testing of wolfberry leaf tea bags provided by this invention has the following advantages compared with the prior art: This invention establishes a complete quality control system applicable to wolfberry leaf tea bags, combining qualitative identification with quantitative determination, thereby improving the scientific rigor and reliability of product quality evaluation. The established method, after methodological verification, exhibits good specificity, linearity, precision, repeatability, stability, and recovery rate. The test results are accurate and reliable, suitable for quality control, production inspection, and product standard formulation of wolfberry leaf tea bags. The quality testing method is simple to operate, has good repeatability, and strong applicability, providing technical support for the industrial production, quality standard formulation, and market supervision of wolfberry leaf tea bags, and has significant potential for widespread application. Attached Figure Description

[0019] Figure 1 Thin-layer chromatogram of dried tangerine peel. (1 is negative control, 2 is dried tangerine peel reference material, 3 is wolfberry leaf tea bag sample, 4 is hesperidin reference standard).

[0020] Figure 2 Thin-layer chromatogram of hawthorn. (1 is negative control, 2 is hawthorn reference material, 3 is wolfberry leaf tea bag sample, 4 is ursolic acid reference standard).

[0021] Figure 3 Chromatogram of betaine in wolfberry leaf tea bags under HPLC-ELSD. A is betaine reference standard, B is wolfberry leaf tea bags, and C is hawthorn-dried tangerine peel (negative control).

[0022] Figure 4 Chromatogram of hesperidin in wolfberry leaf tea bags under HPLC-UV. A is hesperidin reference standard, B is wolfberry leaf tea bags, and C is hawthorn-wolfberry leaf (negative control).

[0023] Figure 5Chromatogram of hyperoside in wolfberry leaf tea bags under HPLC-UV. A is hyperoside reference standard, B is wolfberry leaf tea bags, and C is tangerine peel-wolfberry leaf (negative control). Detailed Implementation

[0024] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are only for illustrating the present invention and are not intended to limit the scope of the present invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art will fall within the scope defined by the appended claims.

[0025] Example 1 A method for preparing wolfberry leaf tea bags, characterized by comprising the following steps: (1) Take 1.0g of wolfberry leaves and grind them to 60-100 mesh, 0.6g of hawthorn and grind them to 60-100 mesh, and 0.4g of dried tangerine peel and grind them to 100-150 mesh respectively; (2) Dry the obtained powder and then mix it evenly; (3) The obtained mixed powder is put into a food-grade filter bag to make wolfberry leaf tea bags.

[0026] Example 2 1. Method for identifying the thin layer of dried tangerine peel in wolfberry leaf tea bags (1) Preparation of the test solution Take 2 g of the tea bag prepared in Example 1, add 15 ml of methanol, sonicate for 25 minutes, filter, take 5 ml of the filtrate, concentrate to 1 ml, and use as the test solution.

[0027] (2) Preparation of reference standards and reference drug solutions Dissolve 1 mg of hesperidin reference standard in methanol and dilute to 1 ml to prepare the reference solution; prepare a solution of 0.4 g of dried tangerine peel reference material according to the method for preparing the test solution to prepare the reference material solution.

[0028] (3) Preparation of negative control solution According to the tea bag prescription in Example 1, take 2g of wolfberry leaves and hawthorn (excluding tangerine peel) and prepare a negative control solution according to the test solution preparation method.

[0029] (4) Determination method Take 4 μL each of the test solution, reference herb solution, reference solution, and negative control solution and spot them on the same 0.5% sodium hydroxide silica gel G thin-layer plate. Develop with ethyl acetate-methanol-water (100:17:13) to approximately 3 cm, remove, air dry, and then develop with the upper layer of toluene-ethyl acetate-formic acid-water (20:10:1:1) to approximately 8 cm. Remove, air dry, spray with aluminum trichloride test solution, and examine under a 365 nm UV lamp. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference solution.

[0030] 2. Thin-layer chromatography method for identifying hawthorn in wolfberry leaf tea bags (1) Preparation of the test solution Take 2g of the powder, add 5ml of ethyl acetate, sonicate for 20 minutes, filter, and use the filtrate as the test solution.

[0031] (2) Preparation of reference standards and reference drug solutions Separately prepare a reference solution by dissolving ursolic acid in methanol to a concentration of 1 mg / ml. Prepare a reference solution by dissolving 0.6 g of hawthorn reference material according to the method for preparing the test solution.

