Application method for improving yield and quality of selfheal medicinal material under drought stress through EBR

Abstract

The invention discloses an application method for improving yield and quality of selfheal medicinal materials under drought stress through EBR, and relates to the field of cultivation of traditional Chinese medicinal materials. The method comprises the step of spraying an EBR solution with the concentration of 0.01-0.20 [mu] mol / L to leaf surfaces of plants in the flowering period of selfheal. The application method has remarkable advantages, the oxidative damage caused by drought is effectively relieved by remarkably enhancing the drought resistance of plants, and meanwhile, the photosynthesis and moisture metabolism conditions are improved. By means of the method, the selfheal can still keep high yield under the drought condition, and the content of medicinal components such as total polyphenol, caffeic acid and rosmarinic acid is remarkably increased. The invention provides a feasible technical scheme for planting the selfheal in the arid region, so that the planting benefit of the selfheal is effectively improved.

Description

Technical Field

[0001] The present invention relates to the field of Chinese medicinal material cultivation, and in particular to an application method of EBR for improving the yield and quality of Prunella vulgaris medicinal materials under drought stress. Background Art

[0002] Drought stress remains a significant factor affecting crop growth in global agricultural production (Chen et al., 2024). With the rapid global warming, global drought conditions are expected to become increasingly severe in the coming decades (Dai, 2013). Therefore, enhancing crop drought resistance and ensuring the yield and quality of agricultural products have become critical challenges that need to be addressed in the development of dryland agriculture.

[0003] Prunella vulgaris L. is a perennial herbaceous plant in the Lamiaceae family. It has high horticultural ornamental value (Wang Yanjie et al., 2022) and is also an important medicinal plant, with its fruit spikes widely used as a key traditional Chinese medicine (Bai et al., 2016). Recent studies have shown that Prunella vulgaris contains multiple active ingredients, including terpenes, phenolics, flavonoids, and organic acids, which impart a wide range of pharmacological activities, including anticancer, antibacterial, anti-inflammatory, and hypoglycemic properties (Qin Xinyi et al., 2023). Currently, market demand continues to grow, and wild resources are nearing depletion. Therefore, developing efficient artificial cultivation techniques, particularly to enhance its drought resistance, has become a key approach to ensuring supply and meeting market demand.

[0004] Brassinosteroids (BRs), sterol plant hormones, are considered the sixth class of classical hormones in plants due to their unique role in regulating plant physiology. These hormones can be synthesized artificially and are safe for plants and animals, earning them the title of novel green plant hormone (Xu Chenyu et al., 2025). 2,4-Epibrassinosteroid (EBR), a synthetic derivative of BRs, plays a crucial role in plant growth, development, and adaptation to stress (Song Jianchao et al., 2023). Studies have shown that BRs have demonstrated promising regulatory potential in response to various abiotic stresses (Chen Huize et al., 2024). However, relatively little research exists on the regulatory effects of BRs on plant growth and medicinal qualities under drought stress. In particular, in the important medicinal plant Prunella vulgaris, the specific mechanisms by which BRs alleviate drought stress and their effects on the accumulation of medicinal components in Prunella vulgaris remain unclear. Therefore, developing a cultivation method that can alleviate the harm of drought stress and effectively improve the yield and medicinal quality of Prunella vulgaris under drought stress has important theoretical significance and application value. Summary of the Invention

[0005] In order to overcome the above shortcomings, the purpose of the present invention is to provide an application method of EBR to improve the yield and quality of Prunella vulgaris materials under drought stress, solve the planting problem in arid environment, and by regulating the growth state of Prunella vulgaris under water stress, not only can its yield be increased, but also its medicinal quality can be improved, which is of great significance for improving the planting efficiency of Prunella vulgaris and increasing farmers' income.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is: In a first aspect, an EBR application method for improving the yield and quality of Prunella vulgaris herbs under drought stress comprises spraying an EBR solution with a concentration of 0.01 to 0.20 μmol / L on the leaves of the Prunella vulgaris plants during the flowering period.

[0007] After further optimization, the concentration of the EBR solution was 0.10 μmol / L.

[0008] Further optimized, the EBR solution is an aqueous solution containing 0.10% Tween-80.

