Method for indoor cultivation of medicinal mulberry and application thereof in improving secondary metabolites of medicinal mulberry

Abstract

The application relates to a method for indoor cultivation of medicinal mulberry and application thereof in improving secondary metabolites of indoor cultivated medicinal mulberry, which comprises transplanting, illumination, fertilization and management steps. The application effectively improves the secondary metabolites of indoor cultivated medicinal mulberry by applying inorganic fertilizer during the growth period of the medicinal mulberry. Especially, high-potassium fertilizer is most obvious for improving the active substances of indoor medicinal mulberry. The high-potassium fertilizer can improve the content of flavones and flavonols in the medicinal mulberry by affecting the biosynthetic metabolic pathways of the flavones and the flavonols.

Description

Technical Field

[0001] This invention belongs to the field of mulberry planting technology, and relates to indoor cultivation of medicinal mulberry, specifically to a method for indoor cultivation of medicinal mulberry and its application in improving the secondary metabolites of medicinal mulberry. Background Technology

[0002] Mulberry trees are primarily known as a primary food source for silkworms, but they also play a crucial role in ecological restoration. Furthermore, the mulberry tree is a treasure trove of medicinal value; its leaves, bark, fruit, and branches all possess medicinal properties. Medicinal mulberries are rich in flavonoids, polysaccharides, polyphenols, alkaloids, minerals, vitamins, and other nutrients, offering benefits such as lowering blood sugar and lipids, anti-oxidation, anti-inflammatory and analgesic effects, and anti-aging properties. The chromosome ploidy of *Morus alba* is 22-fold (22n = 308), a chromosomal characteristic unique among all mulberry germplasm resources. This gives *Morus alba* immense research and economic value. Furthermore, studies have shown that among various mulberry resources, *Morus alba* has the highest content of DNJ ((1-deoxynojirimycin), N-methyl-1-DNJ (N-Me-DNJ), 2-oxo-α-D-galactopyranoside-1-DNJ, fagomine, and other alkaloids). Specifically, the content of rutin is 0.85-1.14 mg / g, isoquercitrin is 0.66-0.85 mg / g, and astragaloside is 0.09-0.29 mg / g. Flavonoids and flavonols have a wide range of biological activities, including antioxidant and free radical scavenging, inhibiting cancer cell growth, inducing apoptosis, anti-tumor effects, reducing cholesterol synthesis, lowering serum cholesterol concentration, preventing atherosclerosis, cardiovascular protection, and also have metabolic regulation and anti-fatty liver effects.

[0003] In conclusion, *Mulberry sphagnum* possesses extremely high development value as both a medicine and food. However, after transplanting *Mulberry sphagnum* to Sichuan and Chongqing, its growth did not reach the expected level. Grafted *Mulberry sphagnum* plants struggled to sustain growth, and even when tissue culture enabled their survival, their biomass was insufficient to meet production needs. Therefore, further exploration of cultivation conditions for *Mulberry sphagnum* is necessary. Many factors influence its growth, including temperature, humidity, light, and fertilizer. Currently, commonly used fertilizers for *Mulberry sphagnum* cultivation include organic and inorganic fertilizers, with the inorganic fertilizer being a high-nitrogen inorganic fertilizer. Summary of the Invention

[0004] Based on this, the purpose of this invention is to provide a method for indoor cultivation of medicinal mulberry. This method not only enables the medicinal mulberry to grow and develop, but also significantly increases the content of secondary metabolites of the cultivated medicinal mulberry, especially the content of active substances such as flavonoids and flavonols.

[0005] The technical solution to achieve the above objectives is as follows.

[0006] The first aspect of the present invention is to provide a method for indoor cultivation of medicinal mulberry, the method comprising the following steps:

[0007] After the tissue culture seedlings of the medicinal mulberry are rooted, they are transplanted into an artificially lit greenhouse for growth and fertilized. The fertilization is carried out by applying an inorganic fertilizer containing nitrogen, phosphorus and potassium during the vigorous growth period of the medicinal mulberry, wherein the mass ratio of nitrogen, phosphorus and potassium is (10-16):(4-10):(36-45).

[0008] In some of these embodiments, the mass ratio of nitrogen, phosphorus and potassium is (12-14):(6-8):(38-42).

[0009] The preferred mass ratio of nitrogen, phosphorus, and potassium is 13:7:(39-41).

