Quality control method for traditional Chinese medicine motherwort in different harvesting periods

Abstract

The invention belongs to the technical field of traditional Chinese medicine quality control methods, and relates to a quality control method for traditional Chinese medicine motherwort in different harvesting periods. The quality control method comprises the following specific steps: preparation of a test solution, preparation of a mixed reference solution and a determination method, wherein a chromatographic column adopts a C18 column; a mobile phase is a methanol-10 [mu] mol / L ammonium acetate aqueous solution, gradient elution is carried out for 0-20 min, and methanol is 45%-100%; the flow velocity is 1.0 mL / min; the detection wavelength is as follows: the wavelength of quercetin, kaempferol and luteolin is 254 nm, and the wavelength of leonurine is 280 nm; the column temperature is 30 DEG C; the sample size is 5 microliters. According to the method disclosed by the invention, drug effects are taken as guidance, the screened chemical components quercetin, kaempferol, luteolin and leonurine not only can reflect the overall drug effects of medicinal materials, but also have part specificity, so that not only can the problem of disordered composition of medicinal parts caused by nonstandard harvesting time be solved, but also accurate association of'component-drug effects-quality 'can be realized, and the method is suitable for popularization and application. The method has important theoretical value and practical significance for guaranteeing the quality stability and the clinical curative effect reliability of the motherwort herb.

Description

Technical Field

[0001] This invention belongs to the technical field of traditional Chinese medicine quality control methods, specifically relating to a quality control method for Leonurus japonicus (Yimucao) at different harvesting periods. Background Technology

[0002] Motherwort refers to the fresh or dried aerial parts of the plant *Leonurus japonicus* Houtt., belonging to the Lamiaceae family. As a commonly used traditional Chinese medicine, it has a long history of clinical application and a wide range of uses. However, with expanding market demand, the problem of inconsistent use of its medicinal parts has become increasingly prominent, becoming one of the key factors restricting the stability of the quality of motherwort medicinal materials.

[0003] Throughout the history of traditional Chinese medicine literature, there are significant differences in the records of the medicinal parts of Leonurus japonicus: the Shennong's Classic of Materia Medica states that "the stems, leaves, and roots can all be used as medicine"; the Tang Dynasty's Newly Revised Materia Medica further specifies the medicinal parts of Leonurus japonicus as "the stems and leaves are harvested and dried in the sun"; while the current Chinese Pharmacopoeia (2025 edition) clearly stipulates that the medicinal parts are "fresh or dried aerial parts" and specifies the harvesting time—fresh products are harvested from the seedling stage in spring to the pre-flowering stage in early summer, while dried products are harvested in summer when the stems and leaves are lush and the flowers have not yet opened or have just opened.

[0004] Despite pharmacopoeia standards as guidance, significant differences remain in the composition of medicinal parts of Leonurus japonicus in actual market circulation. Studies have found a stark contrast in the ratio of stems to leaves among different batches of Leonurus japonicus samples. In some samples, stems account for over 60% while leaves account for less than 20%; in others, stems account for only about 30% while leaves account for 70%. This significant difference in proportion is mainly due to inconsistent harvesting times, and the types and contents of chemical components in different medicinal parts vary considerably. Further research indicates that alkaloids and flavonoids are most abundant in the leaves during the leaf stage; in the early flowering stage, the leaves have the highest content of leonurine hydrochloride and flavonoids, while the flowers have the highest content of stachydrine hydrochloride. This presents considerable challenges to the quality evaluation of Leonurus japonicus.

[0005] The current edition of the Chinese Pharmacopoeia uses only stachydrine hydrochloride and leonurine hydrochloride as indicator components for quality control of Leonurus japonicus. This singular evaluation system has significant limitations. Leonurine hydrochloride is mainly related to its core medicinal effects of anti-inflammatory, analgesic, blood-activating, and menstrual-regulating, and can serve as an important indicator reflecting its gynecological therapeutic effects. Stachydrine hydrochloride is more related to its auxiliary effects such as diuresis and swelling reduction. However, Leonurus japonicus also has many other medicinal effects, such as promoting uterine contraction, improving blood circulation, and regulating immunity. Using only these two alkaloids as indicators cannot reflect the differences in other active components at different harvesting periods and in different parts of the plant, nor can it correlate with the overall medicinal effects.

[0006] Therefore, there is an urgent need to establish a quality control method for Leonurus japonicus that is efficacy-oriented. Summary of the Invention

[0007] In view of the aforementioned technical problems, this invention provides a quality control method for Leonurus japonicus, a traditional Chinese medicine, harvested at different times. This invention, guided by efficacy, screens out chemical components that reflect both the overall efficacy of the herb and the specificity of its parts. This not only solves the problem of inconsistent composition of medicinal parts caused by irregular harvesting times but also achieves a precise correlation between "components-efficacy-quality," which has significant theoretical and practical value for ensuring the quality stability and clinical efficacy reliability of Leonurus japonicus.

