Method for increasing flavone content in day lily through foliage application of nano-selenium

Abstract

The invention provides a method for increasing the flavone content in day lily by applying nano-selenium to leaves. The method comprises the step of spraying 10-40 mg / L of a nano-selenium solution to day lily leaves. According to the method, the total flavonoid content in the day lily can be effectively increased by 7.5% or above, and the selenium content can be increased by 200% or above; the content of flavone with an anti-depression effect, such as quercetin, luteolin and morin, is increased by 40%. According to the method, the content of functional components in the day lily is effectively increased, the nutritional and health-care values of the day lily are improved, and effective assistance is provided for full development of day lily resources.

Description

Technical Field

[0001] The present application relates to the field of food and crop planting. Specifically, the present application provides a method for increasing the flavonoid content in daylily by applying nano-selenium to the leaves. Background Art

[0002] Daylily, also known as daylily and forget-me-not, is a perennial herbaceous plant in the Asphodeloides family. It combines edible, medicinal, and ornamental properties. Its flower buds are the edible part and can be eaten dried or fresh. Daylily is sweet and delicious, rich in nutrients, containing carbohydrates, proteins, vitamins, inorganic salts, and several essential amino acids, and has potential for development as both an edible and medicinal plant. The Compendium of Materia Medica states that it has the properties of calming the mind, enhancing intelligence, relieving chest tightness, beautifying and nourishing the blood, relieving fever and disinfecting, and relieving restlessness and promoting lactation. Modern research indicates that daylily has antidepressant, sleep-promoting, and lactation-enhancing properties, which are closely related to its rich content of active substances, particularly flavonoids. For example, in a rat model of bromocriptine-induced lactational insufficiency, daylily buds improved lactation via the PRLR / JAK2 / STAT5 pathway. Furthermore, freeze-drying daylily can better preserve active ingredients such as flavonoids, thereby enhancing its lactation-promoting effects. However, under the existing cultivation technology conditions, how to effectively and stably increase the content of flavonoids, the core functional ingredients in daylily, to maximize its nutritional and health value and meet the growing demand for healthy food development, remains a technical problem that needs to be solved urgently. Summary of the Invention

[0003] On the one hand, the present application provides a method for increasing the flavonoid content in daylily by applying nano-selenium to the leaves, the method comprising spraying 10-40 mg / L of nano-selenium solution onto the leaves of the daylily.

[0004] Furthermore, the method comprises spraying 20 mg / L of nano-selenium solution onto the leaves of the daylily.

[0005] Furthermore, in the method, spraying is performed once every 10-20 days, for a total of 3-6 spraying times.

[0006] Furthermore, in the method, spraying is performed once every 15 days, for a total of 5 times.

[0007] Furthermore, the flavonoids include aurantiumin (dihydrokaempferol), taxifolin (dihydroquercetin), luteolin, myricetin, 3-O-methylquercetin, kaempferol, quercetin, syringetin (3,5,7,4'-tetrahydroxy-3',5'-dimethoxyflavone), and morin.

[0008] Furthermore, the flavonoids are quercetin, luteolin and morin.

[0009] Furthermore, the method increases the selenium content of daylily.

[0010] Furthermore, the method increases the total flavonoids content in the day lily by more than 7.5% and increases the selenium content in the day lily by more than 200%.

[0011] On the other hand, the present application provides a daylily product, wherein the above method is used during the cultivation process of the daylily product to increase the flavonoid content in the daylily.

[0012] On the other hand, the present application provides the use of the above-mentioned daylily product in the preparation of medicines or health products for anti-depression, promoting lactation, promoting sleep, improving immunity, preventing or treating cancer, and detoxifying heavy metals.

[0013] Nanoselenium, as used herein, refers to nanometer-sized elemental selenium (also known as zero-valent selenium), which is typically a red powder and has low toxicity and high biological activity. It can be prepared through physical treatment, chemical reduction, and biosynthesis. Various commercial products are available.

[0014] Beneficial effects:

[0015] The method described in the present invention can effectively increase the total flavonoid content in daylily by 7.5% and the selenium content by 2 times; this application analyzed the level of flavonoid metabolites in daylily through flavonoid targeted metabolomics, and found that the method described in the present invention can increase the content of flavonoids with antidepressant effects such as quercetin, luteolin and morin by up to 40%. At the same time, through UPLC-MS / MS detection, the main differential metabolites of daylily in the nano-selenium treatment group and the control group were screened, proving the stimulating effect of this method on the flavonoid metabolic pathway of flavonoid metabolism.

[0016] Compared to normal growth and enrichment, the total flavonoid content in daylily after nano-selenium treatment is significantly increased, especially the functional substances such as quercetin, luteolin, and morin, which are of great significance for improving the various medicinal and health functions of daylily, such as anti-depression, lactation promotion, and sleep promotion. After nano-selenium treatment, the selenium content in daylily increased by up to 2 times. As a trace element essential for maintaining human health, selenium plays a very important role in human health. A large number of clinical practices have shown that selenium is involved in various biological and immune regulation in the human body, and has other functions such as anti-cancer and cancer prevention, heavy metal detoxification, and enhancing immunity. The further increase in selenium content in daylily has driven the improvement of its nutritional value. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the PCA diagram of flavonoid metabolites of daylily in NSe group and CK group.

