Comprehensive evaluation method for quality of eucommia male flowers and application

Abstract

The invention discloses a comprehensive evaluation method for the quality of eucommia male flowers and application, belongs to the technical field of quality detection of traditional Chinese medicinal materials, and aims to solve the technical problems of single evaluation means, low efficiency and disjunction of quality and efficacy of the traditional eucommia male flowers. The method comprises the following five core steps: preparing a test solution, constructing and evaluating an UPLC (Ultra Performance Liquid Chromatography) exclusive fingerprint spectrum, quantifying HPLC (High Performance Liquid Chromatography) multi-index functional components, determining the functionality of total polyphenols and total flavonoids and carrying out three-dimensional synergistic judgment. Efficient separation of 19 common characteristic peaks is achieved through customized UPLC chromatographic conditions, the relative retention time range of the characteristic peaks is limited by taking chlorogenic acid as a reference peak, and content limits of four core active ingredients such as aucubin and the like, total polyphenol and total flavone are set based on an efficacy threshold value; and meanwhile, a plurality of groups of defensive experiments are additionally arranged to verify system collaboration, industrial practicability and efficacy relevance. According to the method, three-dimensional management and control of overall qualification, precise quantification and efficacy association of the eucommia ulmoides male flowers are achieved, the UPLC fingerprint detection efficiency is improved by 61% compared with a traditional HPLC method, the authenticity identification accuracy rate reaches 100%, the method is suitable for large-scale application of the whole industrial chain, and a scientific and feasible technical scheme is provided for standardized management and control of the quality of the eucommia ulmoides male flowers.

Description

Technical Field

[0001] This invention relates to the field of quality testing technology for Chinese medicinal materials. Specifically, it relates to a comprehensive quality evaluation method for Eucommia ulmoides male flowers that combines qualitative analysis using ultra-high performance liquid chromatography (UHPLC) with fingerprint spectroscopy, quantitative analysis of multiple efficacy components using HPLC, and determination of functional components such as total polyphenols and total flavonoids. It also relates to the application of this method in the entire industry chain, including the identification of genuine Eucommia ulmoides male flowers, accurate determination of their origin, batch quality consistency control, and prediction of product efficacy potential. Background Technology

[0002] Eucommia male flowers are the male flowers of Eucommia ulmoides Oliv., a plant belonging to the Eucommia genus of the Eucommiaceae family. They are a precious medicinal and edible raw material recognized by the National Health Commission of China. They are rich in iridoids such as aucubin and genipin, phenylpropanoids such as chlorogenic acid, as well as polyphenols and flavonoids. Pharmacological experiments have confirmed that they have core physiological functions such as anti-oxidation, blood lipid regulation, and enhancement of the body's immunity. They are widely used in health food, traditional Chinese medicine decoction pieces, and functional beverages.

[0003] With the increasing market demand for Eucommia ulmoides male flowers year by year, the deficiencies in its quality control system have become increasingly prominent. Existing technologies suffer from three major limitations: First, fingerprint spectroscopy construction often employs traditional high-performance liquid chromatography (HPLC) techniques. For example, in publicly available literature (Zhang Zhi et al., 2022), a single sample test takes as long as 82 minutes, only identifying 15 common peaks, resulting in incomplete chemical information coverage. Furthermore, no system of characteristic peaks specific to the authentic producing areas has been established, failing to meet the precise quality control requirements for identifying Eucommia ulmoides leaves masquerading as male flowers or adulteration from non-authentic producing areas. Second, the quantification of active ingredients is limited to single or a few components, and content limits are based solely on statistical data, without establishing a correlation with core efficacy. For instance, some companies only measure chlorogenic acid content, failing to reflect the "multi-component synergistic effect" characteristic of Eucommia ulmoides male flowers. Third, existing technologies have not formed a synergistic evaluation system of "overall qualitative analysis - precise quantification - functional correlation," often relying on the independent application of single technologies. This leads to a serious disconnect between product quality and actual efficacy, with a market product quality pass rate of only 82%. The difficulty in distinguishing genuine from counterfeit products severely restricts the standardized development of the industry.

[0004] Although ultra-high performance liquid chromatography (UPLC) has been gradually applied to the field of traditional Chinese medicine fingerprinting due to its high separation efficiency and fast detection speed, and the Folin-phenol method and aluminum nitrate-sodium nitrite complexation spectrophotometric method are routine methods for the determination of polyphenols and flavonoids, there are currently no publicly reported technical solutions for customizing UPLC chromatographic conditions for the characteristics of Eucommia ulmoides male flowers, binding the quantification of multiple index components with efficacy thresholds, and constructing a three-dimensional synergistic evaluation system. The industry urgently needs a scientific, efficient, and practical comprehensive evaluation method. Summary of the Invention

[0005] In view of this, the present invention proposes a comprehensive quality evaluation method and application for Eucommia ulmoides male flowers, aiming to solve the technical pain points of existing Eucommia ulmoides male flower quality evaluation methods being singular, inefficient, and disconnected from quality and efficacy, and to achieve dual control of quality and efficacy of Eucommia ulmoides male flowers "from raw materials to products" throughout the entire chain.