[0009] After further optimization, the spraying method is: first evenly spray the front of the plant leaves, and then evenly spray the back of the leaves. Each spraying ensures that the liquid medicine evenly covers the leaf surface. Spray once every two days, for a total of three spraying times.

[0010] A second aspect relates to the use of an EBR solution applied to the leaves of Prunella vulgaris to combat drought stress. The EBR solution is sprayed at a concentration of 0.01 to 0.20 μmol / L. Furthermore, the concentration of the EBR solution is 0.10 μmol / L. Furthermore, the EBR solution is an aqueous solution containing 0.10% Tween-80.

[0011] According to the above technical solution, the beneficial effects of the present invention are: The present invention significantly improves the drought resistance, ear yield and medicinal quality of Prunella vulgaris under drought stress conditions by spraying a 2,4-epibrassinolide (EBR) solution of a specific concentration during the flowering period of Prunella vulgaris. By adopting an application method of evenly spraying EBR on both front and back leaves, spraying once every two days for a total of three times, supplemented by fertilizing with a nutrient solution before spraying, EBR within a concentration range of 0.01 to 0.20 μmol / L can continuously exert an optimal effect in the plant. This application method, frequency and specific concentration work synergistically to jointly improve the drought resistance effect and yield quality.

[0012] Experimental results showed that EBR treatment effectively reduced relative conductivity, the levels of reactive oxygen species (ROS) such as malondialdehyde (MDA), H2O2, and O2·-, thereby alleviating membrane lipid peroxidation damage. Furthermore, EBR treatment significantly increased the activities of superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), catalase (CAT), glutathione peroxidase (GPX), and phenylalanine ammonia lyase (PAL). Furthermore, EBR treatment significantly increased the levels of osmotic regulators such as soluble protein, soluble sugar, and proline, helping to maintain cellular water balance. Regarding photosynthesis, EBR treatment significantly increased the levels of chlorophyll a, chlorophyll b, carotenoids, and total chlorophyll, and improved photosynthetic performance parameters such as net photosynthetic rate, stomatal conductance, and transpiration rate, effectively alleviating the inhibitory effects of drought on photosynthesis. In terms of yield and quality, EBR treatment significantly increased the average weight and total biomass of Prunella vulgaris plants under drought stress and promoted the accumulation of total polyphenols, caffeic acid, ferulic acid, rosmarinic acid, and hyperoside in the panicles. Among the EBR concentrations tested, 0.10 μmol / L was the most effective.

[0013] In summary, the present invention provides reliable technical support for the high-yield and high-quality cultivation of Prunella vulgaris under drought stress, can significantly alleviate the adverse effects of drought on the growth of Prunella vulgaris, and improve its yield and medicinal value. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Effects of foliar spraying of different concentrations of EBR on relative electrical conductivity (A), MDA content (B), H2O2 (C) and O2·- (D) content of Prunella vulgaris under drought stress; Figure 2 Effects of foliar spraying of different concentrations of EBR on SOD activity (A), POD activity (B), APX activity (C), CAT activity (D), GPX activity (E) and PAL activity (F) of Prunella vulgaris under drought stress; Figure 3 Effects of foliar spraying of different concentrations of EBR on the soluble protein content (A), soluble sugar content (B) and proline content (C) of Prunella vulgaris under drought stress; Figure 4 Effects of foliar spraying of different concentrations of EBR on chlorophyll a content (A), chlorophyll b content (B), carotenoid content (C) and chlorophyll a+b content (D) of Prunella vulgaris under drought stress; Figure 5 Effects of foliar spraying of different concentrations of EBR on the net photosynthetic rate (A), stomatal conductance (B), intercellular carbon dioxide concentration (C) and transpiration rate (D) of Prunella vulgaris under drought stress; Figure 6 Effects of foliar spraying of different concentrations of EBR on the ear weight per plant (A) and total weight per plant (B) of Prunella vulgaris under drought stress. DETAILED DESCRIPTION

[0015] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0016] In the following examples, unless otherwise specified, all reagents or materials used are commercially available, and all methods or operations used are conventional technical means.

