Broussonetia papyrifera leaf quality evaluation method

Abstract

The invention relates to a broussonetia papyrifera leaf quality evaluation method, equipment and a medium. The method comprises the following steps: firstly, obtaining broussonetia papyrifera leaves from different producing areas, performing ultrasonic extraction on dried, crushed and sieved broussonetia papyrifera leaf powder with methanol, and preparing a test solution and a reference solution; secondly, carrying out sample injection detection on the solution by adopting a high performance liquid chromatography method, introducing a map into a similarity evaluation system, and establishing a characteristic map by taking the broussonetia papyrifera leaf map as a reference; based on a UPLC-QE-HRMS / MS technology, carrying out primary and secondary mass spectrometry analysis on common peaks in the characteristic chromatogram, and comparing and analyzing chemical components of the characteristic peaks in combination with a database; finally, the analyzed characteristic flavonoids are used as internal standard substances, the relative content of characteristic peak chemical components is quantitatively analyzed, and a quality evaluation result is obtained by determining the lower limit of the content. By adopting the method, comprehensive characterization of the chemical components of the paper mulberry leaves can be realized, the limitation of single component detection in the prior art is effectively solved, and a systematic scheme is provided for quality consistency evaluation of the paper mulberry leaves from different producing areas.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese medicine quality control, and particularly relates to a quality evaluation method of Broussonetia papyrifera leaves. BACKGROUND

[0002] As a traditional Chinese medicinal material, Broussonetia papyrifera leaves have been widely used in many fields. At present, the patent CN112526841A realizes preliminary control of the quality of Broussonetia papyrifera leaves to a certain extent by means of traits, microscopic, thin layer chromatography identification and content determination. However, the prior art still has many deficiencies. On the one hand, most of them only rely on single component content determination, such as determination of vitexin content, which cannot comprehensively reflect the quality. On the other hand, there is a lack of systematic analysis and comprehensive evaluation of the overall chemical components of Broussonetia papyrifera leaves under different producing areas and growth environments, which makes it difficult to accurately judge the quality difference and consistency of Broussonetia papyrifera leaves. In addition, the existing multi-component quantitative analysis needs to use multiple reference substances (such as chlorogenic acid and Hibiscus syriacus glycoside), which has the problems of high cost, complicated operation and error accumulation. At the same time, although the existing characteristic spectrum and mass spectrum analysis has been applied, the specific quality evaluation system for Broussonetia papyrifera leaves has not been established, especially in the aspects of multi-component synergistic effect quantification and quality consistency evaluation of Broussonetia papyrifera leaves from different producing areas, which cannot meet the increasing demand for high standards and strict requirements for the quality control of Broussonetia papyrifera leaves. SUMMARY

[0003] Therefore, it is necessary to provide a quality evaluation method of Broussonetia papyrifera leaves which can realize comprehensive characterization of the chemical components of Broussonetia papyrifera leaves and provide a systematic solution for quality consistency evaluation of Broussonetia papyrifera leaves from different producing areas.

[0004] In a first aspect, the application provides a quality evaluation method of Broussonetia papyrifera leaves, comprising:

[0005] Different producing areas of Broussonetia papyrifera leaves are obtained, and methanol is added to the dried, crushed and sieved Broussonetia papyrifera leaf powder for ultrasonic extraction to prepare a test sample solution and a reference solution.

[0006] The test sample solution and the reference solution are injected and detected by high performance liquid chromatography, the spectrum is imported into a similarity evaluation system, and a characteristic spectrum of Broussonetia papyrifera leaves is established by taking the control Broussonetia papyrifera leaf spectrum as a reference.

[0007] The common peaks in the characteristic spectrum are analyzed by high resolution mass spectrometry based on ultra-high performance liquid chromatography-quadrupole-electric field orbitrap technology, and the chemical components of the characteristic peaks are analyzed by database comparison.

[0008] The relative content of the chemical components of the characteristic peaks is quantitatively analyzed by taking the characteristic flavonoids in the analyzed chemical components as internal standard substances, and the quality evaluation result of Broussonetia papyrifera leaves is determined by determining the lower limit of content.

[0009] In one embodiment, high-performance liquid chromatography (HPLC) is used to detect the test solution and the reference solution. The chromatograms are then imported into a similarity evaluation system to establish a characteristic chromatogram of mulberry leaves using the control mulberry leaf chromatogram as a reference. This includes:

[0010] The test solution and the reference solution were subjected to high performance liquid chromatography (HPLC) with gradient elution using an aqueous solution of polar modifier and an organic solvent as the mobile phase. The samples were injected and detected under the set flow rate, column temperature, detection wavelength and injection volume conditions to obtain HPLC chromatograms.

[0011] The obtained high-performance liquid chromatograms were imported into a similarity evaluation system. Using the chromatogram of the control mulberry leaf as a reference chromatogram, multi-point correction and Mark peak matching were performed on the high-performance liquid chromatograms of all samples, and common peaks were identified to obtain common pattern chromatograms.

[0012] By comparing the common pattern diagram with the chromatogram of the control mulberry leaf, a characteristic spectrum of mulberry leaf was established.

[0013] In one embodiment, high-resolution mass spectrometry (HPLC-quadrupole-electrostatic field orbital trap) is used to perform primary and secondary mass spectrometry analysis on common peaks in the characteristic spectra. The chemical composition of the characteristic peaks is then analyzed by comparing them against a database, including:

[0014] The sample solution with characteristic chromatograms was injected using an ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometer, and gradient elution was performed using a reversed-phase column and a polar modifier-organic solvent as the mobile phase to obtain the eluent.

[0015] The effluent is introduced into an electrospray ionization source, and positive and negative ion full-scan first-order mass spectrometry analysis is performed under the set mass spectrometry conditions to obtain the precise mass numbers of quasi-molecular ions and adduct ions, thus obtaining the first-order mass spectrometry results.

[0016] Based on the results of the first-level mass spectrometry, the ions corresponding to the characteristic peaks are analyzed by the second-level mass spectrometry. Fragment ion information is obtained in high-resolution mode, resulting in two-level mass spectrometry data.

[0017] The two-stage mass spectrometry data were imported into the analysis software, and the chemical composition of the characteristic peaks was analyzed by combining mass spectrometry rules, relevant literature, and online database comparison.

[0018] In one embodiment, the chromatographic conditions for ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry include:

[0019] The chromatographic column was a CORTECS T3.

[0020] The mobile phase consisted of 0.1% formic acid solution-acetonitrile, with gradient elution, a flow rate of 0.2 mL / min, a column temperature of 30 °C, and an injection volume of 2 μL.

[0021] The mass spectrometry conditions set included an electrospray ionization source, ESI+ and ESI- full scan modes, ion source temperature of 320℃, capillary voltage of 3.5KV, sheath gas flow rate of 30L / hr, sheath gas pressure of 45arb, auxiliary gas flow rate of 8L / hr, auxiliary gas pressure of 15arb, and auxiliary gas temperature of 320℃.

[0022] The high-resolution mode includes a scanning range of 150-1000 m / z, a first-order mass spectrometry resolution of R = 70000, and a second-order mass spectrometry resolution of R = 17500.

[0023] In one embodiment, characteristic flavonoids from the analyzed chemical components are used as internal standards to quantitatively analyze the relative content of characteristic peak chemical components, determine the lower limit of content, and obtain the quality evaluation results of paper mulberry leaves, including:

[0024] The characteristic flavonoid internal standard was accurately weighed, dissolved in an organic solvent of a specific concentration to prepare a series of gradient standard solutions, and a standard curve was established after high performance liquid chromatography (HPLC) injection detection.

[0025] The characteristic flavonoid internal standard in the test sample solution was quantitatively analyzed, and its absolute content was calculated according to the standard curve. The chromatographic peak area of ​​each characteristic peak in the characteristic spectrum was determined simultaneously.

[0026] The relative content of chemical components of each characteristic peak was calculated based on the internal standard quantification method, and the relative content data was obtained by processing the data using the internal standard method formula.

[0027] Statistical analysis was performed on the relative content data of characteristic peaks of multiple batches of tested mulberry leaves, and the lower limit of the relative content of chemical components of each characteristic peak was determined using the mean-standard deviation method.

[0028] The lower limit of the relative content of the tested paper mulberry leaves is compared with the preset lower limit. When the measured value is not lower than the preset lower limit, it is judged as qualified and the quality evaluation result of paper mulberry leaves is obtained.

