Hydroponic method for centella asiatica with high total glycoside content

Abstract

The invention relates to the technical field of hydroponic asiatic pennywort herb, in particular to a hydroponic method for asiatic pennywort herb with high total glycosides content, which improves the total glycosides content of asiatic pennywort herb by adjusting the nitrogen content and the nitrogen form, and comprises the following steps: putting asiatic pennywort herb seedlings into a nutrient solution for hydroponic culture, regulating the proportion of red light to blue light in an LED light source for hydroponic asiatic pennywort herb water culture, nutrient solutions with different inorganic nitrogen contents and forms are replaced according to the growth condition of the centella asiatica. According to the hydroponic method for the centella asiatica with the high total glycoside content, the content of inorganic nitrogen in a nutrient solution is dynamically adjusted, meanwhile, the ratio of ammonium nitrogen to nitrate nitrogen is optimized, balanced regulation and control of vegetative growth and secondary metabolism are achieved, and the yield of the centella asiatica is increased. The accurate matching of the nitrogen form and content can effectively activate the key enzyme activity in the synthetic route of the centella asiatica total glycosides, and provides a sufficient material basis for the synthesis of the total glycosides.

Description

Technical Field

[0001] This invention relates to the field of hydroponic Centella asiatica technology, and in particular to a hydroponic method for Centella asiatica with high total glycoside content. Background Technology

[0002] Centella asiatica ( Centella asiatica Centella asiatica is a perennial herb belonging to the genus Centella of the family Apiaceae. It contains total asiaticosides, a class of triterpenoid compounds mainly composed of asiaticoside, hydroxyasiaticoside, asiaticopic acid, and hydroxyasiatic acid. These compounds possess various pharmacological activities, including anti-inflammatory, antioxidant, skin-repair-promoting, and nervous system-protective effects, and are widely used in pharmaceuticals, cosmetics, and health products. With the continuous expansion of market demand, the large-scale, high-quality cultivation of Centella asiatica has become a core requirement for the industry's development.

[0003] Currently, the cultivation methods for Centella asiatica mainly include traditional soil cultivation and hydroponics. Traditional soil cultivation is greatly affected by factors such as soil texture, fertility, and pests and diseases, resulting in an unstable growth environment for Centella asiatica. This leads to significant fluctuations in the total glycoside content of Centella asiatica and poses quality and safety risks such as pesticide residues and heavy metal contamination, making it difficult to meet the demands of large-scale, standardized production. Hydroponics, on the other hand, offers advantages such as controllable growth environment, precise nutrient supply, fewer pests and diseases, and higher product purity, gradually becoming the preferred solution for large-scale Centella asiatica cultivation.

[0004] However, existing hydroponic techniques for Centella asiatica still have many shortcomings: On the one hand, the regulation of nitrogen is not systematic. Most techniques only control nitrogen content without dynamically adjusting it according to the nutritional needs of Centella asiatica at different growth stages, and they also fail to rationally optimize the ratio of ammonium nitrogen to nitrate nitrogen, leading to an imbalance between plant vegetative growth and secondary metabolism, thus hindering the synthesis of total glycosides in Centella asiatica. On the other hand, the hydroponic environment is not well controlled. For example, the influence of the growth environment and secondary metabolism is not fully considered, and there is a lack of targeted supplementation of precursor substances for the synthesis of total glycosides, making it difficult to further increase the total glycoside content of Centella asiatica. In addition, the application of microbial agents in existing hydroponic techniques is relatively extensive, failing to achieve synergistic effects with nutrient regulation and precursor supplementation, and thus failing to fully exert their effects in promoting plant growth and secondary metabolism. At the same time, the synthesis of total glycosides in Centella asiatica is affected by a variety of environmental factors and nutritional conditions, and single-dimensional technical optimization is unlikely to significantly increase the total glycoside content.

[0005] Therefore, developing a hydroponic method that can specifically address the above problems by adjusting the content and form of nitrogen and coordinating multiple factors to increase the total glycoside content of Centella asiatica is of great practical significance and industrial value. Summary of the Invention

[0006] The purpose of this invention is to provide a hydroponic method for producing Centella asiatica with high total glycoside content, dynamically adjusting the inorganic nitrogen content in the nutrient solution, and optimizing the ratio of ammonium nitrogen to nitrate nitrogen to achieve a balanced regulation of vegetative growth and secondary metabolism. The precise matching of nitrogen form and content can effectively activate the activity of key enzymes in the synthesis pathway of total glycosides in Centella asiatica.