[0032] (3) Preparation of negative control solution According to the tea bag prescription in Example 1, take 2g of wolfberry leaves (excluding hawthorn) and 2g of dried tangerine peel, and prepare them according to the test solution preparation method as a negative control solution.

[0033] (4) Determination method Take 4 μl of each of the two solutions mentioned above and spot them separately on the same silica gel G thin-layer plate. Develop the plate using toluene-ethyl acetate-formic acid (20:4:0.5) as the developing solvent. Remove the plate, air dry, spray with sulfuric acid ethanol solution (3→10), and heat at 80℃ until the spots are clearly visible. In the chromatogram of the test sample, the same purplish-red spots appear at the corresponding positions as in the chromatogram of the reference sample; when examined under ultraviolet light (365 nm), the same orange-yellow fluorescent spots appear.

[0034] Example 2 1. Determination of betaine content in wolfberry leaf tea bags (1) Preparation of the test solution Accurately weigh 2 g of tea bag sample (1 g wolfberry leaves, 0.6 g hawthorn, 0.4 g dried tangerine peel), place it in a 50 mL stoppered conical flask, add 25 mL of ultrapure water, sonicate for 40 min, shake well, centrifuge at 13000 rpm for 10 min at low temperature, take the supernatant, filter it through a 0.22 μm microporous membrane, and take the filtrate to obtain the test solution.

[0035] (2) Preparation of reference solution Accurately weigh an appropriate amount of the reference standard, add ultrapure water to prepare a betaine solution with a concentration of 1.00 mg / mL, and dilute different volumes of the above stock solution to prepare a series of reference standard solutions with different concentrations. (3) Negative sample solution According to the tea bag prescription ratio, take the remaining medicinal materials except wolfberry leaves, and make negative samples containing hawthorn and tangerine peel according to the process. Then, prepare the negative sample solution according to the above step (1).

[0036] (4) Content determination method Accurately pipette 10 μL each of the test solution from step (1) and the reference solution of different concentrations from step (2) into the liquid chromatograph to obtain chromatograms. Plot a standard curve with the logarithm of the reference solution concentration as the abscissa and the logarithm of the peak area as the ordinate. Then calculate the content of betaine in the test sample based on the standard curve. The determination results are shown in Table 1.

[0037] The high-performance liquid chromatography (HPLC) determination conditions described above are as follows: Column: Ecosil Carbohydrate ES (250 mm × 4.6 mm, 5 μm); Mobile phase: Deionized water as phase A, acetonitrile as phase B, gradient elution; Flow rate: 1.0 mL / min; Gradient elution conditions: 0–4 min, 15%A → 30%A; 4–12 min, 30%A → 45%A; Drift tube temperature: 50 ℃; Nitrogen flow rate: 2.7 L / min.

[0038] 2. Determination of the content of hesperidin and hyperoside in wolfberry leaf tea bags (1) Preparation of the test solution Accurately weigh 2 g of tea bag sample (1 g wolfberry leaves, 0.6 g hawthorn, 0.4 g dried tangerine peel), place it in a 50 mL stoppered conical flask, accurately add 25 mL of 70% methanol solution, ultrasonically extract for 40 min, shake well, centrifuge at 13000 rpm for 10 min at low temperature, take the supernatant, filter it through a 0.22 μm microporous membrane, and take the filtrate to obtain the test solution.

[0039] (2) Preparation of reference solution Weigh appropriate amounts of hesperidin and hyperoside, add 70% methanol to prepare a reference solution with a concentration of 1 mg / g, and dilute different volumes of the above stock solution to prepare a series of reference solutions with different concentrations for linearity investigation. (3) Negative sample solution According to the tea bag prescription ratio, take the remaining medicinal materials that are missing tangerine peel, and make negative samples containing wolfberry leaves and hawthorn according to the process, and prepare negative sample solutions according to the above step (1); according to the tea bag prescription ratio, take the remaining medicinal materials that are missing hawthorn, and make negative samples containing wolfberry leaves and tangerine peel according to the process, and prepare negative sample solutions according to the above step (1).