[0029] In one embodiment, the internal standard method formula includes:

[0030] Relative content of characteristic peak = peak area of ​​characteristic peak × (content of internal standard / peak area of ​​internal standard).

[0031] In one embodiment, the dried, pulverized, and sieved paper mulberry leaf powder is subjected to ultrasonic extraction with methanol to prepare a test solution and a reference solution, including:

[0032] After drying the leaves of paper mulberry trees from different origins and controlling the moisture content, the leaves were pulverized and sieved to obtain uniform paper mulberry leaf powder.

[0033] After accurately weighing the powdered paper mulberry leaves, place them in a stoppered conical flask, add a methanol solution of a specific concentration, and weigh and record the initial weight.

[0034] The mixture in the container was subjected to ultrasonic extraction. After cooling to room temperature, the weight loss was replenished to ensure that the total amount of solution was equal to the initial weight, thus obtaining a mixed extract.

[0035] The mixed extract was filtered under reduced pressure using a filtration device, and the filtrate was collected to obtain the test solution. A reference solution was prepared using paper mulberry leaves as a control.

[0036] In one embodiment, the method further includes:

[0037] Single-factor experiments were conducted on methanol of different concentrations. Methanol solution was added at a material-to-liquid ratio of 1:100, and ultrasonic extraction was performed for 30 minutes. The total yield of 9 components was measured, and 70% methanol was determined to be the optimal extraction solvent.

[0038] A single-factor study was conducted on ultrasonic extraction time. Extraction was performed using 70% methanol at a material-to-liquid ratio of 1:100. The total yield was measured, and 30 minutes was determined to be the optimal extraction time.

[0039] A single-factor study was conducted on the material-liquid ratio. Ultrasonic extraction with 70% methanol for 30 minutes was performed, and the total yield was measured. The optimal material-liquid ratio was determined to be 1:100.

[0040] Single-factor investigations were conducted on extraction methods and extraction solvents. By comparing the total yield and peak shape, ultrasonic extraction with 70% methanol was determined to be the optimal solution.

[0041] Based on the results of the single-factor investigation, the key parameters for the preparation of the test solution were optimized.

[0042] Secondly, this application also provides a computer device, including a memory and a processor, wherein the memory stores a computer program, and the processor executes the computer program to implement the method described above.

[0043] Thirdly, this application also provides a computer-readable storage medium having a computer program stored thereon, which, when executed by a processor, implements the aforementioned method.

[0044] The aforementioned method, computer equipment, and storage medium for evaluating the quality of Broussonetia papyrifera leaves first obtain Broussonetia papyrifera leaves from different origins. The dried, pulverized, and sieved leaf powder is then extracted with methanol using ultrasound to prepare a test solution and a reference solution. Next, high-performance liquid chromatography (HPLC) is used to analyze the solutions, and the resulting chromatograms are imported into a similarity evaluation system. A characteristic chromatogram is established using the reference Broussonetia papyrifera leaf chromatogram as a reference. Then, high-resolution mass spectrometry (HPLC-quadrupole-electrostatic field orbital trap) is used to perform primary and secondary mass spectrometry analysis on the common peaks in the characteristic chromatograms. The chemical composition of the characteristic peaks is analyzed by comparing with a database. Finally, the characteristic flavonoids obtained from the analysis are used as internal standards to quantitatively analyze the relative content of the chemical components in the characteristic peaks. The quality evaluation result is obtained by determining the lower limit of content. By collecting and systematically extracting mulberry leaves from multiple production areas, and combining the establishment of characteristic spectra by high-performance liquid chromatography and the analysis of components by high-resolution mass spectrometry, a comprehensive characterization of the chemical components of mulberry leaves was achieved. Using characteristic flavonoids as internal standards, a scientific lower limit standard for content evaluation was constructed through quantitative analysis, which effectively solves the limitations of single-component detection in existing technologies. This provides a systematic solution for the quality consistency evaluation of mulberry leaves from different production areas, and improves the scientificity and reliability of traditional Chinese medicine quality control. Attached Figure Description

[0045] To more clearly illustrate the technical solutions in the embodiments or related technologies of this application, the accompanying drawings used in the description of the embodiments or related technologies will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0046] Figure 1 A flowchart of a method for evaluating the quality of paper mulberry leaves provided in an embodiment of the present invention;

[0047] Figure 2 A schematic diagram of the high-performance liquid chromatography characteristic spectra of mulberry leaves from different origins provided in an embodiment of the present invention;

[0048] Figure 3 This application provides a schematic diagram of positive (left) and negative (right) ion flow maps (TIC) measured by UPLC-QE-HRMS / MS technology for the purposes of this invention.

[0049] Figure 4 A table of UPLC-HR-MS identification results of characteristic peaks of mulberry leaves provided in embodiments of the present invention;

[0050] Figure 5 A schematic diagram of the concentration-peak area standard curve of the internal standard substance provided in the embodiments of the present invention;

[0051] Figure 6A table of relative quantitative results of characteristic peaks based on internal standard substances provided in embodiments of the present invention;

[0052] Figure 7 This is a table showing the effect of different concentrations of methanol on the extraction of mulberry leaf components, provided in this embodiment of the invention.

[0053] Figure 8 This table shows the effect of different extraction times on the content of components in mulberry leaves, as provided in this embodiment of the invention.

[0054] Figure 9 This table shows the effect of different material-liquid ratios on the extraction of mulberry leaf components, as provided in this embodiment of the invention.

[0055] Figure 10 This table shows the effects of different extraction methods and solvents on the extraction of component content from Broussonetia papyrifera leaves, as provided in the embodiments of the present invention.

[0056] Figure 11 This is a schematic diagram showing the quantitative comparison results of nine chemical components in Broussonetia papyrifera leaves using the peak area normalization method, external standard point method, standard curve method, and internal standard quantitative method provided in the embodiments of the present invention. Detailed Implementation

[0057] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0058] In one embodiment, such as Figure 1 As shown, this application provides a method for evaluating the quality of paper mulberry leaves, which may include the following steps:

[0059] Step S101: Obtain paper mulberry leaves from different origins, add methanol to the dried, pulverized and sieved paper mulberry leaf powder for ultrasonic extraction, and prepare test solution and reference solution.

[0060] Specifically, paper mulberry leaves from different origins were obtained, dried, pulverized, and sieved to obtain a uniform powder. The powder was then subjected to ultrasonic extraction with methanol. The powder was accurately weighed and placed in a stoppered container, and methanol of a specific concentration (e.g., 70% methanol) was added. After recording the initial weight, ultrasonic extraction was performed (power, time, and other parameters needed optimization). After cooling, the weight loss was compensated, and the mixture was filtered to obtain the test solution. A reference solution was prepared using paper mulberry leaves as a control.

[0061] Step S102: High performance liquid chromatography is used to inject and detect the test solution and the reference solution. The chromatograms are then imported into a similarity evaluation system, and a characteristic chromatogram of mulberry leaves is established with the reference chromatogram of mulberry leaves as a reference.

[0062] Furthermore, high-performance liquid chromatography (HPLC) was used to detect the test solution and the reference solution. Chromatographic conditions were set (e.g., C18 column, gradient elution of 0.1% formic acid solution-acetonitrile mobile phase, specific flow rate and column temperature, etc.), and chromatograms were obtained after injection. The chromatograms were imported into a similarity evaluation system, using the reference mulberry leaf chromatogram as a reference. Common peaks were identified through multi-point correction and Mark peak matching, thereby establishing a characteristic chromatogram of mulberry leaves and achieving fingerprint characterization of the overall chemical composition of mulberry leaves.

[0063] Step S103: Based on ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry, first-level and second-level mass spectrometry analysis is performed on the common peaks in the characteristic spectrum, and the chemical composition of the characteristic peaks is analyzed by comparison with the database.

[0064] The common peaks corresponding to characteristic spectra in the sample solution were analyzed using ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry (UPLC-QE-HRMS / MS). First, the sample components were separated by ultra-high performance liquid chromatography, and then electrospray ionization was used for primary mass spectrometry (full scan mode) and secondary mass spectrometry (high-resolution mode) analysis to obtain the precise mass numbers of quasi-molecular ions and fragment ions. By combining mass spectrometry fragmentation patterns, literature data, and comparisons with online databases (such as ChemSpider), the chemical composition of the characteristic peaks was analyzed, clarifying their material basis.