[0008] To achieve the above objectives, the present invention adopts the following technical solution.

[0009] A quality control method for Leonurus japonicus (a traditional Chinese medicine) harvested at different times includes the following steps:

[0010] Step 1: Preparation of the test solution;

[0011] Step 2, Preparation of mixed reference solution: Accurately weigh appropriate amounts of quercetin, luteolin, kaempferol, and leonurine reference standards, add 90% ethanol to fully dissolve them, and prepare a solution containing 0.098 mg / mL quercetin, 0.109 mg / mL luteolin, 0.103 mg / mL kaempferol, and 0.104 mg / mL leonurine, which is the mixed reference solution;

[0012] Step 3, Determination Method: Accurately pipette the mixed reference solution and the test solution separately, inject them into the liquid chromatograph, and determine the result.

[0013] Further, step 1 specifically involves: pulverizing the motherwort herb and sieving it (using a No. 3 sieve). Take 1.0g of the powder and extract it by reflux with 25 times the amount of 90% ethanol for 2 hours. Repeat the extraction twice, recover the solvent to obtain the extract, dissolve it in 90% ethanol, and prepare a herb with a concentration of 0.8g·mL. -1 The solution was filtered through a 0.22 μm microporous membrane for analysis.

[0014] Further, in step 3, a C18 column (250 mm × 4.6 mm, 5 μm) was used; the mobile phase was methanol-10 μmol / L ammonium acetate aqueous solution (gradient elution: 0–20 min, methanol 45%–100%); the flow rate was 1.0 mL / min; the detection wavelengths were 254 nm for quercetin, kaempferol, and luteolin, and 280 nm for leonurine; the column temperature was 30 °C; and the injection volume was 5 μL.

[0015] Furthermore, in step 3, the quality requirements for the medicinal materials are as follows: The content of quercetin in the motherwort medicinal material shall not be less than 0.014%, luteolin not less than 0.021%, kaempferol not less than 0.017%, and leonurine not less than 0.100%. (Based on the 2025 edition of the Chinese Pharmacopoeia 9101 Analytical Method Validation Guidelines)

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0017] Existing technologies attempt to analyze the differences in components of different parts of Leonurus japonicus using chromatographic techniques (such as HPLC and UPLC), but this only stays at the level of component characterization and does not combine in vivo absorption characteristics and efficacy. At the same time, using only two alkaloids, stachydrine hydrochloride and leonine hydrochloride, as quality control indicators for Leonurus japonicus cannot reflect the differences in other active ingredients in different harvesting periods and different parts, nor can it be linked to the overall efficacy.

[0018] 1. This invention establishes a complete analytical system: for the first time, it realizes a progressive analysis of Leonurus japonicus, namely, “characterization of differential chemical components → in vivo activity screening → target drug binding verification → efficacy confirmation → content determination”, breaking through the limitations of traditional methods that “emphasize components and neglect efficacy”, and clarifying the direct correlation between core differential efficacy components and efficacy and targets.

[0019] 2. The precise core differentially active ingredients identified in this invention are: quercetin, kaempferol, luteolin, and leonurine. These ingredients possess site specificity (enriched in flowers and leaves), in vivo activity (entering the bloodstream), strong target drug binding ability (binding energy < -7.0 kcal / mol), and clear efficacy (antioxidant, anti-inflammatory, and apoptosis regulation). They can serve as key material basis for evaluating the efficacy and quality control of Leonurus japonicus.

[0020] 3. This invention clarifies the dominant medicinal parts: Through component content and in vitro efficacy verification, it confirms the order of medicinal efficacy of Leonurus japonicus as "leaf > flower > whole herb > stem", providing a basis for the standardization of medicinal parts (prioritizing the preservation of flowers and leaves) and the efficient utilization of resources.

[0021] 4. The screening logic of this invention (differentiated components → blood-entering components → network pharmacology → molecular docking → in vitro efficacy → content determination) can provide a technical path for the quality control of other Chinese herbal medicines with multiple parts (such as honeysuckle, chrysanthemum, etc.).