[0010] In some of these embodiments, the volume ratio of the substrate used for transplanting is soil:perlite:vermiculite = (3-5):(0.8-1.2):(1.8-2.2).

[0011] Furthermore, the volume ratio of the substrate used for transplanting is soil: perlite: vermiculite = 4:1:2.

[0012] In some of these embodiments, the mass ratio of the inorganic fertilizer to the transplanting substrate is 1:900-1100, preferably 950-1050.

[0013] In some of these embodiments, inorganic fertilizer is dissolved in water and applied every 15 days.

[0014] In some of the embodiments, the aforementioned method for indoor cultivation of medicinal mulberry is provided with artificial lighting conditions of 25°C, (30-50)%RH, and 15-17 hours of light per day.

[0015] Furthermore, the artificial lighting conditions are 25°C, 40% RH, and the light source is an LED light source with a light intensity of 171 μmol / s PPF, with 16 hours of light per day, from 7:30 to 23:30 per day.

[0016] A second aspect of the present invention is to provide an application of the aforementioned method for indoor cultivation of medicinal mulberry in improving the secondary metabolites of indoor cultivated medicinal mulberry.

[0017] This invention discovers that applying fertilizer can effectively promote the growth of medicinal mulberry when cultivating it indoors. However, research has shown that the growth of medicinal mulberry and the amount of its secondary metabolites are not positively correlated. Further research reveals that using nitrogen, phosphorus, and potassium fertilizers with appropriate compositions, especially high-potassium fertilizers, can effectively increase the secondary metabolites of indoor-cultivated medicinal mulberry. The increase in the active substances of flavonoids in indoor-cultivated medicinal mulberry is most significant. This can increase the content of flavonoids and flavonols in medicinal mulberry by affecting the biosynthetic metabolic pathways of flavonoids and flavonols. Based on this, the method for cultivating medicinal mulberry indoors provided by this invention, through the use of high-potassium fertilizers, enables the indoor-cultivated medicinal mulberry to grow well and increases the content of its secondary metabolites, especially flavonoids, thereby improving the medicinal value of medicinal mulberry. Attached Figure Description

[0018] Figure 1 This is an OPLS-DA analysis under different fertilization treatments. A: Control group, B: High nitrogen fertilizer group, C: High potassium fertilizer group, D: High phosphorus fertilizer group, E: Organic fertilizer group, F: Bio-fertilizer group.

[0019] Figure 2 This study investigated the effects of different fertilizer treatments on the biomass and morphology of mulberry trees grown indoors. A) Leaf fresh weight, B) Leaf dry weight, C) Number of leaves, D) Branch fresh weight, E) Branch dry weight, and F) Plant height. Different lowercase letters on the bar charts indicate significant differences obtained through analysis of variance and multiple range tests (n=8, p<0.05). The treatment groups included: control group (CK), high nitrogen treatment group (HN), high potassium treatment group (HK), high phosphorus treatment group (HP), organic fertilizer treatment group (OF), and bio-fertilizer treatment group (BF).

[0020] Figure 3 This study describes the effects of different fertilizer treatments on chlorophyll fluorescence (ChlF) and relative chlorophyll content (SPAD) of mulberry leaves in an indoor environment. a: Maximum photochemical efficiency of photosystem II (FV / FM). b: Y(II): Actual light energy conversion efficiency of leaves under illumination. c: ETR: Electron transport efficiency of leaves. d: qP: Photochemical quenching coefficient. e: qL: Photochemical quenching coefficient. f: Y(NO): Non-photochemical quenching factor. g: Y(NPQ): Important indicator of light damage. h: qN: Non-photochemical quenching factor. i: Soil and Plant Analysis Development (SPAD) chlorophyll meter value: relative chlorophyll content. Different lowercase letters on the bar chart indicate significant differences obtained through analysis of variance and multiple range tests (n=8, p<0.05).

[0021] Figure 4This figure shows the changes in mulberry leaf metabolites under different fertilizer treatments. The content of the substances is visualized by color in the figure, with red and green representing the increase or decrease of the corresponding parameters, respectively. Among them, CK: control group, HN: high nitrogen fertilizer group, HK: high potassium fertilizer group, HP: high phosphorus fertilizer group, OF: organic fertilizer group, BF: bio-fertilizer group.