[0017] Prunella vulgaris cultivation: Select Prunella vulgaris seeds of uniform size, full grains and no insect infestation. After disinfection, sow the seeds evenly in the turnover box in October of the same year and water them normally. In early April of the following year, when the seedlings grow to about 6 cm, transfer the seedlings to flower pots filled with nutrient soil of the same quality. The nutrient soil is adjusted according to V 土 :V 泥炭土 =7:1, meaning the cultivation medium is a mixture of 7 parts soil to 1 part peat moss. Pots were 24 cm in diameter and 26 cm high, with seven Prunella vulgaris seedlings transplanted into each pot. Watering was done once in mid-April with Hoagland nutrient solution, followed by three waterings every five days. Each pot received 1 L of nutrient solution. Seedling cultivation conditions: Light intensity of 700 μmol·m -2 ·s -1 , cultivated in a plant growth room with a photoperiod of 16 / 8h (day / night), a temperature of 25℃ / 20℃ (day / night), and a relative humidity of 70±10%, and normal water management.

[0018] The drought treatment experiment was carried out during the flowering period of Prunella vulgaris in May 2024. Potted seedlings with uniform growth were selected as experimental materials. A total of 6 treatments were set up, with 10 pots in each group and 6 plants in each pot, for a total of 60 plants.

[0019] The treatment settings were as follows: ① clean water control + 0 μmol / L EBR solution (CK); ② drought treatment + 0 μmol / L EBR solution (DR); ③ drought treatment + 0.01 μmol / L EBR solution (EBR0.01); ④ drought treatment + 0.05 μmol / L EBR solution (EBR0.05); ⑤ drought treatment + 0.10 μmol / L EBR solution (EBR0.1); ⑥ drought treatment + 0.20 μmol / L EBR solution (EBR0.2).

[0020] After the test started, the potted seedlings were moved into a rainproof canopy with a sunshade net and tested. Before drought treatment, different EBR treatments were sprayed with corresponding concentrations of EBR solution. EBR solution spraying was carried out at 18:00 every day. Every pot was evenly sprayed with 50 mL. CK and DR were used to spray an equal amount of distilled water on the leaves. The spraying method was as follows: first, the front of the leaves was sprayed once, and then the back of the leaves was sprayed once to ensure uniformity. Every 2 days, the back of the leaves was sprayed once for a total of 3 times. The soil moisture content was subsequently regulated: the soil moisture content of the CK treatment was maintained at 75 ± 5%, and the soil moisture content of the DR and EBR treatments was maintained at 60 ± 5%. The moisture lost by evaporation was replenished every day by gravimetric method. 0.1% Tween-80 was added when preparing the EBR solution. The EBR solution configured was an aqueous solution containing 0.1% Tween-80 to ensure that EBR adheres to the leaves better.

[0021] After 20 days of drought stress, various physiological parameters were measured. At the end of June, Prunella vulgaris fruit clusters were harvested and oven-dried at 50°C to constant weight. The dried fruit clusters were crushed and passed through a 60-mesh sieve for determination of bioactive components.

[0022] Result analysis: like Figure 1 As shown, drought stress significantly exacerbated cell membrane damage and oxidative stress in Prunella vulgaris leaves. Compared with the CK treatment, the DR treatment increased leaf relative conductivity by 48.68% (P < 0.05), and significantly increased malondialdehyde (MDA), hydrogen peroxide (H2O2), and superoxide anion (O2·-) levels. Treatment with EBR at all concentrations significantly reduced leaf relative conductivity and MDA, H2O2, and O2·- levels, showing a trend of initial decline followed by an increase. The 0.10 μmol / L EBR treatment had the greatest effect, reducing MDA, H2O2, and O2·- levels by 28.37%, 15.77%, and 25.73%, respectively, compared with the DR treatment. The levels of various indicators in the 0.20 μmol / L EBR treatment were higher than those in the 0.10 μmol / L treatment, but still significantly lower than those in the DR treatment, indicating that EBR can effectively maintain cell membrane stability and reduce oxidative damage within the tested concentration range, with 0.10 μmol / L being the optimal concentration. The protective effect weakened after exceeding this concentration, but 0.20 μmol / L was still significantly better than other DR treatments.

