Sculellaria barbata culture medium and preparation method thereof

Abstract

The invention relates to the technical field of sculellaria barbata culture, in particular to a sculellaria barbata culture medium and a preparation method thereof The sculellaria barbata culture medium is prepared from an MS culture medium and a basic raw material according to the mass ratio of (6-7): 1, and the basic raw material is prepared from the following raw materials in parts by weight: 2-4 parts of novel nano silicon dioxide, 0.05-0.08 part of cerous nitrate, 1-2 parts of an organic fungicide, 0.7-0.8 part of 6-benzyladenine, 0.2-0.4 part of alpha-naphthylacetic acid, 0.1-0.3 part of jasmonic acid methyl ester and 2-4 parts of a functional additive. According to the invention, cell wall stability is enhanced and hormone slow release regulation is realized through novel nano silicon dioxide, and cerium nitrate is improved to activate a flavonoid compound synthetase system; the organic fungicide inhibits pathogenic bacteria and promotes nitrogen conversion by secreting lipopeptide antibiotics and nitrogenase systems; and the functional auxiliary agent realizes efficient retention and stable release of the active components.

Description

Technical Field

[0001] This invention relates to the field of Scutellaria barbata cultivation technology, specifically to a culture medium for Scutellaria barbata and its preparation method. Background Technology

[0002] Scutellaria barbata, a herbaceous plant belonging to the genus Scutellaria in the family Lamiaceae, typically grows in warm and humid environments and is widely distributed in Europe, Asia, and North America. The plant is relatively low-growing and usually has opposite leaves and purple or white flowers with compact inflorescences and a labiate corolla. This plant not only has ornamental value in horticulture but is also used in traditional Chinese medicine for its medicinal properties, often for clearing heat and detoxifying, promoting diuresis, and reducing swelling.

[0003] Current culture media for Scutellaria barbata face several challenges. First, traditional media rely solely on basic nutrients and single hormone combinations, resulting in low growth efficiency and insufficient accumulation of active ingredients, making it difficult to balance plant growth with the enhancement of active components. Second, conventional nanomaterials tend to aggregate and settle in the culture medium, leading to low bioavailability and potentially causing toxicity to cells, thus affecting culture outcomes. Existing systems place too much emphasis on bud differentiation and rooting, neglecting the regulation of abiotic stress and the induction of secondary metabolites, resulting in insufficient plant resistance and low efficiency in the synthesis of active ingredients. Finally, browning is a serious problem in Scutellaria barbata tissue culture. Due to the excessively high activity of polyphenol oxidase, traditional methods using single antioxidants are ineffective, easily leading to high browning rates, which in turn affects the retention of active ingredients and plant health.

[0004] Therefore, the present invention provides a culture medium for Scutellaria barbata and a method for preparing the same, in order to solve the aforementioned related technical problems. Summary of the Invention

[0005] The purpose of this invention is to provide a culture medium for Scutellaria barbata and its preparation method. MS medium is used as the basic nutrient carrier. Novel nano-silica is used to enhance cell wall stability and achieve sustained hormone release regulation, while also improving the cerium nitrate-activated flavonoid synthesis enzyme system. Organic bacterial agents inhibit pathogens and promote nitrogen conversion by secreting lipopeptide antibiotics and nitrogenase systems. A combination of traditional hormones, 6-benzyladenine and α-naphthaleneacetic acid, synergistically with a novel hormone, methyl jasmonate, promotes bud differentiation and rooting, while also inducing stress resistance and the accumulation of secondary metabolites. Functional adjuvants achieve efficient retention and stable release of active ingredients.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A culture medium for Scutellaria barbata, wherein the culture medium is prepared from MS medium and basic raw materials at a mass ratio of 6-7:1. The basic raw materials are prepared from the following raw materials in parts by weight: 2-4 parts of novel nano-silica, 0.05-0.08 parts of cerium nitrate, 1-2 parts of organic bacterial agent, 0.7-0.8 parts of 6-benzyladenine, 0.2-0.4 parts of α-naphthaleneacetic acid, 0.1-0.3 parts of methyl jasmonate, and 2-4 parts of functional additives.

[0007] Preferably, 6-benzyladenine is purchased from Shandong Hengke Biotechnology Co., Ltd., cerium nitrate is purchased from Shandong Li'ang New Material Technology Co., Ltd., α-naphthaleneacetic acid is purchased from Beijing Huake Shengda Co., Ltd., and methyl jasmonate is purchased from Hubei Yamaide Biomedical Co., Ltd.

