A quality evaluation method for xanthium seed medicinal materials and application thereof

Abstract

The application discloses a quality evaluation method of Xanthium sibiricum and application thereof, and belongs to the technical field of traditional Chinese medicinal material quality evaluation and detection. The application first proposes quercetin-3-O-glucuronide as a quality marker of Xanthium sibiricum, and provides application of the quality marker in preparation of a detection reagent for evaluating the quality of Xanthium sibiricum. The quality evaluation method takes quercetin-3-O-glucuronide as a detection index, and adopts high performance liquid chromatography for determination. The application solves the technical problem of insufficient specificity of chlorogenic acid as an index in the current standard, and quercetin-3-O-glucuronide has higher species correlation and better chemical stability in Xanthium sibiricum, and can accurately reflect the internal quality of medicinal materials. The method has high precision and good reproducibility, and is suitable for quality evaluation of Xanthium sibiricum, medicinal slices and formula granules.

Description

Technical Field

[0001] This invention belongs to the field of quality evaluation and testing technology of traditional Chinese medicinal materials, specifically involving a method for evaluating the quality of Xanthium sibiricum medicinal material using quercetin-3-O-glucuronide as a quality marker, and the application of this quality marker in the preparation of related testing reagents. Background Technology

[0002] Xanthium sibiricum is a plant belonging to the Asteraceae family (Xanthium sibiricum). Xanthium sibiricum The dried, mature fruit of Xanthium sibiricum (Paperr.) with its involucre is a commonly used pungent and warm exterior-releasing herb in Traditional Chinese Medicine. It has the effects of dispelling wind-cold, clearing nasal passages, and eliminating wind-dampness, and is widely used to treat conditions such as wind-cold headache, nasal congestion and runny nose, sinusitis, urticaria and itching, and dampness-induced arthralgia and contracture. Modern pharmacological research shows that Xanthium sibiricum also possesses various biological activities, including anti-inflammatory, analgesic, antioxidant, antiviral, hypoglycemic, and antitumor effects, and its clinical application value is increasingly recognized.

[0003] The quality of Xanthium sibiricum, a medicinal herb, directly affects the safety and efficacy of clinical medication. Establishing a scientific, accurate, and specific quality evaluation method is crucial to ensuring its quality control. The current 2025 edition of the *Pharmacopoeia of the People's Republic of China* uses chlorogenic acid content as a quantitative control indicator and carboxy-atractylodes glycoside content as a toxicity limit control indicator for Xanthium sibiricum. However, chlorogenic acid belongs to the phenylpropanoid phenolic acid compound class and is widely distributed in dozens of plants such as honeysuckle, chrysanthemum, dandelion, eucommia leaves, and coffee beans. It is not a characteristic component or specific indicator of Xanthium sibiricum. Using chlorogenic acid as a quantitative quality control indicator can only reflect the content level of a common phenolic acid component in the medicinal material, and cannot specifically reflect the authenticity and intrinsic quality of Xanthium sibiricum. In compound preparations containing Xanthium sibiricum or complex sample systems, using chlorogenic acid as an indicator for quality evaluation is more susceptible to interference from other medicinal components, leading to insufficient specificity and accuracy of the evaluation results.

[0004] To overcome the aforementioned shortcomings, alternative quality assessment strategies are being explored in this field. For example, some literature reports the use of ultraviolet spectrophotometry to determine the total flavonoid content in Xanthium sibiricum. However, this method can only provide an overall quantitative analysis of flavonoid components and cannot perform precise quantitative analysis of a specific characteristic flavonoid monomer, nor can it distinguish between structurally similar but differently active flavonoid compounds. Therefore, it still has significant limitations in terms of specificity and accuracy. Thus, finding a marker component in Xanthium sibiricum that possesses high specificity and chemical stability and can truly reflect the intrinsic quality of the medicinal material, and establishing a corresponding quality assessment method based on this component, is a pressing technical problem that needs to be solved in this field.

[0005] Quercetin-3-O-glucuronide (Miquelianin) belongs to the flavonol glycoside class and is one of the main flavonoid components that can be reliably detected in Xanthium sibiricum, and can be obtained from Xanthium sibiricum with a high extraction rate. However, in existing technical understanding, quercetin-3-O-glucuronide is only considered as an ordinary member among the many flavonoid components of Xanthium sibiricum, and its intrinsic relationship with the quality of Xanthium sibiricum, especially its potential value as a specific quality marker, has not been revealed. There is a lack of systematic research in this field on methods for determining the content of quercetin-3-O-glucuronide in Xanthium sibiricum, and there are no reports of its application as a quality marker in the quality evaluation of Xanthium sibiricum. This technological gap has prevented the field from using this component to achieve a specific and accurate evaluation of the quality of Xanthium sibiricum for a long time. Summary of the Invention

[0006] In view of this, the purpose of this invention is to overcome the deficiency of insufficient specificity in the current quality standards for Xanthium sibiricum, and to propose a quality evaluation strategy with quercetin-3-O-glucuronide as the core indicator. Furthermore, this invention provides a method for determining the content of quercetin-3-O-glucuronide in Xanthium sibiricum and its application in quality evaluation, so as to achieve accurate judgment of the quality of Xanthium sibiricum.

[0007] The technical solution of the present invention is achieved in the following ways: This invention provides the application of quercetin-3-O-glucuronide in the preparation of a diagnostic reagent for evaluating the quality of Xanthium sibiricum. This invention is the first to propose and verify the application value and feasibility of quercetin-3-O-glucuronide as a quality marker for Xanthium sibiricum. Compared to chlorogenic acid used in current standards, the distribution of quercetin-3-O-glucuronide in Xanthium sibiricum exhibits higher species specificity. Chlorogenic acid is widely distributed in the plant kingdom, while quercetin-3-O-glucuronide, as a specific flavonol glycoside, has a content level in Xanthium sibiricum that is closely related to the overall quality of the medicinal material, thus more accurately reflecting the intrinsic quality of Xanthium sibiricum. Meanwhile, quercetin-3-O-glucuronide, as a flavonol glycoside, possesses phenolic hydroxyl groups and glycosidic bonds in its chemical structure. Its glycosylation modification gives it better chemical stability than the free aglycone under normal storage and use conditions, making it less susceptible to oxidative degradation and more accurately reflecting the actual quality of the medicinal material during circulation and use. These characteristics give quercetin-3-O-glucuronide significant theoretical advantages and practical value as a quality marker for Xanthium sibiricum.

