A cultivation method for improving active components of prunella vulgaris

Abstract

This invention belongs to the field of medicinal plant cultivation technology, specifically relating to a cultivation method for enhancing the active components of Prunella vulgaris plants. The method involves applying a treatment solution containing a nitric oxide donor at a concentration of 50-200 μM to the above-ground parts of Prunella vulgaris plants, and harvesting the above-ground parts 24-48 hours after treatment. This invention, through the exogenous application of a nitric oxide donor, synergistically regulates endogenous hormones and secondary metabolic pathways in Prunella vulgaris, significantly increasing the content of active components such as rosmarinic acid, ursolic acid, and total flavonoids. It has the advantages of simple operation, low cost, clear mechanism, and suitability for large-scale application.

Description

Technical Field

[0001] This invention belongs to the field of medicinal plant cultivation technology, specifically relating to a cultivation method for improving the active ingredients of Prunella vulgaris plants. Background Technology

[0002] Prunella vulgaris L. is an important economic crop with both medicinal and edible uses, widely used in Asia and Europe for the production of functional teas, pharmaceuticals, and as a natural preservative in food. Its main active ingredients are phenolic acids (such as rosmarinic acid) and triterpenoids (such as ursolic acid), and the content of these components is a key indicator for evaluating the quality of the medicinal material in the Chinese Pharmacopoeia.

[0003] Currently, technologies for increasing the content of active ingredients in Prunella vulgaris mainly focus on two aspects: one is by altering the cultivation environment (such as abiotic stresses like low light and drought); the other is by applying exogenous inducers (such as hydrogen peroxide and methyl jasmonate). Chinese Patent Publication No. 202211442842.3 discloses a method for inducing the accumulation of phenolic acids and triterpenoids in Prunella vulgaris using hydrogen peroxide (H2O2). Spraying the leaves with a 2 mM H2O2 solution during the full bloom period, after 24 hours of treatment, can increase the content of rosmarinic acid and ursolic acid in the leaves. However, this method mainly works through oxidative stress, which carries a certain risk of damage to plant cells and may lead to a decrease in biomass. Furthermore, the synergistic regulatory mechanism between the H2O2 signaling pathway and plant hormones is still unclear, and the complete regulatory network from signaling molecules to transcription factors, key enzyme genes, and final metabolites has not been revealed, limiting the precise optimization and stable reproduction of the technology.

[0004] Furthermore, Tang et al. (2023) reported in *Acta Physiol. Plant.* a method to promote rosmarinic acid accumulation in *Prunella vulgaris* by spraying methyl jasmonate (MeJA), specifically by spraying a 1 mM MeJA solution during the inflorescence growth period and harvesting 24 hours later. However, this method has the following drawbacks: methyl jasmonate is relatively expensive and volatile, resulting in unstable field application effects; this technology mainly promotes the accumulation of phenolic acids, with little effect on triterpenoids such as ursolic acid; and the existing technology has not elucidated the potential link between MeJA and upstream signaling molecules (such as nitric oxide), nor has it provided a more upstream and comprehensive regulatory strategy that can simultaneously and synergistically regulate the accumulation of multiple active ingredients.

[0005] Nitric oxide (NO), as an important signaling molecule, is known to play a crucial role in plant growth, development, and stress responses. Compounds such as sodium nitroprusside (SNP) can serve as NO donors, and existing research indicates that SNP treatment can promote the accumulation of phenolic acids or terpenes in other medicinal plants such as *Salvia miltiorrhiza* and *Artemisia annua*. However, current technology has not elucidated how NO donors treat *Prunella vulgaris* (e.g., concentration, time, location), or through which specific molecular regulatory networks (involving key genes, metabolites, and endogenous hormones) synergistically increase the content of various active ingredients (especially rosmarinic acid and ursolic acid) in *Prunella vulgaris*. This lack of systematic molecular mechanism analysis prevents the development of a targeted, effective, and stable large-scale application technology.

[0006] Therefore, there is an urgent need for a treatment method with a clear mechanism of action, well-defined targets, minimal damage to plants, and ease of standardized operation, in order to simultaneously and synergistically increase the content of multiple key active ingredients such as phenolic acids and triterpenoids in Prunella vulgaris. Summary of the Invention

[0007] Based on the above analysis, the technical solution of this application discloses a cultivation method for improving the active ingredients of Prunella vulgaris plants, which includes applying a treatment solution containing a nitric oxide donor at a concentration of 50-200 μM to the above-ground parts of Prunella vulgaris plants, and harvesting the above-ground parts 24-48 hours after treatment.

[0008] Furthermore, the nitric oxide donor is any one of sodium nitroprusside, S-nitrosoglutathione, or diethylamine / nitric oxide adduct.

[0009] Furthermore, the nitric oxide donor is sodium nitroprusside.

[0010] Furthermore, the concentration of the nitric oxide donor in the treatment solution is 100 μM.

[0011] Furthermore, the application method is foliar spraying.

[0012] Furthermore, the processing time is 24 hours.

[0013] Furthermore, the *Prunella vulgaris* plant is a seedling at the six-leaf stage.

[0014] Furthermore, the harvested above-ground parts include leaves or fruit clusters.

[0015] Furthermore, the treatment solution also contains methyl jasmonate and / or hydrogen peroxide.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1. Significant synergistic enhancement of active ingredients: After treating Prunella vulgaris plants with the method of this invention, the content of rosmarinic acid in the leaves increased by up to 488.0%, the content of ursolic acid increased by up to 488.4%, and the content of total flavonoids increased by 141.7%. This achieved synergistic enhancement of multiple core active ingredients such as phenolic acids and triterpenoids, with comprehensive and significantly better effects than existing single induction techniques.

[0018] 2. Clear mechanism of action, facilitating precise regulation: This invention reveals the complete molecular mechanism by which exogenous nitric oxide donors activate two biosynthetic pathways of phenolic acids and triterpenes by regulating endogenous jasmonic acid signaling and specific transcription factors (such as the MYB and bHLH families). This provides clear gene targets and theoretical basis for subsequent variety improvement, cultivation optimization and precision control.

[0019] 3. Minimal damage to plants and improved medicinal quality: Compared with oxidative stress induction, the method of this invention has a low risk of damaging plant cells and does not significantly inhibit biomass; and due to the significant increase in active ingredients, the in vitro antioxidant activity of Prunella vulgaris extract after treatment is significantly enhanced, directly improving its quality as a raw material for functional foods or medicines.

[0020] 4. Simple operation, low cost, and suitable for industrialization: The cost of nitric oxide donors is much lower than that of inducers such as methyl jasmonate. Foliar spraying is simple and easy to implement, and the treatment concentration and time conditions are clear, which facilitates standardized operation and is suitable for large-scale application in artificial cultivation and understory ecological planting.

[0021] 5. Wide applicability and flexible application: The method of this invention is not only applicable to promoting the accumulation of active ingredients in the leaves of Prunella vulgaris, but also applicable to medicinal parts such as fruit spikes; at the same time, the nitric oxide donor can also be used in combination with low concentrations of methyl jasmonate or hydrogen peroxide, which has good technical scalability and application flexibility. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention 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.

[0023] Figure 1 The results of rosemary content determination for each group of medicinal materials in Example 1;

[0024] Figure 2 The results of ursolic acid content determination of each group of medicinal materials in Example 1;

[0025] Figure 3 The results of the determination of total flavonoid content in each group of medicinal materials in Example 1;

[0026] Figure 4 The results of the test of metabolite components of each group of medicinal materials in Example 1 are as follows: (A) nitric oxide (NO) content; (B) hydrogen peroxide (H2O2) content; (C) peroxidase (POD) activity; (D) superoxide dismutase (SOD) activity; (E) catalase (CAT) activity; (F) ascorbate peroxidase (APX) activity; (G) ascorbic acid (AsA) content; (H) glutathione (GSH) content; (I) ... (J) represents soluble sugar content; (K) represents soluble protein content; (L) represents malondialdehyde (MDA) content; (M) represents proline (Pro) content; (N) represents phenylalanine ammonia-lyase (PAL) activity; (O) represents polyphenol oxidase (PPO) activity; and (O) is a heatmap of log2-transformed FPKM values ​​of differentially expressed genes encoding APX, CAT, and glutathione S-transferase (GST) in leaves of the CK and SNP treatment groups after 24 hours of treatment. Data are expressed as mean ± SD (n=3). Different lowercase letters above the columns indicate significant differences between treatments at the p<0.05 level (one-way ANOVA combined with LSD test).

[0027] Figure 5 The effects of 100 μM exogenous SNP treatment for 24 h on endogenous plant hormone levels and related transcriptional regulation in Prunella vulgaris raw materials were investigated. (A) represents jasmonic acid (JA) content; (B) represents indole-3-acetic acid (IAA) content; and (C) represents salicylic acid (SA) content. Data are expressed as mean ± SD (n = 3). Different lowercase letters above the columns indicate significant differences between treatments at the p < 0.05 level (ANOVA combined with LSD test). (D) is a schematic diagram of transcriptional regulation in JA biosynthesis and signaling pathways; (E) is a heatmap of log2-converted FPKM values ​​for differentially expressed genes related to JA metabolism and signaling; (F) is a schematic diagram of transcriptional regulation in the IAA biosynthesis pathway; and (G) is a heatmap of log2-converted FPKM values ​​for differentially expressed genes related to IAA biosynthesis. In (E) and (G), red and blue boxes represent upregulated and downregulated differentially expressed genes, respectively.

[0028] Figure 6The effects of 100 μM exogenous SNP treatment at 2, 24, and 48 hours on the levels of specific and total bioactive compounds in Prunella vulgaris raw materials were investigated. (A) represents rosmarinic acid (RA) content; (B) represents ursolic acid (UA) content; (C) represents total phenolic acid content; (D) represents total triterpenoid content; and (E) represents total flavonoid content. Data are expressed as mean ± SD (n = 3). Different lowercase letters above the columns indicate significant differences between treatments at the p < 0.05 level (one-way ANOVA combined with LSD test).