[0007] To achieve the above objectives, this invention provides a hydroponic method for producing Centella asiatica with high total glycoside content. This method increases the total glycoside content of Centella asiatica by adjusting the inorganic nitrogen content and nitrogen form, and includes the following steps: S1. Prepare a solution containing 4-8 mmol / L inorganic nitrogen, 4-6 mmol / L potassium sulfate, 2-4 mmol / L calcium chloride, 1-2 mmol / L potassium dihydrogen phosphate, 1-3 mmol / L magnesium sulfate, 0.5-1.5 mmol / L potassium silicate, 0.5-5 mg / L trace elements, 100-200 mg / L seaweed extract, and 50-100 mg / L humic acid. Adjust the pH by adding a pH adjuster, stir well, filter and sterilize to obtain the nutrient solution for later use. S2. The nutrient solution controls the hydroponic temperature of Centella asiatica to 16-28℃. After cleaning and sterilizing the roots of the Centella asiatica seedlings, remove old and diseased roots. Immerse the lower half to two-thirds of the roots of the Centella asiatica seedlings in the nutrient solution in S1. Keep a spacing of 5-8cm between each Centella asiatica seedling. S3. The ratio of red light to blue light in the LED light source for hydroponic Centella asiatica should be 5-7:3-5, and the illumination time of the LED light source should be 12-16 hours / day. S4. Use an oxygenation pump to supply oxygen to the nutrient solution in S2. Regularly carry out pest and disease control for the Centella asiatica in the nutrient solution and remove diseased plants in time. S5. Change the nutrient solution with different inorganic nitrogen contents according to the growth of Centella asiatica. The inorganic nitrogen content in the nutrient solution during the seedling stage of Centella asiatica is 4-5 mmol / L, the inorganic nitrogen content in the nutrient solution during the growth stage of Centella asiatica is 5-6 mmol / L, and the inorganic nitrogen content in the nutrient solution during the vigorous growth stage of Centella asiatica is 6-8 mmol / L.

[0008] Preferably, in S1, the inorganic nitrogen is ammonium nitrogen and nitrate nitrogen, and the concentration ratio of ammonium nitrogen to nitrate nitrogen is 1:2-12.

[0009] Preferably, in S1, the ammonium nitrogen includes one or more of ammonium sulfate, ammonium chloride, ammonium nitrate, and ammonium dihydrogen phosphate; Nitrate nitrogen includes one or more of potassium nitrate, magnesium nitrate, and calcium nitrate.

[0010] Preferably, in S1, the trace elements include one or more of chelated iron, copper sulfate, boric acid, and ammonium molybdate.

[0011] Preferably, in S2, the day-night temperature difference is set to 8-12℃ during hydroponics, and the CO2 concentration in the growth environment is controlled to be 600-1200μmol / mol.

[0012] Preferably, in S1, the pH of the nutrient solution is 5-7 and the EC value is 1.2-2.5 mS / cm.

[0013] Preferably, in S4, the dissolved oxygen content in the nutrient solution is 6-8 mg / L.

[0014] Preferably, in S5, when changing the nutrient solution, 50-100 mg / L of microbial culture solution is added.

[0015] Preferably, in S5, the microbial agent in the microbial solution includes one or more of Bacillus jellyoidus, Bacillus laterosporus, Pseudomonas fluorescens, and Bacillus amyloliquefaciens.

[0016] Preferably, in S5, 50-100 mg / L phenylalanine and 30-50 mg / L mevalonic acid are added to the nutrient solution of Centella asiatica during its vigorous growth period.

[0017] Centella asiatica grown using the hydroponic method described above, which yields high total glycoside content, can be used in the preparation of serums, masks, and creams.