[0008] In some embodiments, the aforementioned evaluation of the quality of Xanthium sibiricum medicinal material refers to determining the quality of the medicinal material. This quality determination can be accomplished by quantitatively measuring the content of quercetin-3-O-glucuronide and comparing it with a preset quality standard threshold.

[0009] In some implementations, the above-mentioned detection reagents are detected using high-performance liquid chromatography (HPLC). HPLC is the most mature and widely used analytical technique platform for the determination of traditional Chinese medicine content, possessing excellent separation capabilities, quantitative accuracy, and widespread instrument availability. Combining quercetin-3-O-glucuronide as an indicator with HPLC balances the specificity of quality evaluation with the method's generalizability, facilitating its widespread application in pharmaceutical manufacturing enterprises and testing institutions.

[0010] In some embodiments, the chromatographic conditions of the above-mentioned high-performance liquid chromatography (HPLC) method include: using octadecylsilane-bonded silica gel as the packing material, acetonitrile as mobile phase A, and 0.4% phosphoric acid aqueous solution as mobile phase B, performing gradient elution, and a detection wavelength of 356 nm. The present invention selects 356 nm as the detection wavelength based on a comprehensive determination of the corresponding chromatographic peaks in the quercetin-3-O-glucuronide reference standard and the Xanthium sibiricum sample. Scanning with a DAD detector in the range of 190–400 nm, the maximum absorption wavelength of the quercetin-3-O-glucuronide reference standard is 355.89 nm, and the maximum absorption wavelength of the corresponding chromatographic peak in the Xanthium sibiricum sample is 352.94 nm, with a difference of only about 3 nm. Selecting the maximum absorption wavelength of the reference standard as the detection wavelength ensures consistent traceability of the quantitative standard, optimal response of the standard curve, and stable system applicability and method reproducibility. The acetonitrile-0.4% phosphoric acid aqueous solution system was selected as the mobile phase. Compared with the methanol-water system and the acetonitrile-low concentration phosphoric acid system, it can more effectively suppress chromatographic peak tailing, improve the separation of quercetin-3-O-glucuronide from adjacent impurity peaks, and obtain chromatographic peaks with good symmetry.

[0011] In some embodiments, the gradient elution program is as follows: within 0-20 minutes, the volume percentage of mobile phase A linearly increases from 10% to 55%, while the volume percentage of mobile phase B linearly decreases from 90% to 45%. This gradient program, employing a linearly increasing acetonitrile ratio, enables the stepwise elution and separation of multiple components with significant polarity differences within 20 minutes. The initial lower acetonitrile ratio facilitates the elution of more polar impurities, avoiding solvent peak interference near the dead time. As the acetonitrile ratio gradually increases to 55%, quercetin-3-O-glucuronide is eluted from the column at an appropriate elution intensity, achieving baseline separation from adjacent peaks, thus balancing analytical efficiency with resolution.

[0012] In some embodiments, the flow rate of the above-described high-performance liquid chromatography (HPLC) is 1.0 ml / min, and the column temperature is 35°C. A flow rate of 1.0 ml / min provides suitable column pressure on a conventional 4.6 mm inner diameter column, ensuring separation efficiency while maintaining system pressure stability and safety. A column temperature of 35°C, slightly higher than room temperature, reduces mobile phase viscosity, improves mass transfer efficiency, and avoids potential damage to the stationary phase or thermal degradation of the analyte due to excessively high temperatures. Systematic investigations at four temperature levels (25°C, 30°C, 35°C, and 40°C) and three flow rate levels (0.9 ml / min, 1.0 ml / min, and 1.1 ml / min) showed that the separation efficiency and peak shape symmetry of the test sample were optimal under these conditions.

[0013] In some embodiments, the content of quercetin-3-O-glucuronide in the aforementioned Xanthium sibiricum medicinal material shall not be less than 0.04% on a dried basis. This content limit is determined based on measured data from 15 batches of Xanthium sibiricum medicinal materials from different origins, and after statistical analysis in accordance with relevant guidelines in the Chinese Pharmacopoeia. The average content of the measured content from the 15 batches of samples is used as a benchmark, and 80% of this average is taken as the recommended limit standard. This comprehensively considers the normal distribution law of medicinal material content determination and the actual testing work, taking into account both the natural fluctuation range of quercetin-3-O-glucuronide content in Xanthium sibiricum medicinal materials from different origins and maintaining a reasonable safety margin for medicinal material quality control. The setting of this limit comprehensively considers the scientific nature and practical operability of the standard, and can effectively identify batches of medicinal materials with quality defects.

[0014] This invention also provides a quality evaluation method for Xanthium sibiricum, which uses quercetin-3-O-glucuronide as the detection index and is determined by high performance liquid chromatography, including the following steps: (1) Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as the packing material; acetonitrile was used as mobile phase A and 0.4% phosphoric acid aqueous solution was used as mobile phase B for gradient elution; the detection wavelength was 356 nm; the theoretical plate number calculated based on the quercetin-3-O-glucuronide peak should not be less than 5000; (2) Preparation of reference solution: Take an appropriate amount of quercetin-3-O-glucuronide reference standard, accurately weigh it, and add 50% ethanol to prepare a solution containing 20 μg per ml. (3) Preparation of test solution: Take about 0.5g of Xanthium sibiricum powder, weigh it accurately, put it in a stoppered conical flask, add 50ml of 50% ethanol accurately, weigh it, reflux and extract for 60 minutes, cool it, weigh it again, make up the lost weight with 50% ethanol, shake well, filter it, and take the filtrate to obtain the test solution. (4) Determination: Accurately pipette 10 μl of the reference solution and the test solution into the high performance liquid chromatograph and determine the result.