[0022] 5. The higher the proportion of flowers and leaves in the medicinal material, the higher the content of the four core differentiating medicinal components, and the better the efficacy and quality of motherwort. This can effectively solve the problem of quality fluctuation caused by the confusion of medicinal parts and non-standard harvesting of motherwort, and provide a scientific and quantifiable basis for the standardized production and quality evaluation of motherwort. Attached Figure Description

[0023] Figure 1Total ion chromatograms (A) of Leonurus japonicus and 90% ethanol extracts of different parts (flowers, stems, and leaves), and total ion chromatograms (B) of rat blank plasma under positive and negative ion modes at 15 min, 30 min, 1 h, 2 h, and 4 h.

[0024] Figure 2 Venn diagram (A) of communication targets between Leonurus japonicus and endothelial dysfunction, GO analysis diagram (B), bubble diagram of signaling pathway enrichment analysis (C), core target diagram of topological analysis (D), and "component-target-pathway" network (E).

[0025] Figure 3 Comparison of the content of core active ingredients from different origins (A); UPLC chromatograms of Leonurus japonicus and mixed reference solution: (B) 280nm; (C) 254nm (1. Leonurus alkaloid, 2. Quercetin, 3. Luteolin, 4. Kaempferol).

[0026] Figure 4 Molecular docking interactions between the core active ingredient and the core target: 3D binding site diagrams of AT1R with the core active ingredient (A), p-p38 with the core active ingredient (B), and p53 with the core active ingredient (C).

[0027] Figure 5 To determine the optimal concentration for modeling (A), the optimal concentration of Leonurus japonicus for administration (B), the optimal concentration for administration of different parts of the plant (C), the optimal therapeutic concentration of quercetin (10 μmol), the optimal therapeutic concentration of kaempferol (15 μmol), the optimal therapeutic concentration of luteolin (10 μmol), the optimal therapeutic concentration of leonurine (10 μmol), the optimal therapeutic concentration of leonurine (10 μmol), and the optimal therapeutic concentration of p38 MAPK inhibitor SB203580 (15 μmol) for cell viability assay using the CCK-8 assay.

[0028] Figure 6 A schematic diagram illustrating the regulatory mechanism of the core component on ANGII-induced endothelial dysfunction through the MAPK signaling pathway.

[0029] Figure 7 ELISA and ROS detection results of HUVECs after intervention with core components: Representative fluorescence images of intracellular ROS levels detected by DCFH-DA staining method (DAPI for nuclear staining, Merge for co-localization) (A); Statistical analysis of relative fluorescence intensity of ROS (B); (C-G) ELISA detection results of inflammatory factors and apoptosis proteins: (C) BCL2, (D) CASP3, (E) IL-6, (F) PTGS2, (G) TNF-α.

[0030] Figure 8To verify the regulatory effects of the core pharmacologically active ingredient on AT1R, p-p38MAPK, and TP53 proteins in HUVEC cells using Western blot, (AC) represent protein blot bands and quantitative analysis of protein expression levels: (A) AT1R, (B) p-p38MAPK, (C) TP53, with GAPDH as an internal control.

[0031] Figure 9 Chromatograms of the 90% ethanol negative control test solution: (A) 280 nm; (B) 254 nm (1. Solvent, 2. Leonurine, 3. Quercetin, 4. Luteolin, 5. Kaempferol). Detailed Implementation

[0032] Unless otherwise specified, the experimental methods described in the following embodiments of the present invention are generally performed under conventional conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the embodiments are commercially available products.

[0033] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0034] The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps is not limited to the steps or modules listed, but may optionally include steps not listed, or may optionally include other steps inherent to such process, method, product, or device.

[0035] Example 1: UPLC-Q-TOF-MS analysis of the differential components of Leonurus japonicus and its flowers, stems and leaves.

[0036] Preparation of test solution:

[0037] The motherwort was purchased from Chaoyang County (44.57°N, 120.53°E), Xinmin City (40.28°N, 123.77°E), Xifeng County (42.15°N, 120.54°E), Benxi City (124°02′E, 40°99′N), Xinbin Manchu Autonomous County (42.55°N, 124.68°E), and Kuandian Manchu Autonomous County (41.06°N, 124.19°E) in Liaoning Province, China. All samples were identified by Professor Zhang Jiankui of the School of Pharmacy, Liaoning University of Traditional Chinese Medicine, as the dried aerial parts of *Leonurus japonicus* Houtt., a plant in the Lamiaceae family. It was divided into flower, stem, and leaf parts. Motherwort and its flowers, stems, and leaves from six different producing areas were collected, pulverized, and sieved (using a No. 3 sieve). 1.0 g of each powder was extracted by reflux with 25 times the volume of 90% ethanol for 2 hours, repeated twice. The solvent was recovered to obtain the extract, which was then dissolved in 90% ethanol to prepare a medicinal material with a concentration of 0.5 mg / mL. -1 The solution was filtered through a 0.22 μm microporous membrane and used for mass spectrometry analysis.