[0018] Therefore, the present invention employs the above-mentioned hydroponic method for Centella asiatica with high total glycoside content, and its beneficial effects are as follows: 1. Based on the different growth characteristics of Centella asiatica during the seedling, growth, and vigorous growth stages, this invention dynamically adjusts the inorganic nitrogen content in the nutrient solution and optimizes the ratio of ammonium nitrogen to nitrate nitrogen to achieve a balanced regulation of vegetative growth and secondary metabolism. The precise matching of nitrogen form and content can effectively activate the activity of key enzymes in the synthesis pathway of total glycosides in Centella asiatica, providing a sufficient material basis for the synthesis of total glycosides. Combined with the phenylalanine and mevalonic acid supplemented during the vigorous growth stage, it further promotes the metabolic flow of total glycoside synthesis and significantly increases the accumulation of total glycosides in Centella asiatica. 2. This invention adds microbial inoculum when changing the nutrient solution. The microorganisms can decompose the organic matter in the nutrient solution, release mineral nutrients, and inhibit the growth of harmful microorganisms. In synergy with seaweed extract and humic acid, it can improve the micro-ecological environment of the nutrient solution, increase nutrient utilization, reduce nutrient waste, achieve stable operation of the hydroponic system, and reduce the cost of large-scale planting. 3. The hydroponic method provided by this invention allows for precise quantitative control throughout the entire process, ensuring the stability of Centella asiatica's growth status and total glycoside content. Furthermore, the technical operation process is simple and controllable, requiring no complex equipment or technical experience, making it easy to achieve industrial-scale and large-scale promotion.

[0019] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0020] Figure 1 The graph shows the data on the content of hydroxyasiaticoside, asiaticoside and total glycosides in Centella asiatica in Examples 4-6 and Comparative Example 2. Detailed Implementation

[0021] The present invention will be further described below with reference to the accompanying drawings and embodiments. Unless otherwise defined, the technical or scientific terms used in this invention should be understood in their ordinary sense by those skilled in the art. The features mentioned above or in the specific examples mentioned in this invention can be combined arbitrarily, and these specific embodiments are only used to illustrate the invention and are not intended to limit the scope of the invention.

[0022] This invention provides a hydroponic method for growing Centella asiatica with high total glycoside content, comprising the following steps: S1. Prepare a solution containing 4-8 mmol / L inorganic nitrogen, 4-6 mmol / L potassium sulfate, 2-4 mmol / L calcium chloride, 1-2 mmol / L potassium dihydrogen phosphate, 1-3 mmol / L magnesium sulfate, 0.5-1.5 mmol / L potassium silicate, 0.5-5 mg / L trace elements, 100-200 mg / L seaweed extract, and 50-100 mg / L humic acid. Adjust the pH by adding a pH adjuster, stir well, filter and sterilize to obtain the nutrient solution for later use. S2. Control the hydroponic temperature of Centella asiatica at 16-28℃. After cleaning and sterilizing the roots of Centella asiatica seedlings, remove old and diseased roots. Immerse the lower half to two-thirds of the roots of Centella asiatica seedlings in the nutrient solution in S1. Keep a spacing of 5-8cm between each Centella asiatica seedling. S3. The ratio of red light to blue light in the LED light source for hydroponic Centella asiatica should be 5-7:3-5, and the illumination time of the LED light source should be 12-16 hours / day. S4. Use an oxygenation pump to supply oxygen, regularly carry out pest and disease control for Centella asiatica in the nutrient solution, and remove diseased plants in a timely manner. S5. Adjust the nutrient solution with different inorganic nitrogen contents according to the growth stage of Centella asiatica. The inorganic nitrogen content in the nutrient solution during the seedling stage is 4-5 mmol / L, during the growing stage it is 5-6 mmol / L, and during the vigorous growth stage it is 6-8 mmol / L. By adjusting the nitrogen content and ammonium-nitrate ratio in stages, the plant is encouraged to shift more carbon skeleton and energy from vegetative growth to the synthesis of defensive secondary metabolites, total glycosides.

[0023] In some embodiments of the present invention, in S1, the inorganic nitrogen is ammonium nitrogen and nitrate nitrogen, and the concentration ratio of ammonium nitrogen to nitrate nitrogen is 1:2-12. Ammonium nitrogen (NH4) + and nitrate nitrogen NO3- Ammonium nitrogen is the primary nitrogen form absorbed by Centella asiatica, and the two nitrogen forms work synergistically to suit the nitrogen absorption characteristics of Centella asiatica at different growth stages. The absorption of ammonium nitrogen acidifies the rhizosphere, consumes carbohydrates, and serves as a physiological stress signal. This mild stress effectively activates secondary metabolic pathways in plants, such as terpene synthesis, thereby promoting the accumulation of total glycosides. Nitrate nitrogen is the safest and primary nitrogen source for plants, ensuring basic growth. The ratio of different nitrogen forms has a significant impact on plant secondary metabolic pathways. Balancing vegetative growth and secondary metabolism, avoiding excessive growth due to ammonium nitrogen or stunted growth due to excessive nitrate nitrogen, and preventing metabolic imbalances caused by excessive single nitrogen form, while simultaneously promoting the synthesis of triterpenoids and total glycosides in Centella asiatica, thus promoting the accumulation of total glycosides.