[0015] In the preparation method of the above-mentioned test solution, all key parameters were systematically optimized. Regarding the extraction method, the effects of ultrasonic extraction and reflux extraction were compared. The content of quercetin-3-O-glucuronide obtained by reflux extraction was significantly higher than that obtained by ultrasonic extraction. Regarding the extraction solvent, the extraction efficiency of nine solvents—water, 30% ethanol, 50% ethanol, 70% ethanol, ethanol, 30% methanol, 50% methanol, 70% methanol, and methanol—was systematically compared. The results showed that 50% ethanol had the highest extraction efficiency. The study on the amount of extraction solvent showed that a solvent volume of 50 ml was sufficient to achieve stable extraction results while ensuring complete extraction. The study on extraction time showed that reflux for 60 minutes was sufficient to ensure complete extraction, and further extending the extraction time did not significantly increase the content. The above optimization process ensured that the preparation of the test solution was both highly efficient and reproducible.

[0016] In some embodiments, in the above quality evaluation method, the gradient elution program in step (1) is as follows: within 0-20 minutes, the volume ratio of mobile phase A linearly increases from 10% to 55%, and the volume ratio of mobile phase B linearly decreases from 90% to 45%; the flow rate is 1.0 ml / min, and the column temperature is 35℃. By combining the above-mentioned optimized chromatographic conditions, the theoretical plate number of the quercetin-3-O-glucuronide peak can be no less than 5000, the peak shape is symmetrical, and the resolution with adjacent impurity peaks meets the requirements for quantitative analysis.

[0017] In some embodiments, in the above-mentioned quality evaluation method, when the measured quercetin-3-O-glucuronide content is less than 0.04% on a dried basis, the Xanthium sibiricum medicinal material is deemed unqualified. By directly linking the content determination results to a preset limit standard, this quality evaluation method can output a clear pass / fail judgment conclusion while providing quantitative data, eliminating the need for operators to perform separate calculations or comparisons, thus improving the standardization and operability of the quality evaluation process.

[0018] The present invention has the following advantages over the prior art: This invention breaks through the conventional approach of using widely distributed phenolic acids such as chlorogenic acid as quality control indicators for Xanthium sibiricum. It proposes and verifies for the first time the application value and feasibility of quercetin-3-O-glucuronide as a quality marker for Xanthium sibiricum. Compared with the current pharmacopoeia standard which uses chlorogenic acid as a quantitative indicator, quercetin-3-O-glucuronide exhibits significantly higher species relevance in Xanthium sibiricum. Its content level can more sensitively reflect the intrinsic quality differences of Xanthium sibiricum from different origins and batches, solving the long-standing key technical bottleneck of "non-specific indicator components" in Xanthium sibiricum quality evaluation. Simultaneously, quercetin-3-O-glucuronide has superior chemical stability compared to chlorogenic acid, and is less prone to degradation during storage and sample processing. This allows the quality evaluation method based on it to more accurately and realistically reflect the intrinsic quality of the medicinal material, providing a more reliable guarantee for the safety and efficacy of clinical use. The quality evaluation method for Xanthium sibiricum based on high performance liquid chromatography (HPLC) has been established based on this methodological verification. It has the characteristics of high precision, good reproducibility, excellent recovery rate, and stability of the test solution within 24 hours. It is also easy to operate and accurate, making it convenient to promote and apply in pharmaceutical manufacturing enterprises and quality inspection institutions. It is expected to provide strong technical support for the improvement and standardization of Xanthium sibiricum quality standards. Attached Figure Description

[0019] 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.

[0020] Figure 1 HPLC localization chromatograms of quercetin-3-O-glucuronide reference standard and Xanthium sibiricum test sample; Figure 2 Full-wavelength scan spectrum of quercetin-3-O-glucuronide reference standard; Figure 3 Full-wavelength scanning spectrum of Xanthium sibiricum medicinal material for testing; Figure 4 The standard curve of quercetin-3-O-glucuronide; Figure 5 HPLC chromatogram of Xanthium sibiricum (origin / source: Tai'an City, Shandong Province); Figure 6 HPLC chromatogram of Xanthium sibiricum (origin / source: Chifeng City, Inner Mongolia Autonomous Region); Figure 7 HPLC chromatogram of Xanthium sibiricum (origin / source: Qichun County, Huanggang City, Hubei Province); Figure 8 HPLC chromatogram of stir-fried Xanthium sibiricum slices; Figure 9 The image shows the HPLC chromatogram of the stir-fried cocklebur granules. Detailed Implementation

[0021] 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.

[0022] Unless otherwise specified, the instruments and reagents used in the following examples are as follows: The electronic balances are Sartorius XPR2 (parts per million) and Mettler Toledo ME204 (parts per ten thousand). The high performance liquid chromatograph was a Thermo Fisher UltiMate 3000 (equipped with a DAD detector). Quercetin-3-O-glucuronide reference standard was purchased from Shanghai Ronghe Pharmaceutical Technology Co., Ltd. (batch number 220623, content 98.54%). Chlorogenic acid reference standard was purchased from the National Institutes for Food and Drug Control (batch number 110753-202018, content 96.1%). Acetonitrile was of chromatographic grade (Fisher chemical). Phosphoric acid was chromatographically pure (Maclean's grade). Both methanol and ethanol were of analytical grade (Sinopharm Chemical Reagent Co., Ltd.). The water is ultrapure water.