[0022] Figure 5 These are differentially synthesized metabolites of mulberry leaves under different fertilization treatments, where A: CK vs HN, B: CK vs HK, C: CK vs HP, D: CK vs OF, and E: CK vs BF. Metabolites with VIP>1, Log2FC≥1 or≤-1, and p<0.05 were identified as differentially synthesized metabolites.

[0023] Figure 6 This is a KEGG enrichment analysis of differentially synthesized metabolites, where A: CK vs HN, B: CK vs HK, C: CK vs HP, D: CK vs OF, and E: CK vs BF; the size and color of the dots represent the number of metabolites and significance (p-value), respectively. Detailed Implementation

[0024] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0025] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0026] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0027] Furthermore, as used herein, the term "or" is an inclusive "or" sign and is equivalent to the term "and / or" unless the context clearly specifies otherwise. The term "based on" is not exclusive and allows for basing on other factors not described unless the context clearly specifies otherwise. Additionally, throughout the specification, the meanings of "an," "a," and "the" include plural indicators. The meaning of "in" includes both "in" and "on."

[0028] The present invention will be further described in detail below with reference to specific embodiments.

[0029] Example 1

[0030] 1. Plant materials, growth and fertilizer sources

[0031] In January 2024, mulberry seedlings from tissue culture were transplanted into 30cm × 28cm pots with a substrate ratio of soil:perlite:vermiculite = 4:1:2, each containing 2.5kg of substrate. They were grown in an artificially lit greenhouse (25℃, 40% RH) under an LED light source of 171μmol / s PPF (ZK3-TB14-VE03 / A, SANANBIO, China), with daily light exposure from 7:30 AM to 11:30 PM. Fertilization was stopped for one month in May 2024. In June 2024, branches were pruned, leaving two buds on each branch, and fertilization was then resumed for 45 consecutive days.

[0032] The inorganic fertilizers are commercial fertilizers, purchased from the Zhongnong Fertilizer flagship store. The NPK ratio in the high-nitrogen fertilizer is 30:10:10 with a small amount of trace elements, the NPK ratio in the high-potassium fertilizer is 13:7:40 with a small amount of trace elements, and the NPK ratio in the high-phosphorus fertilizer is 10:48:10 with a small amount of trace elements.

[0033] Organic fertilizer: made from chicken manure, sheep manure and cow manure composted and fermented in a volume ratio of 1:1:1.

[0034] The bio-fertilizer was purchased from Henan Nanhua Qianmu Co., Ltd., and is made from Bacillus subtilis with an effective live bacteria count of ≥20 billion / g.

[0035] 2. Handling methods

[0036] Control group: Watered but not fertilized.

[0037] Inorganic fertilizer group: Dissolve inorganic fertilizer in water to a concentration of 10g / L, and water each pot with 250ml of inorganic fertilizer solution once every 15 days.

[0038] Organic fertilizer group: 250g / pot, 15 days / time.

[0039] Bio-fertilizer group: Dissolve bio-fertilizer in water to a concentration of 10g / L, and water each pot with 250ml of inorganic fertilizer solution once every 15 days.

[0040] Each group has 8 replicate treatment groups.

[0041] 3. Metabolic analysis

[0042] Forty-five days after fertilization, the third fully expanded leaf from the uppermost morphological end of a mulberry branch was selected, measured, and then quickly immersed in liquid nitrogen before being stored in a -80°C freezer. The mulberry leaves were freeze-dried for 96 hours using a vacuum freeze dryer (ScanSpeed ​​MaxiVac Beta, LaboGene, America). After drying, the samples were milled using a ball mill (MM400, Retsch GmbH, Haan, Germany) at 30 Hz for 5 minutes to ensure thorough pulverization.

[0043] For extraction, 100 mg of dried mulberry sample powder was accurately weighed into a 1.5 mL centrifuge tube, dissolved in 1.25 mL of 70% methanol aqueous solution, and vortexed for 30 s three times, with 10 min intervals between each vortex. The sample was then stored overnight at 4 °C. Subsequently, the sample was centrifuged at 12000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm nylon filter into a sample vial for UPLC-MS / MS analysis.

[0044] 4. Data Analysis

[0045] UPLC-MS / MS data were quantified using Thermo Scientific Xcaliburv2.2. Metabolomics analyses were performed using the Metware platform (https: / / cloud.metware.cn).

