Planting method for increasing content of flavone and terpene lactone in ginkgo leaves

Abstract

The invention provides a planting method for increasing the content of flavone and terpene lactones in ginkgo leaves, which is characterized by comprising the following steps: S1, in a ginkgo seedling field which grows well and is free from plant diseases and insect pests, applying a mixed fertilizer in each hole in a seedling germination stage, namely March of the current year, and then applying a bacterial solution to promote accumulation of flavonoid glycoside; s2, in the seedling leaf fast-growing period, namely May to June of the current year, the mixed fertilizer in the step S1 is applied to each ditch, then the bacterial liquid is applied, and accumulation of flavonoid glycoside is promoted; s3, in the growth period of the seedlings, namely September to October of the current year, a mixed fertilizer which is dissolved with water and diluted into 500 times is sprayed to the leaf surfaces of the seedlings, lactone cyclase is induced, and terpene lactone synthesis is promoted. The ginkgo leaf flavone content is greater than or equal to 26%, the terpene lactone content is greater than or equal to 6.5%, the utilization value of ginkgo leaves is improved, the market demand is met, and greater economic benefits are obtained for the planting industry.

Description

Technical Field

[0001] This invention relates to the field of medicinal plant cultivation technology, specifically to a cultivation method for increasing the content of flavonoids and terpene lactones in ginkgo leaves. Background Technology

[0002] Ginkgo trees, also known as maidenhair trees, belong to the genus Ginkgo in the family Ginkgoaceae. Not only are their ginkgo fruits used for both food and medicine, but their leaves also contain highly valuable medicinal compounds such as ginkgo flavonoids and terpenoid lactones. The flavonoids possess antioxidant and anti-tumor effects, while the terpenoid lactones are strong antagonists of platelet-activating factor, used to prevent cardiovascular diseases and offering strong protection to the central nervous system. Therefore, the market demand for ginkgo leaves is extremely high, with a preference for higher flavonoid and terpenoid content. However, under current technology, increasing the flavonoid and terpenoid content in ginkgo leaves is far from easy due to factors such as natural conditions, seedling cultivation, and planting management. Fertilization management is particularly crucial; excessive fertilization leads to excessive ash content, while insufficient fertilization negatively impacts the healthy growth of the ginkgo tree. Specifically, applying conventional NPK fertilizers can cause excessive vegetative growth in ginkgo leaves, resulting in a 30-40% reduction in flavonoid content compared to wild ginkgo. Improper control of micronutrients in fertilizers, such as excessive zinc, can inhibit the activity of PAL, a key enzyme in flavonoid synthesis, leading to antagonism. Mismatch between the timing of fertilization and the synthesis cycle of ginkgo secondary metabolites can also affect the content of flavonoids and terpene lactones in ginkgo leaves. Therefore, it is necessary to improve existing technologies. Summary of the Invention

[0003] The present invention aims to provide a cultivation method that can significantly increase the content of flavonoids and terpene lactones in ginkgo leaves.

[0004] This invention is achieved through the following technical solution: a cultivation method for increasing the content of flavonoids and terpene lactones in Ginkgo biloba leaves, characterized by comprising the following steps: S1. In a healthy, disease-free ginkgo seedling nursery, apply an appropriate amount of the following mixed fertilizer to each seedling during the seedling budding period, i.e., in March of the same year: Each kilogram of fertilizer contains: nitrogen, phosphorus, and potassium base fertilizer: 600g / kg, sodium selenite: 4.5-5.0mg / kg, ammonium molybdate: 1.0-1.5mg / kg, potassium silicate: 50mg / kg, and a compound root regulator with a ferulic acid: vanillic acid mass ratio of 2:1: 250-350mg / kg; and the fertilization depth is controlled at 15-20cm to activate PAL enzyme and promote mycorrhizal colonization. Within 24 hours of applying the above-mentioned mixed fertilizer, apply bacterial solution at a rate of 5-8 L / mu to promote the accumulation of flavonoids; the volume ratio of the bacterial solution is as follows: *Boletus lactis* YN01: *Trichoderma harzianum* YN02 = 3:2, wherein the spore count of *Boletus lactis* YN01 is ≥1×10⁻⁶.6 CFU / mL; Afterwards, water control, disease prevention, and pest control management were carried out as usual: S2. During the rapid leaf growth period of the seedlings, i.e., May-June of the same year, apply an appropriate amount of the mixed fertilizer described in step S1 to each furrow. 60 days after applying the bacterial solution in step S1, apply the bacterial solution described in step S1 at a rate of 5-8 L / mu to promote the accumulation of flavonoids. Afterwards, carry out routine water control, disease prevention, and pest control management. S3. During the seedling growth period, i.e. September-October of the same year, spray an appropriate amount of the mixed fertilizer described in step S1 diluted with water to 500 times on the leaves of the seedlings to induce lactone cyclase and promote the synthesis of terpene lactones. After that, carry out water control, disease prevention and pest control management as usual until the ginkgo leaves are harvested.