[0008] Furthermore, such as Figure 1 As shown, the preparation method of the functional additive includes the following steps: S1: Wash Scutellaria barbata with deionized water, drain and dry in a hot air drying oven at 35-40℃ until the moisture content is ≤5%, then pulverize and pass through a 50-60 mesh sieve to obtain Scutellaria barbata powder. S2: Prepare the extraction solvent. Mix the Scutellaria barbata powder with the extraction solvent at a mass ratio of 1:18-22. Place the mixture in an ultrasonic extractor and set the temperature to 45-50℃ for 30-35 minutes to obtain a crude extract. Centrifuge the crude extract, collect the supernatant, and filter it to obtain the extract. S3: Mix the extract, cysteine, and disodium EDTA at a mass ratio of 1:0.0008-0.012:0.0002-0.0004 and stir at 23-27℃ for 25-35 minutes to form an inhibitory extract. Then mix the inhibitory extract, ascorbic acid, glutathione, and citric acid at a mass ratio of 1:0.0002-0.0004:0.0008-0.0012:0.0004-0.0006 and add sodium hydroxide solution to adjust the pH to 5.0-5.2 to obtain the antioxidant treatment solution. Among them, cysteine ​​can be sourced from Anhui Qianshun Biotechnology Co., Ltd., disodium ethylenediaminetetraacetate can be sourced from Tianjin Zhonghe Shengtai Chemical Co., Ltd., ascorbic acid can be sourced from Anhui Weimao Biotechnology Co., Ltd., glutathione can be sourced from Xi'an Heshun Biotechnology Co., Ltd., and citric acid can be sourced from Wujiang Xincheng Fine Chemical Co., Ltd.

[0009] S4: The antioxidant treatment solution is filtered and purified to obtain a clear solution, which is then treated to prepare the functional additive.

[0010] Furthermore, the method for preparing the extraction solvent in step S2 is as follows: Prepare citric acid solution and disodium hydrogen phosphate solution separately. Mix the citric acid solution and disodium hydrogen phosphate solution at a volume ratio of 1:1.2-1.4 to obtain a buffer solution. Dissolve ascorbic acid in the buffer solution at a solid-liquid ratio of 0.0015-0.002 g / mL and stir for 10-15 min. Nitrogen gas was introduced into the buffer solution containing ascorbic acid and stirred for 25–35 minutes to obtain the extraction solvent.

[0011] Furthermore, the antioxidant treatment solution is filtered and purified to obtain a clarified solution, and the specific steps are as follows: The antioxidant treatment solution was filtered using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa. The ultrafiltrate was collected and adsorbed using a macroporous adsorption resin column. After adsorption saturation, it was eluted with 70% ethanol to form an eluent. The eluent was evaporated to one-fifth of its original volume to obtain a concentrated solution. It should be noted that it uses an ultrafiltration membrane with a molecular weight cutoff of 3kDa to separate the antioxidant treatment solution, remove large molecular polyphenols and impurities, and retain small molecular active ingredients. The macroporous adsorption resin column is model XAD-16, which can be purchased from the Chemical Reagent Company of East China University of Science and Technology.

[0012] Activated carbon was added to the concentrate at a solid-liquid ratio of 0.0015–0.002 g / mL, and stirred in a water bath at 50–55°C for 40–50 min. After stirring, the mixture was filtered to obtain a clear liquid. The activated carbon used was selected from Shijiazhuang Hongsen Activated Carbon Co., Ltd.

[0013] Furthermore, the specific steps for further processing the clarified liquid are as follows: The clarified liquid was mixed with β-cyclodextrin at a mass ratio of 4.5–5.5:1 and stirred in a water bath at 40–45°C for 1.5–2.5 h to form an inclusion complex solution. The inclusion complex solution was then transferred to a spray dryer to obtain microcapsule powder. The preparation method of β-cyclodextrin is as follows: β-Cyclodextrin was obtained by using corn starch as a substrate and cyclodextrin glucosyltransferase catalyzing the reaction at 55°C and pH 6.5. The corn starch was purchased from Henan Fengwei Biotechnology Co., Ltd., while the cyclodextrin glucosyltransferase was selected from Beijing Tuopu Biotechnology Co., Ltd.

[0014] Gelatin and gum arabic were mixed at a mass ratio of 1:1 and dissolved in deionized water to prepare an 8%–10% wall material solution. Microcapsule powder was mixed with the wall material solution at a mass ratio of 1:4.8–5.2 and stirred at 55–65°C to form an emulsion. The pH of the emulsion was adjusted to 4.4–4.6. After adjustment, 0.4%–0.6% (by mass) of transglutaminase was added to the wall material solution, and the reaction was carried out at 35–45°C for 0.8–1 h. After the reaction was completed, a 5% (by mass) calcium chloride solution was added dropwise to prepare microcapsules. The microcapsules were washed 2–4 times with deionized water to obtain the functional additive.

[0015] The gelatin was sourced from Guangzhou Anrui Food Ingredients Co., Ltd., while the gum arabic was purchased from Shandong Qiangnuo Food Ingredients Co., Ltd.