[0006] The technical solution of this invention is achieved as follows: This invention provides a comprehensive quality evaluation method for Eucommia ulmoides male flowers, including five core steps: preparation of test sample solution, construction and evaluation of UPLC-specific fingerprint spectrum, quantification of multi-index efficacy components by HPLC, determination of total polyphenols and total flavonoids, and three-dimensional synergistic judgment; at the same time, it also provides the application of this method in the whole-industry chain quality control scenario of Eucommia ulmoides male flowers. Its core innovation lies in realizing the three-dimensional linkage evaluation of "overall chemical composition - core efficacy components - functional component group".

[0007] In some embodiments, the specific operation for preparing the test solution is as follows: take Eucommia ulmoides male flower powder that has passed through a No. 3 sieve, accurately weigh it and place it in a stoppered conical flask, add 50%-70% methanol aqueous solution and seal tightly, weigh it, extract it by ultrasonication, cool it, weigh it again and make up the weight lost, shake it well, filter it, and obtain the test solution; the concentration of methanol aqueous solution in this step is selected based on preliminary experimental verification, and the extraction rate of the target component in Eucommia ulmoides male flower by 50% methanol aqueous solution can reach 92%, which is significantly higher than that of 70% methanol (extraction rate 80%) and pure methanol (extraction rate 65%).

[0008] In some embodiments, in the sample solution preparation step, the sample weight of Eucommia ulmoides male flower powder is 0.3 g, the volume fraction of methanol aqueous solution is 50%, the material-liquid ratio is 1:20 (mass-volume ratio), and the ultrasonic extraction conditions are 250 W power, 40 kHz frequency, and 30 min extraction time. This combination of parameters has been validated by methodology and can achieve full extraction of the target components without degradation, with an extraction efficiency 35% higher than that of traditional reflux extraction.

[0009] In some embodiments, the specific operation for constructing and evaluating the UPLC-specific fingerprint spectrum is as follows: The test sample solution is subjected to UPLC analysis with a reference solution containing aucubin, geniposide, chlorogenic acid, geniposide, rutin, isoquercitrin, and quercetin to generate a reference fingerprint spectrum containing 19 common peaks. The similarity between the test sample and the reference fingerprint spectrum must be ≥0.90, and the relative retention time fluctuation of each characteristic peak must be within ±10% of the specified value. This fingerprint spectrum is a specific spectrum for the male flowers of Eucommia ulmoides from the Zhangjiajie authentic producing area. The quality assessment criteria for the male flowers of Eucommia ulmoides from Zhangjiajie authentic producing area are: the similarity between the test sample and the reference fingerprint spectrum generated from 20 batches of authentic samples is ≥0.90, and the relative retention time fluctuation of the 19 common characteristic peaks is within ±10% of the specified value (Tables 2 and 3). Samples that meet this condition can be identified as male flowers of Eucommia ulmoides from the authentic producing area.

[0010] In some embodiments, the chromatographic conditions for the UPLC analysis are as follows: a ZORBAX Eclipse Plus C18 column (2.1 mm × 100 mm), column temperature 36 °C, flow rate 0.2 mL / min, injection volume 2 μL; mobile phase is 0.1% formic acid aqueous solution (A) - acetonitrile (B), gradient elution program is 0–8.5 min, 95%–84% A; 8.5–26 min, 84%–75% A; 26–30 min, 75%–20% A; 30–32 min, 20%–0% A; detection is in variable wavelength mode: 0–2.2 min, 240 nm; 2.2–3.2 min, 203 nm; 3.2–11 min, 240 nm; 11–32 min, 254 nm. These chromatographic conditions have been optimized multiple times and can achieve baseline separation of 19 common peaks with a resolution ≥1.5, which is superior to conventional UPLC conditions (resolution ≤1.2).

[0011] In some embodiments, in the UPLC fingerprint evaluation step, chlorogenic acid peak 5 is used as the reference S peak, and the specified relative retention times of the characteristic peaks are 0.368 (peak 1), 0.565 (peak 2), 0.900 (peak 3), 0.961 (peak 4), 1.069 (peak 6), 1.122 (peak 7), 1.210 (peak 8), 1.248 (peak 9), 1.488 (peak 10), and 1.630 (peak 11). The relative retention time ranges were 1.660 (peak 12), 1.736 (peak 13), 1.834 (peak 14), 1.955 (peak 15), 2.179 (peak 16), 2.336 (peak 17), 2.699 (peak 18), and 3.693 (peak 19), and the concentration of each component in the reference solution was 0.5 mg / mL. This relative retention time range was based on the determination results of 20 batches of authentic samples, with an RSD ≤ 0.17%, indicating excellent method stability.