[0023] from Figure 2It can be seen that drought stress significantly induced an increase in the activities of antioxidant enzymes and phenylalanine ammonia lyase in Prunella vulgaris (P<0.05). Compared with the CK treatment, the activities of superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), catalase (CAT), glutathione peroxidase (GPX), and phenylalanine ammonia lyase (PAL) in the DR treatment were increased, with the APX and GPX activities increasing by 16.14% and 31.68% respectively. Spraying exogenous epibrassinolide (EBR) further enhanced the activities of the above enzymes ( Figure 2 All EBR treatments significantly increased the activities of antioxidant enzymes and PAL in Prunella vulgaris under drought stress, showing an initial increase followed by a decrease with increasing EBR concentration. The 0.10 μmol / L EBR treatment had the most significant effect, with SOD, POD, APX, CAT, GPX, and PAL activities reaching peak values ​​at this concentration, significantly exceeding those of the other treatments and the DR treatment.

[0024] like Figure 3 As shown in the results, drought stress significantly induced the accumulation of osmotic regulatory substances in Prunella vulgaris leaves. Compared with the CK treatment, drought stress increased the contents of soluble protein, soluble sugar, and proline by 18.27%, 34.31%, and 17.88%, respectively. Exogenous epibrassinolide (EBR) spraying significantly increased the contents of soluble protein, soluble sugar, and proline in Prunella vulgaris leaves under drought stress. The 0.10 μmol / L EBR treatment had the most significant effect on increasing the levels of these osmotic regulatory substances, outperforming all other concentrations.

[0025] like Figure 4 As shown, drought stress significantly affected the content of photosynthetic pigments in Prunella vulgaris. Compared with the CK treatment, drought stress reduced chlorophyll a (Chl a) and chlorophyll b (Chl b) contents by 41.63% and 44.52%, respectively. Carotenoid content decreased by 54.15%, and total chlorophyll (Chl a+b) content decreased by 42.33%. Treatment with various concentrations of EBR significantly increased the contents of Chl a, Chl b, carotenoids, and Chl a+b in Prunella vulgaris leaves. Further analysis revealed that photosynthetic pigment content exhibited a clear dose-dependent effect with EBR concentration, showing an initial increase followed by a decrease. Seed soaking with 0.10 μmol / L EBR had the most significant effect on promoting photosynthetic pigment accumulation.

[0026] like Figure 5As shown, drought stress significantly affects the gas exchange process in Prunella vulgaris. Compared with the CK treatment, the net photosynthetic rate, stomatal conductance, and transpiration rate of Prunella vulgaris under DR drought treatment decreased by 45.12%, 48.55%, and 46.98%, respectively, while the intercellular carbon dioxide concentration increased by 14.68%, indicating that stomatal and non-stomatal limitation jointly inhibit photosynthesis. Exogenous epibrassinolide (EBR) spraying can effectively alleviate the inhibition of photosynthetic gas exchange caused by drought. All EBR concentrations increased the net photosynthetic rate, stomatal conductance, and transpiration rate of Prunella vulgaris to varying degrees, with 0.10 μmol / L EBR having the most significant effect on net photosynthetic rate and stomatal conductance. The 0.10 μmol / L EBR treatment significantly improved gas exchange parameters, and its transpiration rate was higher than that of other EBR treatments, but not significantly different from the 0.05 μmol / L EBR treatment. In addition, all EBR treatments reduced the intercellular carbon dioxide concentration, with the 0.05 μmol / L and 0.10 μmol / L EBR treatments showing greater reductions, but there was no significant difference between the two.

[0027] like Figure 6 As shown in the results, drought stress significantly inhibited the weight of individual ear and total plant weight of Prunella vulgaris. Drought stress caused a 38.13% decrease in ear weight and a 34.66% decrease in total plant weight. Application of different concentrations of EBR increased both ear weight and total plant weight under drought stress. The most significant effect was observed with 0.10 μmol / L EBR.

[0028] As shown in Table 1, compared with the simple drought treatment, EBR treatments in the concentration range of 0.01 to 0.10 μmol / L can significantly increase the total phenolic content of Prunella vulgaris. Among them, the improvement effect of the 0.10 μmol / L EBR treatment is the most prominent, and the accumulation of total phenolic substances reaches a peak. When the EBR concentration increases to 0.20 μmol / L, although it is still higher than the drought treatment, it is significantly lower than the 0.10 μmol / L treatment. In terms of secondary metabolite accumulation, the 0.01-0.20 μmol / L EBR treatment can effectively promote the synthesis and accumulation of caffeic acid, ferulic acid, rosmarinic acid and hyperoside. Similarly, the 0.10 μmol / L EBR treatment has the most significant promoting effect on the above components, and although the 0.20 μmol / L treatment is higher than the drought control treatment, the content of each component shows a downward trend.