[0024] In some embodiments of the present invention, in S1, ammonium nitrogen includes one or more of ammonium sulfate, ammonium chloride, ammonium nitrate, and ammonium dihydrogen phosphate. Nitrate nitrogen includes one or more of potassium nitrate, magnesium nitrate, and calcium nitrate. All nitrogen sources used are highly water-soluble and stable inorganic fertilizers that can dissolve rapidly in the nutrient solution, ensuring an effective supply of nitrogen. Some nitrogen raw materials simultaneously provide potassium, magnesium, calcium, and other elements, reducing the variety of raw materials in the nutrient solution.

[0025] In some embodiments of the present invention, in S1, the trace elements include one or more of chelated iron, copper sulfate, boric acid, and ammonium molybdate. Supplementing with trace elements can prevent problems such as iron deficiency chlorosis, molybdenum deficiency growth stagnation, and boron deficiency leaf deformities in Centella asiatica, ensuring normal plant growth and development. Trace elements are components or activators of many key enzymes in Centella asiatica. Chelated iron participates in chlorophyll synthesis and electron transport in photosynthesis, ammonium molybdate participates in nitrogen reduction, and boric acid promotes cell wall synthesis and nutrient transport.

[0026] In some embodiments of the present invention, in step S2, a diurnal temperature range of 8-12°C is set during hydroponics, and the CO2 concentration in the growth environment is controlled at 600-1200 μmol / mol. This diurnal temperature range reduces the intensity of nighttime respiration, decreases the consumption of photosynthetic products such as sugars and amino acids, and promotes the transport of these products to the roots and storage organs, providing sufficient carbon and nitrogen raw materials for the synthesis of total glycosides in Centella asiatica. High CO2 concentration directly improves photosynthetic efficiency, increases the total amount of carbon fixed, and provides a more abundant carbon source for growth and secondary metabolism. Through the increase in CO2 concentration and the regulation of temperature difference, the accumulation of photosynthetic products is promoted in two ways, providing a material basis for the growth of Centella asiatica and the synthesis of total glycosides, enabling a simultaneous increase in plant biomass and total glycoside content.

[0027] In some embodiments of the present invention, in S1, the pH of the nutrient solution is 5-7, and the EC value is 1.2-2.5 mS / cm. The pH value of the nutrient solution is within the optimal range for nutrient absorption by the roots of Centella asiatica. Excessive acidity or alkalinity can affect the permeability of root cell membranes, reduce nutrient absorption efficiency, and even damage the roots. A suitable EC value ensures sufficient nutrient supply, avoiding reverse osmosis damage caused by excessively high ion concentrations or nutrient deficiency caused by excessively low concentrations. By controlling the pH and EC values, the stable presence of various nutrients in the nutrient solution is ensured, preventing precipitation and waste, and improving nutrient utilization efficiency. This avoids root rot and black root problems caused by abnormal pH or EC values, enhances root vitality and nutrient absorption capacity, and provides a guarantee for plant growth.

[0028] In some embodiments of the present invention, in step S4, the dissolved oxygen content in the nutrient solution is 6-8 mg / L. In a hydroponic environment, the roots are directly immersed in the nutrient solution and need dissolved oxygen to meet their respiration requirements. Sufficient dissolved oxygen ensures that the roots can carry out normal aerobic respiration and produce enough energy for nutrient absorption. Oxygen deficiency will lead to anaerobic respiration in the roots, producing alcohol and damaging root cells.

[0029] In some embodiments of the present invention, in step S5, 50-100 mg / L of microbial inoculum is added when changing the nutrient solution. The added microbial inoculum can colonize in the nutrient solution, forming a dominant bacterial community, inhibiting the growth of harmful microorganisms, and simultaneously decomposing residual organic matter in the nutrient solution, releasing mineral nutrients, and realizing nutrient recycling.

[0030] In some embodiments of the present invention, in step S5, the microbial agent in the microbial inoculum includes one or more of *Bacillus jelly-like*, *Bacillus laterosporus*, *Pseudomonas fluorescens*, and *Bacillus amyloliquefaciens*. *Bacillus jelly-like* fixes nitrogen from the air, dissolves phosphorus and potassium fixed in the nutrient solution, directly increasing the N, P, and K content available to the roots, and secretes plant growth hormones to stimulate root development. *Bacillus laterosporus* significantly increases the number of root hairs and lateral roots, expands the absorption area, and improves the plant's stress resistance and absorption capacity. *Pseudomonas fluorescens* efficiently competes for iron ions in the environment by secreting siderophores and directly inhibits iron-dependent pathogens such as wilt pathogens by secreting antibiotics, forming a sterile protective zone around the roots. *Bacillus amyloliquefaciens* can produce various antimicrobial peptides that directly destroy the cell membranes of pathogenic fungi and bacteria, effectively preventing common hydroponic diseases such as root rot and damping-off.