[0012] In some embodiments, the specific operation of HPLC multi-index efficacy component quantification is as follows: the test sample solution and the mixed reference solution are analyzed by HPLC, and the contents of aucubin, geniposide, chlorogenic acid, and geniposide are determined by external standard method; the efficacy correlation of these four components is verified, and the correlation coefficient R between their total content and the antioxidant efficacy of Eucommia ulmoides male flower is... 2 =0.92, which is the core active ingredient.

[0013] In some embodiments, the chromatographic conditions for the HPLC analysis are as follows: a C18 column (4.6 × 250 mm, 5 μm), column temperature 35 °C, flow rate 0.6 mL / min, injection volume 10 μL; mobile phase is 0.1% formic acid aqueous solution (A) - acetonitrile (B), gradient elution program is 0–20 min, 92% A; 20–25 min, 92%–82% A; 25–50 min, 82%–74% A; detection is in variable wavelength mode. The wavelength range is 203 nm from 0 to 10 min and 240 nm from 10 to 50 min. Calculated on a dried basis, the content of aucubin in Eucommia ulmoides male flowers is ≥11.04 mg / g, geniposide ≥13.24 mg / g, chlorogenic acid ≥3.78 mg / g, and geniposide ≥0.79 mg / g. This limit was established based on efficacy experiments of 20 batches of samples and represents the minimum threshold for efficacy compliance. Samples below this limit cannot meet the antioxidant efficacy requirements for health foods.

[0014] In some embodiments, the specific operation for determining the total polyphenol and total flavonoid content is as follows: the total polyphenol and total flavonoid content in the test solution are determined by the Folin-phenol method and the aluminum nitrate-sodium nitrite complexation spectrophotometric method, respectively. The detection wavelength of the Folin-phenol method is 765 nm, and the total polyphenol content is ≥1.9% calculated as gallic acid; the detection wavelength of the aluminum nitrate-sodium nitrite complexation spectrophotometric method is 510 nm, and the total flavonoid content is ≥2.5% calculated as rutin. The spiked recovery rate of this method is 94.31% to 99.92%, which meets the methodological requirements for the detection of traditional Chinese medicinal materials.

[0015] In some embodiments, the specific criteria for the three-dimensional collaborative determination are as follows: if the detection results of UPLC fingerprinting, HPLC multi-index component quantification, and total polyphenol and total flavonoid content determination all meet the above-mentioned preset limits, then it is determined to be Eucommia ulmoides male flower that meets the preset quality and efficacy standards; at the same time, this method can be applied to the identification of authenticity, determination of origin, batch quality consistency control, or prediction of efficacy potential of Eucommia ulmoides male flower; among which, the determination of origin clarifies the geographical coordinate range of Zhangjiajie production area, ensuring the objectivity of the determination criteria.

[0016] The present invention has the following advantages over the prior art: Compared to traditional methods for evaluating the quality of Eucommia ulmoides male flowers, this invention uses customized UPLC technology to replace traditional HPLC technology to construct a unique fingerprint spectrum, reducing the single detection time from 82 minutes to 32 minutes (an efficiency increase of 61%), increasing the number of peaks from 15 to 19 (a 27% increase in chemical information coverage), and establishing a unique characteristic peak system for the authentic producing area. This improves the accuracy of identifying counterfeit products from 80% to 100%, solving the problems of low efficiency and insufficient identification ability of traditional fingerprint spectra. This invention also breaks through the limitations of traditional quantitative analysis that only focuses on content values, directly linking the content limits of four core active ingredients with antioxidant efficacy, clarifying the correlation between "ingredient content = efficacy threshold," making the quality standard more scientific and practical, and aligning with the synergistic effects of multiple components in traditional Chinese medicine. Furthermore, this invention is the first to construct… The invention employs a three-dimensional collaborative evaluation system of "overall qualitative analysis, precise quantitative analysis, and functional correlation." Experimental verification shows that when a single module meets the standard while other modules fail, the core efficacy of the sample will decrease by 16.2% to 37.9%, demonstrating the indivisibility of the system and achieving dual control over the quality and efficacy of Eucommia ulmoides male flowers. Furthermore, the equipment used in this invention consists of industry-standard instruments, and the operating procedures are standardized and repeatable. Verified by two production enterprises, this method has reduced the single-batch quality inspection time from 48 hours to 8 hours and increased the batch quality pass rate from 82% to 98%. It can be adapted to the entire industry chain, including variety selection at the planting end, batch quality control at the processing end, and quality sampling at the regulatory end. This provides a unified and feasible quality evaluation solution for the standardized and high-quality development of the Eucommia ulmoides male flower industry, effectively breaking through the development bottleneck of inconsistent product quality and difficulty in distinguishing genuine from counterfeit products within the industry. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a superimposed UPLC fingerprint similarity graph of 20 batches of male Eucommia ulmoides from Zhangjiajie. The horizontal axis of the graph is the detection time (min), and the vertical axis is the chromatographic peak response value (mAU). The 19 curves are the fingerprint spectra of the 20 batches of samples. It can be seen that the spectra of each batch have a high degree of overlap, and the similarity is ≥0.90. Figure 2 This is a UPLC fingerprint of Eucommia ulmoides male flower samples; the positions of 19 common peaks are marked in the figure, of which peak 1 is aucubin, peak 2 is genipin, peak 5 is chlorogenic acid, peak 9 is genipin, peak 14 is rutin, peak 15 is isoquercitrin, and peak 19 is quercetin. Figure 3 This is the UPLC chromatogram of the mixed reference standard; each chromatographic peak in the figure corresponds to aucubin, geniposide, chlorogenic acid, geniposide, rutin, isoquercitrin, and quercetin, respectively, and can be used to identify the characteristic peaks of the sample. Figure 4 The images show the HPLC chromatograms of Eucommia ulmoides male flower samples and mixed reference standards; where A is the chromatogram of the test solution and B is the chromatogram of the mixed reference standards. The four characteristic peaks correspond to aucubin, geniposide, chlorogenic acid, and geniposide, respectively, enabling precise quantification. Detailed Implementation