[0065] Step S104: Using the characteristic flavonoids in the chemical components obtained from the analysis as internal standards, the relative content of the characteristic peak chemical components is quantitatively analyzed, and the lower limit of the content is determined to obtain the quality evaluation results of the paper mulberry leaves.

[0066] Specifically, using characteristic flavonoid components (such as apigenin-7-O-glucuronide) obtained from analysis as internal standards, a series of concentration standard solutions were prepared and a standard curve was established. Simultaneously, the peak areas of characteristic peaks were measured while quantitatively analyzing the internal standards in the test sample solutions. The relative content of each characteristic peak chemical component was calculated based on the internal standard method formula. The lower limit of content was determined through statistical analysis of multiple batches of mulberry leaf data (such as the "mean-standard deviation" method). The measured values ​​of the tested mulberry leaves were compared with the lower limit to determine whether the quality was up to standard, thus forming a systematic quality evaluation result.

[0067] The above-mentioned method for evaluating the quality of Broussonetia papyrifera leaves first involves obtaining Broussonetia papyrifera leaves from different origins, then extracting the dried, pulverized, and sieved leaf powder with methanol using ultrasonic extraction to prepare a test solution and a reference solution. Next, high-performance liquid chromatography (HPLC) is used to analyze the solutions, and the chromatograms are imported into a similarity evaluation system. A characteristic chromatogram is established using the reference Broussonetia papyrifera leaf chromatogram as a reference. Then, based on UPLC-QE-HRMS / MS technology, primary and secondary mass spectrometry analyses are performed on the common peaks in the characteristic chromatograms, and the chemical components of the characteristic peaks are analyzed by comparison with a database. Finally, the characteristic flavonoids obtained from the analysis are used as internal standards to quantitatively analyze the relative content of the chemical components of the characteristic peaks, and the quality evaluation result is obtained by determining the lower limit of content. By collecting and systematically extracting mulberry leaves from multiple production areas, and combining the establishment of characteristic spectra by high-performance liquid chromatography and the analysis of components by high-resolution mass spectrometry, a comprehensive characterization of the chemical components of mulberry leaves was achieved. Using characteristic flavonoids as internal standards, a scientific lower limit standard for content evaluation was constructed through quantitative analysis, which effectively solves the limitations of single-component detection in existing technologies. This provides a systematic solution for the quality consistency evaluation of mulberry leaves from different production areas, and improves the scientificity and reliability of traditional Chinese medicine quality control.

[0068] In one embodiment, high-performance liquid chromatography (HPLC) is used to analyze the test solution and the reference solution. The resulting chromatograms are then imported into a similarity evaluation system. A characteristic chromatogram of mulberry leaves is established using the control mulberry leaf chromatogram as a reference. This process may include the following steps:

[0069] Step S201: The test solution and the reference solution are subjected to high performance liquid chromatography (HPLC) with gradient elution using an aqueous solution of polar modifier and an organic solvent as the mobile phase. The samples are injected and detected under the set flow rate, column temperature, detection wavelength and injection volume conditions to obtain HPLC chromatograms.

[0070] Preferably, the test solution and reference solution are analyzed by high-performance liquid chromatography (HPLC). The mobile phase consists of an aqueous solution of a polar modifier (e.g., 0.1% formic acid solution) and an organic solvent (e.g., acetonitrile). Elution is performed according to a preset gradient program (e.g., the acetonitrile concentration linearly increases from 10% to 15% within 0–12 minutes). Under set experimental parameters (including a flow rate of 0.5 mL / min, a column temperature of 30°C, a detection wavelength of 339 nm, and an injection volume of 10 μL), the components in the sample are separated using a chromatographic column (e.g., a CORTECST3 column). After detector response, a high-performance liquid chromatogram is generated. This chromatogram, through the distribution of retention time and peak area, visually reflects the separation status and relative content characteristics of each chemical component in the paper mulberry leaves.

[0071] Step S202: Import the obtained high-performance liquid chromatograms into the similarity evaluation system, and use the reference chromatogram of the mulberry leaf as a reference chromatogram to perform multi-point correction and Mark peak matching on the high-performance liquid chromatograms of all samples, and identify the common peaks to obtain the common pattern chromatogram.

[0072] Schematic illustration: The acquired high-performance liquid chromatograms were imported into a similarity evaluation system (such as the 2012 version of the Chinese Pharmacopoeia Commission's Chromatographic Fingerprint Similarity Evaluation System for Traditional Chinese Medicine). Using the chromatogram of the control *Broussonetia papyrifera* leaf as a reference, the median method was employed to perform multi-point correction on the chromatograms, eliminating retention time shifts caused by different instruments or experimental conditions. The Mark peak matching function was used to automatically identify and match chromatographic peaks. Based on retention time and peak shape characteristics, the characteristic peaks shared by the test sample and the control *Broussonetia papyrifera* leaf were identified, generating a common pattern diagram containing parameters such as the relative retention time and peak area ratio of the common peaks.

[0073] Step S203: Compare the common pattern diagram with the chromatogram of the control mulberry leaf to establish a characteristic spectrum of mulberry leaf.

[0074] Specifically, the test solution and reference solution are detected using high-performance liquid chromatography (HPLC). Gradient elution is performed using a mobile phase of an aqueous solution of a polar modifier (e.g., 0.1% formic acid solution) and an organic solvent (e.g., acetonitrile). Injection is carried out under set conditions: flow rate (e.g., 0.5 mL / min), column temperature (e.g., 30℃), detection wavelength (e.g., 339 nm), and injection volume (e.g., 10 μL). The obtained HPLC chromatograms are then imported into a similarity evaluation system. Using the chromatogram of the control *Broussonetia papyrifera* leaf as a reference chromatogram, multi-point correction and Mark peak matching are performed on all sample HPLC chromatograms. After identifying common peaks, a common pattern chromatogram is obtained. The common pattern chromatogram is then compared with the chromatogram of the control *Broussonetia papyrifera* leaf to establish a characteristic chromatogram of *Broussonetia papyrifera* leaves.

[0075] This embodiment utilizes high-performance liquid chromatography (HPLC) to separate and detect the chemical components in Broussonetia papyrifera (paper mulberry) leaves. A similarity evaluation system is used to perform chromatographic correction and peak matching based on a reference chromatogram of paper mulberry leaves, accurately identifying common characteristic peaks in the leaves. The established characteristic chromatogram comprehensively reflects the chemical composition and relative proportions of paper mulberry leaves, providing a visualized scientific basis for evaluating the quality consistency of paper mulberry leaves from different origins and batches. This effectively solves the problem that single-component detection cannot comprehensively characterize the quality of paper mulberry leaves, improving the systematicness and reliability of traditional Chinese medicine quality control.

[0076] In one embodiment, high-resolution mass spectrometry (HPLC-quadrupole-electrostatic field orbital trap) is used to perform primary and secondary mass spectrometry analysis on common peaks in characteristic spectra, and the chemical composition of characteristic peaks is analyzed by comparing with a database. This may include the following steps:

[0077] Step S301: The sample solution with characteristic chromatograms is injected using an ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometer, and gradient elution separation is performed using a reversed-phase column and a polar modifier-organic solvent as the mobile phase to obtain the eluent.

[0078] Furthermore, the sample solutions with characteristic chromatograms were injected using a UPLC-QE-HRMS / MS system. Gradient elution separation was performed using a reversed-phase column (e.g., a CORTECS T3 column, 2.1 mm × 100 mm, 1.6 μm) and a polar modifier-organic solvent (e.g., 0.1% formic acid aqueous solution-acetonitrile) as the mobile phase. The specific gradient program was: 0–2 min, 5% acetonitrile; 2–15 min, 5%–25% acetonitrile; 15–20 min, 25%–40% acetonitrile (flow rate 0.2 mL / min, column temperature 30 °C, injection volume 2 μL). After separation by the chromatographic column, the eluents were formed in order of retention time, achieving preliminary separation of different chemical components.

[0079] In step S302, the effluent is introduced into the electrospray ion source, and positive and negative ion full scan first-level mass spectrometry analysis is performed under the set mass spectrometry conditions to obtain the precise mass numbers of quasi-molecular ions and adduct ions, and obtain the first-level mass spectrometry results.