[0038] Preparation of reference solution:

[0039] Weigh appropriate amounts of quercetin, leonurine hydrochloride, stachydrine hydrochloride, trigonelline, hyperoside, ferulic acid, luteolin, kaempferol, apigenin, caffeic acid, L-phenylalanine, verbascoside, chlorogenic acid, rutin, isoquercitrin, and rhodioloside reference standards respectively. Dissolve each standard in 90% ethanol to prepare a solution with a concentration of 0.5 μg / mL. -1 Reference solution.

[0040] UPLC-Q-TOF-MS analysis conditions:

[0041] Chromatographic separation was performed using an Agilent 1290UPLC-6550Q-TOF chromatography-mass spectrometry system (Agilent Technologies, USA). The chromatographic column was an Infinity Lab Poroshell 120SB-C18 (2.7 μm, 3.0 mm × 100 mm, USA); the injection volume was 0.3 μL; and the flow rate was 0.3 mL / min. -1 Column temperature 30℃; positive ion mobile phase: 0.1% formic acid water (A)-methanol (B); negative ion mobile phase: water (A)-acetonitrile (B); gradient elution program (0-30 min, 5%-100% B; 30-35 min, 100% B); electrospray ionization (ESI), positive and negative ion scanning modes, capillary voltages of 4000V and 3500V respectively, and dry gas flow rate of 13 L·min. -1Dry gas temperature 250℃, atomizer pressure 45psig, sheath gas temperature 350℃, sheath gas flow rate 11L·min -1 Fragmentation voltage 125V, mass range 100-1400m / z.

[0042] Screening for differential components from different parts:

[0043] UPLC-Q-TOF-MS was used to detect metabolites from different parts of Leonurus japonicus, including its flowers, stems, and leaves. After obtaining peak volume data for each component, the p-value (P<0.05) and fold change (FC) (|FC|>2) were used as criteria to screen for characteristic differentially expressed components. Simultaneously, Leonurus japonicus samples from six production areas were selected to validate the screened differentially expressed components, clarifying the differences in component composition among Leonurus japonicus from different origins. During the experiment, strict control was maintained over the consistency of sample collection (e.g., plants with the same growth cycle and similar growing environments), and three parallel experiments were conducted to ensure the stability and reliability of the detection data, providing methodological support for subsequent analysis of component differences in Leonurus japonicus from different origins.

[0044] Chemical composition analysis of motherwort:

[0045] Based on comparison with reference standards and literature reports, a total of 121 compounds were identified according to retention time, precise m / z value, adduct form, and MS / MS fragmentation pattern. These included 9 alkaloids (A), 44 flavonoids (B), 20 terpenes (C), 23 organic acids and their derivatives (D), 13 phenylethanol glycosides (E), and 13 other components such as phenylpropanoids (F), as shown in Table 1. Through screening, 39 components showed significant differences, including 17 in the positive ion mode and 22 in the negative ion mode. These results collectively established a detailed phytochemical map of Leonurus japonicus and its different parts (flowers, stems, and leaves), such as... Figure 1 (A) provides a chemical basis for subsequent bioactivity analysis.

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053] Example 2: Analysis of blood-entering components of Leonurus japonicus.

[0054] Laboratory animals:

[0055] Twelve SPF-grade male rats, weighing (200±20) g, were provided by Liaoning Changsheng Biotechnology Co., Ltd. (Liaoning, China). They were housed in a pathogen-free facility at a temperature of 25±5℃ and humidity of 55±5%, fed standard laboratory feed and water, and acclimatized for 7 days. All animal experiments followed the "Guidelines for the Husbandry and Use of Laboratory Animals," certificate number SCXK(Liaoning)2020-0001. This experiment was approved by the Ethics Committee of Liaoning University of Traditional Chinese Medicine, certificate number 210000620240224.

[0056] Plasma sample preparation:

[0057] Twelve SPF-grade male rats were randomly divided into a control group and a treatment group. Rats in the treatment group were administered the drug via gavage once daily for 7 consecutive days (1 g / kg crude drug concentration). Rats in the treatment group were fasted but allowed free access to water 12 hours before the last administration. Blood samples were collected from the inner canthus of the eye at 15 min, 30 min, 1 h, 2 h, and 4 h after administration. After standing on ice for 1 h, the samples were centrifuged at 4℃ and 3300 rpm for 20 min. The supernatant was collected, and 200 μL of serum and 600 μL of methanol were added. The mixture was vortexed for 1 min and then centrifuged at 4℃ for 10 min (12000 rpm). -1 The supernatant was collected and concentrated using a vacuum centrifuge. It was then reconstituted with 100 μL of methanol and centrifuged at 4°C for 10 min (12000 r·min). -1 The supernatant was collected for mass spectrometry detection.