[0016] Furthermore, the citric acid solution is prepared by mixing citric acid and deionized water at a mass ratio of 1:35-40; The disodium hydrogen phosphate solution is prepared by mixing disodium hydrogen phosphate and deionized water at a mass ratio of 1:20-24.

[0017] Furthermore, the preparation steps of the novel nano-silica are as follows: Nano-sized silica with a particle size of 20 nm, anhydrous ethanol, and 3-aminopropyltriethoxysilane were mixed at a mass ratio of 6–7:13–15:1 and stirred under reflux at 65–75 °C for 5.5–6.5 h to obtain a reaction solution. After the reaction solution was cooled to 25 °C, it was washed 2–4 times by centrifugation with ethanol and dried under vacuum at 55–65 °C for 10–12 h to obtain modified silica. It should be added that the nano-silica was purchased from Tuoyi New Materials (Guangzhou) Co., Ltd., and its particle size is 20nm; while the 3-aminopropyltriethoxysilane was selected from products produced by Dongguan Shanyi Plastics Co., Ltd.

[0018] Modified silica was grafted to obtain grafted silica. The grafted silica, phosphate buffer, and 6-benzyladenine were mixed at a mass ratio of 23-27:80-85:1 and stirred at 23-27°C for 20-25 hours to obtain a loading solution. The loading solution was dialyzed, the dialysate was collected, and then freeze-dried to obtain novel nano-silica.

[0019] Phosphate buffer solution can be a product prepared by Jinan Xiaoshi Chemical Co., Ltd. Furthermore, the specific steps for grafting the modified silica are as follows: Modified silica, deionized water, and polyethylene glycol were mixed in a mass ratio of 1:2.5-3.5:1.5-2.5 to obtain a polymer solution. Ammonium persulfate at a mass of 0.4%-0.6% of polyethylene glycol was added to the polymer solution. The mixture was stirred at 55-65°C for 7.5-8.5 hours under nitrogen protection to form a grafting solution. The polyethylene glycol was purchased from Tianjin Zhonghe Shengtai Chemical Co., Ltd., and the ammonium persulfate was selected from Lianyungang Guansu Industrial Co., Ltd. The grafting solution was dialyzed with deionized water, and after dialyzing, it was freeze-dried to obtain grafted silica.

[0020] Furthermore, the organic microbial agent is selected from any one of Bacillus subtilis, Bacillus belye, and Bacillus amyloliquefaciens, all of which are purchased from Shandong Tainuo Pharmaceutical Co., Ltd.

[0021] A method for preparing a culture medium for Scutellaria barbata includes the following steps: Step 1: Weigh each salt according to the MS medium standard formula, dissolve in deionized water, heat to dissolve, add sucrose and agar, adjust the pH to 5.8-6, and sterilize at 121-125℃ for 15-20 minutes. Step 2: Activate the organic bacteria to obtain organic bacterial agent, and prepare functional adjuvants and novel nano silica according to the steps. After the MS medium is cooled to 45°C, add novel silica, cerium nitrate, organic bacterial agent, 6-benzyladenine, α-naphthaleneacetic acid, methyl jasmonate and functional adjuvant respectively. After adding, stir evenly and let it cool and solidify to obtain a culture medium for Scutellaria barbata.

[0022] Compared with the prior art, the beneficial effects of the present invention are: This invention uses MS medium as the basic nutrient carrier, enhances cell wall stability and achieves sustained hormone release regulation through novel nano-silica, and improves the enzyme system for flavonoid synthesis activated by cerium nitrate; organic bacterial agents inhibit pathogens and promote nitrogen conversion by secreting lipopeptide antibiotics and nitrogenase systems; the traditional hormone combination of 6-benzyladenine and α-naphthaleneacetic acid, synergistically with the novel hormone methyl jasmonate, promotes bud differentiation and rooting, and induces stress resistance and accumulation of secondary metabolites; functional adjuvants achieve efficient retention and stable release of active ingredients; The novel nano-silica enhances biocompatibility and nutrient utilization, while the synergistic effect of microorganisms optimizes the culture environment and reduces the risk of contamination. The innovative hormone combination enhances stress resistance and secondary metabolism. The browning inhibition system uses antioxidants, enzyme inhibitors and encapsulation technology to reduce the browning rate, solving the problems of low extraction rate and easy degradation of active ingredients. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is a flowchart illustrating the preparation of a functional adjuvant for the culture medium of Scutellaria barbata according to the present invention; Figure 2 A bar chart showing the numerical assessment of the reduced browning risk in experiments conducted for this invention. Figure 3 Line graph showing the effect of the experiment conducted in this invention on promoting asexual reproduction. Detailed Implementation

[0025] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Example 1 A culture medium for Scutellaria barbata, wherein the culture medium is prepared by MS medium and basic raw materials at a mass ratio of 6:1. The basic raw materials are prepared by the following parts by weight: 2 parts of novel nano-silica, 0.05 parts of cerium nitrate, 1 part of organic bacterial agent, 0.7 parts of 6-benzyladenine, 0.2 parts of α-naphthaleneacetic acid, 0.1 parts of methyl jasmonate, and 2 parts of functional additives.