[0031] In some embodiments of the present invention, in step S5, 50-100 mg / L phenylalanine and 30-50 mg / L mevalonic acid are added to the nutrient solution of Centella asiatica during its vigorous synthesis period. Centella asiatica total glycosides belong to triterpenoids, and the key precursors in its synthetic pathway are the aromatic compound precursor phenylalanine and the direct terpenoid precursor mevalonic acid. Exogenous supplementation of these two substances during the vigorous synthesis period of total glycosides can directly increase the metabolic flux of the synthetic pathway and promote the improvement of the synthesis efficiency of total glycoside components.

[0032] Example 1 A solution of ammonium nitrogen and nitrate nitrogen with a concentration ratio of 1:5 was prepared. The ammonium nitrogen consisted of ammonium sulfate, ammonium chloride, ammonium nitrate, and ammonium dihydrogen phosphate mixed in a mass ratio of 1:1:1:1. The nitrate nitrogen consisted of potassium nitrate and magnesium nitrate mixed in a mass ratio of 1:1. The resulting inorganic nitrogen content was 4.5 mmol / L. This solution was then prepared with 5 mmol / L potassium sulfate, 3 mmol / L calcium chloride, 1 mmol / L potassium dihydrogen phosphate, 2 mmol / L magnesium sulfate, 0.5 mmol / L potassium silicate, 0.5 mg / L trace elements (chelated iron, copper sulfate, boric acid, and ammonium molybdate in a mass ratio of 2:1:1:1), 100 mg / L seaweed extract, 50 mg / L humic acid, and deionized water. The pH was adjusted to 5.8–6 with a pH adjuster. After stirring thoroughly, the solution was filtered through a 0.22 μm filter membrane for sterilization to obtain the nutrient solution for later use.

[0033] Example 2 A solution of ammonium nitrogen and nitrate nitrogen with a concentration ratio of 1:6 was prepared. The ammonium nitrogen consisted of ammonium sulfate, ammonium chloride, ammonium nitrate, and ammonium dihydrogen phosphate mixed in a mass ratio of 1:1:1:1. The nitrate nitrogen consisted of potassium nitrate and magnesium nitrate mixed in a mass ratio of 1:1. The resulting inorganic nitrogen content was 5.5 mmol / L. This solution was then prepared with 5.5 mmol / L potassium sulfate, 3 mmol / L calcium chloride, 1.5 mmol / L potassium dihydrogen phosphate, 2 mmol / L magnesium sulfate, 1 mmol / L potassium silicate, 2.5 mg / L trace elements (chelated iron, copper sulfate, boric acid, and ammonium molybdate in a mass ratio of 2:1:1:1), 150 mg / L seaweed extract, 80 mg / L humic acid, and deionized water. The pH was adjusted to 5.8–6 with a pH adjuster. After stirring thoroughly, the solution was filtered through a 0.22 μm filter membrane for sterilization to obtain the nutrient solution for later use.

[0034] Example 3 A solution of ammonium nitrogen and nitrate nitrogen with a concentration ratio of 1:7 was prepared. The ammonium nitrogen consisted of ammonium sulfate, ammonium chloride, ammonium nitrate, and ammonium dihydrogen phosphate mixed in a mass ratio of 1:1:1:1. The nitrate nitrogen consisted of potassium nitrate and magnesium nitrate mixed in a mass ratio of 1:1. The resulting inorganic nitrogen content was 6.5 mmol / L. This solution was then prepared with 6 mmol / L potassium sulfate, 4 mmol / L calcium chloride, 2 mmol / L potassium dihydrogen phosphate, 3 mmol / L magnesium sulfate, 1.5 mmol / L potassium silicate, 5 mg / L trace elements (chelated iron, copper sulfate, boric acid, and ammonium molybdate in a mass ratio of 2:1:1:1), 200 mg / L seaweed extract, 100 mg / L humic acid, and deionized water. The pH was adjusted to 5.8–6 with a pH adjuster. After stirring thoroughly, the solution was filtered through a 0.22 μm filter membrane for sterilization to obtain a nutrient solution for later use.