[0023] I. Determination of Detection Indicators and Detection Wavelength Xanthium sibiricum is often used in clinical practice and in the production of traditional Chinese medicine preparations, primarily in the form of decoction. To simulate actual medication use and preliminarily assess the applicability of the indicator components, 0.5 g of Xanthium sibiricum powder was accurately weighed and placed in a stoppered conical flask. 25 ml of water was added, and the mixture was weighed again. The flask was decocted for 1 hour, cooled, and weighed once more. The lost weight was replenished with water, and the mixture was shaken well. The solution was filtered through a 0.45 μm filter membrane, and the filtrate was used as the test solution. Simultaneously, an appropriate amount of quercetin-3-O-glucuronide reference standard was dissolved and diluted in 50% ethanol to prepare a reference solution containing approximately 20 μg per ml.

[0024] The two solutions were injected into a high-performance liquid chromatograph using a ZORBAX Eclipse Plus-C18 column (4.6×250 mm, 5 μm). Gradient elution was performed using acetonitrile-0.4% phosphoric acid aqueous solution as the mobile phase. A DAD detector was used to perform full-wavelength scanning in the wavelength range of 190~400 nm, and the chromatograms and ultraviolet absorption spectra were recorded.

[0025] The results showed that the test solution exhibited a corresponding chromatographic peak at the retention time of the quercetin-3-O-glucuronide reference standard, and the UV absorption curves of the test solution and the reference standard were consistent. Peak purity testing revealed that the peak purity values ​​in the test solution were both greater than 0.99, indicating that under these chromatographic conditions, the quercetin-3-O-glucuronide peak was not interfered with by other impurities, demonstrating good detection specificity. The full-wavelength scan chromatogram of the reference standard showed a maximum absorption wavelength of 355.89 nm, while the corresponding peak in the test solution had a maximum absorption wavelength of 352.94 nm, a difference of approximately 3 nm. The absorption characteristics were highly consistent, confirming the attribution of the chromatographic peak in the test solution.

[0026] Based on the above analysis results, this invention selects quercetin-3-O-glucuronide as the quality evaluation index component of Xanthium sibiricum. The selection of the detection wavelength comprehensively considers the consistency of the maximum absorption wavelength of the reference standard and the test sample, as well as the traceability requirements of quantitative analysis. To ensure the consistency of the traceability link between the quantitative standard and the reference standard, the optimal response of the standard curve, and the stability of system applicability and method reproducibility, the maximum absorption wavelength of the reference standard, 356 nm, is selected as the detection wavelength.

[0027] ( Figure 1 HPLC localization chromatograms of quercetin-3-O-glucuronide reference standard and Xanthium sibiricum test sample; Figure 2 Full-wavelength scan spectrum of quercetin-3-O-glucuronide reference standard; Figure 3 (Full wavelength scanning spectrum of Xanthium sibiricum medicinal material test sample) II. Optimization of chromatographic conditions 1. Selection of mobile phase system The separation effects of four mobile phase systems—acetonitrile-water, acetonitrile-0.1% phosphoric acid aqueous solution, acetonitrile-0.4% phosphoric acid aqueous solution, and methanol-water—on the Xanthium sibiricum sample were investigated. The same sample solution was used for analysis, and the symmetry, theoretical plate number, and resolution with adjacent impurity peaks of quercetin-3-O-glucuronide were compared.

[0028] The results showed that when methanol-water was used as the mobile phase, the chromatographic peaks broadened significantly, but the resolution was poor. When acetonitrile-water was used as the mobile phase, peak tailing occurred. When acetonitrile-0.1% phosphoric acid aqueous solution was used as the mobile phase, the peak shape improved somewhat but was still not ideal. When acetonitrile-0.4% phosphoric acid aqueous solution was used as the mobile phase, the peak symmetry was significantly improved, the peak shape was sharp, the theoretical plate number was high, and baseline separation from adjacent impurity peaks was achieved. The addition of phosphoric acid effectively suppressed peak tailing and improved peak symmetry, with a concentration of 0.4% being optimal. After comprehensive comparison, a mobile phase system with acetonitrile as mobile phase A and 0.4% phosphoric acid aqueous solution as mobile phase B was selected.

[0029] The gradient elution program was optimized through multiple experiments and finally determined to be: 0-20 minutes, with mobile phase A linearly increasing from 10% to 55%, and mobile phase B linearly decreasing from 90% to 45%. Under this gradient condition, impurities are eluted first, and quercetin-3-O-glucuronide elutes at an appropriate elution intensity, achieving optimal separation. The analysis time is 20 minutes, balancing separation effect and detection efficiency. The gradient program is shown in the table below:

[0030] 2. Selection of column temperature and flow rate The effects of column temperatures of 25℃, 30℃, 35℃, and 40℃, and mobile phase flow rates of 0.9 ml / min, 1.0 ml / min, and 1.1 ml / min on the separation efficiency were investigated. The symmetry factor, theoretical plate number, and resolution of the quercetin-3-O-glucuronide peak were used as the main evaluation indicators.

[0031] The results showed that the chromatographic peak symmetry was optimal and the theoretical plate number was highest at a column temperature of 35℃. At column temperatures of 25℃ and 30℃, the mobile phase viscosity was higher, resulting in slightly lower mass transfer efficiency and a decrease in the theoretical plate number. At a column temperature of 40℃, the elution time shifted earlier, and the resolution with adjacent impurity peaks decreased slightly. A flow rate of 1.0 ml / min provided the optimal balance between column pressure and separation performance and system stability. A flow rate of 0.9 ml / min prolonged the analysis time, while a flow rate of 1.1 ml / min increased the column pressure, placing higher demands on the chromatographic system. Therefore, a column temperature of 35℃ and a flow rate of 1.0 ml / min were selected as the optimal chromatographic conditions.