[0027] Furthermore, the preparation method of the functional additive includes the following steps: S1: Wash Scutellaria barbata with deionized water, drain and dry in a 35℃ hot air drying oven until the moisture content is ≤5%, then pulverize and pass through a 50-mesh sieve to obtain Scutellaria barbata powder. S2: Prepare the extraction solvent. Mix the Scutellaria barbata powder with the extraction solvent at a mass ratio of 1:18. Place the mixture in an ultrasonic extractor and set the temperature to 45°C for 30 minutes to obtain a crude extract. Centrifuge the crude extract, collect the supernatant, and filter it to obtain the extract. S3: Mix the extract, cysteine, and disodium ethylenediaminetetraacetate at a mass ratio of 1:0.0008:0.0002 and stir at 23°C for 25 minutes to form an inhibitory extract. Then mix the inhibitory extract, ascorbic acid, glutathione, and citric acid at a mass ratio of 1:0.0002:0.0008:0.0004 and add sodium hydroxide solution to adjust the pH to 5.0 to obtain the antioxidant treatment solution. S4: The antioxidant treatment solution is filtered and purified to obtain a clear solution, which is then treated to prepare the functional additive.

[0028] Furthermore, the method for preparing the extraction solvent in step S2 is as follows: Prepare citric acid solution and disodium hydrogen phosphate solution separately. Mix the citric acid solution and disodium hydrogen phosphate solution at a volume ratio of 1:1.2 to obtain a buffer solution. Dissolve ascorbic acid in the buffer solution at a solid-liquid ratio of 0.0015 g / mL and stir for 10 min. Nitrogen gas was introduced into the buffer solution containing ascorbic acid and stirred for 25 minutes to obtain the extraction solvent.

[0029] Furthermore, the antioxidant treatment solution is filtered and purified to obtain a clarified solution, and the specific steps are as follows: The antioxidant treatment solution was filtered using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa. The ultrafiltrate was collected and adsorbed using a macroporous adsorption resin column. After adsorption saturation, it was eluted with 70% ethanol to form an eluent. The eluent was evaporated to one-fifth of its original volume to obtain a concentrated solution. Activated carbon was added to the concentrate at a solid-liquid ratio of 0.0015 g / mL and stirred in a water bath at 50°C for 40 min. After stirring, the mixture was filtered to obtain a clear liquid. The activated carbon used was selected from Shijiazhuang Hongsen Activated Carbon Co., Ltd.

[0030] Furthermore, the specific steps for further processing the clarified liquid are as follows: The clarified liquid was mixed with β-cyclodextrin at a mass ratio of 4.5:1 and stirred in a water bath at 40°C for 1.5 h to form an inclusion complex solution. The inclusion complex solution was then transferred to a spray dryer to obtain microcapsule powder. Gelatin and gum arabic were mixed at a mass ratio of 1:1 and dissolved in deionized water to prepare an 8% wall material solution. Microcapsule powder was mixed with the wall material solution at a mass ratio of 1:4.8 and stirred at 55°C to form an emulsion. The pH of the emulsion was adjusted to 4.4. After adjustment, 0.4% (by mass) of transglutaminase was added to the wall material solution, and the reaction was carried out at 35°C for 0.8 hours. After the reaction was completed, a 5% (by mass) calcium chloride solution was added dropwise to prepare microcapsules. The microcapsules were washed twice with deionized water to obtain the functional additive.

[0031] The gelatin was sourced from Guangzhou Anrui Food Ingredients Co., Ltd., while the gum arabic was purchased from Shandong Qiangnuo Food Ingredients Co., Ltd.

[0032] Furthermore, the citric acid solution is prepared by mixing citric acid and deionized water at a mass ratio of 1:35; The disodium hydrogen phosphate solution is prepared by mixing disodium hydrogen phosphate and deionized water at a mass ratio of 1:20.

[0033] Furthermore, the preparation steps of the novel nano-silica are as follows: Nano-sized silica with a particle size of 20 nm, anhydrous ethanol, and 3-aminopropyltriethoxysilane were mixed at a mass ratio of 6:13:1 and stirred at 65 °C for 5.5 h to obtain a reaction solution. After the reaction solution was cooled to 25 °C, it was washed twice with ethanol by centrifugation and dried at 55 °C under vacuum for 10 h to obtain modified silica. It should be added that the nano-silica was purchased from Tuoyi New Materials (Guangzhou) Co., Ltd., and its particle size is 20nm; while the 3-aminopropyltriethoxysilane was selected from products produced by Dongguan Shanyi Plastics Co., Ltd.