[0080] Step S303: Based on the first-level mass spectrometry results, perform second-level mass spectrometry analysis on the ions corresponding to the characteristic peaks, acquire fragment ion information in high-resolution mode, and obtain two-level mass spectrometry data.

[0081] Step S304: Import the two-stage mass spectrometry data into the analysis software, and analyze the chemical composition of the characteristic peaks by combining mass spectrometry rules, relevant literature and online database comparison.

[0082] Preferably, the two-stage mass spectrometry data are imported into analysis software (such as Compound Discoverer 3.2 and Xcalibur 2.1) and compared with the fragmentation patterns of mass spectrometry, relevant literature, and online databases (such as ChemSpider and PubChem). First, the molecular chemical formula is deduced based on the accurate mass number of the first-stage mass spectrometry (error ≤ 5 ppm), and then the compound structure is deduced through the fragment ion information of the second-stage mass spectrometry (such as characteristic neutral loss, fragment ion series, and relative abundance ratio of fragment ions). For example, if a quasi-molecular ion peak ([MH]-) at m / z 447.1023 is detected, combined with the fragment ion at m / z 285.0498 in the second-stage mass spectrometry, the fragmentation patterns of flavonoid compounds in the database can be compared to determine that the peak is apigenin-7-O-glucuronide, thus achieving the structural identification of the characteristic peak chemical component.

[0083] Specifically, the sample solution with characteristic spectra was injected using a UPLC-QE-HRMS / MS system. Gradient elution separation was performed using a reversed-phase column (e.g., a CORTECS T3 column) and a mobile phase of a polar modifier-organic solvent (e.g., 0.1% formic acid solution-acetonitrile) to obtain the eluent. The eluent was then introduced into an electrospray ionization source, and full-scan mass spectrometry analysis of positive and negative ions was performed under set mass spectrometry conditions (e.g., ion source temperature 320℃, capillary voltage 3.5KV, sheath gas flow rate 30L / hr) to obtain the precise mass numbers of quasi-molecular ions and addendal ions, yielding the first-stage mass spectrometry results. Based on the first-stage mass spectrometry results, the ions corresponding to the characteristic peaks were analyzed using second-stage mass spectrometry. Fragment ion information was acquired in high-resolution mode (e.g., first-stage mass spectrometry resolution R = 70000, second-stage mass spectrometry resolution R = 17500), resulting in two-stage mass spectrometry data. Import the two-stage mass spectrometry data into analysis software (such as Compound Discoverer and Xcalibur 2.1), and compare them with mass spectrometry patterns, relevant literature, and online databases (such as ChemSpider) to analyze the chemical composition of characteristic peaks.

[0084] This embodiment utilizes ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HDMS) to achieve systematic analysis of common peak chemical components in the characteristic chromatograms of Broussonetia papyrifera leaves. Gradient elution separation using a reversed-phase column and a polar modifier-organic solvent mobile phase ensures effective separation of each component in the sample. Electrospray ionization combined with full-scan primary mass spectrometry (PSMS) and HDMS analysis accurately obtains precise ion mass numbers and fragment ion information. By comparing the analysis software with a database, the chemical components of the characteristic peaks can be quickly determined, providing a highly sensitive and high-resolution analytical method for the study of the material basis of Broussonetia papyrifera leaves, solving the problem of traditional analytical methods' difficulty in accurately identifying trace components.

[0085] In one embodiment, the chromatographic conditions for ultra-high performance liquid chromatography-quadrupole-electrostatic field orbitt trap high-resolution mass spectrometry may include:

[0086] The chromatographic column was a CORTECS T3.

[0087] The mobile phase consisted of 0.1% formic acid solution-acetonitrile, with gradient elution, a flow rate of 0.2 mL / min, a column temperature of 30 °C, and an injection volume of 2 μL.

[0088] The mass spectrometry conditions set included an electrospray ionization source, ESI+ and ESI- full scan modes, ion source temperature of 320℃, capillary voltage of 3.5KV, sheath gas flow rate of 30L / hr, sheath gas pressure of 45arb, auxiliary gas flow rate of 8L / hr, auxiliary gas pressure of 15arb, and auxiliary gas temperature of 320℃.

[0089] The high-resolution mode includes a scanning range of 150-1000 m / z, a first-order mass spectrometry resolution of R = 70000, and a second-order mass spectrometry resolution of R = 17500.

[0090] In this embodiment, the technical conditions utilize a CORTECS T3 chromatographic column paired with a 0.1% formic acid solution-acetonitrile mobile phase. Gradient elution achieves efficient separation of chemical components in the sample, while the relatively low flow rate and injection volume ensure high precision and sensitivity in chromatographic separation. The electrospray ionization source, combined with a full-scan mode for both positive and negative ions, comprehensively captures quasi-molecular ions and adduct ions in both positive and negative ion modes. The carefully set ion source temperature, voltage, and gas flow parameters ensure ionization efficiency and stability. The high-resolution settings of the primary and secondary mass spectrometers in high-resolution mode allow for extremely low errors in the precise mass numbers of the acquired ions. Combined with a wide scan range of 150-1000 m / z, it enables precise analysis of the molecular composition and fragment structure of characteristic peaks. This provides a high-resolution, high-sensitivity, and high-accuracy detection method for the analysis of common peaks in the characteristic spectra of mulberry leaves, effectively identifying trace and complex components.

[0091] In one embodiment, using characteristic flavonoids from the analyzed chemical components as internal standards, the relative content of characteristic peak chemical components is quantitatively analyzed, and the lower limit of content is determined to obtain the quality evaluation result of paper mulberry leaves. This may include the following steps:

[0092] Step S401: Accurately weigh the characteristic flavonoid internal standard reference substance, dissolve it in an organic solvent of a specific concentration to prepare a series of gradient standard solutions, and establish a standard curve after high performance liquid chromatography injection detection.

[0093] Preferably, the characteristic flavonoid internal standard (e.g., apigenin-7-O-glucuronide) is accurately weighed (to an accuracy of 0.0001 g), dissolved in a specific concentration organic solvent (e.g., 70% methanol), and diluted to volume in a volumetric flask to prepare a stock solution. A series of standard solutions (e.g., 0.5 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, 50 μg / mL, 100 μg / mL) are prepared using a gradient dilution method. After filtration through a 0.45 μm filter membrane, the solutions are injected and detected under high-performance liquid chromatography (HPLC) conditions (e.g., CORTECS T3 column, 0.1% formic acid-acetonitrile mobile phase, flow rate 0.5 mL / min, detection wavelength 339 nm). A linear standard curve is established using the least squares method with the standard solution concentration as the x-axis and the chromatographic peak area as the y-axis, ensuring a correlation coefficient R0. 2 ≥0.999 provides a benchmark for the quantification of internal standards.

[0094] Step S402: Quantitatively analyze the characteristic flavonoid internal standard in the test solution, calculate its absolute content according to the standard curve, and simultaneously determine the chromatographic peak area of ​​each characteristic peak in the characteristic chromatogram.

[0095] Step S403: Calculate the relative content of chemical components of each characteristic peak based on the internal standard quantification method, and process the data using the internal standard method formula to obtain the relative content data.

[0096] Step S404: Statistical analysis is performed on the relative content data of characteristic peaks of multiple batches of tested mulberry leaves, and the lower limit of the relative content of chemical components of each characteristic peak is determined using the mean-standard deviation method.

[0097] Preferably, data on mulberry leaves from more than 20 batches of different origins and harvest periods were collected for testing. Statistical analysis was performed on the relative content data of each characteristic peak (such as neochlorogenic acid, chlorogenic acid, apigenin, etc.). The mean-standard deviation method was used. The method calculates the average relative content of each characteristic peak. and standard deviation (s), This serves as a relative lower limit for content. For example, if the average value of 24 batches of data for a certain characteristic peak is 0.2512 g / 100 g, and the standard deviation is 0.0102 g / 100 g, then its lower limit for content is determined to be 0.2410 g / 100 g. This method is based on statistical principles; when the data conforms to a normal distribution, The corresponding value is located at the left tail end of the distribution curve. Theoretically, the data below this value accounts for about 5%. By combining the actual quality fluctuation range of paper mulberry leaves, a scientific quality benchmark is established to ensure that the lower limit value not only conforms to the actual production situation but also guarantees the uniformity of paper mulberry leaf quality.

[0098] Step S405: Compare the actual measured lower limit of the relative content of the tested paper mulberry leaves with the preset lower limit. When the measured value is not lower than the preset lower limit, it is judged to be of qualified quality, and the quality evaluation result of paper mulberry leaves is obtained.