[0029] Figure 7 Transcriptional reprogramming of key secondary metabolic pathways in Prunella vulgaris raw materials 24 hours after treatment with 100 μM SNP-1; (A) is a schematic diagram of the phenylpropane / rosmarinic acid biosynthesis pathway, with differentially expressed genes marked, and red and blue boxes representing upregulated and downregulated genes, respectively; (B) is a log2-converted FPKM value expression heatmap of differentially expressed genes related to polyphenol biosynthesis.

[0030] Figure 8 Transcriptional reprogramming of key secondary metabolic pathways in Prunella vulgaris raw material 24 hours after treatment with 100 μM SNP-2; (A) is a schematic diagram of the terpene skeleton / ursolic acid biosynthesis pathway, with differentially expressed genes marked; (B) is a log2 conversion FPKM value expression heatmap of differentially expressed genes related to triterpene biosynthesis.

[0031] Figure 9 Effect of exogenous 100 μM SNP treatment on the in vitro antioxidant capacity of ethanol extract from common bean leaves (2, 24, and 48 hours after treatment) (A) DPPH free radical scavenging activity, at half-maximal inhibitory concentration (IC50). 50 (B) ABTS free radical scavenging activity, expressed as IC50. 50 (C) Correlation heatmap, showing the Pearson correlation coefficients among antioxidant activities (DPPH, ABTS), key active ingredients, and endogenous plant hormones at 24-hour time points. RA: Rosmarinic acid; UA: Ursolic acid; TF: Total flavonoids; TP: Total phenolic acids; TT: Total triterpenes; JA: Jasmonic acid; IAA: Indole-3-acetic acid. Data in (A) and (B) are mean ± standard deviation (n=3). Different lowercase letters above the columns indicate significant differences between treatments at the same time point (p < 0.05, LSD test after one-way ANOVA). * indicates the significance level of the correlation coefficient: * p < 0.05.

[0032] Figure 10Differential expression of transcription factors in response to SNP treatment; where (A) represents the number of differentially expressed transcription factors between the CK and SNP (100 μM) treatment groups at 24 hours (FDR < 0.01, |log2FC| ≥ 1); (B–E) are heatmaps of expression of key transcription factor families (MYB / MYB related, bHLH, WRKY, and NAC) based on log2 (FPKM) values;

[0033] Figure 11 Metabolomics analysis and KEGG pathway enrichment analysis of Prunella vulgaris raw materials 24 hours after treatment with 100 μM exogenous SNPs. (A) Classification of differentially abundant metabolites (DAMs); (B) Number of upregulated and downregulated flavonoid, terpene, and phenolic acid DAMs in the CK_24 and SNP_24 comparison groups; (C) KEGG pathway enrichment analysis of DAMs. The left axis lists the pathway names. Dots represent DAMs; larger dots indicate more DAMs enriched in that pathway. The color gradient from red to blue corresponds to decreasing significance (i.e., p-value from small to large). Red boxes highlight key pathways related to isoflavone biosynthesis, terpene skeleton biosynthesis, and diterpene biosynthesis (from top to bottom); (D) KEGG enrichment pathway analysis integrating DEGs and DAMs. Triangles represent DEGs, and dots represent DAMs; larger symbols indicate more DEGs or DAMs enriched in that pathway. The color gradient from red to blue corresponds to a decrease in significance (i.e., p-values ​​from small to large). The red boxes highlight the key pathways: diterpene biosynthesis; phenylalanine, tyrosine, and tryptophan biosynthesis; flavonoid biosynthesis; and terpene skeleton biosynthesis (from top to bottom).

[0034] Figure 12 This study analyzed the correlation network of key DAMs and DEGs related to the biosynthesis of flavonoids, terpenoids, and phenolic acids in Prunella vulgaris raw materials 24 hours after SNP treatment. Significant correlations were defined as r² ≥ 0.8 and p < 0.05; only significant correlations are shown in the figure.

[0035] Figure 13 This is a regulatory model for SNP-induced metabolic reprogramming of Prunella vulgaris seedlings. Detailed Implementation

[0036] To make the technical problems solved, the technical solutions, and the beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0037] The embodiments of this application disclose a cultivation method for improving the active ingredients of Prunella vulgaris plants, which includes applying a treatment solution containing a nitric oxide donor at a concentration of 50-200 μM to the above-ground parts of Prunella vulgaris plants, and harvesting the above-ground parts 24-48 hours after treatment.

[0038] The term "nitric oxide donor (NO donor)" refers to a compound capable of releasing nitric oxide (NO) or NO-related active species in aqueous solutions or within the plant environment. Nitric oxide donors include, but are not limited to, sodium nitroprusside (SNP), S-nitrosoglutathione (GSNO), and diethylamine / nitric oxide adduct (DEA / NONOate). Sodium nitroprusside is a preferred donor in this invention due to its good water solubility, moderate NO release rate, low cost, and ease of acquisition.

[0039] The "treatment solution" refers to a homogeneous aqueous solution prepared with nitric oxide donor as the active ingredient and a solvent. The solvent is preferably sterile distilled water to avoid interference from microorganisms or impurities with the stability of the NO donor and the normal physiological state of the plant leaves. The treatment solution should be prepared and used immediately and stored away from light to prevent premature decomposition and release of NO by the NO donor under light conditions, which could lead to loss of the active ingredient and concentration deviation.

[0040] The concentration of the nitric oxide donor treatment solution is 50-200 μM: this concentration range is determined based on the signal transduction kinetics of NO in plants and cell tolerance. Within this range, exogenous NO can effectively trigger signal cascade reactions in plants, activate the expression of secondary metabolism-related genes, thereby promoting the accumulation of phenolic acids and triterpenoid active ingredients; at the same time, this concentration range is below the threshold for causing significant oxidative damage or growth inhibition in *Prunella vulgaris* plants, thus balancing treatment efficacy and plant safety. According to a preferred embodiment of the present invention, the concentration of the nitric oxide donor is further preferably 100 μM, which achieves an optimal balance between the increase in active ingredient content, treatment cost, and operational safety.

[0041] Harvesting is carried out 24-48 hours after the above treatment: this time window is determined based on the time-kinetic characteristics of NO signal transduction, endogenous hormone reprogramming, and the synthesis and accumulation of secondary metabolites. Within this time interval after treatment, the expression of key enzyme genes related to the biosynthesis of phenolic acids and triterpenes in *Prunella vulgaris* plants has been significantly upregulated, and the corresponding metabolites have been fully synthesized and accumulated to a detectably increased level. According to a preferred embodiment of the present invention, the treatment time is further preferably 24 hours, at which time the accumulation of key active ingredients such as rosmarinic acid and ursolic acid reaches its peak.

[0042] The term "above-ground parts" refers to the organs of the plant above the soil surface, including but not limited to leaves, stems, inflorescences, and fruit spikes. In this invention, the above-ground parts are preferably leaves and / or fruit spikes, as they are the main components bearing the medicinal and edible value of Prunella vulgaris.

[0043] In a further embodiment, "application" refers to treating the solution in any way that allows the aboveground parts of the *Prunella vulgaris* plant to effectively absorb or sense it. The application methods include, but are not limited to, foliar spraying, stem application, root irrigation, or injection of the solution into the cultivation substrate. Foliar spraying is the preferred application method of this invention; specifically, a misting nozzle can be used to evenly spray the solution onto both sides of the leaves until droplets are about to fall. This method allows the NO donor to act directly on the metabolically active mesophyll cells, resulting in rapid absorption and simple operation. If root irrigation or substrate injection is used, the treatment time can be appropriately extended or the concentration slightly increased to ensure sufficient NO signal transduction to the aboveground parts.

[0044] In a preferred embodiment, this application uses *Prunella vulgaris* seedlings that have grown to the six-leaf stage. Plants at this growth stage have vigorous metabolism, and their secondary metabolic pathways are highly sensitive to exogenous signals, enabling a significant increase in active ingredients within a short period. However, the method of this invention is also applicable to *Prunella vulgaris* plants at other growth stages, such as the vegetative growth stage or the inflorescence growth stage; only the amount of treatment solution applied needs to be adjusted appropriately according to the plant size and physiological state.

[0045] In a further embodiment, after the treatment solution is applied, the *Prunella vulgaris* plants can continue to be cultivated under conventional greenhouse conditions, such as maintaining the relative moisture content of the substrate at 60%–70% of field capacity, a day / night temperature of 25°C / 10°C, a photoperiod of 12 / 12 hours, a relative humidity of 50%–60%, and a light intensity of 700–1300 μmol·m⁻¹. -2 ·s -1 During harvesting, all above-ground parts can be collected, or leaves, fruit clusters, and other parts can be selectively harvested according to medicinal needs. The harvested material can be used to extract active ingredients or directly as high-quality medicinal raw materials.

[0046] In a further embodiment, the treatment solution may further contain methyl jasmonate (MeJA) and / or hydrogen peroxide (H2O2). In a specific embodiment of the present invention, 0.1-1 mM of MeJA and / or 1-5 mM of H2O2 can be combined in a 100 μM SNP treatment solution. This combined treatment can produce a synergistic effect based on the cross-interaction mechanism between NO signaling and jasmonate signaling or oxidative stress signaling, further promoting the accumulation of active ingredients. However, it should be noted that the concentration of each component should be adjusted accordingly during the combined treatment to avoid causing excessive physiological stress to the plant.

[0047] The technical means and effects of this application will be described in detail below through specific embodiments.

[0048] Example 1: A cultivation method for enhancing the active ingredients in Prunella vulgaris plants

[0049] 1. Material preparation: The seeds of *Prunella vulgaris* L. were collected in June 2023 from Shejian Town, Zhaohua District, Guangyuan City, Sichuan Province (latitude 32°25′N, longitude 105°43′E). The seeds were air-dried naturally before use.