[0034] Modified silica was grafted to obtain grafted silica. The grafted silica, phosphate buffer, and 6-benzyladenine were mixed at a mass ratio of 23:80:1 and stirred at 23°C for 20 h to obtain a loading solution. The loading solution was dialyzed, the dialysate was collected, and then freeze-dried to obtain novel nano silica.

[0035] Phosphate buffer solution can be a product prepared by Jinan Xiaoshi Chemical Co., Ltd. Furthermore, the specific steps for grafting the modified silica are as follows: Modified silica, deionized water, and polyethylene glycol were mixed in a mass ratio of 1:2.5:1.5 to obtain a polymer solution. Ammonium persulfate at a mass of 0.4% of polyethylene glycol was added to the polymer solution. The mixture was stirred at 55°C for 7.5 hours under nitrogen protection to form a grafting solution. The polyethylene glycol was purchased from Tianjin Zhonghe Shengtai Chemical Co., Ltd., and the ammonium persulfate was selected from Lianyungang Guansu Industrial Co., Ltd. The grafting solution was dialyzed with deionized water, and after dialyzing, it was freeze-dried to obtain grafted silica.

[0036] Furthermore, the organic microbial agent is selected from Bacillus subtilis.

[0037] A method for preparing a culture medium for Scutellaria barbata includes the following steps: Step 1: Weigh each salt according to the MS medium standard formula, dissolve in deionized water, heat to dissolve, add sucrose and agar, adjust pH to 5.8, and autoclave at 121℃ for 15 min. Step 2: Activate the organic bacteria to obtain organic bacterial agent, and prepare functional adjuvants and novel nano silica according to the steps. After the MS medium is cooled to 45°C, add novel silica, cerium nitrate, organic bacterial agent, 6-benzyladenine, α-naphthaleneacetic acid, methyl jasmonate and functional adjuvant respectively. After adding, stir evenly and let it cool and solidify to obtain a culture medium for Scutellaria barbata.

[0038] Example 2 The preparation method of the culture medium for Scutellaria barbata provided in this embodiment is basically the same as that in Example 1. The main difference between the two lies in the specific composition and ratio of the raw materials used. The specific composition of the raw materials used in this embodiment is as follows: the culture medium for Scutellaria barbata is prepared by MS culture medium and basic raw materials at a mass ratio of 6.5:1. The basic raw materials are made from the following parts by weight: 3 parts of novel nano silica, 0.06 parts of cerium nitrate, 1.5 parts of organic bacterial agent, 0.75 parts of 6-benzyladenine, 0.3 parts of α-naphthaleneacetic acid, 0.2 parts of methyl jasmonate, and 3 parts of functional additives.

[0039] Furthermore, the preparation method of the functional additive includes the following steps: S1: Wash Scutellaria barbata with deionized water, drain and dry in a 38℃ hot air drying oven until the moisture content is ≤5%, then pulverize and pass through a 55-mesh sieve to obtain Scutellaria barbata powder. S2: Prepare the extraction solvent. Mix the Scutellaria barbata powder with the extraction solvent at a mass ratio of 1:20. Place the mixture in an ultrasonic extractor and set the temperature to 47°C for 33 minutes to obtain a crude extract. Centrifuge the crude extract, collect the supernatant, and filter it to obtain the extract. S3: Mix the extract, cysteine, and disodium ethylenediaminetetraacetate at a mass ratio of 1:0.01:0.0003 and stir at 25°C for 30 minutes to form an inhibitory extract. Then mix the inhibitory extract, ascorbic acid, glutathione, and citric acid at a mass ratio of 1:0.0003:0.001:0.0005 and add sodium hydroxide solution to adjust the pH to 5.1 to obtain the antioxidant treatment solution. S4: The antioxidant treatment solution is filtered and purified to obtain a clear solution, which is then treated to prepare the functional additive.

[0040] Furthermore, the method for preparing the extraction solvent in step S2 is as follows: Prepare citric acid solution and disodium hydrogen phosphate solution separately. Mix the citric acid solution and disodium hydrogen phosphate solution at a volume ratio of 1:1.3 to obtain a buffer solution. Dissolve ascorbic acid in the buffer solution at a solid-liquid ratio of 0.0018 g / mL and stir for 13 min. Nitrogen gas was introduced into the buffer solution containing ascorbic acid and stirred for 30 minutes to obtain the extraction solvent.

[0041] Furthermore, the antioxidant treatment solution is filtered and purified to obtain a clarified solution, and the specific steps are as follows: The antioxidant treatment solution was filtered using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa. The ultrafiltrate was collected and adsorbed using a macroporous adsorption resin column. After adsorption saturation, it was eluted with 70% ethanol to form an eluent. The eluent was evaporated to one-fifth of its original volume to obtain a concentrated solution. Activated carbon was added to the concentrate at a solid-liquid ratio of 0.0018 g / mL and stirred in a water bath at 53°C for 45 min. After stirring, the mixture was filtered to obtain a clear liquid. The activated carbon used was selected from Shijiazhuang Hongsen Activated Carbon Co., Ltd.