[0018] Figure 2 OPLS-DA analysis and permutation test of the differential flavonoid metabolites of daylily between NSe group and CK group

[0019] Figure 3A Volcano plot of differential metabolites of daylily between NSe group and CK group.

[0020] Figure 3B These are the differential metabolites of daylily between NSe group and CK group.

[0021] Figure 4 Figure 2 is the differential metabolite pathway enrichment map between the NSe group and the CK group. DETAILED DESCRIPTION

[0022] Example 1 Materials and Methods

[0023] The daylilies used in this experiment were cultivated at the daylily base in Sanshilipu, Yunzhou District, Datong City. The cultivated variety was "Datong Yellow Flower". Starting from April 2024, the experimental group (Nse) and the control group (CK) were sprayed with 20 mg·L -1 Nano-selenium solution (nano-selenium is produced by Yantai Jialong Nano Industry Co., Ltd.) or an equal amount of distilled water was sprayed each time between 9 and 10 in the morning when the weather was clear and windless, and sprayed 5 times in total. In July 2024, samples from each group that were free of pests and diseases, had no obvious mechanical damage, and were uniform in color and size were randomly and evenly harvested, then quickly frozen in liquid nitrogen and stored at -80°C for the determination of total flavonoids and selenium content and flavonoid metabolomics analysis.

[0024] Example 2 Determination of total flavonoids content in daylily samples

[0025] Fresh daylily flowers were freeze-dried in a vacuum oven, ground into powder using a tissue grinder, and stored at 4°C. 0.10g of daylily flower sample was accurately weighed and added to 1.5mL of 70% anhydrous ethanol solution. Ultrasonic extraction was performed at 60°C for 30 minutes, followed by centrifugation at 6000r / min for 10 minutes. The volume was then adjusted to 5mL for analysis. 500μL of the sample solution was pipetted into a test tube, mixed with 150μL of 5% NaNO₂ solution, and allowed to stand for 6 minutes. 150μL of a 10% Al(NO₃)₃ solution was added, mixed, and allowed to stand for 6 minutes. Then, 1500μL of a 4% NaOH solution was added, mixed, and allowed to stand at room temperature for 15 minutes. 200μL of the solution was transferred to a 96-well plate, and the absorbance of the sample was measured at a wavelength of 510nm. For the blank control, the daylily flower sample was replaced with distilled water, while all other parameters remained unchanged. Rutin solution was used as the standard solution in place of the sample. The standard curve was drawn with rutin mass concentration (mg RE / ml) as the horizontal axis and absorbance as the vertical axis, and the regression equation was obtained. Then, the sample concentration (C, mg RE / ml) was calculated. As shown in Table 1, the total flavonoid content in the Nse group was significantly increased compared with the CK group (p < 0.01).

[0026] Table 1 Total flavonoids and total selenium contents in daylily with nano-selenium and control group

[0027]

[0028] Example 3 Total selenium content of daylily samples

[0029] Weigh 0.1-1.0 g of daylily (Daylily lily) into a polytetrafluoroethylene jar, add 5 mL of nitric acid and 1 mL of perchloric acid, mix thoroughly, and heat on a hot plate at a temperature between 130 and 150°C. After the abundant brown smoke disappears, increase the temperature to 180°C and continue digestion. If the digestion solution turns brown-black, add a small amount of nitric acid as needed until white smoke is emitted and the digestion solution becomes colorless, transparent, or slightly yellow. Cool the solution and transfer it to a 50 mL volumetric flask. Wash the beaker several times with small amounts of water, combine the washings in the volumetric flask, bring to volume, mix thoroughly, and filter. A reagent blank was also prepared. The filtrate was analyzed by HPLC-ICP-MS. As shown in Table 1, the total selenium content in the Nse group was significantly increased compared to the CK group (p < 0.001).

[0030] Example 4 Metabolomics Analysis

[0031] Daylily sample pretreatment:

[0032] Weigh 50 mg of daylily powder and add 1200 μL of 70% methanol-water internal standard extract pre-cooled at -20°C. Vortex once every 30 minutes for 30 seconds each time, for a total of 6 times. Finally, centrifuge at 12000 rpm for 3 minutes, aspirate the supernatant, filter the sample with a 0.22 μm pore size microporous filter membrane, and store it in an injection vial for UPLC-MS / MS analysis.

[0033] Flavonoid targeted metabolomics detection:

[0034] Ultra-high performance liquid chromatography and tandem mass spectrometry were used for detection. Liquid phase conditions included: chromatographic column: Agilent SB-C18 1.8 μm, 2.1 mm x 100 mm; mobile phase: phase A: ultrapure water (containing 0.1% formic acid), phase B: acetonitrile (containing 0.1% formic acid); elution gradient: 0.00 min, 5% phase B; 0-9.00 min, 5%-95% phase B, linear increase; 9.00-10.00 min, 95% phase B; 10.00-11.10 min, 95%-5% phase B; 5% phase B, 11.10 min-14.00 min; flow rate: 0.35 mL / min; column temperature: 40°C; injection volume: 2 μL.