[0046] 5. Results:

[0047] 5.1 The OPLS-DA model is stable and reliable.

[0048] OPLS-DA analysis was performed on the control group and each experimental group, and the results are as follows: Figure 1 As shown in the left figure, the sample points of each experimental group are clearly separated and fall into different intervals, indicating significant differences between different treatment groups. The R-squared values ​​of the OPLS-DA regression model parameters are also shown. 2 Y is close to 1, while Q 2 A value greater than 0.5 indicates that the model is stable and reliable (right figure).

[0049] 5.2 Growth Analysis

[0050] Fertilization can significantly promote the accumulation of biomass in mulberry plants. Figure 2 The bio-fertilizer (BF) treatment showed the most significant effect, increasing leaf fresh weight to 414.73% of the control group (CK), dry weight to 379.55%, and leaf number to 204.88% (p<0.05), which was significantly better than the inorganic fertilizer treatment group (IF) and the organic fertilizer treatment group (OF). Figure 2Although inorganic fertilizer treatments (IF) and organic fertilizer treatments (OF) with different nitrogen, phosphorus, and potassium ratios only resulted in limited increases in leaf fresh weight (leaf fresh weight: 186.4%-236.26%, dry weight: 181.11%-227.23%), the increase brought about by bio-fertilizer (BF) treatment was 2.2-2.7 times higher than the maximum increase of inorganic fertilizer (IF) and organic fertilizer (OF). The response of stem biomass exhibited a treatment-dependent pattern: high potassium treatment (HK) increased stem fresh weight to 229.12% and dry weight to 218.84%. High phosphorus treatment (HP) increased these parameters to 216.28% and 210.16%, respectively. Bio-fertilizer (BF) treatment showed superior results (fresh weight: 372.99%; dry weight: 371.80%). High nitrogen treatment (HN) and organic fertilizer treatment (OF) failed to achieve statistically significant increases in stem biomass. Further measurements of plant height confirmed these trends: the plant heights under the high potassium treatment (HK) and the bio-fertilizer treatment (BF) reached 177.76% and 228.80% of the control group plant heights, respectively, reflecting that bio-fertilizer (BF) has a synergistic promoting effect on the development of both source organs (leaves) and sink organs (stems).

[0051] After 45 days of fertilization treatment, the chlorophyll fluorescence kinetics and SPAD values ​​of the mulberry plant were comprehensively measured. Figure 3 (ai). Compared with the control group (CK), fertilization significantly regulated photosynthetic parameters. Positive regulation was manifested in the following ways: the maximum photochemical efficiency (FV / FM) of photosystem II increased to 177.06%–187.77% (inorganic fertilizer treatment group, IF), 161.78% (organic fertilizer treatment group, OF), and 175.65% (bio-fertilizer treatment group, BF). The actual photochemical efficiency (Y(II)) of photosystem II increased to 357.55%–468.33% (inorganic fertilizer group), 428.58% (organic fertilizer group), and 484.22% (bio-fertilizer group). The electron transport rate (ETR) increased to 136.68%–170.5% (inorganic fertilizer group), 155.98% (organic fertilizer group), and 176.18% (bio-fertilizer group). The photochemical quenching coefficient (qP) increased to 242.90%–300.10% (inorganic fertilizer group), 327.82% (organic fertilizer group), and 321.03% (bio-fertilizer group). The non-photochemical quenching coefficient (qL) increased to 280.8%–395.97% (inorganic fertilizer group), 492.95% (organic fertilizer group), and 443.32% (bio-fertilizer group). The relative chlorophyll content (SPAD) increased to 124.04%–143.6% (inorganic fertilizer group), 139.62% (organic fertilizer group), and 150.62% (bio-fertilizer group). Figure 3 ae、i).

[0052] Specific treatment results showed that the relative chlorophyll content (SPAD) in the bio-fertilizer treatment group was the highest (150.62% of the control group). Figure 3 i), which is related to leaf biomass dominance (i), Figure 2 The organic fertilizer treatment group (OF) achieved the maximum values ​​for both the photochemical quenching coefficient (qP, 327.82% of the control group) and the non-photochemical quenching coefficient (qL, 492.95% of the control group). Figure 3 (de). The high-nitrogen treatment group showed the least effect in terms of improving photosynthetic performance and alleviating stress.