[0050] Before sowing, the seeds were surface-sterilized: seeds were placed in a 2% (v / v) hydrogen peroxide solution, gently shaken and soaked for 2 minutes, then rinsed repeatedly with sterile distilled water 3-4 times (30 seconds each time). The sterilized seeds were then soaked in pure water at 25±1℃ for 12 hours to promote water absorption and germination. Subsequently, 100 seeds were evenly sown in 90 mm sterile petri dishes lined with double layers of moist sterile filter paper, and germinated in an incubator at 25±1℃. After 20 days, seedlings with uniform growth at the two-leaf stage were selected and transplanted into plastic seedling trays (50 holes / tray, single hole dimensions: length × width = 5 cm × 5 cm, height 10 cm). Each hole was filled with 25±2 g of sterilized cultivation substrate (sandy loam: vermiculite = 20:1, v / v, pH 7.23), and the substrate's physicochemical properties were determined according to Chinese agricultural and forestry industry standards.

[0051] After transplanting, water regularly with sterile distilled water to maintain the relative moisture content of the substrate at 60%–70% of field capacity. Seedlings were cultivated in an experimental greenhouse under the following environmental conditions: a photoperiod of 12 h light / 12 h darkness, day / night temperature of 25℃ / 10℃, relative humidity of 50%–60% (monitored daily at 10:00 and 16:00), and photosynthetically active radiation (PAR) of 700–1300 μmol·m⁻¹. -2 ·s -1 (Measured at noon using a TES-1335 PAR sensor). After approximately 70 days of cultivation, exogenous application of the herbicide was performed when the *Prunella vulgaris* plants reached the six-leaf stage.

[0052] 2. Preparation of treatment solutions: Sodium nitroprusside (SNP, analytical grade, purity ≥99%, Shanghai Maclean Biochemical Technology Co., Ltd.) was used as the active ingredient. Solutions were prepared fresh with sterile distilled water and stored protected from light. SNP aqueous solutions with concentrations of 50 μmol / L (μM), 100 μM, and 200 μM were prepared. The control group (CK) used an equal volume of sterile distilled water (0 μM SNP).

[0053] 3. Exogenous application: Choose a sunny morning at 10:00 AM (ambient temperature 25±2℃, PAR 800±50 μmol·m⁻¹).-2 ·s -1 Foliar spraying was performed. Using a misting nozzle (0.5 mm diameter), the SNP aqueous solution prepared in step 2 was evenly sprayed onto both sides of the leaves of the Prunella vulgaris seedlings until droplets were about to drip from the leaf surface (approximately 200 mL of solution was sprayed per seedling tray to ensure that the leaves were fully and evenly covered with the drug without causing mechanical damage). The control group was sprayed with an equal volume of sterile distilled water (CK, 0 μM SNP).

[0054] 4. Cultivation and Harvesting: The treated Prunella vulgaris seedlings were further cultivated under the same greenhouse conditions as described above. The above-ground parts (mainly leaves) of Prunella vulgaris were harvested at 2 h, 24 h, 48 h, and 72 h after treatment for the extraction of active ingredients or as high-quality medicinal raw materials. At each time point and for each treatment, 5 seedlings were randomly selected from each replicate tray (15 seedlings in total for each treatment), and the top pair of fully expanded true leaves at the top of each plant were collected.

[0055] Samples for active ingredient extraction: Fresh leaves were dried in a 60℃ forced-air drying oven for 5 hours until constant weight, then pulverized and passed through a 60-mesh sterile sieve to obtain dried powder, which was then sealed and stored for later use.

[0056] Samples used for physiological, biochemical, or omics analysis: Fresh leaves were rapidly placed in liquid nitrogen for 5 minutes to terminate the physiological reaction, wrapped in aluminum foil, and stored in an ultra-low temperature freezer at -80°C for subsequent determination of the content of active ingredients such as total phenolic acids, total triterpenes, total flavonoids, rosmarinic acid, and ursolic acid, as well as analysis of antioxidant activity.

[0057] Experimental Example 1: Determination of the content of key bioactive components

[0058] The contents of rosemary, ursolic acid and total flavonoids in each group of medicinal materials obtained in Example 1 were determined. The contents of rosmarinic acid and ursolic acid were determined by high performance liquid chromatography-diode array detector (HPLC-DAD), and the contents of total flavonoids were determined by sodium nitrite-aluminum nitrate colorimetric method.

[0059] The measurement results are as follows Figure 1 (Rosemary content) Figure 2 (ursolic acid content) Figure 3 (Total flavonoid content) is shown below:

[0060] Depend on Figure 1 It was found that, compared with the control group (CK), treatment with different concentrations of SNP (50, 100, and 200 μM) all increased the rosmarinic acid content, exhibiting a certain time-dose dependence. At 2 h of treatment, the rosmarinic acid content in each SNP concentration group was slightly increased, with the 200 μM SNP group reaching 5.863 mg·g. -1The result was approximately the same as the control group (2.536 mg / g). -1 The concentration of SNPs was 2.31 times that of the control group. After 24 h of treatment, the concentrations in each SNP group increased significantly, with the 100 μM and 200 μM SNP groups reaching 14.815 mg·g⁻¹. -1 and 16.329 mg·g -1 The values ​​were 2.519 mg / g, respectively, compared to the control group at the same time point. -1 The levels were 5.88 and 6.48 times higher than the control, with the most significant increase. At 48 h, the content decreased slightly compared to 24 h, but was still much higher than the control; the 200 μM SNP group was 14.665 mg / g. -1 At 72 h, except for the 50 μM SNP group which maintained a high level (8.188 mg·g⁻¹), -1 In addition, the 100 μM and 200 μM SNP groups decreased to 6.325 mg·g, respectively. -1 and 8.894 mg·g -1 The results showed that SNP treatment significantly promoted the accumulation of rosmarinic acid, with the best effect achieved by treating with 100–200 μM SNP for 24 h.

[0061] Depend on Figure 2 It can be seen that the ursolic acid content also changed significantly after SNP treatment. At 2 h, the content in each SNP group was slightly higher than that in the control (0.232 mg / g). -1 The 200 μM group had a concentration of 0.247 mg·g. -1 At 24 h, the 50 μM SNP group and the control (0.244 mg / g) showed significant differences. -1 The levels remained basically unchanged, while the levels in the 100 μM and 200 μM SNP groups increased sharply to 1.436 mg·g⁻¹. -1 and 1.008 mg·g -1 The levels were approximately 5.89 times and 4.13 times higher than the control, respectively. At 48 h, the 100 μM and 200 μM SNP groups still maintained high levels (1.114 mg / g). -1 and 0.595 mg·g -1 The 50 μM SNP group also increased to 0.852 mg / g. -1 At 72 h, the concentrations in all treatment groups decreased compared to 48 h, but the 100 μM SNP group still reached 0.962 mg / g. -1 Overall, treatment with 100 μM SNP for 24 h was most conducive to the accumulation of ursolic acid.

[0062] Depend on Figure 3 It was found that the total flavonoid content was most sensitive to the SNP treatment. At 2 h, the total flavonoid content in the 200 μM SNP group increased to 133.997 mg·g⁻¹.-1 The concentration was higher than the control (109.198 mg / g). -1 At 24 h, the total flavonoid content in all SNP treatment groups increased significantly, reaching 246.396, 283.734, and 276.284 mg·g in the 50, 100, and 200 μM groups, respectively. -1 The values ​​were 117.369 mg / g, respectively, compared to the control group at the same time point (117.369 mg / g). -1 The levels were 2.10, 2.42, and 2.35 times higher than those of the control group. At 48 h, although the levels in each group decreased slightly, they remained at a relatively high level (253.6–265.2 mg / g). -1 At 72 h, the levels in each group decreased further, but remained significantly higher than the control (126.686 mg / g). -1 The 50 μM SNP group had a concentration of 159.419 mg·g⁻¹. -1 The 100 μM group had a concentration of 146.713 mg·g. -1 This indicates that SNP treatment can significantly promote the synthesis and accumulation of total flavonoids, with the strongest promoting effect observed when treated with 100 μM SNP for 24 h.

[0063] Experimental Example 2: Determination of the content of metabolite components

[0064] Experimental Methods: Oxidative stress markers, antioxidant defense systems, osmotic regulators, and the activities of secondary metabolism-related enzymes were tested in leaf samples of *Prunella vulgaris* seedlings obtained in Example 1 (CK group and 100 μM SNP group, 2 h, 24 h, and 48 h after treatment). All assays were performed using commercially available kits provided by Nanjing Jiancheng Bioengineering Research Institute, following the instructions. Each sample was tested in triplicate, and results are expressed as mean ± standard deviation.

[0065] The NO content was determined using the nitrate reductase method at a detection wavelength of 550 nm, and the H₂O₂ content was determined using the ammonium molybdate colorimetric method at a detection wavelength of 405 nm. Results were expressed as μmol·g⁻¹. -1 protein and mmol·g -1 Protein representation.

[0066] Superoxide dismutase (SOD) activity: xanthine oxidase method, detection wavelength 450 nm, unit U·mg -1 protein.

[0067] Peroxidase (POD) activity: guaiacol method, detection wavelength 420 nm, unit U·mg -1 protein.

[0068] Catalase (CAT) activity: ammonium molybdate method, detection wavelength 405 nm, unit U·mg -1protein.

[0069] Ascorbate peroxidase (APX) activity: detection wavelength 290 nm, unit U·mg -1 protein.

[0070] Ascorbic acid (AsA) was detected using the phenanthroline colorimetric method at a wavelength of 536 nm, with units of μg·mg. -1 Protein; glutathione (GSH) was detected using the dithiodinitrobenzoic acid (DTNB) method at a wavelength of 420 nm, with units of mg GSH·g. -1 protein.

[0071] Soluble sugar content: anthrone colorimetric method, detection wavelength 620 nm, unit μg·g -1 Fresh weight (FW).

[0072] Soluble protein content: Coomassie Brilliant Blue G-250 staining method, detection wavelength 595 nm, unit g·L -1 .

[0073] Proline (Pro) content: Acidic ninhydrin colorimetric method, detection wavelength 520 nm, unit μg·g -1 FW.