[0058] Analysis of blood-entering components of motherwort:

[0059] A total of 46 components were analyzed from the drug-treated plasma samples, including 37 unchanged components (P1–P37) and 9 metabolized components (M1–M9). Detailed data are shown in Table 2, and the mass spectra are shown below. Figure 1 (B)

[0060] Differentiating components of motherwort that enter the bloodstream:

[0061] Comparative analysis of the chemical components of different medicinal parts of Leonurus japonicus showed that there were 39 different chemical components. The intersection of these with the 46 components that enter the bloodstream yielded 25 different components that enter the bloodstream, which were used for subsequent experimental analysis. The specific data are shown in Table 2.

[0062]

[0063]

[0064]

[0065] Example 3: Network pharmacology predicts core active ingredients, targets and pathways.

[0066] Target acquisition: The intersection of the identified Leonurus japonicus components entering the bloodstream and the differentially expressed components obtained through screening yielded 25 target components. Swiss target prediction and TCMSP databases were used to retrieve relevant targets for these differentially expressed components. Targets with a probability greater than 0 were selected to obtain their chemical composition-target gene information. Using GeneCards, OMIM, drugbank, TTD, and pharmgkb databases, with "endothelial dysfunction" as the search term, the collected targets were merged and deduplicated to obtain disease-related targets. A total of 331 potential targets and 2383 gene targets related to vascular endothelial dysfunction were predicted.

[0067] Intersection target screening: A Venn diagram of the intersection of Leonurus japonicus target components and targets related to endothelial dysfunction was constructed using the VENNY 2.1.0 online website. Figure 2 A) 163 intersection target points were obtained.

[0068] GO and KEGG Analysis: GO and KEGG pathway enrichment analyses were performed on the intersection targets obtained using the Metascape database. The top-ranked results were visualized, and a bubble analysis chart was created. Figure 2 C). GO analysis involved 984 biological functions, with 730 identified in BP, covering vascular endothelial cell damage repair, regulation of oxidative stress response, secretion of inflammatory factors, and positive regulation of the MAPK cascade and negative regulation of apoptosis. CC identified 79 functions, mainly involving structures closely related to function, such as the vascular endothelial cell membrane and mitochondria; meanwhile, MF included 175 functions (P<0.05), mainly protein binding and kinase activity regulation. The top 10 functions in each category were used for microbioinformatics GO enrichment analysis (…). Figure 2 B), and based on the P value, the top 15 KEGG enriched pathways were listed, and the enriched KEGG pathways were combined with the core key targets to highlight the MAPK signaling pathway as the key mechanism.

[0069] Core Components and Target Identification: Protein-protein network analysis was performed on intersecting targets using the STRING platform. The minimum interaction threshold was set to "medium confidence (0.4)," and other parameters remained at their default settings. A PPI network was constructed, and network topology analysis was performed using Cytoscape 3.9.1 software. Based on the degree value, the top ten targets were selected as core targets: AT1, IL-6, TNF, INS, IL1B, CASP3, TP53, ESR1, BCL2, and PTGS2. Meanwhile, components with a degree ≥ 10 were identified as core differentially expressed components: quercetin, apigenin, kaempferol, rutin, isorhamnetin, leonurine, stachydrine, projugantin, and luteolin. Figure 2 D); then, import the motherwort, target components and their corresponding targets, and endothelial dysfunction-related targets into the software to construct a "traditional Chinese medicine-component-target-disease" network. Figure 2 E).

[0070] Identification of core differentially active ingredients: In the "Traditional Chinese Medicine-Ingredient-Target-Disease" network constructed using Cytoscape 3.9.1, quercetin ranked first among the core ingredients with the highest degree value, indicating that it has the strongest connection with the target in the network and may be a key pivotal ingredient for exerting its efficacy. Leonuridine showed the highest content in different origins and parts of Leonurus japonicus, and as one of the most important active ingredients in the medicinal material, its efficacy verification has important material basis support. Kaempferol and luteolin showed significant site-specific distribution differences, with extremely low or even undetectable content in some parts. The correlation between this distribution characteristic and its potential efficacy deserves further investigation. Based on the above network pharmacological analysis results and component content characteristics, quercetin, leonuridine, kaempferol, and luteolin were selected as core differentially active ingredients for subsequent efficacy verification, which can comprehensively cover the efficacy value of the network's core effects, main material basis, and specifically distributed components.

[0071] Example 4: Verification of the differences in components of motherwort from different origins.