[0005] The mass ratio of the nitrogen, phosphorus, and potassium base fertilizer is: N∶P2O5∶K2O = 8∶12∶15.

[0006] The molecular formula of sodium selenite is Na₂SeO₃, the molecular formula of ammonium molybdate is (NH₄)₂MoO₄, the molecular formula of potassium silicate is K₂SiO₃, and the molecular formula of ferulic acid is C₂SiO₃. 10 H 10 O4, the molecular formula of vanillic acid is C8H8O4.

[0007] The lactobacillus YN01 is a commercially available product.

[0008] The Trichoderma harzianum YN02 is a commercially available product.

[0009] Through the above technical solutions, the present invention achieves the following technical effects: The combined application of sodium selenite, ammonium molybdate, and potassium silicate, along with the application of a ferulic acid-vanillic acid compound root regulator, promotes mycorrhizal colonization, increasing the infection rate to 87%. Targeted fertilization measures at three different stages—seedling germination, rapid leaf growth, and seedling development—precisely match the synthesis cycle of ginkgo leaf flavonoids and ginkgo terpene lactones, resulting in a ginkgo leaf flavonoid content ≥26% and a terpene lactone content ≥6.5%, thereby increasing the utilization value of ginkgo leaves, meeting market demand, and generating greater economic benefits for the planting industry. Attached Figure Description

[0010] Figure 1 The optimized curve for the selenium-molybdenum ratio; Figure 2 FTIR spectrum of ferulic acid-vanillic acid complex; Figure 3 Dynamic graphs of flavonoid and terpene lactone content under three fertilization stages; Figure 4The images show microscopic images of hyphal chemotaxis, where: the left image is a microscopic image of hyphal chemotaxis using conventional methods, and the right image is a microscopic image of hyphal chemotaxis according to an embodiment of the present invention; Figure 5 This is an X-ray fluorescence (SR-XRF) spectrum. Detailed Implementation

[0011] The technical solution of the present invention will be described in detail below with reference to the embodiments. The following examples were all completed at the applicant's experimental base in Kunming, Yunnan Province. The base is at an altitude of 1890m, with red soil and a pH of 5.8. The ginkgo seedlings used were three-year-old ginkgo seedlings, which were propagated from the GINK-SE-09 mother plant. Example 1

[0012] A cultivation method for increasing the content of flavonoids and terpene lactones in ginkgo leaves includes the following steps: S1. In a well-grown, pest-free three-year-old ginkgo seedling nursery, apply an appropriate amount of the following mixed fertilizer to each seedling during the seedling budding period, i.e., in March of the same year: Each kilogram of fertilizer contains: nitrogen, phosphorus, and potassium base fertilizer: 600g / kg, sodium selenite: 4.5mg / kg, ammonium molybdate: 1.0mg / kg, potassium silicate: 50mg / kg, and a compound root regulator with a ferulic acid: vanillic acid mass ratio of 2:1: 250mg / kg; the fertilization depth is controlled at 15cm to activate PAL enzyme and promote mycorrhizal colonization. Among them, in the nitrogen, phosphorus, and potassium basic fertilizer, N∶P2O5∶K2O = 8∶12∶15 (mass ratio); 22 hours after applying the above-mentioned mixed fertilizer, apply bacterial solution at a rate of 5 L / mu to promote the accumulation of flavonoids; the volume ratio of the bacterial solution is as follows: Boletus lactis YN01: Trichoderma harzianum YN02 = 3:2, wherein the spore count of Boletus lactis YN01 is ≥1×10 6 CFU / mL; Afterwards, water control, disease prevention, and pest control management were carried out as usual: S2. During the rapid leaf growth period of the seedlings, i.e., in May of the same year, apply an appropriate amount of the mixed fertilizer described in step S1 to each furrow. On the 62nd day after applying the bacterial solution in step S1, apply the bacterial solution described in step S1 at a rate of 5L / acre to promote the accumulation of flavonoids. Afterward, carry out routine water control, disease prevention, and pest control management. S3. During the seedling growth period, i.e. September of the same year, spray an appropriate amount of the mixed fertilizer described in step S1 diluted with water to 500 times on the leaves of the seedlings to induce lactone cyclase and promote the synthesis of terpene lactones. After that, carry out water control, disease prevention and pest control management as usual until the ginkgo leaves are harvested.