[0035] Example 4 S1. Prepare the nutrient solution from Examples 1-3 for later use.

[0036] S2. Control the hydroponic temperature of Centella asiatica at 28℃ during the day and 20℃ at night, so that the day-night temperature difference is 8℃. Control the CO2 concentration in the growth environment to 1000μmol / mol. After cleaning and sterilizing the roots of Centella asiatica seedlings, remove old and diseased roots. Immerse the lower half to two-thirds of the roots of the Centella asiatica seedlings in the nutrient solution of Example 1, with an EC value of 2.5mS / cm. Maintain a spacing of 7cm between each Centella asiatica seedling.

[0037] S3. The ratio of red light to blue light in the LED light source for hydroponic Centella asiatica is 5:5, and the illumination time of the LED light source is 15 hours / day.

[0038] S4. Use an oxygenation pump to supply oxygen to the nutrient solution, ensuring a dissolved oxygen content of 8 mg / L. Regularly control pests and diseases in the Centella asiatica plant, and promptly remove diseased plants.

[0039] S5. Change the nutrient solution with different nitrogen contents according to the growth status of Centella asiatica. Use the nutrient solution from Example 1 during the seedling stage. Use the nutrient solution from Example 2 during the growth period. When changing between the two nutrient solutions, add 80 mg / L of microbial inoculum (Bacillus subtilis, Bacillus laterosporus, Pseudomonas fluorescens, and Bacillus amyloliquefaciens in a mass ratio of 1:1:1:1) to the nutrient solution from Example 2. Use the nutrient solution from Example 3 during the vigorous growth stage of Centella asiatica, adding 80 mg / L of phenylalanine and 40 mg / L of mevalonic acid to the nutrient solution during this stage.

[0040] Example 5 S1. Prepare the nutrient solution from Examples 1-3 for later use.

[0041] S2. Control the hydroponic temperature of Centella asiatica at 28℃ during the day and 20℃ at night, so that the day-night temperature difference is 8℃. Control the CO2 concentration in the growth environment to 1000μmol / mol. After cleaning and sterilizing the roots of Centella asiatica seedlings, remove old and diseased roots. Immerse the lower half to two-thirds of the roots of the Centella asiatica seedlings in the nutrient solution of Example 1, with an EC value of 2.5mS / cm. Maintain a spacing of 7cm between each Centella asiatica seedling.

[0042] S3. The ratio of red light to blue light in the LED light source for hydroponic Centella asiatica is 5:5, and the illumination time of the LED light source is 15 hours / day.

[0043] S4. Use an oxygenation pump to supply oxygen to the nutrient solution, ensuring a dissolved oxygen content of 8 mg / L. Regularly control pests and diseases in the Centella asiatica plant, and promptly remove diseased plants.

[0044] S5. Change the nutrient solution with different inorganic nitrogen contents according to the growth status of *Centella asiatica*. Use the nutrient solution from Example 1 during the seedling stage. Use the nutrient solution from Example 2 during the growing season. When changing between the two nutrient solutions, add 50 mg / L of microbial inoculum (Bacillus subtilis, Bacillus laterosporus, Pseudomonas fluorescens, and Bacillus amyloliquefaciens in a mass ratio of 1:1:1:1) to the nutrient solution from Example 2. Use the nutrient solution from Example 3 during the vigorous growth stage of *Centella asiatica*, adding 50 mg / L phenylalanine and 30 mg / L mevalonic acid to the nutrient solution during this stage.

[0045] Example 6 S1. Prepare the nutrient solution from Examples 1-3 for later use.

[0046] S2. Control the hydroponic temperature of Centella asiatica at 28℃ during the day and 20℃ at night, so that the day-night temperature difference is 8℃. Control the CO2 concentration in the growth environment to 1000μmol / mol. After cleaning and sterilizing the roots of Centella asiatica seedlings, remove old and diseased roots. Immerse the lower half to two-thirds of the roots of the Centella asiatica seedlings in the nutrient solution of Example 1, with an EC value of 2.5mS / cm. Maintain a spacing of 7cm between each Centella asiatica seedling.

[0047] S3. The ratio of red light to blue light in the LED light source for hydroponic Centella asiatica is 5:5, and the illumination time of the LED light source is 15 hours / day.

[0048] S4. Use an oxygenation pump to supply oxygen to the nutrient solution, ensuring a dissolved oxygen content of 8 mg / L. Regularly control pests and diseases in the Centella asiatica plant, and promptly remove diseased plants.