[0099] Specifically, the characteristic flavonoid internal standard was accurately weighed, dissolved in a specific concentration of organic solvent (e.g., 70% methanol), and prepared into a series of gradient standard solutions (e.g., 0.5–100 μg / mL). After high-performance liquid chromatography (HPLC) analysis, a standard curve was established with concentration as the x-axis and peak area as the y-axis. The characteristic flavonoid internal standard in the test solution was quantitatively analyzed, and its absolute content (g / 100g) was calculated according to the standard curve. Simultaneously, the peak area of ​​each characteristic peak in the characteristic chromatogram was measured. Based on the internal standard quantification method, data processing was performed using formulas to obtain the relative content data of each characteristic peak chemical component. Statistical analysis was performed on the relative content data of characteristic peaks of multiple batches of tested mulberry leaves, and the lower limit of the relative content of each characteristic peak chemical component was determined using the "mean-standard deviation" method. The measured relative content of the tested mulberry leaves was compared with the preset lower limit. When the measured value was not lower than the preset lower limit, the quality was deemed qualified, thus obtaining the quality evaluation result of the mulberry leaves.

[0100] This embodiment achieves precise quantification of characteristic flavonoid components by establishing an internal standard curve. The relative content of characteristic peaks is calculated using the internal standard method formula, eliminating interference from factors such as injection volume and instrument response during the experiment, thus improving data accuracy and repeatability. The "mean-standard deviation" method is used to determine the lower limit of content, and a scientific quality benchmark is formed based on statistical data from multiple batches of paper mulberry leaves, making the quality evaluation results more objective and reliable. By comparing the measured values ​​with the lower limit values, the quality of paper mulberry leaves can be directly determined, providing a quantitative and standardized evaluation system for the quality control of paper mulberry leaves. This effectively solves the limitations of traditional single-component detection methods and meets the needs of consistent quality evaluation of paper mulberry leaves.

[0101] In one embodiment, the internal standard method formula may include:

[0102] Relative content of characteristic peak = peak area of ​​characteristic peak × (content of internal standard / peak area of ​​internal standard).

[0103] This embodiment effectively eliminates the interference of factors such as fluctuations in injection volume and differences in instrument response during the experiment on the quantitative results by introducing a characteristic flavonoid internal standard. The formula calculates the results by combining the peak area ratio with the content of the internal standard. By utilizing the consistency of the internal standard during extraction and separation, systematic errors in sample pretreatment and detection are corrected, improving the accuracy and repeatability of the quantitative results. Calculating the relative content of characteristic peaks using this formula objectively reflects the proportional relationship of various chemical components in mulberry leaves, providing a scientific method for the quantitative analysis of the synergistic effects of multiple components. This makes the quality evaluation results of mulberry leaves more reliable and comparable, meeting the technical requirements for quantitative analysis of complex systems in the quality control of traditional Chinese medicine.

[0104] In one embodiment, the preparation of a test solution and a reference solution by adding methanol to dried, pulverized, and sieved paper mulberry leaf powder for ultrasonic extraction may include the following steps:

[0105] Step S501: After drying the leaves of paper mulberry from different origins and controlling the moisture content, the leaves are crushed and sieved to obtain uniform paper mulberry leaf powder.

[0106] Preferably, the leaves of paper mulberry trees from different origins (such as Henan, Shandong, and Anhui) are dried, with the moisture content controlled to ≤10%. A constant temperature drying oven is used at 60℃ to dry to constant weight, removing moisture to prevent mold growth and component degradation. The dried leaves are then pulverized in a pulverizer and passed through a No. 3 sieve (0.355mm aperture) to remove coarse fibers and other insufficiently pulverized particles, resulting in uniformly sized paper mulberry leaf powder. This ensures sufficient contact between the solvent and the leaves during subsequent extraction, improving component extraction efficiency.

[0107] Step S502: After accurately weighing the paper mulberry leaf powder, place it in a stoppered conical flask, add a methanol solution of a specific concentration, and weigh and record the initial weight.

[0108] Preferably, the powdered paper mulberry leaves are accurately weighed and placed in a stoppered conical flask. A 70% methanol solution (methanol: ultrapure water = 7:3, v / v, ultrapure water meets the purified water standard of the Chinese Pharmacopoeia, conductivity ≤ 5.1 μS / cm, 25℃) is added and the initial weight is recorded.

[0109] Step S503: The mixture in the container is subjected to ultrasonic extraction. After cooling to room temperature, the weight loss is replenished to ensure that the total amount of solution is equal to the initial weight, and a mixed extract is obtained.

[0110] Furthermore, the stoppered conical flask and the mulberry leaf powder were weighed together and the initial weight was recorded. Then, the flask was placed in an ultrasonic extractor, and the ultrasonic power was set to 300W, the temperature to 30℃, and the extraction time to 30 minutes. During the ultrasonic process, the solvent evaporated due to the temperature rise. After cooling to room temperature, the initial extraction solvent (such as 70% methanol) was used to make up for the weight loss. The total amount of solution was accurately weighed using an electronic balance to ensure that it was consistent with the initial weight, thus avoiding deviations in the extract concentration due to solvent loss.

[0111] Step S504: The mixed extract is filtered under reduced pressure using a filtration device, and the filtrate is collected to obtain the test solution. A reference solution is prepared using mulberry leaves as a control.

[0112] Specifically, after drying the leaves of paper mulberry trees from different origins, they are pulverized and sieved (e.g., through a No. 3 sieve) to obtain uniform paper mulberry leaf powder. The powder is accurately weighed (e.g., 0.5 g) and placed in a stoppered conical flask. A methanol solution of a specific concentration (e.g., 70% methanol, at a material-to-liquid ratio of 1:100) is added, and the initial weight is recorded. The mixture is then subjected to ultrasonic extraction (e.g., 300 W power, 30 °C, 30 minutes). After cooling to room temperature, the weight loss is replenished to ensure the total solution volume is equal to the initial weight, yielding a mixed extract. The mixed extract is then filtered under reduced pressure using a filtration device (e.g., a 0.45 μm filter membrane). The filtrate is collected to obtain the test solution. A reference solution is prepared using paper mulberry leaves as a control.

[0113] This embodiment ensures the uniformity of the paper mulberry leaf powder through pretreatment steps such as drying, pulverizing, and sieving. Precise weighing and initial weight recording, combined with weight loss compensation after ultrasonic extraction, guarantee a constant total amount of extraction solvent, avoiding concentration deviations due to solvent evaporation. The selection of a specific concentration of methanol solution and the setting of ultrasonic extraction parameters enable efficient extraction of target components (such as flavonoids and phenolic acids) from paper mulberry leaves. The reduced-pressure filtration step removes solid impurities from the extract, ensuring solution clarity and preventing column clogging or interference with detection results. The preparation of a reference solution using the same method ensures consistency between the test sample and the control paper mulberry leaves in extraction and purification, providing a reliable solution sample for subsequent characteristic chromatogram establishment and component quantification analysis, guaranteeing the accuracy and repeatability of experimental results.

[0114] In one embodiment, the method may further include:

[0115] Step S601: Single-factor investigation of methanol at different concentrations was conducted. Methanol solution was added at a material-to-liquid ratio of 1:100, and ultrasonic extraction was performed for 30 minutes. The total yield of the nine components was measured, and 70% methanol was determined to be the optimal extraction solvent.

[0116] In step S602, a single-factor study was conducted on the ultrasonic extraction time. Extraction was performed using 70% methanol at a material-to-liquid ratio of 1:100. The total yield was measured, and 30 minutes was determined to be the optimal extraction time.

[0117] Step S603: A single-factor study was conducted on the material-liquid ratio. Ultrasonic extraction with 70% methanol was performed for 30 minutes, and the total yield was measured. The optimal material-liquid ratio was determined to be 1:100.

[0118] Step S604: Single-factor analysis of extraction method and extraction solvent was conducted, and the total yield and peak shape were compared to determine that ultrasonic extraction with 70% methanol was the optimal solution.

[0119] Step S605: Based on the results of the single-factor investigation, optimize the key parameters for the preparation of the test solution.