[0072] Samples of Leonurus japonicus from six production areas in Liaoning Province were selected, and qualitative analysis of chemical components was performed using UPLC-Q-TOF-MS technology. Figure 3 A). The results showed that the chemical composition and distribution characteristics of Leonurus japonicus from different origins were consistent: flavonoids (quercetin, kaempferol, luteolin, etc.) and alkaloids (leonurine, stachydrine, etc.) were significantly enriched in the flowers and leaves, while terpenes were mainly distributed in the stems. However, the relative contents of each component showed significant differences depending on the origin. Quantitative analysis of the core differentially active components by UPLC further confirmed that leonurine had the highest content in Leonurus japonicus (1.308 mg / g). Figure 3B), followed by luteolin (0.336 mg / g) and kaempferol (0.215 mg / g), with quercetin (0.185 mg / g) having the lowest content. Figure 3 C) is consistent with the mass spectrometry analysis results.

[0073] Example 5: Molecular docking verification of core differentially active ingredients and core targets.

[0074] To clarify the interaction mechanism between the core differentially active ingredients (quercetin, kaempferol, luteolin, and leonurine) and key targets of the MAPK signaling pathway (AT1R, p-p38MAPK, and p53), molecular docking simulations were used to evaluate the binding affinity between the ingredients and the targets. The results showed that all four ingredients formed stable binding bonds with three targets, with binding energies all below -7.0 kcal / mol (indicating strong affinity). Among them, leonurine had the lowest binding energy with AT1R (-8.3 kcal / mol). Figure 4 A) primarily binds competitively to the AT1R active pocket via hydrogen bonding, thus inhibiting its overactivation; quercetin binds to p-p38MAPK at a rate of -8.5 kcal / mol, specifically inhibiting its phosphorylation activity. Figure 4 B); Luteolin binds to P53 at an energy of -8.2 kcal / mol, and can weaken the transcriptional activity of P53 through hydrogen bonding. Figure 4 C) The above molecular docking results confirm that quercetin, kaempferol, luteolin, and leonurine interact with the target as active ingredients, providing direct evidence that they exert endothelial protective effects by regulating the AT1R / p-p38MAPK / P53 pathway.

[0075] Example 6: In vitro efficacy verification of the core differentially active ingredients.

[0076] The protective effect of the core differentially active ingredient against vascular endothelial dysfunction: intervention of HUVEC cells with gradient concentrations (0.01 μmol, 0.1 μmol, 1 μmol, 10 μmol) of AngII. Figure 5 A) The optimal concentration for modeling was determined to be 1 μmol, at which point the cell viability was close to 50%, the number of cells shrinking and becoming rounded increased, a small amount of debris was present, and some cells exhibited filamentous phenomena. HUVEC cells were treated with culture media containing different concentrations of Leonurus japonicus for 24 h, and the optical density (OD) value at 450 nm was measured in each well. Cell viability was calculated, and the optimal concentration of Leonurus japonicus was determined to be 1 mg / mL. Figure 5 B), and the therapeutic effects of Leonurus japonicus and its different parts on endothelial dysfunction were obtained as follows: leaf > flower > whole herb > stem ( Figure 5 C), further screening yielded quercetin 10 μmol ( Figure 5 D), Kaempferol 15μmol ( Figure 5 E), luteolin 10μmol ( Figure 5 F), Leonurus alkaloid 10μmol ( Figure 5 The optimal therapeutic concentration for G and the p38 MAPK inhibitor SB203580 is 15 μmol. Figure 5 H). Compared with the AngII model group (48.6% ± 3.2%), the cell survival rate of the above-mentioned component treatment groups was significantly increased (P<0.05), suggesting that these active components may play an endothelial protective role.

[0077] Measurement of reactive oxygen species (ROS): The levels of reactive oxygen species (ROS) in an Ang II-induced HUVEC cell endothelial dysfunction model were measured to show... Figure 7 A) Treatment with 1 μmol Ang II significantly increased the fluorescence intensity of ROS in cells. After treatment with quercetin, kaempferol, luteolin, and leonurine, ROS levels were significantly reduced, with quercetin showing the most significant inhibitory effect. This suggests that these components may improve endothelial cell oxidative stress damage by reducing Ang II-induced excessive ROS production. Figure 7 B).