[0053] All fertilizer treatments reduced photoinhibition indices: Y(NO), Y(NPQ), and qN: significantly inhibited by the high-potassium treatment group (p<0.05, reduction ranging from 8.07% to 35.35%), and significantly inhibited by the bio-fertilizer treatment (p<0.05, reduction ranging from 8.64% to 39.75%). Figure 3 fh).

[0054] Key analytical features: Parameter correlation: The parallel trend between Y(II) and ETR (r>0.95) confirms the validity and functional consistency of the measurement; the dual efficacy of the biofertilizer treatment group (BF) in relative chlorophyll content (SPAD, source intensity) and fluorescence index (photosynthetic efficiency) reflects its effectiveness in... Figure 2 Synergistic effect of promoting growth. Stress threshold: The unfertilized control group showed chronic photoinhibition (basal Y(NO)>0.35), while the high potassium treatment group and the bio-fertilizer treatment group showed the most significant improvement.

[0055] 5.3 Differences in the metabolic substances of indoor medicinal mulberry under different fertilization treatments

[0056] Based on UPLC-MS / MS and a self-built database, a total of 82 metabolites were detected, including 13 alkaloids, 12 amino acids, 2 fatty acids, 18 flavonoids, 7 lysophospholipids, 4 nucleic acids and nucleic acid derivatives, and C. 19 H 39 N2O3 + One type of phenolic acid, one type of polypeptide, one type of tryptophan metabolite, eight types of vitamins, and ten other substances. The content of these substances is displayed in the form of a heatmap. Figure 4 As shown, from Figure 4The results show that fertilizer application can upregulate or downregulate some active substances in medicinal mulberry. Cluster analysis grouped the high-nitrogen fertilizer group, high-potassium fertilizer group, and high-phosphorus fertilizer group together, indicating that the application of chemical fertilizers has a more significant impact on active substances compared to organic fertilizers, bio-fertilizers, and the control group. Among chemical fertilizers, the application of high-potassium fertilizer can increase most of the active substances in indoor medicinal mulberry, which is significantly better than other chemical fertilizer groups. The results show that the effect of improving the active substances in indoor medicinal mulberry is: high-potassium fertilizer > high-nitrogen fertilizer > high-phosphorus fertilizer > bio-fertilizer = organic fertilizer > control group.

[0057] Metabolites from indoor medicinal mulberry under different fertilization treatments were compared and analyzed with a control group to screen differentially synthesized metabolites with VIP>1, |log2FC|>1, and p<0.05. Results are as follows... Figure 5 As shown, compared with the control group, the high-nitrogen fertilizer group showed upregulation of 11 metabolites, mainly flavonoids, and downregulation of 1 substance, tyrosine. Figure 5 A). Compared with the control group, the high-potassium fertilizer showed an upregulation of 21 metabolites, mainly flavonoids ( Figure 5 B). Compared with the control group, the high-phosphorus fertilizer showed an upregulation of four metabolites, mainly flavonoids, and a downregulation of one metabolite, niacin. Figure 5 C). Compared to the control group, the use of organic fertilizer led to an increase in two metabolites in mulberry leaves, namely L-piperacic acid and L-carnitine. One metabolite decreased to L-aspartic acid. Figure 5 D). Compared to the control group, the use of bio-fertilizer upregulated two substances in mulberry leaves, namely fagomin O-hexoside-I and L-aspartic acid, and downregulated one substance, a salicylic acid derivative. Figure 5 E).

[0058] 5.4. Differential KEGG pathway enrichment analysis of metabolites from indoor medicinal mulberry under different fertilization treatments