[0013] The following table compares the content of NPK compound fertilizer in harvested ginkgo leaves with that of a control group that was only treated with NPK compound fertilizer and planted using conventional methods:

[0014] The detection methods in the table are as follows: flavonoids and ginkgolides: UPLC-PDA detection was performed according to the 2020 edition of the Pharmacopoeia; soil selenium was detected according to GB 5009.93-2017; mycorrhizal infection rate was obtained by staining sections using the grid cross method; statistical analysis was performed using the t-test (p<0.01), and data are expressed as mean ± standard deviation. Example 2

[0015] A cultivation method for increasing the content of flavonoids and terpene lactones in Ginkgo biloba leaves, characterized by comprising the following steps: S1. In a well-grown, disease- and pest-free two-year-old ginkgo seedling nursery, apply an appropriate amount of the following mixed fertilizer to each seedling during the budding stage of the seedlings, i.e., in March of the same year: Each kilogram of fertilizer contains: nitrogen, phosphorus, and potassium base fertilizer: 600g / kg, sodium selenite: 5.0mg / kg, ammonium molybdate: 1.5mg / kg, potassium silicate: 50mg / kg, and a compound root regulator with a ferulic acid: vanillic acid mass ratio of 2:1: 350mg / kg; the fertilization depth is controlled at 20cm to activate PAL enzyme and promote mycorrhizal colonization. Among them, in the nitrogen, phosphorus, and potassium basic fertilizer, N∶P2O5∶K2O = 8∶12∶15 (mass ratio); Twenty hours after applying the above-mentioned mixed fertilizer, apply bacterial solution at a rate of 8 L / mu to promote the accumulation of flavonoids; the bacterial solution is as follows: Boletus lactis YN01: Trichoderma harzianum YN02 = 3:2, wherein the spore count of Boletus lactis YN01 is ≥1×10 6 CFU / mL; Afterwards, water control, disease prevention, and pest control management were carried out as usual: S2. During the rapid leaf growth period of the seedlings, i.e., in June of the same year, apply an appropriate amount of the mixed fertilizer described in step S1 to each furrow. On the 65th day after applying the bacterial solution in step S1, apply the bacterial solution described in step S1 at a rate of 8L / acre to promote the accumulation of flavonoids. Afterward, carry out routine water control, disease prevention, and pest control management. S3. During the seedling growth period, i.e. October of the same year, spray an appropriate amount of the mixed fertilizer described in step S1, dissolved in water and diluted 500 times, on the leaves of the seedlings to induce lactone cyclase and promote the synthesis of terpene lactones. After that, carry out water control, disease prevention and pest control management as usual until the ginkgo leaves are harvested.

[0016] The following table compares the content of NPK compound fertilizer in harvested ginkgo leaves with that of a control group that was only treated with NPK compound fertilizer and planted using conventional methods:

[0017] The detection methods in the table are the same as in Example 1. Example 3

[0018] A cultivation method for increasing the content of flavonoids and terpene lactones in Ginkgo biloba leaves, characterized by comprising the following steps: S1. In a well-grown, disease-free, three-year-old ginkgo seedling nursery, apply an appropriate amount of the following mixed fertilizer to each seedling during the budding stage of the seedlings, i.e., in March of the same year: Each kilogram of fertilizer contains: nitrogen, phosphorus, and potassium base fertilizer: 600g / kg, sodium selenite: 4.8mg / kg, ammonium molybdate: 1.2mg / kg, potassium silicate: 50mg / kg, and a compound root regulator with a ferulic acid: vanillic acid mass ratio of 2:1: 300mg / kg; the fertilization depth is controlled at 18cm to activate PAL enzyme and promote mycorrhizal colonization. Among them, in the nitrogen, phosphorus, and potassium basic fertilizer, N∶P2O5∶K2O = 8∶12∶15 (mass ratio); Twenty-one hours after applying the above-mentioned mixed fertilizer, apply bacterial solution at a rate of 7 L / acre to promote the accumulation of flavonoids; the bacterial solution is as follows: *Boletus lactis* YN01: *Trichoderma harzianum* YN02 = 3:2, wherein the spore count of *Boletus lactis* YN01 is ≥1×10⁻⁶. 6 CFU / mL; Afterwards, water control, disease prevention, and pest control management were carried out as usual: S2. During the rapid leaf growth period of the seedlings, i.e., in June of the same year, apply an appropriate amount of the mixed fertilizer described in step S1 to each furrow. On the 63rd day after applying the bacterial solution in step S1, apply the bacterial solution described in step S1 at a rate of 7 L / acre to promote the accumulation of flavonoids. Afterward, carry out routine water control, disease prevention, and pest control management. S3. During the seedling growth period, i.e. October of the same year, spray an appropriate amount of the mixed fertilizer described in step S1, dissolved in water and diluted 500 times, on the leaves of the seedlings to induce lactone cyclase and promote the synthesis of terpene lactones. After that, carry out water control, disease prevention and pest control management as usual until the ginkgo leaves are harvested.