[0032] Based on the optimization results above, the final chromatographic conditions are determined as follows: a column packed with octadecylsilane-bonded silica gel (4.6 × 250 mm, 5 μm); acetonitrile as mobile phase A and 0.4% phosphoric acid aqueous solution as mobile phase B, eluted according to the above gradient program; detection wavelength of 356 nm; flow rate of 1.0 ml / min; column temperature of 35℃. The theoretical plate number, calculated based on the quercetin-3-O-glucuronide peak, should be no less than 5000.

[0033] III. Optimization of the preparation method of the test sample solution 1. Selection of extraction method The extraction efficiency of ultrasonic extraction and reflux extraction was investigated separately. Approximately 0.5 g of Xanthium sibiricum powder (passed through a No. 3 sieve), in two portions, was accurately weighed and placed in stoppered conical flasks. 25 ml of 50% methanol was accurately added to each flask, and the weight was verified. One portion was ultrasonically treated (300 W, 40 kHz) for 40 minutes, while the other portion was extracted by reflux for 40 minutes. Both flasks were cooled, weighed again, and the weight loss was replenished with 50% methanol. The mixture was shaken well, filtered, and the filtrate was injected for analysis.

[0034] The results showed that the content of quercetin-3-O-glucuronide in the test sample obtained by reflux extraction (0.4255 mg / g) was significantly higher than that obtained by ultrasonic extraction (0.3794 mg / g), and the reproducibility of ultrasonic extraction was poor (RSD 8.10%). Reflux extraction, through continuous heating and solvent reflux circulation, can more effectively disrupt the cell structure of Xanthium sibiricum, promote the full dissolution of the target components, and achieve higher and more stable extraction efficiency. Therefore, reflux extraction was chosen as the method for preparing the test sample solution.

[0035] 2. Selection of extraction solvent The extraction efficiencies of nine solvents—water, 30% ethanol, 50% ethanol, 70% ethanol, anhydrous ethanol, 30% methanol, 50% methanol, 70% methanol, and anhydrous methanol—were investigated. Approximately 0.5 g of Xanthium sibiricum powder was accurately weighed into nine portions and placed in stoppered conical flasks. 25 ml of each of the nine solvents was accurately added to each flask, and the portions were weighed. The flasks were sonicated for 40 minutes, cooled, the weight loss was replenished, the mixture was shaken well, filtered, and the filtrate was used for analysis.

[0036] The results showed that in the ethanol system, the highest content of quercetin-3-O-glucuronide (0.4082 mg / g) was obtained when 50% ethanol was used as the extraction solvent. The extraction efficiency, from highest to lowest, was 50% ethanol > 30% ethanol > 70% ethanol > water > anhydrous ethanol. In the methanol system, the highest content (0.3794 mg / g) was obtained when 50% methanol was used as the extraction solvent. Comparing the extraction efficiencies of 50% ethanol and 50% methanol, 50% ethanol was superior. Quercetin-3-O-glucuronide is a moderately polar flavonol glycoside, and the polarity of 50% ethanol best matches it, which is conducive to the full dissolution of the target component. Considering extraction efficiency, solvent safety, and environmental friendliness, 50% ethanol was chosen as the extraction solvent.

[0037] 3. Selection of solvent dosage The extraction effects of three solvent volumes (25 ml, 50 ml, and 100 ml) were investigated. Approximately 0.5 g of Xanthium sibiricum powder was accurately weighed into three portions, and different volumes of 50% ethanol were precisely added. The mixtures were then refluxed for 40 minutes. The results showed that the RSD value of quercetin-3-O-glucuronide content in the test solution was 1.78% under all three solvent volumes, which was less than the reproducibility requirement (RSD < 6%), indicating that the extraction effect met the requirements. To balance extraction efficiency, ease of operation, and experimental cost, 50 ml was selected as the optimal solvent volume.

[0038] 4. Selection of extraction time The extraction effects at three reflux times (40 minutes, 60 minutes, and 90 minutes) were investigated. Approximately 0.5 g of Xanthium sibiricum powder was accurately weighed, and an appropriate amount of 50% ethanol was added for each extraction. The samples were then refluxed for different times. The results showed that the RSD of quercetin-3-O-glucuronide content was 2.10% at all three extraction times, which was lower than the reproducibility requirement (RSD < 4%). The content increased slightly after 60 minutes of reflux compared to 40 minutes; further extension to 90 minutes resulted in a more gradual increase in content. To save time while ensuring sufficient extraction, 60 minutes was chosen as the extraction time.

[0039] Based on the above optimization results, the preparation method of the test solution is determined as follows: Take about 0.5 g of Xanthium sibiricum powder (passed through a No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 50 ml of 50% ethanol, weigh it, heat and reflux for 60 minutes, cool it, weigh it again, replenish the lost weight with 50% ethanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0040] IV. Methodological Validation In accordance with the requirements of the "Guiding Principles for Validation of Analytical Methods" in Part IV of the Chinese Pharmacopoeia 9101, the established content determination method was systematically validated.

[0041] The powder of Xanthium sibiricum from the following 15 batches of samples was tested and found to contain quercetin-3-O-glucuronide at a content of 0.4606 mg / g, which was used for subsequent verification tests.

[0042] 1. Examination of linear relationships Accurately weigh 13.005 mg of quercetin-3-O-glucuronide reference standard, place it in a 100 ml volumetric flask, dissolve and dilute to the mark with 50% ethanol, and shake well to obtain a reference standard stock solution with a concentration of 128.1513 mg / L. Accurately measure an appropriate amount of the stock solution and serially dilute it with 50% ethanol to prepare six reference standard solutions of different concentrations. Accurately inject 10 μl of each solution into the high-performance liquid chromatograph (HPLC) and record the peak area. Perform linear regression analysis with concentration on the x-axis and peak area on the y-axis, obtaining the regression equation Y = 0.2438X - 0.0094, and the correlation coefficient R. 2 =0.9997.