[0042] Furthermore, the specific steps for further processing the clarified liquid are as follows: The clarified liquid was mixed with β-cyclodextrin at a mass ratio of 5:1 and stirred in a water bath at 43°C for 2 hours to form an inclusion complex solution. The inclusion complex solution was then transferred to a spray dryer to obtain microcapsule powder. Gelatin and gum arabic were mixed at a mass ratio of 1:1 and dissolved in deionized water to prepare a 9% wall material solution. Microcapsule powder was mixed with the wall material solution at a mass ratio of 1:5 and stirred at 60°C to form an emulsion. The pH of the emulsion was adjusted to 4.5. After adjustment, 0.5% (by mass) of transglutaminase was added to the wall material solution and reacted at 40°C for 0.9 hours. After the reaction was completed, a 5% (by mass) calcium chloride solution was added dropwise to prepare microcapsules. The microcapsules were washed three times with deionized water to obtain the functional additive.

[0043] The gelatin was sourced from Guangzhou Anrui Food Ingredients Co., Ltd., while the gum arabic was purchased from Shandong Qiangnuo Food Ingredients Co., Ltd.

[0044] Furthermore, the citric acid solution is prepared by mixing citric acid and deionized water at a mass ratio of 1:38; The disodium hydrogen phosphate solution is prepared by mixing disodium hydrogen phosphate and deionized water at a mass ratio of 1:22.

[0045] Furthermore, the preparation steps of the novel nano-silica are as follows: Nano-sized silica with a particle size of 20 nm, anhydrous ethanol, and 3-aminopropyltriethoxysilane were mixed at a mass ratio of 6.5:14:1 and stirred at reflux for 6 h to obtain a reaction solution. After the reaction solution was cooled to 25 °C, it was washed three times by centrifugation with ethanol and dried at 60 °C under vacuum for 11 h to obtain modified silica. Modified silica was grafted to obtain grafted silica. The grafted silica, phosphate buffer, and 6-benzyladenine were mixed at a mass ratio of 25:83:1 and stirred at 25°C for 24 hours to obtain a loading solution. The loading solution was dialyzed, the dialysate was collected, and then freeze-dried to obtain novel nano silica.

[0046] Phosphate buffer solution can be a product prepared by Jinan Xiaoshi Chemical Co., Ltd. Furthermore, the specific steps for grafting the modified silica are as follows: Modified silica, deionized water, and polyethylene glycol were mixed in a mass ratio of 1:3:2 to obtain a polymer solution. Ammonium persulfate, which was 0.5% of the mass of polyethylene glycol, was added to the polymer solution. The mixture was stirred at 60°C for 8 hours under nitrogen protection to form a grafting solution. The polyethylene glycol was purchased from Tianjin Zhonghe Shengtai Chemical Co., Ltd., while the ammonium persulfate was selected from Lianyungang Guansu Industrial Co., Ltd. The grafting solution was dialyzed with deionized water, and after dialyzing, it was freeze-dried to obtain grafted silica.

[0047] Furthermore, the organic microbial agent is selected from Bacillus belye.

[0048] Example 3 The preparation method of the culture medium for Scutellaria barbata provided in this embodiment is basically the same as that in Example 1. The main difference between the two lies in the specific composition and ratio of the raw materials used. The specific composition of the raw materials used in this embodiment is as follows: the culture medium for Scutellaria barbata is prepared by MS culture medium and basic raw materials at a mass ratio of 7:1. The basic raw materials are made from the following parts by weight: 4 parts of novel nano silica, 0.08 parts of cerium nitrate, 2 parts of organic bacterial agent, 0.8 parts of 6-benzyladenine, 0.4 parts of α-naphthaleneacetic acid, 0.3 parts of methyl jasmonate, and 4 parts of functional additives.

[0049] Furthermore, the preparation method of the functional additive includes the following steps: S1: Wash Scutellaria barbata with deionized water, drain and dry in a 40℃ hot air drying oven until the moisture content is ≤5%, then pulverize and pass through a 60-mesh sieve to obtain Scutellaria barbata powder. S2: Prepare the extraction solvent. Mix the Scutellaria barbata powder with the extraction solvent at a mass ratio of 1:22. Place the mixture in an ultrasonic extractor and set the temperature to 50°C for 35 minutes to obtain a crude extract. Centrifuge the crude extract, collect the supernatant, and filter it to obtain the extract. S3: Mix the extract, cysteine, and disodium ethylenediaminetetraacetate at a mass ratio of 1:0.012:0.0004 and stir at 27°C for 35 minutes to form an inhibitory extract. Then mix the inhibitory extract, ascorbic acid, glutathione, and citric acid at a mass ratio of 1:0.0004:0.0012:0.0006 and add sodium hydroxide solution to adjust the pH to 5.2 to obtain the antioxidant treatment solution. S4: The antioxidant treatment solution is filtered and purified to obtain a clear solution, which is then treated to prepare the functional additive.