[0074] Malondialdehyde (MDA) content: Thiobarbituric acid (TBA) method, detection wavelength 532 nm, unit nmol·g -1 protein.

[0075] Phenylalanine ammonia-lyase (PAL) activity: detection wavelength 290 nm, unit U·mg -1 protein.

[0076] Polyphenol oxidase (PPO) activity: catechol colorimetric method, detection wavelength 420 nm, unit U·g -1 FW.

[0077] All the above indicators were measured using a UV-5880 UV-Vis spectrophotometer (Shanghai Younico Instruments Co., Ltd.). For specific calculation formulas, please refer to the instructions for each reagent kit.

[0078] Experimental Results: Physiological and biochemical indicators of Prunella vulgaris seedling leaves in the control group (CK) and the 100 μM SNP treatment group in Example 1 were measured at 2 h, 24 h, and 48 h. The results are as follows: Figure 4 As shown ( Figure 4 Subplots A–N for each indicator represent NO, H₂O₂, POD, SOD, CAT, APX, AsA, GSH, soluble sugar, soluble protein, MDA, Pro, PAL, and PPO, respectively. The main results are as follows:

[0079] (1) NO and H2O2 content

[0080] Compared with the control (CK), the SNP treatment significantly increased the NO content in the leaves ( Figure 4 A). After treatment, NO content increased by approximately 243.6%, 88.0%, and 26.8% at 2 h, 24 h, and 48 h, respectively. Conversely, SNP treatment significantly reduced H2O2 content ( Figure 4 B), which showed reductions of approximately 19.2%, 12.0%, and 11.6% at the corresponding time points, respectively. This indicates that exogenous NO can effectively alleviate oxidative stress.

[0081] (2) Antioxidant enzyme activity

[0082] SNP treatments have different effects on antioxidant enzyme activity. Figure 4 C–F). POD activity increased slightly (+2.0%) at 2 h, but decreased significantly by 53.4% ​​and 18.2% at 24 h and 48 h, respectively. Figure 4 C). SOD activity increased significantly at all time points, by 13.6%, 9.0%, and 32.5%, respectively. Figure 4 D). CAT activity increased most significantly, increasing by 121.0%, 175.4%, and 50.7%, respectively. Figure 4 E). APX activity also increased significantly, by 93.3%, 161.3%, and 105.3%, respectively. Figure 4 F).

[0083] (3) AsA and GSH content

[0084] Two hours after SNP treatment, the AsA content decreased slightly (-3.7%), but significantly increased by 10.4% and 6.4% at 24 and 48 hours, respectively. Figure 4 G). GSH levels increased significantly at all time points, increasing by 7.3%, 24.7%, and 53.0%, respectively. Figure 4 H).

[0085] (4) Osmotic regulators

[0086] The soluble sugar content increased significantly at all time points after SNP treatment, increasing by 95.9%, 9.2%, and 19.2%, respectively. Figure 4 I). The soluble protein content showed a trend of first decreasing and then increasing: it decreased by 8.4% at 2 h, and increased by 15.5% and 4.6% at 24 h and 48 h, respectively. Figure 4 J). MDA content increased significantly, by 49.8%, 91.0%, and 44.3%, respectively. Figure 4 K). Proline (Pro) content also increased significantly, rising by 79.0%, 67.9%, and 120.3%, respectively. Figure 4 L).

[0087] (5) PAL and PPO activity

[0088] PAL activity increased by 41.7% and 38.8% at 2 h and 24 h, respectively, but decreased by 10.3% compared to the control at 48 h. Figure 4 M). PPO activity increased significantly at all time points, by 66.9%, 35.5%, and 196.2%, respectively. Figure 4 N).

[0089] (6) Comprehensive analysis and determination of optimal conditions

[0090] The results of combined experiment 1 (determination of active ingredient content) and experiment 2 (determination of physiological and biochemical indicators) are as follows:

[0091] Regarding active ingredients: After treatment with 100 μM SNP for 24 h, rosmarinic acid (14.815 mg·g) was reduced. -1 ), ursolic acid (1.436 mg·g) -1 ) and total flavonoids (283.734 mg·g -1 The content of all three reached peak values ​​or significantly high levels, being 5.88 times, 5.89 times, and 2.42 times that of the control, respectively.

[0092] In terms of physiology and biochemistry: after 24 h of treatment with 100 μM SNP, CAT and APX activities increased by 175.4% and 161.3%, respectively, GSH increased by 24.7%, soluble protein increased by 15.5%, and MDA and proline also accumulated significantly, indicating that the plant's antioxidant capacity was enhanced, secondary metabolism was active, and there was a certain degree of stress response. PAL activity remained at a high level (+38.8%) after 24 h of treatment, which is conducive to the synthesis of phenolic acids.

[0093] In summary, the optimal concentration of SNP aqueous solution was 100 μM, and the optimal treatment time was 24 h. Subsequent experiments (such as transcriptomics and metabolomics) were all conducted under these conditions.

[0094] Experiment Example 3: Validation Experiment on Endogenous Plant Hormone Levels and Related Transcriptional Regulation

[0095] Based on Experiment 1 and Experiment 2, experiments were conducted on the endogenous plant hormone levels and related transcriptional regulation of Prunella vulgaris raw materials treated with 100 μM sodium nitroprusside (SNP) aqueous solution for 24 h.

[0096] Experimental methods:

[0097] (1) Extraction of endogenous plant hormones

[0098] Fresh leaf samples of Prunella vulgaris stored at -80℃ were placed in a pre-cooled grinding jar and homogenized into powder using a ball mill (30 Hz, 1 min). 50.0 ± 0.1 mg of powder was accurately weighed and added to 10 μL of deuterated internal standard solution (100 ng·mL⁻¹). -1 Then add 1 mL of pre-cooled (4℃) extraction solvent (methanol / water / formic acid = 15:4:1, v / v / v, containing 0.1% BHT antioxidant). Mix thoroughly using a multi-tube vortex mixer (3000 rpm, 10 min), and centrifuge at 12,000 rpm for 5 min at 4℃ (Eppendorf 5430R). Collect the supernatant and concentrate to dryness using a vacuum concentration system. Redissolve in 100 μL of 80% methanol (containing 0.1% formic acid), filter through a 0.22 μm filter membrane, and transfer to a pre-cooled sample vial (4℃ for analysis).

[0099] (2) Preparation of reference standard

[0100] Accurately weigh 10.00 mg each of jasmonic acid (JA), salicylic acid (SA), and indoleacetic acid (IAA) reference standards (purity ≥98%), dissolve them in chromatographic methanol, and dilute to 10 mL to prepare a 1.0 mg / mL solution. -1 Mother liquor. Constructed from 0.01–500 ng / mL using a serial dilution method. -1 The series of standard working solutions exhibited good linearity (R > 0.999) in each standard curve. The standard working solutions were aliquoted into amber sample vials and stored at -18°C protected from light.

[0101] (3) UHPLC-MS / MS analytical conditions

[0102] Instrument: ExionLC TM The AD ultra-high performance liquid chromatography system (Sciex) is coupled with a QTRAP® 6500+ triple quadrupole mass spectrometer (electrospray ionization source, positive and negative ion switching mode).

[0103] Chromatographic conditions: Column: Waters ACQUITY UPLC HSS T3 C18 column (1.8 μm, 100 × 2.1 mm); Mobile phase: A: ultrapure water (containing 0.04% acetic acid), B: chromatographic grade acetonitrile (containing 0.04% acetic acid); Flow rate: 0.35 mL·min -1 Column temperature: 40 ± 0.5℃; Injection volume: 2 μL.

[0104] The MRM parameters for each compound are shown in Table 1.

[0105] Table 1. Compound scanning results

[0106] .

[0107] (4) Standard curve

[0108] Plot the concentration of the standard as the x-axis (X, ng·mL) -1 Plot a standard curve with the peak area as the ordinate (Y):

[0109] Jasmonic acid: Y = 69821X - 11275, r = 0.9997, linear range 0.05~50 ng;

[0110] Salicylic acid: Y = 125546X + 57503, r = 0.9999, linear range 0.1~100 ng;

[0111] Indoleacetic acid: Y = 186340X + 6026.4, r = 0.9998, linear range 0.1~100 ng.

[0112] (5) Analysis of transcriptional regulatory gene expression

[0113] The expression levels of genes related to jasmonic acid (JA) and indoleacetic acid (IAA) metabolism and signal transduction were detected using quantitative real-time PCR (qRT-PCR). Genes related to the JA pathway included: DAD1 (anther dehiscence defect 1, phospholipase A1), LOX2S (lipoxygenase 2S), AOC (propene oxide cyclase), OPR (12-oxophytic dienoic acid reductase), ACX (acyl-CoA oxidase), MFP2 (multifunctional protein 2), JMT (jasmonic acid carboxymethyltransferase), MYC2 (transcription factor MYC2), JAZ (jasmonic acid ZIM domain protein), and COI1 (coronavirus-insensitive protein 1). Genes related to the IAA pathway included: TAA1 (tryptophan aminotransferase 1), YUCCA (YUCCA flavin monooxygenase), and ALDH (aldehyde dehydrogenase). The relative expression level of *Prunella vulgaris* actin gene (PvActin) was calculated using the 2⁻ΔΔCt method as an internal control.

[0114] Experimental Results: The endogenous hormone content and transcriptional regulatory gene expression were measured in the leaves of *Prunella vulgaris* treated with 100 μM SNP for 24 h compared to the control group (CK). The results are as follows: Figure 5 As shown ( Figure 5 A–C represents the content of JA, IAA, and SA; Figure 5 D–G represents a heatmap or bar chart of related gene expression. The main results are as follows:

[0115] (1) Changes in endogenous plant hormone content

[0116] Compared with the control group, after 24 h of SNP treatment, the jasmonic acid (JA) content in Prunella vulgaris leaves increased significantly by approximately 23.5%. Figure 5 A); the content of indoleacetic acid (IAA) decreased significantly by approximately 27.8% ( Figure 5 B); Salicylic acid (SA) content showed no significant change ( Figure 5 C). This result indicates that exogenous NO treatment can specifically activate the JA signaling pathway and inhibit the accumulation of IAA.