[0019] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] The instruments and materials used in the examples are as follows: 1. Instruments: Agilent 1290 Infinity II ultra-high performance liquid chromatograph (Agilent Technologies, including diode array detector), Z126 Infinity II Binary high performance liquid chromatograph (Agilent Technologies), FA2004 0.001 g electronic balance (Mettler-Toledo AG), AB135-S 0.001 g electronic balance (Mettler-Toledo AG), SK8200LHC ultrasonic extractor (Shanghai Kedao Ultrasonic Instrument Co., Ltd., power calibration value 250W).

[0021] 2. Reagents: Aucupin (purity 98.6%), Genipin (purity 99.1%), Chlorogenic acid (purity 98.9%), Rutin (purity 99.2%), Isoquercitrin (purity 98.3%), Quercetin (purity 99.0%) (all purchased from the National Institutes for Food and Drug Control); Genipin (purity 98.2%, purchased from Chengdu Prefabricated Chemical Co., Ltd.); Folin-Ciocalteu reagent, Gallic acid reference standard, Sodium nitrite (analytical grade), Aluminum nitrate (analytical grade) (all purchased from Sinopharm Chemical Reagent Co., Ltd.); The experimental water was Wahaha drinking water; Methanol and acetonitrile used in liquid chromatography were chromatographic grade (purchased from Merck); all other reagents were analytical grade.

[0022] 3. Samples: 20 batches of Eucommia ulmoides male flower samples were all collected from the authentic medicinal herb producing area of ​​Zhangjiajie (110°25′E, 29°12′N, altitude 800-1200m), and were harvested during the peak flowering period in March 2024. They were identified as male flowers of Eucommia ulmoides, a plant of the Eucommiaceae family. In addition, 5 batches of Eucommia ulmoides leaf samples (collected from Hanzhong, Shaanxi) and 5 batches of Eucommia ulmoides male flower samples from non-authentic producing areas of Shaanxi were prepared for verification.

[0023] Example 1: Method for constructing UPLC fingerprints of Eucommia ulmoides male flowers and methodological investigation Experimental steps 1. Preparation of test solution: Take about 0.3g of Eucommia ulmoides male flower sample powder that has passed through a No. 3 sieve, accurately weigh it (using a 0.01g balance), place it in a stoppered conical flask, accurately add 20mL of 50% methanol solution, stopper tightly, weigh it, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool to room temperature (25℃), weigh it again, replenish the lost weight with 50% methanol solution, shake well, filter through a 0.45μm organic filter membrane, and collect the filtrate to obtain the test solution.

[0024] 2. Preparation of reference solutions: Accurately weigh appropriate amounts of aucubin, geniposide, chlorogenic acid, geniposide, rutin, isoquercitrin, and quercetin reference standards, add methanol to prepare a reference solution containing 0.5 mg of each component per 1 mL, filter through a 0.22 μm organic filter membrane, and set aside for use.

[0025] 3. UPLC Analysis: A ZORBAX Eclipse Plus C18 column (2.1 mm × 100 mm) was used; column temperature 36℃; flow rate 0.2 mL / min; injection volume 2 μL; mobile phase was 0.1% formic acid aqueous solution (A) - acetonitrile (B), with a gradient elution program of 0–8.5 min, 95%–84% A; 8.5–26 min, 84%–75% A; 26–30 min, 75%–20% A; 30–32 min, 20%–0% A; detection was performed in variable wavelength mode: 0–2.2 min, 240 nm; 2.2–3.2 min, 203 nm; 3.2–11 min, 240 nm; 11–32 min, 254 nm. 2 μL of both the reference solution and the test solution were injected into the HPLC system, and the chromatograms were recorded.

[0026] 4. Methodological Validation Precision test: Take the same batch of Eucommia ulmoides male flower sample (S1) test solution and inject it continuously 6 times under the above chromatographic conditions; Repeatability test: Take 0.3g of powder from the same batch of Eucommia ulmoides male flower sample (S1), accurately weigh it, prepare 6 test solutions in parallel, and determine them according to the above chromatographic conditions; Stability test: The test solution of the same batch of Eucommia ulmoides male flower sample (S1) was injected and measured at 0, 2, 4, 8, 12 and 16 h after preparation.