[0078] ELISA assay: ELISA results showed that in HUVEC cells induced by Ang II (1 μmol), the inflammatory factor IL-6 (… Figure 7 E), PTGS2 ( Figure 7 F), TNF-α Figure 7 The content of G) was significantly increased, while among apoptosis-related proteins, the anti-apoptotic factor BCL2 (G) was significantly increased. Figure 7 The content of C) decreased, and the pro-apoptotic factor CASP3 (C) was reduced. Figure 7 D) Increased levels. After intervention with each component, the levels of inflammatory factors and apoptotic proteins were significantly improved, with regulatory trends consistent with the SB203580 group. Quercetin showed the most significant effect in downregulating inflammatory factors, while kaempferol showed the best effect in upregulating BCL2 and downregulating CASP3 protein. This suggests that the above components may alleviate Ang II-induced endothelial dysfunction by inhibiting the p38 MAPK pathway, synergistically reducing the inflammatory response and improving the imbalance of apoptosis.

[0079] Western blot analysis: Western blot results showed that in HUVEC cells induced by Ang II (1 μmol), the expression levels of AT1R, p53, and phosphorylated p38 (p-p38) proteins were significantly upregulated. However, after intervention with various components, the expression of the above proteins was downregulated to varying degrees, with the leonurine group showing the most significant downregulation of AT1R. Figure 8 A) Quercetin significantly inhibited the phosphorylation activation of p38MAPK, and its trend of action was consistent with that of SB203580. Figure 8B), while luteolin has the best effect in downregulating P53 protein ( Figure 8 C). This suggests that these components may exert endothelial protective effects by targeting the AT1R / p38 MAPK / p53 pathway.

[0080] Example 7: Determination of the content of core differentially active ingredients.

[0081] Chromatographic conditions: C18 column (250 mm × 4.6 mm, 5 μm) was used; mobile phase was methanol-10 μmol / L ammonium acetate aqueous solution (gradient elution: 0–20 min, methanol 45%–100%); flow rate was 1.0 mL / min; detection wavelength was 254 nm for quercetin, kaempferol, and luteolin, and 280 nm for leonurine; column temperature was 30 ℃; injection volume was 5 μL.

[0082] Preparation of mixed reference solution: Accurately weigh appropriate amounts of quercetin, luteolin, kaempferol, and leonurine reference standards, add 90% ethanol to fully dissolve them, and prepare a solution containing 0.098 mg / mL quercetin, 0.109 mg / mL luteolin, 0.103 mg / mL kaempferol, and 0.104 mg / mL leonurine, which is the mixed reference solution.

[0083] Preparation of the test solution: Motherwort herbs from six producing areas in Liaoning Province (Chaoyang County, Chaoyang City; Xinmin City, Shenyang City; Xifeng County, Tieling City; Benxi City; Xinbin Manchu Autonomous County, Fushun City; and Kuandian Manchu Autonomous County, Dandong City) were pulverized and sieved (using a No. 3 sieve). 1.0 g of powder from each area was extracted by reflux with 25 times the volume of 90% ethanol for 2 hours, repeated twice. The solvent was recovered to obtain the extract, which was then dissolved in 90% ethanol to prepare a solution with a concentration of 0.8 g / mL. -1 The solution was filtered through a 0.22 μm microporous membrane for analysis.

[0084] Methodological validation:

[0085] 1. Specificity test: Accurately pipette 5 μL each of the blank solvent (90% ethanol), the reference solution and the test solution from the precision test section, and the negative control test solution prepared according to the "Preparation of Test Solution" method, and inject them into the liquid chromatograph. Record the chromatograms. See [link to relevant documentation]. Figure 9 .Depend on Figure 9 As can be seen, the negative control showed no interference, indicating that the content determination method has good specificity and meets the requirements for content determination.

[0086] 2. Linearity Study: Accurately weigh appropriate amounts of quercetin, luteolin, kaempferol, and leonurine reference standards, dissolve them in 90% ethanol, and dilute to 50 mL to prepare mixed reference standard stock solutions with concentrations of 1.728 mg / mL, 1.792 mg / mL, 0.832 mg / mL, and 1.552 mg / mL, respectively. Then, accurately pipette 0.156 mL, 0.312 mL, 0.625 mL, 1.25 mL, 2.5 mL, 5 mL, and 10 mL of the stock solutions and dilute to 10 mL in volumetric flasks, respectively, and dilute to volume with 90% ethanol to obtain mixed reference standard solutions with seven concentration gradients. Filter the solutions through a 0.22 μm filter membrane for linearity study.

[0087] Table 3 Linear Relationship Table

[0088]

[0089]

[0090] 3. Precision test: The same sample solution from Kuandian Manchu Autonomous County, Dandong City, was injected six times repeatedly. The peak area was measured and the RSD was calculated. The results showed that the RSD of the peak areas of quercetin, luteolin, kaempferol, and leonurine were all less than 1.00%, which met the methodological requirements and indicated that the method had good precision. The results are detailed in Table 4.