[0040] (4) Content determination method Accurately pipette 10 μL each of the test solution from step (1) and the reference solution of different concentrations from step (2), inject them into the liquid chromatograph, obtain the chromatogram, plot the standard curve with the concentration of the reference solution as the abscissa and the peak area as the ordinate, and then calculate the content of hesperidin and hyperoside in the test sample based on the standard curve. The determination results are shown in Table 1.

[0041] The high-performance liquid chromatography (HPLC) determination conditions described above are as follows: Chromatographic column: 4.6 mm × 250 mm, 5 μm C18 column; Mobile phase: 0.1% formic acid aqueous solution as phase A, acetonitrile as phase B, gradient elution; Flow rate: 1 mL / min; Gradient elution conditions: 0–7 min, 76% A → 70% A; 7–9 min, 70% A → 70% A; Detection wavelengths: hesperidin was detected at 280 nm; hyperoside was detected at 360 nm. The results are shown in Table 1.

[0042] Table 1 Results of sample content determination 3. Methodological Validation (1) Methodological validation of the betaine determination method Accurately pipette the prepared reference solution, perform serial dilutions, and inject and determine under the same liquid chromatography conditions. Plot a standard curve with the logarithm of the reference solution concentration (ng / mL) on the x-axis and the logarithm of the peak area on the y-axis. Calculate the linear regression equation and correlation coefficient r. Betaine: y = 1.3394x + 6.4487(r) 2 =0.999); its linear range is 0.03125–1.000 mg / mL, and all standard curves show good linearity within the linear range. At S / N=3, the limit of detection (LOD) of the reference standard is 0.1532 μg / mL, and at S / N=10, the limit of quantitation (LOQ) of the control group is 0.5107 μg / mL.

[0043] The same reference solution was injected six times consecutively under the same liquid phase conditions. The RSD value of the betaine peak area was calculated. The precision RSD was 2.42% (n=6), which is within the standard range, indicating that the instrument precision is good.

[0044] Six tea bag samples were taken, and test solutions were prepared in parallel. The samples were injected and analyzed under the same liquid phase conditions. The RSD value of betaine content was calculated. The repeatability RSD was 3.13% (n=6), which was within the standard range, indicating that the method has good repeatability.

[0045] A tea bag sample was taken and injected at 6 time points for analysis. The stability was assessed by the RSD value of the betaine peak area. The RSD of the stability within 48 hours was 4.21% (n=6), which was within the standard range, indicating that the sample stability of this method is good.

[0046] Six tea bag samples were accurately weighed, and reference standards with the same proportions of components in the samples were added to each sample. The samples were then analyzed under the same liquid chromatography conditions. The recovery rate and RSD were calculated. The recovery rate was 101.2%, and the RSD was 3.18%. Both the recovery rate and RSD were within the standard range, indicating that the method has good recovery.

[0047] (2) Methodological validation of the hesperidin determination method The prepared reference solutions were precisely pipetted, serially diluted, and injected under the same conditions. A standard curve was plotted with the concentration of the reference solution on the x-axis and the peak area on the y-axis. The linear regression equation and correlation coefficient r were calculated: hesperidin: y = 1161.2x - 35955 (r² = 0.9984); its linear range was 0.03125 mg / mL to 1.000 mg / mL. All standard curves showed good linearity within the linear range. At S / N = 3, the amount of each reference standard was used as the limit of detection (LOD), which was 0.1024 μg / mL. At S / N = 10, the amount of the control group was used as the limit of quantitation (LOQ), which was 0.3412 μg / mL.

[0048] The reference solution was injected six times consecutively under the same liquid phase conditions. The RSD value of the peak area of ​​hesperidin was calculated. The precision RSD was 2.13% (n=6), which is less than 5%, indicating that the instrument precision is good.

[0049] Six tea bag samples were taken, and test solutions were prepared in parallel. The samples were injected and analyzed under the same liquid phase conditions. The RSD value of hesperidin content was calculated. The repeatability RSD was 2.13% (n=6), which was within the standard range, indicating that the method has good repeatability.

[0050] A tea bag sample was taken and injected at 6 time points for analysis. The stability was assessed by the RSD value of the hesperidin peak area. The RSD of stability within 48 hours was 4.13% (n=6), which was within the standard range, indicating that the sample stability of this method is good.