[0050] Furthermore, the method for preparing the extraction solvent in step S2 is as follows: Prepare citric acid solution and disodium hydrogen phosphate solution separately. Mix the citric acid solution and disodium hydrogen phosphate solution at a volume ratio of 1:1.4 to obtain a buffer solution. Dissolve ascorbic acid in the buffer solution at a solid-liquid ratio of 0.002 g / mL and stir for 15 min. Nitrogen gas was introduced into the buffer solution containing ascorbic acid and stirred for 35 minutes to obtain the extraction solvent.

[0051] Furthermore, the antioxidant treatment solution is filtered and purified to obtain a clarified solution, and the specific steps are as follows: The antioxidant treatment solution was filtered using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa. The ultrafiltrate was collected and adsorbed using a macroporous adsorption resin column. After adsorption saturation, it was eluted with 70% ethanol to form an eluent. The eluent was evaporated to one-fifth of its original volume to obtain a concentrated solution. Activated carbon was added to the concentrate at a solid-liquid ratio of 0.002 g / mL and stirred in a water bath at 55°C for 50 min. After stirring, the mixture was filtered to obtain a clear liquid. The activated carbon used was selected from Shijiazhuang Hongsen Activated Carbon Co., Ltd.

[0052] Furthermore, the specific steps for further processing the clarified liquid are as follows: The clarified liquid was mixed with β-cyclodextrin at a mass ratio of 5.5:1 and stirred in a water bath at 45°C for 2.5 hours to form an inclusion complex solution. The inclusion complex solution was then transferred to a spray dryer to obtain microcapsule powder. Gelatin and gum arabic were mixed at a mass ratio of 1:1 and dissolved in deionized water to prepare a 10% wall material solution. Microcapsule powder was mixed with the wall material solution at a mass ratio of 1:5.2 and stirred at 65°C to form an emulsion. The pH of the emulsion was adjusted to 4.6. After adjustment, 0.6% (by mass) of transglutaminase was added to the wall material solution and reacted at 45°C for 1 hour. After the reaction was completed, a 5% (by mass) calcium chloride solution was added dropwise to prepare microcapsules. The microcapsules were washed four times with deionized water to obtain the functional additive.

[0053] The gelatin was sourced from Guangzhou Anrui Food Ingredients Co., Ltd., while the gum arabic was purchased from Shandong Qiangnuo Food Ingredients Co., Ltd.

[0054] Furthermore, the citric acid solution is prepared by mixing citric acid and deionized water at a mass ratio of 1:40; The disodium hydrogen phosphate solution is prepared by mixing disodium hydrogen phosphate and deionized water at a mass ratio of 1:24.

[0055] Furthermore, the preparation steps of the novel nano-silica are as follows: Nano-sized silica with a particle size of 20 nm, anhydrous ethanol, and 3-aminopropyltriethoxysilane were mixed at a mass ratio of 7:15:1 and stirred at reflux for 6.5 h to obtain a reaction solution. After the reaction solution was cooled to 25 °C, it was washed four times by centrifugation with ethanol and dried under vacuum at 65 °C for 12 h to obtain modified silica. It should be added that the nano-silica was purchased from Tuoyi New Materials (Guangzhou) Co., Ltd., and its particle size is 20nm; while the 3-aminopropyltriethoxysilane was selected from products produced by Dongguan Shanyi Plastics Co., Ltd.

[0056] Modified silica was grafted to obtain grafted silica. The grafted silica, phosphate buffer, and 6-benzyladenine were mixed at a mass ratio of 27:85:1 and stirred at 27°C for 25 h to obtain a loading solution. The loading solution was dialyzed, the dialysate was collected, and then freeze-dried to obtain novel nano silica.

[0057] Phosphate buffer solution can be a product prepared by Jinan Xiaoshi Chemical Co., Ltd. Furthermore, the specific steps for grafting the modified silica are as follows: Modified silica, deionized water, and polyethylene glycol were mixed in a mass ratio of 1:3.5:2.5 to obtain a polymer solution. Ammonium persulfate at a mass of 0.6% of polyethylene glycol was added to the polymer solution. The mixture was stirred at 65°C for 8.5 hours under nitrogen protection to form a grafting solution. The polyethylene glycol was purchased from Tianjin Zhonghe Shengtai Chemical Co., Ltd., and the ammonium persulfate was selected from Lianyungang Guansu Industrial Co., Ltd. The grafting solution was dialyzed with deionized water, and after dialyzing, it was freeze-dried to obtain grafted silica.

[0058] Furthermore, the organic bacterial agent is selected from Bacillus amyloliquefaciens.