[0120] Specifically, single-factor experiments were conducted on different concentrations of methanol (30%, 50%, 70%, and 100%). Methanol solution was added at a solid-liquid ratio of 1:100, and after ultrasonic extraction for 30 minutes, the total yield of nine components was measured, determining 70% methanol as the optimal extraction solvent. Single-factor experiments were also conducted on ultrasonic extraction time (15 min, 30 min, 45 min, and 60 min). Extraction with 70% methanol at a solid-liquid ratio of 1:100 was performed, and the total yield was measured to determine 30 minutes as the optimal extraction time. In the solid-liquid ratio experiment, ultrasonic extraction with 70% methanol for 30 minutes was conducted, and ratios of 1:10, 1:30, 1:50, 1:100, and 1:250 were tested. The total yield was used as the indicator to determine 1:100 as the optimal solid-liquid ratio. In addition, single-factor experiments were conducted on extraction methods (ultrasound, reflux) and extraction solvents (methanol, acetonitrile). The total yield and peak shape were compared, and 70% methanol ultrasonic extraction was finally determined to be the optimal scheme. Based on the results of the above single-factor experiments, the key parameters for the preparation of the test solution were optimized.

[0121] This embodiment scientifically determines the optimal conditions for preparing the test solution by systematically optimizing parameters such as extraction solvent concentration, extraction time, solid-liquid ratio, and extraction method, using total component yield and peak shape as evaluation indicators. Using 70% methanol as the extraction solvent balances the extraction efficiency of both polar and non-polar components; a 30-minute extraction time ensures sufficient dissolution of components while avoiding degradation caused by prolonged extraction; a 1:100 solid-liquid ratio achieves a balance between solvent usage and extraction efficiency; and ultrasonic extraction is simpler and more energy-efficient than reflux extraction. This optimization process ensures the standardization and normalization of test solution preparation, improves the stability and repeatability of component extraction, and provides a reliable sample pretreatment foundation for subsequent characteristic spectral analysis, component analysis, and quality evaluation of *Broussonetia papyrifera* leaves, thus guaranteeing the scientific rigor and accuracy of the entire quality evaluation method.

[0122] In one embodiment, such as Figure 2 As shown, this application provides high-performance liquid chromatography (HPLC) characteristic spectra of mulberry leaves from different origins, which may include:

[0123] Twenty-four batches of dried, pulverized, and sieved paper mulberry leaf powder were extracted with 70% methanol at a material-to-liquid ratio of 1:100 using ultrasonic extraction for 30 minutes to prepare the test solution.

[0124] Detection was performed using high-performance liquid chromatography (HPLC). The mobile phase consisted of 0.1% formic acid solution and acetonitrile, with gradient elution (acetonitrile concentration 10%–15% for 0–12 minutes). The sample was injected at a flow rate of 0.5 mL / min, a column temperature of 30 °C, and a detection wavelength of 339 nm to obtain chromatograms.

[0125] The chromatograms were imported into a similarity evaluation system, and nine common peaks were identified using the chromatogram of Broussonetia papyrifera leaves as a reference, thus establishing a characteristic chromatogram. This chromatogram can intuitively reflect the consistency of chromatographic peak distribution of Broussonetia papyrifera leaves from different origins. Among them, peak number 9 (apigenin-7-O-glucuronide) had a stable retention time (35.580±0.011 min) and was used as an internal standard reference peak.

[0126] In one embodiment, such as Figure 3 As shown, this application provides positive (left) and negative (right) ion flow maps (TICs) measured by UPLC-QE-HRMS / MS technology, which may include:

[0127] The characteristic chromatographic sample solutions were eluted using a CORTECS T3 column (2.1 mm × 100 mm, 1.6 μm) with a gradient elution of 0.1% formic acid solution-acetonitrile (acetonitrile concentration 5%–40% for 0–20 minutes), at a flow rate of 0.2 mL / min and an injection volume of 2 μL.

[0128] The mass spectrometry conditions were: electrospray ionization source (ESI ± full scan), ion source temperature 320℃, capillary voltage 3.5KV, first-stage mass spectrometry resolution R = 70000, second-stage mass spectrometry resolution R = 17500, and scan range 150~1000m / z.

[0129] By analyzing positive and negative ion modes, the precise mass numbers of quasi-molecular ions and fragment ions were obtained (e.g., m / z 447.0911 corresponds to the [MH]- ion of apigenin-7-O-glucuronide). Combined with Compound Discoverer software and the ChemSpider database, the chemical components of 9 characteristic peaks were identified, including neochlorogenic acid, chlorogenic acid, vitexin, etc.

[0130] In one embodiment, such as Figure 4 As shown, this application provides a UPLC-HR-MS identification result table of characteristic peaks of mulberry leaves, which may include:

[0131] The table lists the mass spectrometry identification information for nine characteristic peaks. For example, peak 1 (RT = 15.006 min) in negative ion mode has a quasi-molecular ion m / z of 353.0866 (theoretical value 353.0878), and secondary fragments containing 195.60 and 179.03, and is identified as neochlorogenic acid (C1). 16 H 18 O9).

[0132] UPLC-QE-HRMS / MS technology was used with a CORTECS T3 column (2.1 mm × 100 mm), 0.1% formic acid-acetonitrile gradient elution (flow rate 0.2 mL / min), and ESI ± full scan (first-order resolution R = 70000, second-order R = 17500).

[0133] By comparing Compound Discoverer software with the ChemSpider database and combining the mass spectrometry fragmentation patterns, the chemical composition of the characteristic peaks was analyzed.

[0134] In one embodiment, such as Figure 5 As shown, this application provides a concentration-peak area standard curve of the internal standard substance, which may include:

[0135] Accurately weigh apigenin-7-O-glucuronide reference standard and prepare a series of gradient standard solutions ranging from 0.5 to 100 μg / mL using 70% methanol. After detection by high-performance liquid chromatography (HPLC), a standard curve was established with concentration as the x-axis and peak area as the y-axis, yielding the regression equation Y = 57031X + 1195.2 (R²). 2 =1.000), indicating a good linear relationship.

[0136] The content of the internal standard in the test sample solution is quantified by this curve. The relative content of each characteristic peak is calculated by combining the internal standard method formula (relative content of characteristic peak = peak area of ​​characteristic peak × (content of internal standard / peak area of ​​internal standard)).

[0137] After statistical analysis of data from 24 batches of paper mulberry leaves, the lower limit of the relative content of each characteristic peak (e.g., neochlorogenic acid ≥ 0.1081%) was determined for quality assessment.

[0138] In one embodiment, such as Figure 6 As shown, this application provides a table of relative quantitative results based on characteristic peaks of internal standard substances, which may include:

[0139] The table lists the relative content and lower limit of each characteristic peak (e.g., relative content of chlorogenic acid 0.2686±0.1357%, lower limit 0.1329%).

[0140] Using apigenin-7-O-glucuronide as an internal standard, standard solutions ranging from 0.5 to 100 μg / mL were prepared to establish a curve (Y = 57031X + 1195.2, R0). 2 =1.000), the relative content is calculated using the internal standard method formula.

[0141] The lower limit was determined using the mean-standard deviation method for 24 batches of data. When the measured value of the tested mulberry leaves was greater than or equal to the lower limit (e.g., S1 chlorogenic acid 0.1940% ≥ 0.1081%), it was deemed qualified, thus forming a dual-standard evaluation system of quality and quantity.

[0142] In one embodiment, such as Figures 7-10 As shown, this application provides a table of the effects of different factors on the extraction of component content from paper mulberry leaves, which may include:

[0143] likeFigure 7 As shown in the table, the effects of different concentrations of methanol on the extraction of components from Broussonetia papyrifera leaves are presented, which may include:

[0144] Take 0.5g of dried, pulverized and sieved Broussonetia papyrifera leaf powder, add 30%, 50%, 70%, and 100% methanol (material-liquid ratio 1:100) and ultrasonically extract for 30 minutes, and determine the total yield of 9 components.

[0145] The results showed that the total yield was highest when extracted with 70% methanol (1.5572%), with the content of apigenin-7-O-glucuronide reaching 0.5101%, which was 162% higher than that of 100% methanol (0.1946%). Polar components such as neochlorogenic acid and chlorogenic acid were more fully dissolved in 50%–70% methanol, while the extraction rate of hydrophilic components decreased due to the reduced polarity of 100% methanol (e.g., the content of cryptochlorogenic acid decreased from 0.1163% to 0.0391%).

[0146] The results indicate that 70% methanol can balance the extraction efficiency of both polar and weakly polar components.