[0117] (2) Expression of JA pathway-related genes: To verify the increase in JA content, the expression of genes related to JA biosynthesis and signal transduction was detected.

[0118] ( Figure 5 The results showed that key JA synthesis enzyme genes such as LOX2S (lipoxygenase), AOC (alendene oxide cyclase), and ACX (acyl-CoA oxidase) were significantly upregulated. Regarding JA signal transduction, the transcription factor MYC2 was activated (upregulated), but upregulation of the transcriptional repressor JAZ (jasmonic acid ZIM domain protein) family genes was also observed, while the receptor gene COI1 (coronavirus-insensitive protein 1) was significantly downregulated. This pattern of "MYC2 upregulation + JAZ upregulation + COI1 downregulation" suggests that the JA signaling pathway may have feedback regulation or adaptive regulation mechanisms after initial activation.

[0119] (3) Expression of genes related to the IAA pathway

[0120] Consistent with the decrease in IAA levels, the expression of the rate-limiting enzyme gene for IAA biosynthesis was significantly downregulated (Figure).

[0121] (5F–G). Specifically, the expression of TAA1 (tryptophan aminotransferase 1) and several YUCCA family genes (flavin monooxygenases) was inhibited, and ALDH (aldehyde dehydrogenase) family genes also showed a downregulation trend. These results indicate that SNP treatment reduces endogenous IAA levels by inhibiting the transcription of key genes in the IAA synthesis pathway.

[0122] (4) SA pathway-related genes

[0123] Consistent with the lack of significant changes in SA content, the core SA biosynthesis and signal transduction-related genes remained unchanged.

[0124] No significant difference in expression was observed 24 hours after treatment.

[0125] Overall conclusion: Treatment with exogenous 100 μM SNPs for 24 h significantly activated the JA signaling pathway in Prunella vulgaris leaves (increased JA content and upregulated synthase genes), while inhibiting IAA synthesis (decreased IAA content and downregulated genes such as TAA1 / YUCCA), while the SA pathway remained largely unaffected. This regulatory pattern of "JA increase and IAA decrease" in hormone levels may be closely related to the accumulation of SNP-induced secondary metabolites (such as phenolic acids and flavonoids).

[0126] Experiment 4: Verification Experiment on Transcriptional Regulation Related to the Metabolism and Synthesis of Phenolic Acids and Triterpenoids

[0127] Experimental methods:

[0128] (1) Determination of active ingredient content

[0129] Following the method described in Example 1, the contents of rosmarinic acid (RA), ursolic acid (UA), total phenolic acids, total triterpenes, and total flavonoids in Prunella vulgaris leaves were determined. RA and UA were determined using HPLC-DAD (chromatographic conditions as before), total phenolic acids were determined using the Folin-Ciocalteu colorimetric method, total triterpenes were determined using the vanillin-perchloric acid colorimetric method, and total flavonoids were determined using the sodium nitrite-aluminum nitrate colorimetric method. Each sample was tested in triplicate, and the results are expressed as mean ± standard deviation.

[0130] (2) Transcriptomics analysis and qRT-PCR validation

[0131] Transcriptome sequencing of *Prunella vulgaris* leaves treated with 100 μM SNP for 24 h was performed using RNA-seq technology to screen differentially expressed genes (DEGs). KEGG pathway enrichment analysis identified significantly enriched pathways related to the biosynthesis of phenolic acids, terpenes, and flavonoids. Real-time quantitative PCR (qRT-PCR) was used to verify the expression levels of key enzyme genes, including: PvPAL, PvC4H, Pv4CL, PvTAT, and PvRAS in the phenylpropanoid metabolism pathway; MEP pathway genes (PvDXS, PvHDS, PvHDR), MVA pathway genes (PvHMGS, PvHMGR), and downstream key genes (PvSQS, PvSQE, Pvβ-AS) in the terpenoid biosynthesis pathway. The relative expression level of *Prunella vulgaris* actin gene (PvActin) was calculated using the 2⁻ΔΔCt method as an internal control.

[0132] Experimental Results: Based on Experiment 1, the contents of RA, UA, total phenolic acids, total triterpenes, and total flavonoids in Prunella vulgaris leaves were further determined at 2 h, 24 h, and 48 h after treatment with 100 μM SNP. The molecular mechanism was then analyzed using transcriptomics. The results are as follows: Figure 6 – Figure 8 As shown.

[0133] (1) Dynamic changes in the content of active ingredients

[0134] Compared with the control group, 100 μM SNP treatment significantly promoted the accumulation of multiple active ingredients in Prunella vulgaris leaves. Figure 6 A–E):

[0135] Rosmarinic acid (RA): After treatment for 2 h, 24 h, and 48 h, the RA content increased by approximately 53.6%, 488.0%, and 361.5%, respectively. Figure 6 A). The increase was most significant at 24 h, consistent with the results of Experiment 1.

[0136] Ursolic acid (UA): The content increased by approximately 10.0%, 488.4%, and 314.3% at the corresponding time points, respectively. Figure 6 (B) The effect is best after 24 hours.

[0137] Total phenolic acids increased by approximately 6.6%, 75.4%, and 67.7% at 2 h, 24 h, and 48 h, respectively. Figure 6 C).

[0138] Total triterpenes increased by approximately 10.0%, 7.9%, and 17.9% at 2 h, 24 h, and 48 h, respectively. Figure 6 D).

[0139] Total flavonoids increased by approximately 3.6%, 141.7%, and 109.5% at 2 h, 24 h, and 48 h, respectively. Figure 6 E).

[0140] The above results indicate that SNP treatment has the most significant promoting effect on RA and UA (both increased by nearly 5 times at 24 h), followed by the promoting effect on total flavonoids, and also has a stable increase on total phenolic acids and total triterpenes.

[0141] (2) Transcriptional regulatory mechanisms of phenolic acid components

[0142] To elucidate the molecular basis of phenolic acid accumulation such as RA, KEGG pathway enrichment analysis was performed on samples treated for 24 h. The results showed that the phenylalanine / tyrosine metabolic pathway (including the RA biosynthetic pathway) was significantly enriched. Figure 7 A). In this pathway, multiple genes encoding rate-limiting enzymes are synergistically upregulated ( Figure 7B), specifically includes: 4 PvPAL genes (phenylalanine ammonia-lyase), 6 PvC4H genes (cinnamic acid-4-hydroxylase), 2 Pv4CL genes (4-coumarate-coenzyme A ligase), 2 PvTAT genes (tyrosine aminotransferase), and 1 PvRAS gene (rosmarinic acid synthase). The transcriptional activation of these genes provides a sufficient enzymatic basis for the large-scale accumulation of RA and total phenolic acids.

[0143] (3) Transcriptional regulatory mechanisms of triterpenoid components

[0144] Transcriptome analysis of the accumulation of UA and total triterpenes revealed significant reprogramming of the terpene skeletal biosynthetic pathway. Figure 8 A). The methyl erythritol phosphate (MEP) pathway was significantly enhanced: one PvDXS gene, three PvHDS genes, and seven PvHDR genes were all significantly upregulated. Conversely, the mevalonate (MVA) pathway exhibited feedback inhibition: five PvHMGR genes were downregulated, while only one PvHMGS gene was upregulated. This redistribution of metabolic flux may facilitate the provision of more precursors for UA synthesis.

[0145] Further analysis revealed the activation of key downstream genes in UA biosynthesis: one PvSQS (squalene synthase) and two PvSQE (squalene epoxidase) genes were significantly upregulated. Simultaneously, three Pvβ-AS (β-amyrin synthase) genes in the competitive branching pathway were significantly downregulated, which helps direct metabolic flux towards UA rather than oleanolic acid (OA) production. Figure 8 B).

[0146] In summary, exogenous 100 μM SNP treatment promotes the synthesis of RA and total phenolic acids by synergistically upregulating key enzyme genes (PAL, C4H, 4CL, TAT, RAS) in the phenylpropanoid metabolic pathway at the transcriptional level. Simultaneously, it achieves metabolic flux reprogramming by strengthening the MEP pathway, inhibiting MVA pathway feedback, upregulating SQS / SQE, and downregulating β-AS, thereby driving the efficient accumulation of UA and total triterpenes. These transcriptional regulatory mechanisms are highly consistent with the dynamic changes in the content of active ingredients (peak at 24 h), further validating the scientific validity of the optimal treatment conditions (100 μM SNP, 24 h) determined in Experiments 1 and 2.

[0147] Experimental Example 5: In vitro antioxidant activity and correlation analysis of alcohol extracts

[0148] Experimental methods:

[0149] (1) Preparation of alcohol extract samples

[0150] Accurately weigh 1.00 g of dried Prunella vulgaris leaf powder (CK group and 100 μM SNP group) harvested at each time point (2 h, 24 h, 48 h) in Example 1, place it in an Erlenmeyer flask, add 30 mL of 70% ethanol, and extract ultrasonically at 70 °C for 30 min. After extraction, filter, and repeat the extraction twice under the same conditions, combining the three filtrates. Concentrate the combined filtrate under reduced pressure to an appropriate volume, and dilute with anhydrous ethanol to prepare a series of working solutions with varying concentration gradients for subsequent antioxidant activity determination.

[0151] (2) Determination of DPPH free radical scavenging ability

[0152] Accurately weigh DPPH and dissolve it in methanol to prepare a solution of 0.04 mg / mL. -1 Prepare fresh DPPH-methanol solutions immediately. Take 1.0 mL of sample working solutions of different concentrations, add 4.0 mL of freshly prepared DPPH-methanol solution, mix well, and react at room temperature in the dark for 30 min. After the reaction, measure the absorbance at 517 nm. Use anhydrous ethanol instead of the sample solution as a blank control and vitamin C as a positive control. Perform three replicates for each concentration. The clearance rate is calculated using the following formula:

[0153] Clearance rate (%) = [(A0 - A1) / A0] × 100%

[0154] In the formula: A0 is the absorbance of the blank control, and A1 is the absorbance of the sample reaction solution.