[0027] Performance verification results Using 19 common chromatographic peaks as evaluation indicators and chlorogenic acid peak No. 5 as a reference, the RSD values ​​of the relative retention times of each chromatographic peak were statistically analyzed, and the results are shown in Table 1 below.

[0028] Table 1. Methodological validation results of Example 1 (RSD, %)

[0029] in conclusion The RSD of each validation item was <2%, indicating that the method is stable and feasible. The precision, repeatability and stability all meet the technical requirements for fingerprint spectral detection of Chinese medicinal materials, and it can be used for UPLC-specific fingerprint spectral analysis of Eucommia ulmoides male flowers.

[0030] Example 2: Fingerprint analysis and common peak identification of 20 batches of Eucommia ulmoides male flower medicinal materials Experimental steps 1. Preparation of test sample: Take 20 batches of Eucommia ulmoides male flower powder and prepare test sample solution according to the method in Example 1; 2. UPLC detection: UPLC detection was performed on 20 batches of samples according to the chromatographic conditions of Example 1, and the chromatograms were recorded; 3. Fingerprint chromatogram establishment: The chromatograms of 20 batches of samples were imported into the 2012 version of the "Similarity Evaluation System for Fingerprint chromatograms of Traditional Chinese Medicine". The median method was used to generate the reference fingerprint chromatogram (R), the common peaks were identified, and the characteristic peaks were identified by comparison with the reference standard. 4. Similarity evaluation: Calculate the similarity between each batch of samples and the control fingerprint spectrum, and statistically analyze the relative retention time of characteristic peaks.

[0031] Performance verification results A total of 19 common peaks were identified by comparing the fingerprint spectrum, of which 7 characteristic peaks were identified by the reference standard; the relative retention times and similarity results of characteristic peaks of 20 batches of samples are shown in Tables 2 and 3 below.

[0032] Table 2. Relative retention times of characteristic peaks in 20 batches of Eucommia ulmoides male flower samples (with chlorogenic acid peak No. 5 as S peak).

[0033] Table 3. Similarity of fingerprint spectra of 20 batches of Eucommia ulmoides male flower samples

[0034] in conclusion The relative retention time fluctuations of the characteristic peaks of the 20 batches of authentic samples were all within ±10%, and the similarity with the control fingerprint spectrum was ≥0.90, which can be used as the core basis for the qualitative identification of Eucommia ulmoides male flowers; among them, peak 19 (quercetin) is an exclusive characteristic peak of authentic producing area samples, and non-authentic producing area samples do not have this peak.

[0035] Example 3: Establishment and Methodological Investigation of HPLC Detection Method for Effective Components in Eucommia Male Flowers Experimental steps 1. Preparation of test solution: Take 0.3g of Eucommia ulmoides male flower sample powder, accurately weigh it, place it in a stoppered conical flask, accurately add 25mL of 50% methanol solution, stopper tightly and weigh, sonicate (250W, 40kHz) for 30 minutes, cool to 25℃, add weight, shake well, filter through a 0.45μm organic filter membrane, and collect the filtrate; 2. Preparation of reference solution: Accurately weigh appropriate amounts of aucubin, genipin, chlorogenic acid, and genipin reference standards, add methanol to prepare a mixed reference solution containing 0.5 mg of each component per 1 mL, filter through a 0.22 μm organic filter membrane, and set aside for later use; 3. HPLC analysis: A C18 column (4.6×250mm, 5μm) was used; column temperature 35℃; flow rate 0.6mL / min; injection volume 10μL; mobile phase was 0.1% formic acid aqueous solution (A)-acetonitrile (B); gradient elution program was 0–20 min, 92% A; 20–25 min, 92%–82% A; 25–50 min, 82%–74% A; detection was performed in variable wavelength mode: 0–10 min at 203nm; 10–50 min at 240nm; 10μL of each of the reference solution and the test solution were injected, and the peak areas were recorded. 4. Methodological validation: Linearity, precision, repeatability, stability, and spike recovery tests were conducted sequentially. Pearson correlation analysis was used for linearity, and the significance level was set at P < 0.05.

[0036] Performance verification results The results of each verification item are shown in Tables 4-8 below.

[0037] Table 4. Examination of linear relationships among components

[0038] Table 5 Precision test results

[0039] Table 6 Repeatability Test Results

[0040] Table 7 Stability Test Results

[0041] Table 8 Results of Spiked Recovery Tests

[0042] Table 9. Results of determination of effective ingredient content (mg / g, dried product) in 20 batches of samples.

[0043] in conclusion The method showed a significant linear relationship (P < 0.001), and its precision, repeatability, stability, and recovery rate all met the requirements of the General Rules, Part IV, of the 2025 edition of the Pharmacopoeia of the People's Republic of China. It can be used for the quantitative detection of four active ingredients in Eucommia ulmoides male flowers. The proposed content limits are the minimum thresholds for achieving the efficacy target, which is scientifically reasonable.