[0051] Six portions of tea bags were accurately weighed, and reference standards with the same proportions of components as those in the sample were added to each. The samples were then analyzed under the same liquid chromatography conditions. The recovery rate and RSD were calculated. The recovery rate was 98.2%, and the RSD was 4.28%. Both the recovery rate and RSD were within the standard range, indicating that the method has good recovery.

[0052] (3) Methodological validation of hyperoside determination method The prepared reference solutions were precisely pipetted and serially diluted. They were then injected and analyzed under the same conditions. A standard curve was plotted with the concentration of the reference solution on the x-axis and the peak area on the y-axis. The linear regression equation and correlation coefficient r were calculated: Hyperoside: y = 3E + 0.6x + 5358.5 (r² = 0.9995); its linear range was 5.208 μg / mL to 166.7 μg / mL. All standard curves showed good linearity within the linear range. At S / N = 3, the amount of each reference standard was used as the limit of detection (LOD), which was 0.0512 μg / mL. At S / N = 10, the amount of each control group was used as the limit of quantitation (LOQ), which was 0.1705 μg / mL.

[0053] The reference solution was injected six times consecutively under the same liquid phase conditions. The RSD value of the hyperoside peak area was calculated. The precision RSD was 1.62% (n=6), which is within the standard range, indicating that the instrument precision is good.

[0054] Six tea bag samples were taken, and test solutions were prepared in parallel. The samples were injected and analyzed under the same liquid phase conditions. The RSD values ​​of each component were calculated. The repeatability RSD was 2.53% (n=6), which was within the standard range, indicating that the method has good repeatability.

[0055] A tea bag sample was taken and injected at 6 time points for analysis. The stability was examined by the RSD value of the peak area of ​​each component. The RSD of the stability within 48 hours was 3.21% (n=6), which was within the standard range, indicating that the sample stability of this method is good.

[0056] Six portions of tea bags were accurately weighed, and reference standards with the same proportions of components as those in the sample were added to each. The samples were then analyzed under the same liquid chromatography conditions. The recovery rate and RSD were calculated. The recovery rate was 101.3%, and the RSD was 3.12%. Both the recovery rate and RSD were within the standard range, indicating that the method has good recovery.

[0057] Example 3 1. Establishment of quality evaluation standards for wolfberry leaf tea bags The thin-layer chromatography identification results of tangerine peel and hawthorn obtained in Example 1, the betaine content, hesperidin content, hyperoside content measured in Example 2, and the total content of the three indicator components were used as evaluation indicators to formulate evaluation standards for the quality of wolfberry leaf tea bags.

[0058] Accordingly, based on the test results, the present invention can provide the following reference standards: tangerine peel and hawthorn should meet the requirements of thin-layer chromatography identification; the content of betaine in each bag should not be less than 1.3%, the content of hesperidin should not be less than 1.7%, and the content of hyperoside should not be less than 0.75 mg.

[0059] The above experimental results demonstrate that the quality testing method for wolfberry leaf tea bags provided by this invention establishes a quality evaluation standard by using thin-layer chromatography identification of tangerine peel and hawthorn as qualitative evaluation indicators, and the content of three chemical components—betaine, hesperidin, and hyperoside—as quantitative evaluation indicators. Compared with existing methods for wolfberry leaf tea bags that lack unified quality standards or only use a single component for quality evaluation, this invention can more comprehensively and objectively reflect the raw material composition and intrinsic quality of wolfberry leaf tea bags, achieving a comprehensive evaluation of the product's authenticity, stability, and effectiveness.

[0060] The thin-layer chromatography identification method established in this invention has strong specificity and good repeatability, and can accurately identify tangerine peel and hawthorn in prescriptions. The high-performance liquid chromatography (HPLC) content determination method has good linearity, precision, repeatability, stability and recovery rate, and the detection results are accurate and reliable, enabling accurate determination of three indicator components: betaine, hesperidin and hyperoside. The quality standard established based on the test results of multiple batches of samples is scientific and reasonable, and can be effectively used for raw material selection, production process quality control, finished product quality evaluation and product quality standard formulation for wolfberry leaf tea bags. It is of great significance for improving the consistency of wolfberry leaf tea bag product quality, ensuring product quality and promoting its standardization and industrialization.