[0155] With sample concentration (mg·mL) -1 The dose-response curve was fitted using a four-parameter Logistic (4PL) regression model with clearance rate (%) on the x-axis and the half-maximal inhibitory concentration (IC50) on the y-axis. 50 mg·mL -1 IC 50 The smaller the value, the stronger the sample's ability to scavenge DPPH free radicals and the higher its antioxidant activity.

[0156] (3) Determination of ABTS free radical scavenging ability

[0157] 7 mmol·L -1 ABTS solution with 2.45 mmol·L -1 Equal volumes of potassium persulfate were mixed and placed in the dark for 16 hours to allow for complete oxidation and the formation of ABTS. + Free radical working solution. ABTS is prepared using anhydrous ethanol. + The working solution is diluted to an absorbance of 0.70 ± 0.02 at a wavelength of 734 nm, which is the working solution for ABTS determination.

[0158] Take 1.0 mL of sample working solution of different concentrations, add 1.0 mL of ABTS assay working solution, mix well, and react at room temperature in the dark for 6 min. Measure the absorbance at 734 nm. Use anhydrous ethanol instead of the sample solution as a blank control, and vitamin C as a positive control. Each concentration is repeated three times. The clearance rate is calculated using the same formula as the DPPH method, and a four-parameter logistic regression model is used to calculate the IC50. 50 value.

[0159] (4) Correlation analysis and statistical methods

[0160] To investigate the intrinsic relationships among various indicators in *Prunella vulgaris* leaves 24 h after SNP treatment, the following indicators were measured at this time point: endogenous NO content, active ingredients (rosmarinic acid RA, ursolic acid UA, total flavonoids TF, total phenolic acids TPA, total triterpenoids TT), endogenous hormones (jasmonic acid JA, indoleacetic acid IAA), and in vitro antioxidant activity indicators (DPPH IC). 50 ABTS IC 50 Using variables as variables, the Pearson correlation coefficient is used to assess the linear relationship between pairs of variables. The closer the absolute value of the correlation coefficient (r) is to 1, the stronger the correlation; the positive or negative sign indicates a positive or negative correlation.

[0161] Experimental data are expressed as mean ± standard deviation (Mean ± SD). T-tests were used for comparisons between two groups, one-way ANOVA was used for comparisons among multiple groups, and LSD post-hoc tests were used for multiple comparisons among groups. The statistical significance level was set at p < 0.05. All data analyses were performed using SPSS 26.0 software.

[0162] Experimental Results: The in vitro antioxidant activity of the ethanol extract of Prunella vulgaris leaves in the control group (CK) and the 100 μM SNP treatment group was determined at 2 h, 24 h, and 48 h. Correlation analysis of the various indicators at 24 h was also performed. The results are as follows: Figure 9 As shown ( Figure 9 A–B are ICs for DPPH and ABTS. 50 change, Figure 9 C represents the correlation heatmap.

[0163] (1) DPPH free radical scavenging ability

[0164] like Figure 9 As shown in Figure A, DPPH IC of the SNP group and the CK group 2 h after treatment 50 There was no significant difference in values ​​(SNP group 0.614 mg / mL). -1 vs CK group 0.624 mg·mL -1 However, at 24 h and 48 h, the IC of the SNP group was lower.50 The values ​​decreased to approximately 0.287 mg·mL. -1 and 0.300 mg·mL -1 Compared with the CK group (0.597 mg·mL) at the same time point -1 and 0.500 mg·mL -1 The IC values ​​decreased significantly by approximately 52.0% and 40.3%, respectively. 50 The lower the value, the stronger the antioxidant activity, indicating that the DPPH free radical scavenging ability of the Prunella vulgaris leaf alcohol extract was greatly improved after SNP treatment for 24 h and 48 h, with the most significant effect at 24 h.

[0165] (2) ABTS free radical scavenging ability

[0166] like Figure 9 As shown in Figure B, compared with the CK group, the SNP treatment group showed significantly enhanced ABTS radical scavenging ability at all time points. Specifically, the SNP group showed significantly enhanced IC50 at 2 h, 24 h, and 48 h. 50 The values ​​were 0.289, 0.117, and 0.124 mg·mL, respectively. -1 Compared with the CK group (0.321, 0.297, 0.292 mg·mL) at the same time point, -1 The levels of SNPs decreased by approximately 9.8%, 60.5%, and 57.4%, respectively. These results further confirm that exogenous SNP treatment can significantly enhance the in vitro antioxidant activity of the ethanol extract of Prunella vulgaris leaves, with the greatest increase observed at 24 h.

[0167] (3) Correlation analysis

[0168] To investigate the intrinsic relationship between endogenous NO, active ingredients, plant hormones, and antioxidant activity in Prunella vulgaris leaves 24 h after exogenous SNP treatment, Pearson correlation analysis was performed on each indicator. The results are shown in [Figure 1]. Figure 9 C.

[0169] The relationship between NO and active ingredients and hormones: NO content was significantly positively correlated with RA, UA, TF, TPA, TT, and JA (r > 0.88, close to 1), while it was significantly negatively correlated with IAA (r = -0.778). This indicates that exogenous NO can synergistically promote the accumulation of multiple active ingredients, activate the JA signaling pathway, and inhibit IAA synthesis.

[0170] Relationship between active ingredients and antioxidant activity: RA, UA, TF, TPA, TT and DPPH IC 50 and ABTS IC 50 All showed a significant negative correlation (r values ​​ranged from -0.999 to -1.000). Due to IC... 50The lower the value, the stronger the antioxidant activity. This strong negative correlation indicates that the accumulation of the above-mentioned active ingredients is the direct material basis for the enhanced in vitro antioxidant capacity of Prunella vulgaris extract.

[0171] Relationship between hormones and active ingredients and antioxidants: JA was positively correlated with all active ingredients (RA, UA, TF, TPA, TT) (r approximately 0.88–0.89), and also with DPPH / ABTS IC. 50 A negative correlation was observed (r approximately -0.89 to -0.90), suggesting that JA may mediate SNP-induced co-synthesis of active ingredients, thereby enhancing antioxidant activity. Conversely, IAA showed a negative correlation with active ingredients (r approximately -0.78) and with DPPH / ABTS IC. 50 A positive correlation was observed (r approximately 0.77), suggesting that a decrease in endogenous IAA levels is beneficial for the accumulation of polyphenols and the improvement of antioxidant capacity.

[0172] Overall Conclusion: The Pearson correlation analysis results are highly consistent with the findings of Experiments 1–4: exogenous SNPs, by increasing endogenous NO levels, activate the JA signaling pathway, upregulate key genes for phenolic acid and terpene synthesis, and promote the accumulation of active components such as RA, UA, and total flavonoids. The enrichment of these components directly endows the Prunella vulgaris alcohol extract with stronger in vitro antioxidant activity (especially at 24 h). Simultaneously, the downregulation of IAA may have relieved the inhibition of secondary metabolism. These results provide direct chemical and biological evidence for SNP treatment to improve the quality of Prunella vulgaris.

[0173] Experimental Example 6: Differential Expression Analysis of Transcription Factors

[0174] Experimental methods: The expression differences of transcription factors in the CK group and the SNP treatment group were compared, and the number, family distribution and up / down regulation of differentially expressed transcription factors were statistically analyzed.

[0175] Experimental Results: Based on experiments 1–5, to investigate the transcriptional regulatory network of exogenous SNP treatment regulating the accumulation of active components in Prunella vulgaris, full-length transcriptome sequencing was performed on leaves of the control group (CK) and those treated with 100 μM SNP for 24 h. The differential expression of transcription factors (TFs) was analyzed in detail. The results are as follows: Figure 10 As shown ( Figure 10 A represents the top 20 families of differentially expressed transcription factors. Figure 10 B–F represents the up / down regulation of transcription factors in the promoters of key secondary metabolic pathway genes.

[0176] (1) Overview of differentially expressed transcription factors

[0177] Comparative analysis revealed 295 differentially expressed transcription factors, belonging to 54 transcription factor families, between the CK and SNP treatment groups. Of these, 136 transcription factors were upregulated and 159 were downregulated. Figure 10 A shows the top 20 transcription factor families with the most significant expression changes.

[0178] (2) Major differentially expressed transcription factor families

[0179] Among the differentially expressed transcription factor families, the most significant changes were observed in: MYB-related family: 24 upregulated and 3 downregulated; MYB family: 3 upregulated and 4 downregulated; bHLH family: 7 upregulated and 8 downregulated; WRKY family: 2 upregulated and 5 downregulated; and NAC family: 3 upregulated and 2 downregulated.

[0180] The above results indicate that exogenous SNP treatment (100 μM, 24 h) induced differential expression of multiple transcription factor families in *Prunella vulgaris* leaves, with the most significant changes observed in members of the MYB, bHLH, WRKY, and NAC families. These transcription factors may synergistically regulate the transcriptional activation of downstream target genes by binding to the promoters of key enzyme genes in the biosynthetic pathways of phenolic acids, flavonoids, and triterpenes, thereby promoting the accumulation of active components such as RA, UA, and total flavonoids. This finding provides direct transcriptional evidence for revealing the NO-mediated secondary metabolic regulatory network and lays a theoretical foundation for subsequent functional verification of key transcription factors.

[0181] Experiment 7: RT-qPCR validation of RNA-Seq data

[0182] Experimental methods:

[0183] (1) Total RNA extraction and reverse transcription

[0184] Leaf samples of *Prunella vulgaris* treated with 100 μM SNP for 24 h from the control group (CK) were collected, and total RNA was extracted using TRIzol® reagent. RNA purity and integrity were assessed using a Nanodrop spectrophotometer and agarose gel electrophoresis. 500 ng of total RNA was used, and gDNA removal was performed first. Then, cDNA was synthesized by reverse transcription in a 20 μL reaction system using the HiScript II qRT SuperMix kit. The reverse transcription product was diluted and used for real-time quantitative PCR analysis.