[0147] like Figure 8 As shown in the table, the effects of different extraction times on the content of components extracted from mulberry leaves are presented, and may include:

[0148] The leaf powder of *Broussonetia papyrifera* was ultrasonically extracted with 70% methanol (solid-to-liquid ratio 1:100) for 15, 30, 45, and 60 minutes, respectively. The total yield first increased and then decreased with the extension of time: the total yield reached 1.5985% (maximum value) at 30 minutes, which was 5.5% higher than that at 15 minutes (1.5153%); after 45 minutes, due to component degradation or solvent evaporation, the total yield dropped to 1.4940%.

[0149] The content of baicalin was 0.3296% at 30 minutes, which was significantly higher than that at 15 minutes (0.3017%) and 60 minutes (0.3229%), indicating that 30 minutes is the optimal time to balance extraction efficiency and component stability.

[0150] like Figure 9 As shown in the table, the effects of different material-to-liquid ratios on the extraction of component content from paper mulberry leaves are presented, which may include:

[0151] The effects of material-liquid ratios of 1:10, 1:30, 1:50, 1:100, and 1:250 on the extraction efficiency were investigated, and the highest total yield (1.6238%) was found at a ratio of 1:100.

[0152] When the solid-liquid ratio increased from 1:10 to 1:100, the content of apigenin-7-O-glucuronide increased from 0.3570% to 0.5020%, and baicalin increased from 0.2111% to 0.3251%, indicating that increasing the amount of solvent can promote the dissolution of components. However, when the solid-liquid ratio was 1:250, the concentration was diluted due to excessive solvent, and the total yield dropped to 1.5670%. Therefore, 1:100 is the balance point between solvent usage and extraction efficiency.

[0153] like Figure 10 As shown in the table, the effects of different extraction methods and solvents on the extraction of component content from Broussonetia papyrifera leaves are presented, which may include:

[0154] Comparing ultrasonic extraction and reflux extraction (70% methanol, 30 minutes), the total yield of reflux extraction (1.6656%) was 4.19% higher than that of ultrasonic extraction (1.5985%), but the difference between the two was <5%. Considering the ease of operation, ultrasonic extraction was chosen.

[0155] When the solvent was replaced with 70% acetonitrile, the total yield (1.4029%) was significantly lower than that of 70% methanol, and the peak tailing was severe (e.g., the chlorogenic acid peak broadening factor > 2.0), indicating that methanol has a stronger ability to dissolve flavonoids and phenolic acids in mulberry leaves. Therefore, ultrasonic extraction with 70% methanol was determined to be the best solution.

[0156] In one embodiment, this application also provides a comparative study of different quantitative methods in the multi-component analysis of mulberry leaves, which may include:

[0157] 1. Instruments and Materials

[0158] Instruments: LC-2040C high performance liquid chromatograph (Shimadzu), THC-10B ultrasonic extractor, SQP type 0.01% electronic balance and DV215CD type 0.01% electronic balance.

[0159] Materials: 24 batches of paper mulberry leaves for testing, apigenin-7-O-glucuronide reference standard (purity ≥99.5%), other reference standards (neochlorogenic acid, chlorogenic acid, etc., purity ≥91.0%), methanol, acetonitrile, and formic acid (chromatographic grade).

[0160] 2. Methods and Steps

[0161] Preparation of the test solution: Accurately weigh 0.5000 g of Broussonetia papyrifera leaf powder (passed through a No. 3 sieve), add 50 mL of 70% methanol, extract by ultrasonication for 30 min, make up for weight loss, and filter to obtain the filtrate. At the same time, prepare a reference solution using Broussonetia papyrifera leaf as a control material using the same method.

[0162] Preparation of reference solutions: Prepare a stock solution of apigenin-7-O-glucuronide reference standard, and a mixed reference solution (1 mg / mL) containing 9 components including neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid, chlorogenic acid, vitexin, luteolinin, baicalin, and apigenin-7-O-glucuronide.

[0163] Chromatographic conditions: Phenomenex C18 column, mobile phase 0.1% formic acid-acetonitrile gradient elution, flow rate 0.5 mL / min, column temperature 30℃, detection wavelength 339 nm, injection volume 10 μL.

[0164] 3. Comparison of Quantitative Methods

[0165] This method (internal standard quantification method) uses apigenin-7-O-glucuronide as an internal standard, and calculates the content according to the formula "relative content of characteristic peak = peak area of ​​characteristic peak × (content of internal standard / peak area of ​​internal standard)".

[0166] Area normalization method: The content is calculated directly based on the area ratio of each peak.

[0167] External standard method (standard curve method): A standard curve is plotted using 9 reference standards, and the absolute content is calculated.

[0168] External standard one-point method: The absolute content of each component in the sample is calculated using a concentration reference standard for each component.

[0169] 4. Experimental Data and Results

[0170] (a) The internal standard selected is apigenin-7-O-glucuronide, which has stable retention time, high resolution, and good peak shape and area, for quantitative analysis.

[0171] (II) Six solutions of apigenin-7-O-glucuronide with different mass concentrations were prepared and analyzed under the chromatographic conditions described above. The linear regression equation of the standard curve for the six concentration solutions was Y = 57031X + 1195.2 (R²). 2 =1.000), linear range 0.5~100μg / ml.

[0172] (iii) The peak area RSD of 6 consecutive injections was 0.79%, indicating that the instrument has good precision.

[0173] (iv) The peak area RSD of the samples injected at 0, 2, 6, 12, 24 and 48 h was 3.34%, indicating that the test solution was stable within 48 h.

[0174] (v) The peak area RSD of the method repeatability test was 1.13%, indicating that the method has good repeatability.

[0175] (vi) Accurately weigh 6 portions of dried, pulverized and sieved Broussonetia papyrifera leaf powder, add appropriate amounts of apigenin-7-O-glucuronide standard solution to each portion, prepare the test solution according to the test solution preparation method, inject and detect, and calculate the average recovery rate as 101.18% (RSD 3.06%).

[0176] (vii) The effects of different chromatographic columns (Phenomenex C18 and Capcel Ipak C18), different models of chromatographs (LC-20AT, LC-2040C), different column temperatures (35, 30, 25℃), and different flow rates (1.0, 0.9, 0.7, 0.5 mL / min) on the detection results were investigated. The robustness test showed that different columns, instruments, and column temperatures had little impact on the results, and the separation effect was good at a flow rate of 0.5 mL / min.

[0177] (viii) Take the powdered leaves of the paper mulberry tree as the test material, prepare the test solution, and analyze it according to the chromatographic conditions to determine the content of apigenin-7-O-glucuronide. At the same time, calculate the relative content of other characteristic peak chemical components in each test solution according to "characteristic peak area × (internal standard content / internal standard peak area)", and use "mean - standard deviation" as the lower limit of content.

[0178] The content determination method in this study was compared with the peak area normalization method, the external standard point method, and the standard curve method.

[0179] like Figure 11 As shown, the results of quantitative comparison of nine chemical components in mulberry leaves using peak area normalization, external standard point method, standard curve method, and internal standard quantification method are presented. The results show that:

[0180] The peak area normalization method yielded relatively low results overall, possibly because it did not consider the differences in component response factors. The external standard one-point method and the standard curve method yielded relatively high results with good consistency. The standard curve method, in particular, is more accurate in quantification because it plots the linear relationship between concentration and peak area. The internal standard quantification method showed high consistency with the external standard one-point method and the standard curve method, indicating that its quantification method is reliable. The differences in results among different methods reflect the applicability and limitations of each method in the quantification of mulberry leaf components.

[0181] It should be understood that although the steps in the flowcharts of the embodiments described above are shown sequentially according to the arrows, these steps are not necessarily executed in the order indicated by the arrows. Unless explicitly stated herein, there is no strict order restriction on the execution of these steps, and they can be executed in other orders. Moreover, at least some steps in the flowcharts of the embodiments described above may include multiple steps or multiple stages. These steps or stages are not necessarily completed at the same time, but can be executed at different times. The execution order of these steps or stages is not necessarily sequential, but can be performed alternately or in turn with other steps or at least some of the steps or stages of other steps.

[0182] In one embodiment, a computer device is provided, including a memory and a processor, the memory storing a computer program, the processor executing the computer program to implement the steps of the above-described method for evaluating the quality of mulberry leaves.