[0019] The following table compares the content of NPK compound fertilizer in harvested ginkgo leaves with that of a control group that was only treated with NPK compound fertilizer and planted using conventional methods:

[0020] The detection methods in the table are the same as in Example 1.

[0021] In addition, the OD of hyphal migration using conventional methods 600 =0.15; while the hyphal migration OD of the embodiment of the present invention 600 =0.49, an increase of 2.3 times.

[0022] This invention significantly increases the content of flavonoids and terpene lactones in ginkgo leaves through a three-stage fertilization process, such as... Figure 3 .

[0023] After the implementation of this invention, the hyphae all extend in a directional manner, such as Figure 4 The right image in the text.

[0024] Aseptic roots obtained by conventional methods: The amount of Se / Mo absorbed by the root system is 0.05 mg / kg; The method of this invention involves mycorrhizae: the root system's absorption of Se / Mo is 0.38 mg / kg, an increase of 75%, as shown below. Figure 5 .

[0025] The quercetin glycoside content of the embodiment of the present invention was 1.52 mg / g when detected by UPLC, while the quercetin glycoside content of conventional methods was only 0.73 mg / g.

[0026] Furthermore, the total selenium content of the fertilizer applied in this invention is ≤10mg / kg, which meets the requirement of ≤15mg / kg in GB 1903.28-2018.

[0027] The viable spore rate of the bacterial solution applied in this invention is ≥95%, which meets the NY / T 1109-2017 microbial inoculant standard.

Claims

1. A planting method for increasing the content of ginkgo leaf flavonoids and terpene lactones, characterized in that The method comprises the following steps: S1. In a good growth and no disease and insect pests of ginkgo seedling land, at the seedling germination period, i.e. March of the current year, a proper amount of the following mixed fertilizer is applied to each hole: Each kilogram of the fertilizer contains: nitrogen, phosphorus and potassium base fertilizer: 600g / kg, sodium selenite: 4.5-5.0mg / kg, ammonium molybdate: 1.0-1.5mg / kg, potassium silicate: 50mg / kg, and a compound root system regulator of ferulic acid: vanillic acid = 2:1 mass ratio: 250-350mg / kg; the fertilization depth is controlled to be 15-20cm to activate PAL enzyme and promote mycorrhizal colonization at the same time; Within 24 hours after applying the mixed fertilizer, apply the bacterial solution in an amount of 5-8 L / acre to promote the accumulation of flavonoid glycosides; the volume ratio of the bacterial solution is as follows: Suillus bovinus YN01:H. zygorium YN02 = 3:2, wherein the spore amount of Suillus bovinus YN01 is ≥1×10 6 CFU / mL; the spore amount of H. zygorium YN02 is ≥1×10 6 CFU / mL. The application also provides a method for improving the quality of a crop, comprising the following steps: applying a mixed fertilizer to a crop; and applying a bacterial solution to the crop within 24 hours after applying the mixed fertilizer, wherein the mixed fertilizer comprises the following components: 10-20 kg / acre of nitrogen, Then, normal water control, disease prevention and insect prevention management are carried out; S2. At the leaf rapid growth period of the seedling, i.e. May-June of the current year, a proper amount of the mixed fertilizer of step S1 is applied to each ditch, and 60 days after the application of the bacterial liquid in step S1, the bacterial liquid of step S1 is applied at an amount of 5-8L per mu to promote the accumulation of flavonoid glycosides, and then normal water control, disease prevention and insect prevention management are carried out; S3. At the growth period of the seedling, i.e. September-October of the current year, the mixed fertilizer of step S1 diluted with water to 500 times is sprayed on the leaf surface of the seedling to induce lactone cyclase and promote terpene lactone synthesis, and then normal water control, disease prevention and insect prevention management are carried out until the ginkgo leaves are harvested.

2. The method for increasing the content of ginkgo leaf flavonoids and terpene lactones according to claim 1, characterized in that The mass ratio of the nitrogen, phosphorus and potassium base fertilizer is N:P2O5:K2O = 8:12:15.

Images