[0185] (2) Primer design

[0186] Twenty-one differentially expressed genes (DEGs) were selected for RT-qPCR validation. Gene-specific primers were designed using Primer Premier 5 software; primer sequences are shown in Table 2. The *Prunella vulgaris* β-actin gene was used as an internal control.

[0187] Table 2 Primer sequences for RT-qPCR detection

[0188] .

[0189] (3) RT-qPCR reaction system and procedure

[0190] The real-time quantitative PCR reaction system (20 μL) consisted of: 4 μL diluted cDNA, 0.4 μM each of forward and reverse primers (final concentration), 10 μL SYBR Green Master Mix, 0.4 μL 50×ROX Reference Dye 2, and 4.8 μL nuclease-free water. Amplification was performed using a real-time quantitative PCR instrument. The reaction program was: 95℃ pre-denaturation for 10 min; followed by 40 cycles of 95℃ denaturation for 15 s and 60℃ annealing / extension for 60 s. Melting curve analysis was performed after each cycle to ensure the specificity of the amplified products. Three biological replicates were performed for each sample.

[0191] (4) Calculation of relative expression levels

[0192] Use 2 - The relative expression levels of each gene were calculated using the ΔΔCt method. With the CK group as the control, the expression levels of the SNP-treated groups were expressed as fold changes relative to the CK group.

[0193] Experimental Results: To verify the reliability and accuracy of the transcriptome sequencing (RNA-Seq) data, 21 representative differentially expressed genes (DEGs) were selected, and their expression patterns were validated using RT-qPCR. The results are as follows: Figure 10 As shown, the qPCR expression trends of all validation genes are highly consistent with the RNA-Seq results.

[0194] (1) Verification of gene selection

[0195] The 21 selected genes cover the following functional categories:

[0196] Key enzyme genes for rosmarinic acid (RA) biosynthesis (6 genes): PAL (phenylalanine ammonia-lyase), C4H (cinnamic acid-4-hydroxylase), 4CL (4-coumaric acid coenzyme A ligase), TAT (tyrosine aminotransferase), CYP98A (p-coumaric acid-3-hydroxylase), and RAS (rosmarinic acid synthase).

[0197] Terpenoid biosynthesis-related genes (7): HMGS (hydroxymethylglutaryl-CoA synthase), HMGR (hydroxymethylglutaryl-CoA reductase), SQS (squalene synthase), SQE (squalene epoxygenase), DXS (1-deoxy-D-xylitol-5-phosphate synthase), HDS (2-C-methyl-D-erythritol-2,4-cyclic pyrophosphate synthase), HDR (4-hydroxy-3-methylbut-2-enyl diphosphate reductase).

[0198] Jasmonic acid (JA) regulatory network related genes (3): AOC (propene oxide cyclase), COI1 (coronavirus insensitive protein 1), and JAZ (jasmonic acid ZIM domain protein).

[0199] Defense signaling pathway genes (2): CNGC2 (cyclic nucleotide-gated channel protein 2) and CDPK (calcium-dependent protein kinase).

[0200] Transcription factor genes (3): bHLH (basic helical-loop-helical transcription factor), MYB-related (MYB-related transcription factor).

[0201] (2) RT-qPCR validation results

[0202] like Figure 10 As shown, for the 21 genes mentioned above, the upregulation or downregulation trend of SNP expression in the RT-qPCR treatment group relative to the CK group is completely consistent with the FPKM change trend of RNA-Seq. Although there may be slight differences in the absolute fold change values ​​between the two (due to different detection platform sensitivities), the expression direction (upregulation / downregulation) of all genes is consistent with the transcriptome data, indicating that the RNA-Seq results have high accuracy and reproducibility.

[0203] In conclusion, RT-qPCR validation further confirmed the transcriptional regulatory effects of exogenous SNP treatment (100 μM, 24 h) on phenolic acid synthesis, terpene synthesis, JA signaling pathway, and defense response-related genes in *Prunella vulgaris* leaves, corroborating the transcriptome analysis results in Experiments 4 and 6. These validation results provide reliable gene expression data to support subsequent molecular mechanism studies.

[0204] Experimental Example 8: Differential Metabolite Analysis

[0205] Experimental methods:

[0206] (1) Sample preparation and metabolite extraction

[0207] Frozen samples of *Prunella vulgaris* leaves treated with 100 μM SNP for 24 h were taken from the control group (CK) and freeze-dried using a Scientz-100F vacuum freeze dryer (-80℃, vacuum ≤5 Pa) for 63 ± 0.5 h (moisture content ≤5%, verified by Karl Fischer method). The dried samples were then ground into a fine powder (particle size ≤50 μm) using an MM 400 grinder (Retsch, 30 Hz, 3 cycles × 30 s intermittent grinding, with a 2 min ice bath interval) under liquid nitrogen conditions. 50.0 ± 0.1 mg of the powder was accurately weighed (MS105DM microbalance, conforming to ISO 17025 standard) and added to 1.2 mL of pre-cooled (-20℃) 70:30 (v / v) methanol / water extract (containing 0.1% formic acid and 250 μg·mL⁻¹). -1 Methyl jasmonate was used as an internal standard. After vortexing (3000 rpm, 30 s), periodic shaking extraction was performed at 4℃ (vortexing at 2500 rpm for 30 s every 30 min, for a total extraction time of 3 h). The extract was centrifuged at 12,000 rpm for 3 min at 4℃, and the supernatant was filtered through a 0.22 μm Phenex-GH membrane (Pall) and stored at 4℃ for later use.

[0208] (2) LC-MS analysis conditions

[0209] Metabolite separation was performed using a Waters ACQUITY Premier HSS T3 column (1.8 μm, 2.1 mm × 100 mm) at a column temperature of 40.0 ± 0.1℃. Mobile phase A was 0.1% formic acid (LC-MS grade, purity ≥99.9%), and mobile phase B was 0.1% formic acid (HPLC grade) at a flow rate of 0.40 mL·min. -1 (Pressure fluctuation <5 psi). The gradient elution program is shown in Supplementary Table S3, with a total run time of 10 min. Mass spectrometry was performed using a Q Exactive HF-X mass spectrometer (Thermo Scientific, resolution 240,000 @ m / z 200), with alternating scans in positive and negative electrospray ionization (ESI) modes (PAL RTC autosampler, 4°C).

[0210] (3) Data processing and multivariate statistical analysis

[0211] Raw mass spectrometry data were converted to mzXML format (Centroid mode) using ProteoWizard v3.0.20321, and then peak detection, retention time correction (RT bias <0.2 min, Obiwarp algorithm), and peak alignment (m / z window 5 ppm) were performed using XCMS v3.16.1. Peaks with a missing rate >50% were removed, and background subtraction (KNN, k=5) and batch effect correction (SVR, RBF kernel) were applied. Metabolite annotation was performed using precise mass matching (error ≤2 ppm), MS / MS spectra, and isotope distribution, combined with SIRIUS 5.6.1 and Mass Frontier 8.0 software and comparison with public databases (HMDB, KEGG) (confidence score ≥0.5). Precision control was performed using QC samples (peak CV <30%), and redundant peaks were removed using CAMERA 1.52. The data matrix was normalized by probability quotient (PQN) and transformed using log2.

[0212] Principal component analysis (PCA, scale=TRUE) was performed using the prcomp function in R to observe sample clustering and outliers, and orthogonal partial least squares discriminant analysis (OPLS-DA) was applied to maximize between-group separation. Differentially accumulating metabolites (DAMs) were screened based on a variable projection importance (VIP) > 1.0 and p < 0.05. Functional annotation and KEGG pathway enrichment analysis of DAMs were performed using MetExplore 3.0 and hypergeometric tests. To elucidate the association between DEGs and DAMs, an integrated KEGG pathway analysis was conducted on both.

[0213] Experimental Results: Non-targeted metabolomics analysis was performed on the leaves of *Prunella vulgaris* treated with 100 μM SNPs for 24 h, comparing the control group (CK) with those treated with 100 μM SNPs, to reveal the impact of exogenous SNPs on the metabolomic profile. The classification, quantity, and KEGG enrichment analysis results of differentially expressed metabolites (DAMs) are as follows: Figure 11 As shown ( Figure 11 A represents the DAMs classification and flavonoid / terpene subdivision. Figure 11 B represents the up-regulation / down-regulation amounts of flavonoids, terpenes, and phenolic acids. Figure 11 C represents KEGG pathway enrichment. Figure 11 D represents the integration pathway analysis of DEGs and DAMs.

[0214] (1) Overview of differential metabolites

[0215] A total of 508 differentially expressed metabolites (DAMs) were identified between the SNP-treated group and the control group, of which 336 were upregulated and 172 were downregulated. These DAMs belonged to multiple categories ( Figure 11A): Amino acids and their derivatives (23.23%), organic acids (16.73%), benzene and its substituted derivatives (9.84%), flavonoids (6.10%), terpenes (4.33%), lipids (3.94%), phenolic acids (3.54%), nucleotides and their derivatives (2.56%), alkaloids (2.56%), lignans and coumarins (1.38%), steroids (0.39%), quinones (0.39%), and others (12.60%).

[0216] (2) Subclass analysis of secondary metabolites

[0217] Among secondary metabolites, flavonoids are the most abundant. Figure 11 Terpenes (A) comprise nine main types: flavonoids (29.03%), other flavonoids (22.58%), isoflavones (9.68%), flavanols (9.68%), anthocyanins (9.68%), flavonols (6.45%), flavanones (6.45%), chalcones (3.23%), and dihydroflavonols (3.23%). Terpenes also exhibit diversity. Figure 11 A), which contains 7 main types: triterpenes (27.27%), diterpenes (22.73%), monoterpenes (13.64%), sesquiterpenes (9.09%), terpenes (9.09%), tetraterpenes (4.55%) and triterpenoid saponins (13.64%).