[0061] The above description is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, various modifications, substitutions and improvements can be made to the present invention without departing from the spirit and principle of the present invention, and such modifications, substitutions and improvements should all fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing wolfberry leaf tea bags, characterized in that, Includes the following steps: (1) Take 10-30 parts of wolfberry leaves and grind them to 60-100 mesh, 6-18 parts of hawthorn and grind them to 60-100 mesh, and 4-12 parts of dried tangerine peel and grind them to 100-150 mesh respectively; (2) Dry the obtained powder and then mix it evenly; (3) The obtained mixed powder is put into a food-grade filter bag to make wolfberry leaf tea bags.

2. The quality testing method for wolfberry leaf tea bags according to claim 1, characterized in that, Includes the following steps: (1) Thin-layer chromatography was used to qualitatively identify tangerine peel and hawthorn in wolfberry leaf tea bags; (2) The content of betaine in wolfberry leaf tea bags was determined by high performance liquid chromatography-evaporative light scattering detection method; (3) The contents of hesperidin and hyperoside in wolfberry leaf tea bags were determined by high performance liquid chromatography-ultraviolet detection method; (4) The thin-layer chromatography identification results of tangerine peel and hawthorn obtained in step (1), the betaine content obtained in step (2), the hesperidin and hyperoside content obtained in step (3), or the total content of the three indicator components are used as evaluation indicators to establish quality evaluation standards for wolfberry leaf tea bags and to carry out product quality control.

3. The quality testing method for wolfberry leaf tea bags according to claim 2, characterized in that, The thin-layer chromatography determination steps described in step (1) are as follows: (1) Take 2 g of tea bags, add 15 ml of methanol, sonicate for 25 minutes, filter, take 5 ml of filtrate, concentrate to 1 ml, and use as the test solution; Separately prepare a saturated solution of hesperidin reference standard in methanol as the hesperidin reference standard solution. Take 0.4 g of dried tangerine peel reference material and prepare it according to the test solution preparation method to prepare the dried tangerine peel reference material solution; According to the tea bag prescription ratio, take 2g of wolfberry leaves and hawthorn herbs (excluding dried tangerine peel) and prepare them according to the test sample solution preparation method as a negative control solution; Take 2 μl of each of the above solutions and spot them separately on the same silica gel G thin-layer plate prepared with 0.5% sodium hydroxide solution. Use ethyl acetate-methanol-water (100:17:13) as the developing solvent and develop to about 3 cm. Remove and air dry. Then use the upper layer of toluene-ethyl acetate-formic acid-water (20:10:1:1) as the developing solvent and develop to about 8 cm. Remove and air dry. Spray with aluminum trichloride test solution and examine under a UV lamp at 365 nm. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample. (2) Take 2 g of tea bags, add 5 ml of ethyl acetate, sonicate for 20 minutes, filter, and take the filtrate as the test solution; Separately prepare a ursolic acid reference solution by adding methanol to a solution containing 1 mg per 1 ml. Take 0.6 g of hawthorn reference material and prepare it according to the method for preparing test solution to serve as hawthorn reference material solution; According to the tea bag prescription ratio, take 2g of wolfberry leaves (excluding hawthorn) and dried tangerine peel, and prepare them according to the test solution preparation method as a negative control solution; Take 4 μl of each of the above solutions and spot them separately on the same silica gel G thin-layer plate. Develop the plate using toluene-ethyl acetate-formic acid (20:4:0.5) as the developing solvent. Remove the plate, air dry it, spray it with sulfuric acid ethanol solution (3→10), and heat it at 80℃ until the spots are clearly visible. In the chromatogram of the test sample, the same purplish-red spots appear at the corresponding positions as in the chromatogram of the reference sample. When examined under a UV lamp at 365 nm, the same orange-yellow fluorescent spots appear.