[0035] The mass spectrometry conditions mainly included: electrospray ionization source temperature of 500°C; ion spray voltage of 5500V (positive ion mode) / -4500V (negative ion mode); ion source gas I, gas II, and curtain gas were set to 50, 60, and 25 psi, respectively, and the collision-induced ionization parameter was set to high. The QQQ scan used the MRM mode, and the collision gas (nitrogen) was set to medium. The declustering voltage (DP) and collision energy (CE) of each MRM ion pair were further optimized. A specific set of MRM ion pairs was monitored in each period based on the metabolites eluting in each period.

[0036] Processing and analysis of flavonoid-targeted metabolomics data:

[0037] Based on the self-built MWDB (metware database), mass spectrometry data were processed using Analyst 1.6.3 software for qualitative and quantitative mass spectrometric analysis of the sample metabolites. Characteristic ions for each substance were screened using a triple quadrupole detector, and the signal intensity (CPS) of the characteristic ions was obtained in the detector. The sample's off-camera mass spectrometry file was opened using MultiQuant software for chromatographic peak integration and correction. The peak area (Area) of each chromatographic peak represents the relative content of the corresponding substance.

[0038] Identification of differential metabolites between nano-selenium-treated and control-treated daylilies:

[0039] First, PCA analysis was performed on the daylily samples treated with NSe and CK to determine the overall metabolite differences of the daylily samples treated with different methods and the degree of variation between samples within the group. Figure 1 As shown in Figure 3, the PCA results showed that the grouping ellipses of flavonoid metabolites in the CK group and the NSe group were significantly separated, indicating that there were significant differences in the flavonoid metabolite compositions between the two groups.

[0040] Next, OPLS-DA analysis was used to further study the metabolite differences between the different treatments of daylily samples. The prediction parameters of the OPLS-DA evaluation model are R2X, R2Y and Q2. R2X and R2Y represent the explanatory rate of the established model for the X and Y matrices respectively, and Q2 represents the predictive ability of the model. The closer these three indicators are to 1, the more stable and reliable the model is. When Q2>0.5, it can be considered an effective model, and when Q2>0.9, it is a stable model. Figure 2 As shown in the figure, the flavonoid metabolic profiles of daylily in the NSe group and the CK group were significantly distinguishable, and the Q2 value of the NSe group was 0.95, indicating that the explanatory and predictive abilities of the model were reliable.

[0041] Differential flavonoid metabolites were screened between the NSe and control groups based on variable importance projection (VIP) values ​​greater than 1, FC values ​​≥ 2, and FDR < 0.05, derived from the OPLS-DA model. As shown in Figure 3 and Table 2, a total of 90 differential metabolites were screened. Compared with the control group, 60 differential metabolites were upregulated and 30 metabolites were downregulated in the NSe group.

[0042] Table 2 Differential flavonoid metabolites between CK group and NSe group

[0043]

[0044]

[0045]

[0046]

[0047] Subsequently, KEG pathway enrichment was performed on the differential metabolites between the groups, and it was found that the differential metabolites between the NSe group and the CK group were mainly enriched in the flavonoid and flavonol biosynthesis and flavonoid biosynthesis pathways ( Figure 4 , Table 3 ), which contained 8 key differential metabolites, namely, citronellin (dihydrokaempferol), taxifolin (dihydroquercetin), luteolin, myricetin, 3-O-methylquercetin, kaempferol, quercetin, and thujatin (3,5,7,4′-tetrahydroxy-3′,5′-dimethoxyflavone).

[0048] Table 3 Flavonoid metabolites enriched in differential metabolic pathways in the CK and NSe groups

[0049]

Claims

1. A method for increasing the flavonoid content in daylily by applying nano-selenium to the leaves, characterized in that: The method comprises spraying 10-40 mg / L of nano-selenium solution onto leaves of the daylily.

2. The method according to claim 1, comprising spraying 20 mg / L of nano-selenium solution onto daylily leaves.

3. The method according to claim 1, wherein the spraying is performed once every 10-20 days, for a total of 3-6 sprayings.

4. method according to claim 3, in described method, spray once every 15 days, spray 5 times in total.

5. The method according to claim 1, wherein the flavonoids include citrus aurantifolia, taxifolin, luteolin, myricetin, 3-O-methylquercetin, kaempferol, quercetin, syringetin, and morin. The method according to claim 5 , wherein the flavonoids are quercetin, luteolin and morin.

7. The method according to claim 1, wherein the method increases the selenium content of daylily.

8. The method according to claim 7, wherein the method increases the total flavonoids content in day lily by more than 7.5% and the selenium content in day lily by more than 200%.

9. Daylily product, characterized in that The method according to any one of claims 1 to 8 is used during the cultivation of the daylily product to increase the flavonoid content in the daylily.

10. Use of the daylily product according to claim 9 in the preparation of medicines or health products for anti-depression, promoting lactation, promoting sleep, improving immunity, preventing or treating cancer and / or detoxifying heavy metals.

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