[0218] (3) Up-regulation / down-regulation of flavonoids, terpenes and phenolic acids

[0219] like Figure 11 As shown in Figure B, there are differences in the accumulation of flavonoids, terpenes, and phenolic acids between the CK_24 and SNP_24 groups:

[0220] Flavonoids: including 9 flavonoids (7 upregulated, 2 downregulated), 7 other flavonoids (4 upregulated, 3 downregulated), 3 isoflavones (2 upregulated, 1 downregulated), 3 flavanols (all upregulated), 3 anthocyanins (all downregulated), 2 flavonols (1 upregulated, 1 downregulated), 2 flavanones (1 upregulated, 1 downregulated), 1 chalcone (upregulated), and 1 dihydroflavonol (downregulated).

[0221] Terpenes: including 6 triterpenes (1 upregulated, 5 downregulated), 5 diterpenes (3 upregulated, 2 downregulated), 3 monoterpenes (2 upregulated, 1 downregulated), 3 triterpenoid saponins (2 upregulated, 1 downregulated), 2 sesquiterpenes (1 upregulated, 1 downregulated), 2 terpenes (both downregulated) and 1 tetraterpenes (downregulated).

[0222] Phenolic acids: The content of 13 phenolic acids was increased, and the content of 5 phenolic acids was decreased.

[0223] (4) KEGG pathway enrichment analysis

[0224] To further elucidate the differences in metabolic pathways between the CK and SNP treatment groups, DAMs were annotated into the KEGG database for association analysis. Figure 10 C). A total of 42 DAMs were annotated to 44 metabolic pathways. The main pathways commonly annotated by the DAMs included isoflavone biosynthesis, terpene skeletal biosynthesis, and diterpene biosynthesis. The results showed that flavonoid and terpene biosynthetic pathways were significantly enriched 24 h after SNP treatment, suggesting that these metabolic pathways may play a key role in the response of Prunella vulgaris seedling leaves to exogenous SNP treatment.

[0225] (5) Analysis of the integration of DEGs and DAMs with KEGG pathway

[0226] To reveal the association between differentially expressed genes (DEGs) and differentially expressed metabolites (DAMs) after 24 h of CK and SNP treatment, an integrated KEGG pathway analysis was performed. Figure 10 D). A total of 2033 DEGs and 42 DAMs were annotated to 42 metabolic pathways. The major pathways commonly enriched by both included: flavonoid biosynthesis, flavonoid and flavonol biosynthesis, isoflavone biosynthesis, terpene skeleton biosynthesis, diterpene biosynthesis, and phenylalanine, tyrosine, and tryptophan biosynthesis. Therefore, 24 h after SNP treatment, pathways related to flavonoid (flavonoids, flavonols, isoflavones), terpene (diterpenes), and phenylpropanane metabolism were significantly enriched, indicating that they collectively participate in the response of *Prunella vulgaris* seedling leaves to exogenous SNP treatment.

[0227] Overall Conclusion: This study, through non-targeted metabolomics analysis, clarified that exogenous 100 μM SNP treatment for 24 h significantly altered the metabolic profile of *Prunella vulgaris* leaves. A total of 508 metabolites (DAMs) were screened, with flavonoids and terpenoids showing the most significant changes. Most flavonoids (such as flavonols, flavanols, and isoflavones) and some diterpenes and monoterpenes were upregulated. KEGG enrichment analysis and DEGs-DAMs integration analysis both pointed to significant activation of flavonoid biosynthesis, terpenoid skeletal biosynthesis, and phenylpropane metabolic pathways. This result is highly consistent with the increased content of active ingredients in Example 1, the changes in transcriptional regulation in Example 4, and the differential expression of transcription factors in Example 6, further validating the molecular mechanism by which SNP treatment promotes the accumulation of active ingredients in *Prunella vulgaris* at the metabolic level.

[0228] Example 10: Correlation Network Analysis of Key Metabolites and Genes

[0229] Experimental methods:

[0230] Based on Experiment 8 (differential metabolite analysis) and Experiment 4 (transcriptome analysis), a correlation network analysis was performed on key differentially expressed metabolites (DAMs) and differentially expressed genes (DEGs) in the biosynthetic pathways of flavonoids, terpenes and phenolic acids in Prunella vulgaris leaves after 24 h of SNP treatment.

[0231] (1) Data integration

[0232] The relative abundance data of DAMs obtained from metabolomics were paired and integrated with the expression levels of DEGs (FPKM) obtained from transcriptomics to screen for DAMs and DEGs that are involved in the biosynthesis pathways of flavonoids, terpenes or phenolic acids.

[0233] (2) Correlation analysis

[0234] Pearson correlation analysis was used to calculate the correlation coefficient (r) and significance level (p-value) between each DAM and DEG pair. Metabolite-gene pairs with significant correlations were screened using a threshold of |r| ≥ 0.8 and p < 0.05. Only associations meeting these criteria were retained for subsequent network construction.

[0235] (3) Network construction and visualization

[0236] A correlation network diagram was constructed using Cytoscape software, where square nodes represent differentially expressed metabolites (DAMs) and circular nodes represent differentially expressed genes (DEGs). Node size represents the degree of connectivity, i.e., the number of edges connecting that node. Solid lines indicate positive correlations, and dashed lines indicate negative correlations; line thickness represents the absolute value of the correlation coefficient. Only significant correlations satisfying r² ≥ 0.8 and p < 0.05 were displayed in the network. The network diagram visually illustrates the regulatory relationship between key metabolites and their significantly associated genes.

[0237] Experimental results:

[0238] To further elucidate the regulatory relationships between key metabolites and related genes in *Prunella vulgaris* leaves after 24 h of exogenous SNP treatment, a correlation network analysis was performed on DAMs and DEGs in the biosynthetic pathways of flavonoids, terpenes, and phenolic acids. The results are as follows: Figure 12 As shown ( Figure 12 This is a correlation network diagram showing the association between key metabolites and their significantly related genes.

[0239] (1) Overall characteristics of the network

[0240] Using |r| ≥ 0.8 and p < 0.05 as thresholds, significantly correlated metabolite-gene pairs were screened. Network analysis showed that different metabolites exhibited specific gene association patterns.

[0241] (2) Correlation of flavonoid metabolites - luteolin

[0242] Luteolin (a flavonoid compound) was significantly positively correlated with 9 DEGs ( Figure 12 These genes primarily encode key enzymes in the phenylpropanoid metabolic pathway, including: Pv_QC_transcript_20510 (cinnamic acid-4-hydroxylase, C4H), r = 0.908; Pv_QC_transcript_27819 (C4H), r = 0.921; Pv_QC_transcript_3677 (C4H), r = 0.924; Pv_QC_transcript_20504 (C4H), r = 0.875; Pv_QC_transcript_18570 (C4H), r = 0.827; and Pv_QC_transcript_20023 (cytochrome P450 98A2 analog, CYP98A2), r = 0.857. These results indicate that SNP treatment promotes luteolin biosynthesis by upregulating the expression of multiple C4H genes and the CYP98A2 gene.

[0243] (3) Correlation of terpenoid metabolites—abietic acid

[0244] Rosin acid (a diterpenoid compound) showed a significant positive correlation with two DEGs ( Figure 12 Pv_QC_transcript_8901 (terpene synthase 13, TPS13), r = 0.853; Pv_QC_transcript_6792 (terpene synthase, TPS), r = 0.907. The strong positive correlation between abietic acid and the TPS gene indicates that SNP treatment-induced upregulation of the TPS gene is an important molecular basis for abietic acid accumulation.

[0245] (4) Correlation of phenolic acid metabolites—3,4-dihydroxybenzaldehyde

[0246] 3,4-Dihydroxybenzaldehyde (a phenolic acid compound) showed a significant correlation with five DEGs ( Figure 12 (This includes 3 positive correlations and 2 negative correlations).

[0247] These results indicate that the accumulation of 3,4-dihydroxybenzaldehyde is synergistically regulated by multiple genes, exhibiting both activating and inhibitory effects.

[0248] Overall conclusion: Correlation network analysis further validated the transcriptional regulatory effects of exogenous SNP treatment (100 μM, 24 h) on the biosynthetic pathways of flavonoids, terpenes, and phenolic acids in Prunella vulgaris leaves. The model is as follows: Figure 13 As shown, luteolin is highly positively correlated with multiple C4H and CYP98A2 genes, abietic acid is highly positively correlated with the TPS gene, and 3,4-dihydroxybenzaldehyde exhibits a complex association pattern with multiple genes, collectively revealing the key gene targets for SNP-induced accumulation of active ingredients. These results corroborate the conclusions of Experiments 4, 6, and 8, providing a theoretical basis for subsequent functional identification and metabolic engineering of key genes.

[0249] The above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be included within the protection scope of the present invention.

Claims

1. A cultivation method for enhancing the active components of Prunella vulgaris plants, characterized in that, This includes applying a treatment solution containing a nitric oxide donor at a concentration of 50-200 μM to the aboveground parts of the selfheal plant, and harvesting the aboveground parts 24-48 hours after treatment.

2. The method according to claim 1, characterized in that, The nitric oxide donor is any one of sodium nitroprusside, S-nitrosoglutathione, or diethylamine / nitric oxide adduct.

3. The method according to claim 2, characterized in that, The nitric oxide donor is sodium nitroprusside.

4. The method according to claim 1, characterized in that, The concentration of the nitric oxide donor in the treatment solution is 100 μM.

5. The method according to claim 1, characterized in that, The application method is foliar spraying.

6. The method according to claim 1, characterized in that, The processing time is 24 hours.

7. The method according to claim 1, characterized in that, The *Prunella vulgaris* plant was a seedling at the six-leaf stage.

8. The method according to claim 1, characterized in that, The harvested above-ground parts include leaves or fruit clusters.

9. The method according to claim 1, characterized in that, The treatment solution also contains methyl jasmonate and / or hydrogen peroxide.

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