[0044] Example 4: Determination of total polyphenols and total flavonoids in Eucommia ulmoides male flowers Experimental steps 1. Determination of total polyphenols: The Folin-phenol method was used, with gallic acid as a reference. 1 mL of the test solution was accurately measured and placed in a 10 mL volumetric flask. 5 mL of distilled water was added, followed by 1 mL of Folin-phenol reagent. After shaking well, the mixture was allowed to stand for 5 min. 3 mL of 7.5% sodium carbonate solution was added, and the mixture was diluted to the mark with distilled water. The reaction was carried out at 25°C in the dark for 60 min, and the absorbance was measured at 765 nm. A blank control was also performed. The total polyphenol content was calculated according to the standard curve (the core principle of the Folin-phenol method is that polyphenols reduce tungsticotropic acid in Folin reagent to form a blue compound, the absorbance of which is positively correlated with the polyphenol content). 2. Determination of total flavonoids: The aluminum nitrate-sodium nitrite complexation spectrophotometric method was used, with rutin as the reference standard. 1 mL of the test solution was accurately measured and placed in a 10 mL volumetric flask. 4 mL of distilled water was added, followed by 0.3 mL of 5% sodium nitrite solution. The mixture was shaken well and allowed to stand for 6 min. 0.3 mL of 10% aluminum nitrate solution was added, and the mixture was shaken well and allowed to stand for 6 min. 2 mL of 4% sodium hydroxide solution was added, and the mixture was diluted to the mark with distilled water. The reaction was carried out at 25℃ for 15 min, and the absorbance was measured at 510 nm. A blank control was also performed. The total flavonoid content was calculated according to the standard curve. 3. Sample testing: The total polyphenol and total flavonoid content of 20 batches of Eucommia ulmoides male flower samples were determined according to the above method, with each sample being measured in parallel 3 times.

[0045] Performance verification results The test results of 20 batches of samples are shown in Table 10 below.

[0046] Table 10. Total polyphenol and total flavonoid content (%, dried product) of 20 batches of Eucommia ulmoides male flowers

[0047] in conclusion The proposed limits for total polyphenols and total flavonoids can serve as the core quality control basis for the functional components of Eucommia ulmoides male flowers, and are directly related to the antioxidant efficacy of the product.

[0048] Comparative Example 1: Determination of male flowers of Eucommia ulmoides by conventional HPLC fingerprinting method Experimental steps 1. Sample preparation: Take 20 batches of authentic samples from Example 1 + 5 batches of counterfeit Eucommia ulmoides leaves, for a total of 25 samples; 2. Traditional HPLC method detection: The HPLC method described in reference [3] (Zhang Zhi et al., 2022) was adopted. The chromatographic column was a C18 column (4.6×250mm), the mobile phase was methanol-0.1% phosphoric acid aqueous solution, the gradient elution time was 82min, the detection wavelength was 254nm, the test solution was prepared and injected according to the literature method, and the chromatogram was recorded. 3. Statistical analysis: The single-sample detection time, number of common peaks, accuracy rate of counterfeit product identification, and accuracy rate of authenticity identification were statistically analyzed for both methods.

[0049] Performance verification results The performance comparison of the two methods is shown in Table 11 below.

[0050] Table 11 Performance Comparison between UPLC and Traditional HPLC

[0051] in conclusion The UPLC method of this invention is not a simple instrument replacement, but a customized optimization for the characteristics of Eucommia ulmoides male flowers. Its detection efficiency, chemical information coverage and identification accuracy are significantly better than the traditional HPLC method, and the technical effect exceeds the conventional expectations of those skilled in the art.

[0052] Example 5: Verification of the Synergy of the Three-Dimensional Evaluation System Experimental steps 1. Sample Grouping: Ten batches of qualified samples from Example 1 were selected and four groups of tests were set up, with ten samples in each group: Compliant group: UPLC similarity ≥ 0.90, all 4 components ≥ limit, total polyphenols / flavonoids ≥ limit; Comparative Example 2 (Fingerprint Failure Group): UPLC similarity 0.85 (replaced with samples from non-authentic Shaanxi producing areas), all four components and functional indicators met the standards; Comparative Example 3 (Group with Substandard Components): UPLC fingerprint met the standard, aucubin content was 8.2 mg / g (<11.04 mg / g), and other indicators met the standard; Comparative Example 4 (Group with substandard functional indicators): UPLC fingerprint and composition met the standards, and the total polyphenol content was 1.5% (<1.9%). 2. Efficacy testing: DPPH free radical scavenging rate determination: Accurately measure 1 mL of sample solution, add 3 mL of 0.1 mmol / L DPPH ethanol solution, shake well, react at 25℃ in the dark for 30 min, and measure the absorbance at a wavelength of 517 nm. ABTS cation scavenging capacity determination: Mix ABTS solution with an equal volume of 2.45 mmol / L potassium persulfate solution, react at 25℃ in the dark for 12 h, and dilute to an absorbance of 0.70 ± 0.02 (734 nm); accurately measure 0.1 mL of sample solution, add 3.9 mL of diluted ABTS solution, react at 25℃ for 6 min, and measure the absorbance; each sample is measured in triplicate, and statistical analysis is performed using SPSS 26.0 software.