4. The quality testing method for wolfberry leaf tea bags according to claim 2, characterized in that, The determination steps of the high performance liquid chromatography-evaporative light scattering detection method described in step (2) are as follows: (1) Preparation of the test solution Accurately weigh 2 g of tea bag sample, place it in a 50 mL stoppered conical flask, add 25 mL of ultrapure water, sonicate for 40 min, shake well, centrifuge at 13000 rpm for 10 min, take the supernatant, filter it through a 0.22 μm microporous membrane, and take the filtrate to obtain the test solution. (2) Preparation of reference solution Accurately weigh an appropriate amount of the reference standard and add it to ultrapure water to prepare a betaine reference standard solution with a concentration of 1.00 mg / mL. Dilute different volumes of the betaine reference standard solution to prepare a series of betaine reference standard solutions with different concentrations. (3) Negative sample solution According to the tea bag prescription ratio, take 2g of hawthorn and tangerine peel (excluding wolfberry leaves) and prepare a negative sample solution containing hawthorn and tangerine peel according to the test sample solution preparation method in step (1). (4) Content determination method Accurately pipette 10 μL each of the test solution from step (1) and the reference solution of different concentrations from step (2), inject them into the liquid chromatograph, obtain the chromatogram, plot the standard curve with the logarithm of the reference solution concentration as the abscissa and the logarithm of the peak area as the ordinate, and then calculate the content of betaine in the test sample based on the standard curve.

5. The quality testing method for wolfberry leaf tea bags according to claim 4, characterized in that, The high-performance liquid chromatography (HPLC) determination conditions described above are as follows: Chromatographic column: Ecosil Carbohydrate ES, 250 mm × 4.6 mm, 5 μm; Mobile phase: Deionized water as phase A, acetonitrile as phase B, gradient elution: 0–4 min, 15% A → 30% A; 4–12 min, 30% A → 45% A; Flow rate: 1.0 mL / min; Drift tube temperature: 50℃; Nitrogen flow rate: 2.7 L / min.

6. The method for quality testing of wolfberry leaf tea bags according to claim 2, characterized in that, The high performance liquid chromatography-ultraviolet detection method determination steps described in step (3) are as follows: (1) Preparation of the test solution Accurately weigh 2 g of tea bag sample, place it in a 50 mL stoppered conical flask, accurately add 25 mL of 70% methanol solution, ultrasonically extract for 40 min, shake well, centrifuge at 13000 rpm for 10 min, take the supernatant, filter it through a 0.22 μm microporous membrane, and take the filtrate to obtain the test solution. (2) Preparation of reference solution Weigh appropriate amounts of hesperidin and hyperoside, respectively, and add 70% methanol to prepare reference solutions of hesperidin and hyperoside with a concentration of 1 mg / g. Take different volumes of the above reference solutions of hesperidin and hyperoside and dilute them to prepare a series of reference solutions of different concentrations for linearity investigation. (3) Negative sample solution According to the tea bag prescription ratio, take 2g of wolfberry leaves and hawthorn (lacking tangerine peel) and prepare a negative sample solution containing wolfberry leaves and hawthorn according to the test sample solution preparation method in step (1); According to the tea bag prescription ratio, take 2g of dried tangerine peel and wolfberry leaves (lacking hawthorn) and prepare a negative sample solution containing wolfberry leaves and dried tangerine peel according to the test sample solution preparation method in step (1); (4) Content determination method Accurately pipette 10 μL each of the test solution from step (1) and the reference solution of different concentrations from step (2), inject them into the liquid chromatograph, obtain the chromatogram, plot the standard curve with the concentration of the reference solution as the abscissa and the peak area as the ordinate, and then calculate the content of hesperidin and hyperoside in the test sample based on the standard curve.

7. The method for quality testing of wolfberry leaf tea bags according to claim 6, characterized in that, The high-performance liquid chromatography (HPLC) determination conditions described above are as follows: Column: 4.6 mm × 250 mm, 5 μm C 18 column; Mobile phase: 0.1% formic acid aqueous solution as phase A, acetonitrile as phase B, gradient elution: 0-7 min, 76% A → 70% A; 7-9 min, 70% A → 70% A; Flow rate: 1 mL / min; Detection wavelengths: hesperidin is detected at 280 nm; hyperoside is detected at 360 nm.

8. The method for quality testing of wolfberry leaf tea bags according to claim 2, characterized in that, The established quality evaluation standards are as follows: both dried tangerine peel and hawthorn should meet the requirements of thin-layer chromatography identification; the betaine content should not be less than 1.3%, the hesperidin content should not be less than 1.7%, and the hyperoside content should not be less than 0.75 mg.

9. The method for quality testing of wolfberry leaf tea bags according to claim 2, characterized in that, The quality testing method described herein is applicable to the evaluation of raw material quality, quality control of the production process, quality inspection of finished products, and establishment of product quality standards for wolfberry leaf tea bags.

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