[0049] S5. Change the nutrient solution with different nitrogen contents according to the growth status of Centella asiatica. Use the nutrient solution from Example 1 during the seedling stage. Use the nutrient solution from Example 2 during the growth period. When changing between the two nutrient solutions, add 100 mg / L of microbial inoculum (Bacillus colloidis, Bacillus laterosporus, Pseudomonas fluorescens, and Bacillus amyloliquefaciens in a mass ratio of 1:1:1:1) to the nutrient solution from Example 2. Use the nutrient solution from Example 3 during the vigorous growth stage of Centella asiatica, adding 100 mg / L of phenylalanine and 50 mg / L of mevalonic acid to the nutrient solution during this stage.

[0050] Comparative Example 1 6.5 mmol / L inorganic nitrogen is a mixture of potassium nitrate and magnesium nitrate in a 1:1 mass ratio, which is then combined with 6 mmol / L potassium sulfate, 4 mmol / L calcium chloride, 2 mmol / L potassium dihydrogen phosphate, 3 mmol / L magnesium sulfate, 1.5 mmol / L potassium silicate, 5 mg / L chelated iron, 200 mg / L seaweed extract, 100 mg / L humic acid, and deionized water to form a solution. The pH of the nutrient solution is adjusted to 5.8-6 by adding a pH adjuster. After stirring evenly, the solution is filtered through a 0.22 μm filter membrane for sterilization to obtain the nutrient solution for later use.

[0051] Comparative Example 2 S1. Prepare the nutrient solution for Comparative Example 1.

[0052] S2. Control the hydroponic temperature of Centella asiatica at 28℃ during the day and 20℃ at night, so that the diurnal temperature range is 8℃. Control the CO2 concentration in the growth environment to 1000μmol / mol. After cleaning and sterilizing the roots of Centella asiatica seedlings, remove old and diseased roots. Immerse the lower half to two-thirds of the roots of the Centella asiatica seedlings in the nutrient solution of Comparative Example 1, with an EC value of 2.5mS / cm. Maintain a spacing of 7cm between each Centella asiatica seedling.

[0053] S3. The ratio of red light to blue light in the LED light source for hydroponic Centella asiatica is 5:5, and the illumination time of the LED light source is 15 hours / day.

[0054] S4. Use an oxygenation pump to supply oxygen to the nutrient solution, ensuring a dissolved oxygen content of 8 mg / L. Regularly control pests and diseases in the Centella asiatica plant, and promptly remove diseased plants.

[0055] S5. When replacing the nutrient solution in Comparative Example 1 with a new one based on the growth of Centella asiatica, add 80 mg / L of microbial culture solution (Bacillus colloidis, Bacillus laterosporus, Pseudomonas fluorescens and Bacillus amyloliquefaciens in a mass ratio of 1:1:1:1) to the new nutrient solution.

[0056] Test case The Centella asiatica plants were cultured for 30 days using the methods in Examples 4-6 and Comparative Example 2, respectively. The total weight (fresh weight), root length, number of roots, and number of leaves (including fallen leaves) of the Centella asiatica plants were measured before and after the start of cultivation, as shown in Table 1.

[0057] Table 1. Growth of Centella asiatica in Examples 4-6 and Comparative Example 2

[0058] After drying the 30-day-old Centella asiatica in a 50℃ oven, the content of asiaticoside was determined. Referring to the Pharmacopoeia of the People's Republic of China (2022 edition), high-performance liquid chromatography (HPLC) was used to determine the contents of hydroxyasiaticoside and asiaticoside in each group of dried Centella asiatica, and the total glycoside content was calculated. Figure 1 As shown.

[0059] As shown in Table 1, Examples 4-6 significantly outperformed Comparative Example 2 in all growth indicators, indicating that the nutrient solution and hydroponic method provided by this invention can effectively promote the biomass accumulation and vegetative growth of Centella asiatica. Figure 1 As can be seen from Table 1, the hydroponic methods in Examples 4-6 can simultaneously achieve a significant increase in total glycoside yield and effective component content.