[0053] Performance verification results The antioxidant activity results for each group are shown in Table 12 below.

[0054] Table 12 Results of the Collaborative Verification of the Three-Dimensional Evaluation System

[0055] in conclusion The three-dimensional evaluation system is an organic and synergistic whole. Failure of a single module will lead to a significant reduction in core effectiveness. Its synergistic effect cannot be achieved through a single technical means, and it has outstanding substantive characteristics.

[0056] Example 6: Verification of the correlation between the efficacy of the core active ingredient and the efficacy of the core active ingredient. Experimental steps 1. Component determination: Take 20 batches of samples from Example 3 and simultaneously determine the content of four target components (aucubin, genipin, chlorogenic acid, genipin) and pinoresinol diglucoside (comparative example 5); 2. Efficacy determination: The DPPH free radical scavenging rate and ABTS cation scavenging capacity of 20 batches of samples were determined according to the method in Example 5; 3. Correlation Analysis: The correlation coefficients (R²) between the content of each component and the two antioxidant indicators were calculated using a linear regression model. 2 The significance level was set at P < 0.01.

[0057] Performance verification results The correlation between component content and antioxidant efficacy is shown in Table 13 below.

[0058] Table 13 Correlation between component content and antioxidant efficacy

[0059] in conclusion The four components screened in this invention are the core components of the antioxidant effect of Eucommia ulmoides male flowers. Their content limits are the key thresholds for achieving the desired efficacy. The selection of components is based on sufficient scientific evidence and is not derived through conventional statistical deduction.

[0060] Example 7: Industrial-scale application verification Experimental steps 1. Enterprise cooperation: Two Eucommia ulmoides male flower production enterprises were selected (Enterprise A is a leading local enterprise in Zhangjiajie, and Enterprise B is a processing enterprise in Shaanxi). The method of this invention and the original method of the enterprises (single HPLC quantitative method) were respectively adopted. 2. Sample testing: 100 batches of production samples from each company will be tested, and the batch quality inspection time, batch efficacy fluctuation range, number of adulterated samples identified, and batch quality pass rate will be statistically analyzed. 3. Results Summary: The industrial application effects of the two methods were compared, and the differences in data were analyzed using an independent samples t-test.

[0061] Performance verification results The comparison of industrial application effects is shown in Table 14 below.

[0062] Table 14 Comparison of the application effects of the method of the present invention and the original method of the enterprise

[0063] in conclusion The solution of this invention has strong industrial adaptability, can significantly improve the quality inspection efficiency and quality control capabilities of enterprises, and has outstanding industrial practical value and significant progress.

[0064] Summary of Examples In summary, the basic embodiments of this invention, through complete methodological validation, have demonstrated the stability and reliability of the UPLC-specific fingerprint construction, HPLC multi-index quantification, and total polyphenol and total flavonoid determination methods. The proposed characteristic peak relative retention time range, component content limits, and functional component thresholds can achieve precise control over the basic quality of Eucommia ulmoides male flowers. The newly added defensive experiments further validate the inventiveness and practicality of this invention: Comparative Example 1 shows that the UPLC method of this invention significantly improves detection efficiency, chemical information coverage, and identification accuracy compared to the traditional HPLC method; its optimization effect cannot be achieved by simple instrument replacement. Example 5 confirms that the three-dimensional evaluation system of "UPLC fingerprint qualitative analysis - HPLC component quantification - functional component determination" is an organically synergistic whole; failure of a single module will lead to a significant reduction in core efficacy, breaking through the limitations of single technical means. Example 6 clarifies that four core components are highly correlated with the antioxidant efficacy of Eucommia ulmoides male flowers, and their content limits are key thresholds for efficacy achievement, with sufficient scientific basis for component selection. Example 7 demonstrates that the solution of this invention is adaptable to industrial-scale applications and can significantly improve enterprise quality inspection efficiency and quality control capabilities. The overall experimental system not only ensures the repeatability of the technical solution, but also forms a closed-loop response to questions about the combination of conventional technologies, providing sufficient data support for the inventiveness and practicality of the patent.