[0043] The results showed that the quercetin-3-O-glucuronide reference standard exhibited good linearity in the concentration range of 2.5630–128.1513 mg / L, with a correlation coefficient greater than 0.999, meeting the requirements for quantitative analysis.

[0044] ( Figure 4 (Standard curve graph) 2. Repeatability test Six portions of 0.5 g of Xanthium sibiricum powder from the same batch were accurately weighed and prepared in parallel according to the test solution preparation method. Each sample was then injected for analysis. The RSD value of quercetin-3-O-glucuronide content in the six test solutions was 1.24% (n=6), less than 3%, indicating good repeatability of the method.

[0045] 3. Precision test Take the same sample solution from the repeatability test and inject it six times consecutively, recording the peak area. The RSD value of the peak area of ​​the six injections is 0.58%, which is less than 3%, indicating that the instrument system has good precision.

[0046] 4. Stability test Take the same sample solution from the repeatability test, place it at room temperature, and inject it for measurement at 0, 2, 4, 8, 12, and 24 hours after preparation. The RSD value of the peak area at each time point within 24 hours is 0.94%, which is less than 6%, indicating that the sample solution has good stability at room temperature within 24 hours.

[0047] 5. Accuracy test (spiking recovery rate) Take approximately 0.25 g of Xanthium sibiricum powder with a known content (quercetin-3-O-glucuronide content is 0.4606 mg / g), make 6 portions, accurately weigh them, and accurately add 1 ml of quercetin-3-O-glucuronide reference solution with a concentration of 128.1513 mg / L (0.1282 mg spiked) to each portion. Prepare 6 spiked test solutions according to the test solution preparation method, inject them for determination, and calculate the recovery rate.

[0048] The recoveries of the six spiked test solutions were 94.86%, 99.45%, 98.27%, 94.63%, 95.43%, and 96.58%, respectively, with an average recovery of 96.54% and an RSD of 2.03%. All spiked recoveries were within the 90%–108% standard range specified in the Chinese Pharmacopoeia, and the RSD was less than 6%, indicating good accuracy of the method.

[0049] 6. System suitability and column durability The same test solution was analyzed using three different brands and models of C18 columns. Column 1: ZORBAX Eclipse Plus-C18 (4.6×250 mm, 5 μm, Agilent); Column 2: Agilent 5 TC-C18(2) (4.6×250 mm, 5 μm, Agilent); Column 3: Diamonsil Plus C18 (4.6×250 mm, 5 μm, Dima).

[0050] The theoretical plate numbers for the determination of quercetin-3-O-glucuronide using three different chromatographic columns were 61,882, 105,374, and 51,924, respectively, all significantly higher than the minimum requirement for system suitability (5,000). The RSD of the determination results was 1.87% (less than 6%), indicating that the method has good robustness to C18 columns of different brands or batches.

[0051] 7. Instrument durability The same test solution was analyzed using three different brands and models of high-performance liquid chromatographs (HPLC). Instrument 1: Thermo UltiMate 3000; Instrument 2: Shimadzu LC-2030 Plus; Instrument 3: Waters 2695. The content was determined under each instrument condition, and the RSD was 1.39% (less than 6%), indicating that the method has good applicability and durability to different brands of HPLC.

[0052] V. Actual Sample Determination and Content Limit Establishment The content of Xanthium sibiricum samples from 15 batches of medicinal materials from 3 production areas was determined using the quality evaluation method established in this invention.

[0053]

[0054] Take each batch of medicinal material powder, prepare test solutions according to the test solution preparation method, and inject them under the determined chromatographic conditions to calculate the content of quercetin-3-O-glucuronide (calculated on a dried basis). The test results are as follows:

[0055] Among 15 batches of Xanthium sibiricum medicinal materials, the content of quercetin-3-O-glucuronide ranged from 0.0289% to 0.0613%, with an average content of 0.0458%. The content varied among different producing areas: materials from Qichun, Hubei Province, had a generally higher content (0.0501% to 0.0613%), materials from Chifeng, Inner Mongolia Province had a moderate content (0.0357% to 0.0549%), and materials from Tai'an, Shandong Province had a relatively lower content (0.0289% to 0.0419%). This reflects the influence of different ecological environments and harvesting times on the quality of the medicinal materials, and also indicates that the content of quercetin-3-O-glucuronide can be used as a sensitive indicator for evaluating the quality of Xanthium sibiricum medicinal materials.

[0056] Based on the above test results, a content limit is proposed. Referring to the statistical methods for setting content limits in the "Guiding Principles for Validation of Analytical Methods for Drug Quality Standards" of the Chinese Pharmacopoeia, Volume IV, the average value (0.0458%) of the measured results from 15 batches of samples is used as the benchmark, and 80% of the average value (i.e., 0.0366%) is taken as the recommended limit value. This method of setting limits comprehensively considers the estimation of the population mean from the sample mean and the statistical law that the content of medicinal materials usually follows a normal distribution. A 20% reduction from the average value can cover the content level of most normal batches of medicinal materials. Based on this, and considering practical operability and the principle of strict standards, the proposed content limit is: This product, calculated on a dried basis, contains quercetin-3-O-glucuronide (C... 21 H 18 O 13 () It shall not be less than 0.04%.

[0057] Based on this limit, 15 batches of samples were judged to be qualified. Three batches (S01, S02, and S04) had a content below 0.04% and were judged to be unqualified, with a failure rate of 20.0%. All of these samples originated from Tai'an, Shandong. All samples from Chifeng, Inner Mongolia, and Qichun, Hubei were qualified. This limit can effectively identify Xanthium sibiricum medicinal materials from different origins and of different quality grades, and has good applicability and distinguishability.