[0059] KEGG pathway enrichment analysis was performed on differentially expressed metabolites, focusing on metabolic pathways with p < 0.05. Differentially expressed metabolites from the CK vs HN groups were annotated into 38 pathways; however, no pathways were significantly enriched. Figure 6 A). In the CK vs HP group, the biosynthetic metabolic pathway of flavonoids and flavonols (ko00944) was significantly enriched. Figure 6 B). Kaempferol 3-O-glucoside; luteolin 7-O-glucoside; quercetin 3-O-glucoside; quercetin 3-O-malonyl glucoside; and rutin are the five substances involved in this metabolic pathway, and all of them show an upregulation trend. Figure 5 B). The CK vs HP group showed significant enrichment of the metabolic pathways of nicotinic acid and nicotinamide (ko00760). Figure 6 C). Niacin and L-aspartic acid are involved in this metabolic pathway, and the level of niacin is downregulated. Figure 5 C). In the CK vs OF group, two metabolic pathways showed significant enrichment: the biosynthesis of ornithine, lysine, and nicotinic acid-derived alkaloids (ko01064) and microbial metabolism under different environments (ko01120). Figure 6 D). L-piperidinic acid and aspartic acid are involved in this metabolic pathway, with aspartic acid showing a decreasing trend and L-piperidinic acid showing an increasing trend. Figure 5 D). A total of 34 metabolic pathways were designed to differentiate metabolites in the CK vs BF groups, but no metabolic pathways were significantly enriched among them. Figure 6 E).

[0060] 5.5 The results analysis shows that only 8 flavonoids and flavonols were upregulated in the high-nitrogen fertilizer group. Among them, rutin increased by 2.2 times, astragaloside by 9.5 times, quercetin 3-O-glucoside by 2.3 times, and kaempferol by 3 times compared with the control group.

[0061] In the high-potassium fertilizer group, 15 kinds of flavonoids and flavonols were upregulated, including rutin, which increased by 4.8 times, astragaloside, which increased by 30 times, quercetin 3-O-glucoside, which increased by 5.9 times, and kaempferol, which increased by 7 times compared with the control group.

[0062] In summary, fertilization can effectively promote the growth of medicinal mulberry, especially the bio-fertilizer group, which has the most significant promoting effect. However, further research found that the bio-fertilizer group cannot significantly increase the content of active substances in medicinal mulberry. On the contrary, although the growth-promoting effect of the inorganic fertilizer group is not as good as that of the bio-fertilizer group, the application of inorganic fertilizer can increase the content of active substances in indoor medicinal mulberry, mainly concentrated in flavonoids. In particular, high-potassium fertilizer has the most significant effect on increasing the content of active substances in indoor medicinal mulberry. At the same time, it can affect the biosynthetic metabolic pathways of flavonoids and flavonols, thereby increasing the content of flavonoids and flavonols in medicinal mulberry.

[0063] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method of growing a medicinal mulberry plant in a room, characterized by: The tissue culture seedlings of the medicinal mulberry are transplanted to an artificial illumination greenhouse for growth after rooting, and are subjected to fertilization treatment, the fertilization being inorganic fertilizer containing nitrogen, phosphorus and potassium applied during the vigorous growth period of the medicinal mulberry, wherein the mass ratio of nitrogen, phosphorus and potassium is (12-14):(6-8):(38-42), the illumination condition of the artificial illumination is 25℃, (30-50)% RH, daily illumination for 15-17 hours, and the light source is LED light source with 171 μmol / s PPF.

2. The method for cultivating the medicinal mulberry in a room according to claim 1, wherein: The mass ratio of nitrogen, phosphorus and potassium is 13:7:(39-41).

3. The method for cultivating the mulberry in a room according to claim 1, wherein: The volume ratio of the substrate used for the transplanting is soil: perlite: vermiculite = (3-5):(0.8-1.2):(1.8-2.2).

4. The method of claim 3, wherein the indoor cultivation of the mulberry is performed by the steps of: The volume ratio of the substrate used for the transplanting is soil: perlite: vermiculite = 4:1:

2. ​ 5. The method of cultivating mulberry in a room according to any one of claims 1 to 4, wherein: The mass ratio of the inorganic fertilizer to the transplanting substrate is 1:900-1100.

6. The method of claim 5, wherein the indoor cultivation of the mulberry is characterized by: The mass ratio of the inorganic fertilizer to the transplanting substrate is 1:950-1050.

7. The method of cultivating mulberry in a room according to any one of claims 1 to 4, wherein: The inorganic fertilizer is dissolved in water, and the fertilization is performed once every 15 days.

8. The method for cultivating mulberry in a room according to claim 1, wherein: The illumination condition of the artificial illumination is 25℃, 40% RH, daily illumination for 16 hours, and the daily illumination time is 7:30 to 23:

30.

9. Use of the method according to any one of claims 1 to 8 in improving secondary metabolites of indoor cultivation of medicinal mulberry.

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