[0029] Table 1 Effects of different EBR concentrations on the contents of total phenols, caffeic acid, ferulic acid, rosmarinic acid, and hyperoside in Prunella vulgaris under drought stress (mg / g) In the table, CK: clear water control treatment; DR: drought treatment; EBR0.01, EBR0.05, EBR0.1, and EBR0.2 represent spraying of 0.01 μmol / L EBR solution, 0.05 μmol / L EBR solution, 0.10 μmol / L EBR solution, and 0.20 μmol / L EBR solution under drought treatment, respectively.

[0030] Analysis of data from the CK and DR treatments revealed that drought stress significantly impacted the physiological and biochemical processes of Prunella vulgaris. Drought caused a significant accumulation of reactive oxygen species in Prunella vulgaris leaves, triggering increased membrane lipid peroxidation. Although plants responded to drought stress by upregulating antioxidant enzyme activity and increasing the synthesis of osmotic regulators and secondary metabolites, these defense mechanisms were insufficient to offset the inhibitory effects of drought stress on photosynthesis. Drought stress significantly reduced the content of photosynthetic pigments in Prunella vulgaris, thereby weakening its photosynthetic capacity, ultimately leading to a significant decrease in biomass and ear yield.

[0031] Epibrassinolide (EBR), a plant hormone analog, has shown promising results in alleviating drought stress in Prunella vulgaris. EBR application significantly increased antioxidant enzyme activity and osmotic regulatory substance content in drought-stressed Prunella vulgaris leaves across all treatments, strengthening the plant's self-protection mechanisms. Furthermore, EBR treatment effectively increased photosynthetic pigment content, significantly boosted net photosynthetic rate, reduced reactive oxygen species levels, and mitigated drought-induced oxidative damage to leaves.

[0032] In terms of yield formation, EBR treatment significantly increased the weight of individual Prunella vulgaris ears and total biomass. Regarding the accumulation of secondary metabolites, EBR treatment significantly promoted the accumulation of total polyphenols, caffeic acid, ferulic acid, and hyperoside in the ears. Different EBR concentrations exhibited varying effects, with the 0.1 μmol / L EBR treatment demonstrating the most significant improvement in drought resistance, yield, and quality. This provides important technical support for high-quality Prunella vulgaris production in arid regions.

[0033] It should be noted that the above embodiments are only used to illustrate the present invention, but the present invention is not limited to the above embodiments. Any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention fall within the scope of protection of the present invention.

Claims

1. The method for applying EBR to improve the yield and quality of Prunella vulgaris herbs under drought stress is characterized by: During the flowering period of Prunella vulgaris, spray the leaves of the plants with an EBR solution with a concentration of 0.01-0.20 μmol / L.

2. The method for applying EBR to improve the yield and quality of Prunella vulgaris herbs under drought stress according to claim 1, characterized in that: The concentration of the EBR solution is 0.10 μmol / L.

3. The method for applying EBR to improve the yield and quality of Prunella vulgaris herbs under drought stress according to claim 1, characterized in that: The EBR solution is an aqueous solution containing 0.1% Tween-80.

4. The method for applying EBR to improve the yield and quality of Prunella vulgaris herbs under drought stress according to claim 1, characterized in that: The spraying method is: first spray the front of the plant leaves evenly, then spray the back of the leaves evenly. Make sure the liquid covers the leaf surface evenly each time you spray. Spray once every 2 days, for a total of 3 times.

5. Application of EBR solution on the leaves of Prunella vulgaris to resist drought stress.

6. The use according to claim 5, characterized in that: The spraying concentration of the EBR solution is 0.01-0.20 μmol / L.

7. The use according to claim 6, characterized in that: The concentration of the EBR solution is 0.10 μmol / L.

8. The use according to claim 6, characterized in that: The EBR solution is an aqueous solution containing 0.10% Tween-80.

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