[0058] ( Figure 5 HPLC chromatogram of Xanthium sibiricum, origin: Tai'an, Shandong; Figure 6 HPLC chromatogram of Xanthium sibiricum, origin: Chifeng, Inner Mongolia; Figure 7 (HPLC chromatogram of Xanthium sibiricum, origin: Qichun, Hubei) VI. Applicability Verification of Xanthium sibiricum Processed Products Take clean Xanthium sibiricum seeds, stir-fry until the surface is yellowish-brown, remove the thorns, sift clean, pulverize, and pass through a No. 3 sieve to obtain stir-fried Xanthium sibiricum powder. Take 100-200 g of the above-mentioned stir-fried Xanthium sibiricum powder, add water and decoct 2-3 times, adding 10-12 times the amount of water each time, decoct for 90-120 minutes (soak for 60 minutes before the first decoction), filter, combine the filtrates, concentrate under reduced pressure to a clear extract, add appropriate excipients, dry, pulverize, add appropriate excipients to granulate, and obtain stir-fried Xanthium sibiricum formula granules.

[0059] Approximately 0.5 g each of roasted Xanthium sibiricum powder and roasted Xanthium sibiricum granules were accurately weighed and prepared according to the test solution preparation method. The results showed that the retention time of the quercetin-3-O-glucuronide peak in the HPLC chromatograms of both the roasted Xanthium sibiricum powder and granules was basically consistent with that of the raw Xanthium sibiricum, with good peak shape and resolution meeting the requirements for content determination, allowing for accurate quantification. Specifically, the quercetin-3-O-glucuronide content in the roasted Xanthium sibiricum powder was 0.0582%, and the content in the roasted Xanthium sibiricum granules was 0.0325%. This indicates that the quality evaluation method established in this invention is applicable not only to raw Xanthium sibiricum but also to its processed products such as powdered Xanthium sibiricum and granules.

[0060] ( Figure 8 HPLC chromatogram of stir-fried Xanthium sibiricum slices; Figure 9 (HPLC chromatogram of the stir-fried cocklebur granules) VII. Validation of the specificity of quercetin-3-O-glucuronide as a quality marker To verify the species specificity of quercetin-3-O-glucuronide as a quality marker for Xanthium sibiricum, this embodiment presents a systematic comparative analysis of genuine Xanthium sibiricum and its closely related plants.

[0061] Sample collection: Genuine Xanthium sibiricum ( Xanthium sibiricum Patr. 3 batches of medicinal materials (numbers S06, S10, S11, respectively from Chifeng, Inner Mongolia; Chifeng, Inner Mongolia; Qichun, Hubei); 2 species of closely related fruits, 2 batches of each: Northeast Xanthium sibiricum ( Xanthium mongolicum Kitag.) fruit (numbers M01, M02), burdock ( Xanthium spinosum L. fruits (numbered S01 and S02) were collected from wild distribution areas and identified as species.

[0062] Preparation of test solutions: Each sample was prepared according to the test solution preparation method (approximately 0.5 g of powder, precisely added to 50 ml of 50% ethanol, refluxed for 60 minutes).

[0063] Chromatographic analysis: Under the determined chromatographic conditions, accurately pipette 10 μl of each test solution and inject it into the high-performance liquid chromatograph (HPLC), recording the chromatograms. The focus is on comparing the presence and retention time consistency of the quercetin-3-O-glucuronide peak in each sample.

[0064] Content determination: Quercetin-3-O-glucuronide was quantitatively determined, and the content differences between the genuine product and closely related plants were compared. The determination results are shown in the table below:

[0065] Results Analysis: In the HPLC chromatograms of the three batches of genuine Xanthium sibiricum samples, clear chromatographic peaks were observed at the corresponding retention time (approximately 13.2 minutes) of the quercetin-3-O-glucuronide reference standard. The peak shapes were symmetrical, the peak area response values ​​were stable, and the contents were all higher than the proposed limit standard of 0.04%. However, in the HPLC chromatograms of the test solutions from both Northeast Xanthium sibiricum and Xanthium sibiricum, no obvious chromatographic peaks were detected at the corresponding retention time of the quercetin-3-O-glucuronide reference standard, or the peak area response values ​​were below the limit of quantitation. Specifically, two batches of Northeast Xanthium sibiricum (M01 and M02) showed no identifiable chromatographic peaks at the corresponding retention time, with contents below 0.001%; ​​one batch of Xanthium sibiricum (SP01) was also undetectable, and another batch (SP02), although showing a weak response signal at the corresponding position, had a content of only 0.0012%, a difference of more than 40 times compared to the genuine Xanthium sibiricum.

[0066] The results of the above comparative experiments show that quercetin-3-O-glucuronide in Xanthium sibiricum ( Xanthium sibiricum This component exhibits a significantly enriched distribution in *Xanthium sibiricum* (Patr.), but its content is extremely low or absent in closely related species such as *Xanthium sibiricum* and *Xanthium sibiricum*. This indicates that the distribution of this component varies significantly among different species within the *Xanthium* genus, making it a characteristic chemical marker for distinguishing genuine *Xanthium sibiricum* seeds from closely related species. Using it as a quality marker can effectively reflect the species authenticity and quality of *Xanthium sibiricum* medicinal materials, providing a reliable chemical basis for the quality evaluation of medicinal materials.

[0067] VIII. Stability Comparison of Quercetin-3-O-glucuronide and Chlorogenic Acid as Quality Control Indicators To further verify the technical advantages of quercetin-3-O-glucuronide over chlorogenic acid, a quantitative indicator currently used in the pharmacopoeia, this embodiment presents a systematic parallel comparative study of the chemical stability of the two under accelerated testing conditions.