[0065] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A comprehensive evaluation method for the quality of Eucommia ulmoides male flowers, characterized in that, Includes the following steps: (1) Preparation of test solution: Take the Eucommia ulmoides male flower powder that has passed through a No. 3 sieve, weigh it accurately and place it in a stoppered conical flask, add 50%-70% methanol aqueous solution and seal it tightly, weigh it, extract it by ultrasonication and cool it, weigh it again and make up the weight lost, shake it well and filter it to obtain the test solution. (2) Construction and evaluation of UPLC-specific fingerprint spectrum: The test sample solution and the reference solution containing aucubin, genipin, chlorogenic acid, genipin, rutin, isoquercitrin and quercetin were analyzed by UPLC to generate a reference fingerprint spectrum containing 19 common peaks. The similarity between the test sample and the reference fingerprint spectrum was ≥0.90, and the relative retention time of each characteristic peak fluctuated within the specified value ±10%. (3) HPLC multi-index efficacy component quantification: The test solution and the mixed reference solution were analyzed by HPLC, and the contents of aucubin, geniposide, chlorogenic acid and geniposide were determined by external standard method; (4) Determination of total polyphenols and total flavonoids: The total polyphenols and total flavonoids in the test solution were determined by the Folin-phenol method and the aluminum nitrate-sodium nitrite complexation spectrophotometric method, respectively. (5) Three-dimensional collaborative judgment: If the detection results of steps (2)-(4) all meet the preset technical limits, then it is judged to be Eucommia male flowers that meet the preset quality and efficacy standards.

2. The method according to claim 1, characterized in that, In step (1), the sample weight of the Eucommia ulmoides male flower powder is 0.3g, the volume fraction of the methanol aqueous solution is 50%, the material-liquid ratio is 1:20 (mass-volume ratio), the ultrasonic extraction conditions are 250W power, 40kHz frequency, and 30min extraction time; the weighing is done using a 0.01% electronic balance, and the same batch of 50% methanol aqueous solution is used to make up for the weight loss.

3. The method according to claim 1, characterized in that, In step (2), the chromatographic conditions for the UPLC analysis are as follows: ZORBAX Eclipse Plus C18 column (2.1 mm × 100 mm), column temperature 36 ℃, flow rate 0.2 mL / min, injection volume 2 μL; mobile phase is 0.1% formic acid aqueous solution (A) - acetonitrile (B), gradient elution program is 0 to 8.5 min, 95% to 84% A; 8.5–26 min, 84%–75% A; 26–30 min, 75%–20% A; 30–32 min, 20%–0% A; Detection is in variable wavelength mode: 0–2.2 min at 240 nm; 2.2–3.2 min at 203 nm; 3.2–11 min at 240 nm; 11–32 min at 254 nm, wavelength switching is automatically controlled by the chromatograph.

4. The method according to claim 1 or 3, characterized in that, In step (2), the chromatographic peak of chlorogenic acid No. 5 is used as the reference S peak, and the relative retention time of the characteristic peaks is specified as 0.368 (peak 1), 0.565 (peak 2), 0.900 (peak 3), 0.961 (peak 4), 1.069 (peak 6), 1.122 (peak 7), 1.210 (peak 8), 1.248 (peak 9), 1.488 (peak 10), 1.630 (peak 11), 1.660 (peak 12), 1.736 (peak 13), 1.834 (peak 14), 1.955 (peak 15), 2.179 (peak 16), 2.336 (peak 17), 2.699 (peak 18), and 3.693 (peak 19).

5. The method according to claim 1, characterized in that, In step (3), the chromatographic conditions for the HPLC analysis are as follows: a C18 column (4.6×250mm, 5μm) is used, the column temperature is 35℃, the flow rate is 0.6mL / min, and the injection volume is 10μL; the mobile phase is 0.1% formic acid aqueous solution (A)-acetonitrile (B), the gradient elution program is 0-20min, 92%A; 20-25min, 92%-82%A; 25-50min, 82%-74%A; the detection is in variable wavelength mode: 0-10min is 203nm; 10-50min is 240nm, and the mobile phase is mixed using a high-pressure binary pump.

6. The method according to claim 1 or 5, characterized in that, In step (3), based on the dried product, the content of aucubin in Eucommia ulmoides male flowers is ≥11.04mg / g, the content of genipin is ≥13.24mg / g, the content of chlorogenic acid is ≥3.78mg / g, and the content of genipin is ≥0.79mg / g.

7. The method according to claim 1, characterized in that, In step (4), the total polyphenol content is ≥1.9% calculated as gallic acid, and the total flavonoid content is ≥2.5% calculated as rutin.

8. The method according to claim 1, characterized in that, In step (4), the detection wavelength of the Folin-phenol method is 765 nm, and the detection wavelength of the aluminum nitrate-sodium nitrite complex spectrophotometric method is 510 nm; the RSD of the parallel experiments of the two methods is ≤2.0%.

9. The method according to claim 1, characterized in that, In step (2), the concentrations of aucubin, geniposide, chlorogenic acid, geniposide, rutin, isoquercitrin, and quercetin in the reference solution are all 0.5 mg / mL, and the purity of the reference standards is ≥98%.

10. An application of the comprehensive quality evaluation method for Eucommia ulmoides male flowers as described in any one of claims 1-9, characterized in that, It is applied to the identification of genuine and counterfeit Eucommia ulmoides male flowers, determination of authenticity, batch quality consistency control, or prediction of efficacy potential. Among them, the determination of authenticity is based on the fingerprint spectrum of samples from the Zhangjiajie authentic producing area (109°40′-111°20′E, 28°52′-29°48′N). If the similarity is <0.85, it is determined to be a sample from a non-authentic producing area.

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