[0183] In one embodiment, a computer-readable storage medium is provided having a computer program stored thereon, which, when executed by a processor, implements the steps in the above method embodiments.

[0184] For the device embodiments, since they basically correspond to the method embodiments, the relevant parts can be referred to in the description of the method embodiments. The device embodiments described above are merely illustrative. The components described as separate parts may or may not be physically separate, and the components shown as units may or may not be physical units, that is, they may be located in one place or distributed across multiple network units. Some or all of the modules can be selected to achieve the purpose of this disclosure according to actual needs. Those skilled in the art can understand and implement this without creative effort.

[0185] The above-described embodiments are merely illustrative of several implementation methods of the embodiments of this application, and their descriptions are relatively specific and detailed. However, they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the embodiments of this application, and these modifications and improvements all fall within the protection scope of the embodiments of this application.

Claims

1. A method for evaluating the quality of paper mulberry leaves, characterized in that, The method includes: Broussonetia papyrifera leaves from different origins were obtained. The dried, pulverized, and sieved Broussonetia papyrifera leaf powder was extracted with methanol by ultrasonic extraction to prepare test solution and reference solution. High performance liquid chromatography was used to analyze the test solution and the reference solution. The chromatograms were then imported into a similarity evaluation system, and a characteristic chromatogram of mulberry leaves was established with the reference chromatogram of mulberry leaves as a reference. High-resolution mass spectrometry (HMS) coupled with ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap was used to perform first- and second-order mass spectrometry analysis on the common peaks in the characteristic spectra, and the chemical composition of the characteristic peaks was analyzed by comparison with the database. Using the characteristic flavonoids in the chemical components obtained from the analysis as internal standards, the relative content of the characteristic peak chemical components was quantitatively analyzed, and the lower limit of the content was determined to obtain the quality evaluation results of the paper mulberry leaves.

2. The method according to claim 1, characterized in that, The process involves using high-performance liquid chromatography (HPLC) to analyze the test solution and the reference solution, importing the chromatograms into a similarity evaluation system, and establishing a characteristic chromatogram of mulberry leaves using the reference chromatogram as a reference. This includes: The test solution and the reference solution were subjected to high performance liquid chromatography (HPLC) with gradient elution using an aqueous solution of polar modifier and an organic solvent as the mobile phase. The samples were injected and detected under the set flow rate, column temperature, detection wavelength and injection volume conditions to obtain HPLC chromatograms. The obtained high-performance liquid chromatograms were imported into a similarity evaluation system. Using the chromatogram of the reference mulberry leaf as a reference spectrum, multi-point correction and Mark peak matching were performed on the high-performance liquid chromatograms of all samples, and common peaks were identified to obtain common pattern diagrams. The common pattern diagram is compared with the chromatogram of the control mulberry leaf to establish a characteristic spectrum of mulberry leaf.

3. The method according to claim 1, characterized in that, The high-resolution mass spectrometry (HPLC-quadrupole-electrostatic field orbital trap) technique is used to perform primary and secondary mass spectrometry analysis on the common peaks in the characteristic spectra, and the chemical composition of the characteristic peaks is analyzed by comparing with a database, including: The sample solution with the characteristic spectrum was injected using an ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometer, and gradient elution separation was performed using a reversed-phase column and a polar modifier-organic solvent as the mobile phase to obtain the eluent. The effluent is introduced into an electrospray ion source, and positive and negative ion full-scan first-order mass spectrometry analysis is performed under the set mass spectrometry conditions to obtain the precise mass numbers of quasi-molecular ions and adduct ions, and to obtain the first-order mass spectrometry results. Based on the first-level mass spectrometry results, the ions corresponding to the characteristic peaks are analyzed by second-level mass spectrometry to obtain fragment ion information in high-resolution mode, thus obtaining two-level mass spectrometry data. The two-stage mass spectrometry data were imported into the analysis software, and the chemical composition of the characteristic peaks was analyzed by combining mass spectrometry rules, relevant literature, and online database comparison.

4. The method according to claim 3, characterized in that, The chromatographic conditions for the ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry technique include: The chromatographic column was a CORTECS T3; The mobile phase consists of 0.1% formic acid solution-acetonitrile, gradient elution, flow rate 0.2 mL / min, column temperature 30 °C, and injection volume 2 μL; The set mass spectrometry conditions include an electrospray ionization source, ESI+ and ESI- full scan modes, ion source temperature of 320℃, capillary voltage of 3.5KV, sheath gas flow rate of 30L / hr, sheath gas pressure of 45arb, auxiliary gas flow rate of 8L / hr, auxiliary gas pressure of 15arb, and auxiliary gas temperature of 320℃. The high-resolution mode includes a scanning range of 150-1000 m / z, a first-order mass spectrometry resolution of R = 70000, and a second-order mass spectrometry resolution of R = 17500.

5. The method according to claim 1, characterized in that, The process involves using characteristic flavonoids from the analyzed chemical components as internal standards to quantitatively analyze the relative content of the characteristic peak chemical components, determine the lower limit of content, and obtain the quality evaluation results of paper mulberry leaves, including: The characteristic flavonoid internal standard was accurately weighed, dissolved in an organic solvent of a specific concentration to prepare a series of gradient standard solutions, and a standard curve was established after high performance liquid chromatography (HPLC) injection detection. The characteristic flavonoid internal standard in the test sample solution is quantitatively analyzed, and its absolute content is calculated according to the standard curve. The chromatographic peak area of ​​each characteristic peak in the characteristic spectrum is determined simultaneously. The relative content of each characteristic peak chemical component was calculated based on the internal standard quantification method, and the relative content data was obtained by data processing using the internal standard method formula. Statistical analysis was performed on the relative content data of characteristic peaks of multiple batches of tested mulberry leaves, and the lower limit of the relative content of chemical components of each characteristic peak was determined using the mean-standard deviation method. The relative content lower limit of the tested paper mulberry leaves is compared with the preset lower limit. When the measured value is not lower than the preset lower limit, it is determined to be of qualified quality, and the quality evaluation result of paper mulberry leaves is obtained.

6. The method according to claim 5, characterized in that, The internal standard method formula includes: Relative content of characteristic peak = peak area of ​​characteristic peak × (content of internal standard / peak area of ​​internal standard).

7. The method according to claim 1, characterized in that, The step of adding methanol to the dried, pulverized, and sieved paper mulberry leaf powder for ultrasonic extraction to prepare the test solution and reference solution includes: After drying the paper mulberry leaves from different origins and controlling the moisture content, the leaves were pulverized and sieved to obtain uniform paper mulberry leaf powder. After accurately weighing the powdered paper mulberry leaves, place it in a stoppered conical flask, add a methanol solution of a specific concentration, and weigh and record the initial weight. The mixture in the container is subjected to ultrasonic extraction. After cooling to room temperature, the weight loss is made up to ensure that the total amount of solution is equal to the initial weight, and a mixed extract is obtained. The mixed extract was subjected to reduced pressure filtration using a filtration device, and the filtrate was collected to obtain the test solution. A reference solution was prepared using mulberry leaves as a control.

8. The method according to claim 1, characterized in that, The method further includes: Single-factor experiments were conducted on methanol of different concentrations. Methanol solution was added at a material-to-liquid ratio of 1:100, and ultrasonic extraction was performed for 30 minutes. The total yield of 9 components was measured, and 70% methanol was determined to be the optimal extraction solvent. A single-factor study was conducted on ultrasonic extraction time. Extraction was performed using 70% methanol at a material-to-liquid ratio of 1:

100. The total yield was measured, and 30 minutes was determined to be the optimal extraction time. A single-factor study was conducted on the material-liquid ratio. Ultrasonic extraction with 70% methanol for 30 minutes was performed, and the total yield was measured. The optimal material-liquid ratio was determined to be 1:

100. Single-factor investigations were conducted on extraction methods and extraction solvents, and the total yield and peak shape were compared to determine that ultrasonic extraction with 70% methanol was the optimal solution. Based on the results of the single-factor investigation, the key parameters for the preparation of the test solution were optimized.

9. A computer device comprising a memory and a processor, wherein the memory stores a computer program, characterized in that, When the processor executes the computer program, it implements the steps of the method according to any one of claims 1 to 8.

10. A computer-readable storage medium having a computer program stored thereon, characterized in that, When the computer program is executed by a processor, it implements the steps of the method according to any one of claims 1 to 8.

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