[0068] Sample Preparation: Five portions of Xanthium sibiricum powder from the same batch (No. S06, quercetin-3-O-glucuronide content 0.0473%, chlorogenic acid content 0.2835%), approximately 0.5 g each, were accurately weighed and placed in open weighing bottles. One portion served as a control sample for day 0, sealed, protected from light, and refrigerated at 4℃. The remaining four portions were treated under the following conditions: high temperature group (60℃ constant temperature drying oven, open for 10 days), high humidity group (90%±5% relative humidity in a constant humidity desiccator, 25℃ room temperature for 10 days), strong light group (4500 Lux±500 Lux light intensity in a light test chamber, 25℃ room temperature for 10 days), and comprehensive group (60℃ high temperature + 90% relative humidity + 4500 Lux light intensity, open for 10 days). Each treatment group had three replicates.

[0069] Content determination: After treatment, test solutions were prepared according to the method for preparing test solutions for each treatment group. The content of quercetin-3-O-glucuronide was determined under the chromatographic conditions of this invention. Simultaneously, the content of chlorogenic acid in the same test solution was determined according to the method for content determination of Xanthium sibiricum in the 2025 edition of the current Chinese Pharmacopoeia (using acetonitrile-0.4% phosphoric acid aqueous solution as the mobile phase and detection wavelength 327 nm). Using the content of each component in the control sample (day 0) as a baseline (relative content 100%), the relative residual contents of quercetin-3-O-glucuronide and chlorogenic acid under various treatment conditions were calculated and compared. The determination results are shown in the table below:

[0070] Results analysis: Under high temperature, high humidity and strong light conditions, the contents of quercetin-3-O-glucuronide and chlorogenic acid both showed a decreasing trend to varying degrees, but they showed significant differences in stability. After being treated at 60℃ for 10 days, the relative remaining content of quercetin-3-O-glucuronide was 96.82%, a decrease of only about 3%, while the relative remaining content of chlorogenic acid had decreased to 81.35%, a decrease of nearly 19%. Under high humidity conditions, quercetin-3-O-glucuronide showed almost no significant degradation (relative remaining content 98.14%), while chlorogenic acid experienced a content loss of about 10% (relative remaining content 90.26%). Under strong light conditions, the relative remaining content of quercetin-3-O-glucuronide was 94.53%, and that of chlorogenic acid was 83.71%. Under the most stringent comprehensive accelerated conditions, the relative remaining content of quercetin-3-O-glucuronide remained at 91.27%, while that of chlorogenic acid had decreased to 68.49%, a content loss of more than 30%.

[0071] The above results clearly demonstrate that quercetin-3-O-glucuronide exhibits significantly better chemical stability than chlorogenic acid under conditions of high temperature, high humidity, and light exposure that may be encountered during routine storage and transportation. Using quercetin-3-O-glucuronide as a quality marker for Xanthium sibiricum, compared to chlorogenic acid used in the current pharmacopoeia standard, can more accurately and stably reflect the actual quality status of the medicinal material during circulation, storage, and use. This effectively avoids misjudgments of quality due to the degradation of the marker component itself, thus providing a more reliable guarantee for the safety and efficacy of clinical medication.

[0072] 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. Application of quercetin-3-O-glucuronide in the preparation of a test reagent for evaluating the quality of Xanthium sibiricum medicinal material.

2. The application according to claim 1, characterized in that, The evaluation of Xanthium sibiricum quality refers to the determination of the quality of Xanthium sibiricum.

3. The application according to claim 1, characterized in that, The detection in this application is performed using high-performance liquid chromatography.

4. The application according to claim 3, characterized in that, The chromatographic conditions of the high performance liquid chromatography method include: using octadecylsilane-bonded silica gel as the stationary phase, acetonitrile as mobile phase A, and 0.4% phosphoric acid aqueous solution as mobile phase B, performing gradient elution, and the detection wavelength is 356 nm.

5. The application according to claim 4, characterized in that, The gradient elution program is as follows: within 0 to 20 minutes, the volume percentage of mobile phase A increases linearly from 10% to 55%, and the volume percentage of mobile phase B decreases linearly from 90% to 45%.

6. The application according to claim 4, characterized in that, The flow rate of the high-performance liquid chromatography was 1.0 ml / min, and the column temperature was 35℃.

7. The application according to claim 1, characterized in that, The content of quercetin-3-O-glucuronide in the Xanthium sibiricum medicinal material shall not be less than 0.04% on a dried basis.

8. A method for quality evaluation of Xanthium sibiricum medicinal material, characterized in that, Quercetin-3-O-glucuronide was used as the detection index, and its determination was performed by high performance liquid chromatography, including the following steps: (1) Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as the packing material; acetonitrile was used as mobile phase A and 0.4% phosphoric acid aqueous solution was used as mobile phase B for gradient elution; the detection wavelength was 356 nm; the theoretical plate number calculated based on the quercetin-3-O-glucuronide peak should not be less than 5000; (2) Preparation of reference solution: Take an appropriate amount of quercetin-3-O-glucuronide reference standard, accurately weigh it, and add 50% ethanol to prepare a solution containing 20 μg per ml. (3) Preparation of test solution: Take about 0.5g of Xanthium sibiricum powder, weigh it accurately, put it in a stoppered conical flask, add 50ml of 50% ethanol accurately, weigh it, reflux and extract for 60 minutes, cool it, weigh it again, make up the lost weight with 50% ethanol, shake well, filter it, and take the filtrate to obtain the test solution. (4) Determination: Accurately pipette 10 μl of the reference solution and the test solution into the high performance liquid chromatograph and determine the result.

9. The quality evaluation method according to claim 8, characterized in that, In step (1), the gradient elution program is as follows: within 0 to 20 minutes, the volume ratio of mobile phase A increases linearly from 10% to 55%, and the volume ratio of mobile phase B decreases linearly from 90% to 45%; the flow rate is 1.0 ml / min, and the column temperature is 35℃.

10. The quality evaluation method according to claim 8, characterized in that, When the measured content of quercetin-3-O-glucuronide is less than 0.04% on a dried basis, the quality of the Xanthium sibiricum is deemed substandard.

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