[0091] Table 4. Precision test results

[0092]

[0093] 4. Stability Study: The same sample solution from Kuandian Manchu Autonomous County, Dandong City, was injected at 0h, 2h, 4h, 6h, 8h, 12h, and 24h. The peak areas of quercetin, luteolin, kaempferol, and leonurine were measured, and the RSD was calculated. The results are detailed in Table 5. The results show that the RSD of the peak areas of quercetin, luteolin, kaempferol, and leonurine in the test solution within 24h was all less than 2.00%, indicating that the test solution was stable within 24h.

[0094] Table 5. Stability Test Results

[0095]

[0096] 5. Repeatability Test: Approximately 1 g of *Leonurus japonicus* sample from Kuandian Manchu Autonomous County, Dandong City, was accurately weighed and placed in a stoppered conical flask. Six test solutions were prepared according to the prescribed method. 5 μL of each solution was accurately pipetted into the liquid chromatograph, and analysis was performed under the aforementioned chromatographic conditions. Peak areas were measured, and the results are detailed in Table 6. The results showed that the RSDs of quercetin, luteolin, kaempferol, and leonurine in the six samples were all less than 3.00%, meeting the methodological requirements and indicating good repeatability of the method.

[0097] Table 6 Results of Repeatability Tests

[0098]

[0099]

[0100] 6. Recovery test: Weigh about 0.5g of Leonurus japonicus sample powder from Kuandian Manchu Autonomous County, Dandong City, and add quercetin, luteolin, kaempferol, and leonurine reference solutions in proportion to the original amount to each sample. Then, extract and process according to the above-mentioned "Preparation of Test Solution", and determine the peak area of ​​each component according to the established chromatographic conditions. Calculate the recovery rate (recovery rate = (measured amount - original amount) / added amount × 100%) and relative standard deviation (RSD). See Table 7 for specific data.

[0101] Table 7 Results of the recovery rate test

[0102]

[0103]

[0104] 7. Sample testing: The method of this invention was used to test the motherwort herbs from 6 production areas. The test results are shown in Table 8.

[0105] Table 8. Results of Content Determination

[0106]

[0107] 8. Content limits: In accordance with the 2025 edition of the Chinese Pharmacopoeia 9101 Analytical Method Validation Guidelines, it is stipulated that the content of quercetin in Leonurus japonicus shall not be less than 0.014%, luteolin not less than 0.021%, kaempferol not less than 0.017%, and leonurine not less than 0.100%.

Claims

1. A quality control method for Leonurus japonicus (a traditional Chinese medicine) harvested at different times, characterized in that... Includes the following steps: Step 1: Preparation of the test solution; Step 2, Preparation of mixed reference solution: Accurately weigh appropriate amounts of quercetin, luteolin, kaempferol, and leonurine reference standards, add 90% ethanol to fully dissolve them, and prepare a solution containing 0.098 mg / mL quercetin, 0.109 mg / mL luteolin, 0.103 mg / mL kaempferol, and 0.104 mg / mL leonurine, which is the mixed reference solution; Step 3, Determination Method: Accurately pipette the mixed reference solution and the test solution separately, inject them into the liquid chromatograph, and determine the result.

2. The quality control method for Leonurus japonicus (a traditional Chinese medicine) at different harvesting periods according to claim 1, characterized in that, Step 1 specifically involves: pulverizing the motherwort herb through a No. 3 sieve, taking 1.0 g of the powder, and refluxing it with 25 times the amount of 90% ethanol for 2 hours. This extraction is repeated twice. The solvent is recovered to obtain the extract, which is then dissolved in 90% ethanol to prepare a herb concentration of 0.8 g·mL. -1 The solution was filtered through a 0.22 μm microporous membrane for analysis.

3. The quality control method for Leonurus japonicus (a traditional Chinese medicine) at different harvesting periods according to claim 1, characterized in that, In step 3, a C18 column was used; the mobile phase was methanol-10 μmol / L ammonium acetate aqueous solution, with gradient elution: 0~20 min, methanol 45%-100%; the flow rate was 1.0 mL / min; the detection wavelengths were 254 nm for quercetin, kaempferol, and luteolin, and 280 nm for leonurine; the column temperature was 30℃; and the injection volume was 5 μL.

4. The quality control method for Leonurus japonicus (a traditional Chinese medicine) at different harvesting periods according to claim 1, characterized in that, In step 3, the determination requirements are as follows: the content of quercetin in the motherwort herb shall not be less than 0.014%, luteolin not less than 0.021%, kaempferol not less than 0.017%, and leonurine not less than 0.100%.

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