[0060] Therefore, this invention employs the aforementioned hydroponic method for producing Centella asiatica with high total glycoside content. Based on the different growth characteristics of Centella asiatica during its seedling, growth, and vigorous growth stages, the inorganic nitrogen content in the nutrient solution is dynamically adjusted, while the ratio of ammonium nitrogen to nitrate nitrogen is optimized to achieve a balanced regulation of vegetative growth and secondary metabolism. The precise matching of nitrogen form and content can effectively activate the activity of key enzymes in the total glycoside synthesis pathway of Centella asiatica, providing a sufficient material basis for the synthesis of total glycoside components. Combined with the phenylalanine and mevalonic acid supplemented during the vigorous growth stage, the metabolic flow of total glycoside synthesis is further directionally promoted, significantly increasing the accumulation of total glycoside components in Centella asiatica.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A hydroponic method for growing Centella asiatica with high total glycoside content, characterized in that: The total glycoside content of Centella asiatica can be increased by adjusting the nitrogen content and nitrogen form, including the following steps: S1. Prepare a solution containing 4-8 mmol / L inorganic nitrogen, 4-6 mmol / L potassium sulfate, 2-4 mmol / L calcium chloride, 1-2 mmol / L potassium dihydrogen phosphate, 1-3 mmol / L magnesium sulfate, 0.5-1.5 mmol / L potassium silicate, 0.5-5 mg / L trace elements, 100-200 mg / L seaweed extract, and 50-100 mg / L humic acid. Adjust the pH by adding a pH adjuster, stir well, filter and sterilize to obtain the nutrient solution for later use. S2. Control the hydroponic temperature of Centella asiatica at 16-28℃. After cleaning and sterilizing the roots of Centella asiatica seedlings, remove old and diseased roots. Immerse the lower half to two-thirds of the roots of Centella asiatica seedlings in the nutrient solution in S1. Keep a spacing of 5-8cm between each Centella asiatica seedling. S3. The ratio of red light to blue light in the LED light source for hydroponic Centella asiatica should be 5-7:3-5, and the illumination time of the LED light source should be 12-16 hours / day. S4. Use an oxygenation pump to supply oxygen to the nutrient solution in S2. Regularly carry out pest and disease control for the Centella asiatica in the nutrient solution and remove diseased plants in time. S5. Change the nutrient solution with different inorganic nitrogen contents according to the growth of Centella asiatica. The inorganic nitrogen content in the nutrient solution during the seedling stage of Centella asiatica is 4-5 mmol / L, the inorganic nitrogen content in the nutrient solution during the growth stage of Centella asiatica is 5-6 mmol / L, and the inorganic nitrogen content in the nutrient solution during the vigorous growth stage of Centella asiatica is 6-8 mmol / L.

2. The hydroponic method for Centella asiatica with high total glycoside content according to claim 1, characterized in that: In S1, inorganic nitrogen consists of ammonium nitrogen and nitrate nitrogen, with a concentration ratio of 1:2-12.

3. The hydroponic method for Centella asiatica with high total glycoside content according to claim 2, characterized in that: In S1, ammonium nitrogen includes one or more of ammonium sulfate, ammonium chloride, ammonium nitrate, and ammonium dihydrogen phosphate; Nitrate nitrogen includes one or more of potassium nitrate, magnesium nitrate, and calcium nitrate.

4. The hydroponic method for Centella asiatica with high total glycoside content according to claim 1, characterized in that: In S1, trace elements include one or more of chelated iron, copper sulfate, boric acid, and ammonium molybdate.

5. The hydroponic method for Centella asiatica with high total glycoside content according to claim 1, characterized in that: In S2, during hydroponics, the day-night temperature difference is set to 8-12℃, and the CO2 concentration in the growth environment is controlled at 600-1200μmol / mol.

6. The hydroponic method for Centella asiatica with high total glycoside content according to claim 1, characterized in that: In S1, the pH of the nutrient solution is 5-7, and the EC value is 1.2-2.5 mS / cm.

7. The hydroponic method for Centella asiatica with high total glycoside content according to claim 1, characterized in that: In S4, the dissolved oxygen content in the nutrient solution is 6-8 mg / L.

8. The hydroponic method for Centella asiatica with high total glycoside content according to claim 1, characterized in that: In S5, when changing the nutrient solution, add 50-100 mg / L of microbial solution.

9. A hydroponic method for Centella asiatica with high total glycoside content according to claim 8, characterized in that: In S5, the microbial agents in the microbial solution include one or more of the following: Bacillus jellyoidus, Bacillus laterosporus, Pseudomonas fluorescens, and Bacillus amyloliquefaciens.

10. A hydroponic method for Centella asiatica with high total glycoside content according to claim 1, characterized in that: In S5, 50-100 mg / L phenylalanine and 30-50 mg / L mevalonic acid are added to the nutrient solution during the vigorous growth period of Centella asiatica.

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