[0059] Comparative Example 1: The preparation method and specific ratio of raw materials of the culture medium of Scutellaria barbata provided in this example are roughly the same as those in Example 1. The main difference is that an equal amount of Scutellaria barbata powder is used to replace the functional additives in this example.

[0060] Comparative Example 2: The preparation method and specific ratio of raw materials of the culture medium for Scutellaria barbata provided in this example are roughly the same as those in Example 1. The main difference is that this example does not contain functional additives.

[0061] Comparative Example 3: The preparation method and specific ratio of raw materials of the culture medium for Scutellaria barbata provided in this example are roughly the same as those in Example 1. The main difference is that an equal amount of nano-silica is used instead of the novel nano-silica. Comparative Example 4: The preparation method and specific ratio of raw materials for the culture medium of Scutellaria barbata provided in this example are roughly the same as those in Example 1. The main difference is that in the preparation step of the novel nano-silica, an equal amount of unmodified nano-silica is used for grafting.

[0062] Effect test The culture media of Scutellaria barbata prepared by Examples 1 to 3 of the present invention are referred to as Experimental Examples 1 to 3; the culture media of Scutellaria barbata prepared by Comparative Examples 1 to 4 are referred to as Comparative Examples 1 to 4; and then the performance of equal amounts of each group of Scutellaria barbata culture media is tested.

[0063] Experimental setup: Sampling: Each experimental group / control group was inoculated with stem segments of uniform size (1.5 cm long, with 1 bud). Each group had 3 replicates, with 10 explants per replicate. The explants were cultured for 30 days at 25±2℃ and 16 h / d light (2000 lx light intensity). On the 30th day of culture, fresh cultures (including callus and regenerated buds) were collected from each group.

[0064] Effects of promoting the accumulation of flavonoids: Stem segments were inoculated onto the culture media of Examples 1-3 and Comparative Examples 1-4, respectively. On day 30 of culture, fresh cultures (including callus and regenerated shoots) were collected from each group. The total flavonoid content was determined by ultrasound-assisted ethanol extraction, using rutin as a standard, and absorbance was measured at 510 nm using the aluminum nitrate-sodium nitrite colorimetric method. The total flavonoid content (mg / gDW, milligrams per gram of dry weight) was calculated. The experimental results are detailed in Table 1. Table 1: Flavonoid Content Table This test aimed to evaluate the promoting effect of the culture medium on the synthesis of secondary metabolites of Scutellaria barbata. The total flavonoid content in Examples 1-3 was significantly higher than that in all comparative examples, and showed an increasing trend with the increase of the proportion of functional components in the formulation, indicating a significant synergistic effect between the functional adjuvant and the novel nano-silica. Comparative Example 1 showed better results than Comparative Example 2, indicating that the microencapsulation and compound antioxidant treatment of the functional adjuvant effectively protected and promoted the action of the active ingredients. Comparative Examples 3 and 4 showed the worst results, confirming that the surface modification of nano-silica and the loading of 6-benzyladenine are key to enhancing its biological effects and promoting flavonoid synthesis.

[0065] Assessment of reduced browning risk: After inoculation of stem segments, observations and samples were taken on days 7, 14, and 21 of culture. The experimental results are shown in Table 2. Figure 2 Its measurement indicators are: Browning rate: The percentage (%) of explants that have browned (turned dark brown or black); Polyphenol oxidase (PPO) activity: Take 0.1g of fresh sample, extract the enzyme solution with phosphate buffer, use catechol as substrate, and measure the change in absorbance per minute at a wavelength of 420nm. The result is expressed as U / gFW (enzyme activity units per gram of fresh weight).

[0066] Table 2: Browning Assessment Table Data from 21 days of cultivation showed that the browning rate was lowest in the example group, and PPO activity was significantly inhibited. The complex antioxidant system (cysteine, ascorbic acid, etc.) and microencapsulated sustained-release properties contained in the functional adjuvants can continuously scavenge free radicals and inhibit the oxidation of phenolic substances. The novel nano-silica may indirectly enhance the stress resistance of explants by improving the cellular microenvironment and promoting rapid cell division through the loading of 6-benzyladenine. Comparative Example 1 showed antioxidant activity, but it was not long-lasting; Comparative Example 2 completely lacked antioxidant protection; and Comparative Examples 3 and 4 had limited anti-browning effects due to the biocompatibility or single function of the nanomaterials.

[0067] Effects of promoting asexual reproduction: After inoculation of stem segments, statistics were collected on day 30 of culture, as detailed in Table 3 and [other tables]. Figure 3 Its measurement indicators are: Bud induction rate: The percentage (%) of explants that produce bud clusters (bud clusters) out of the total number of inoculated explants.

[0068] Average number of buds: The average number of buds produced per explant that produces clustered buds.

[0069] Average bud length: The length of 30 newly formed buds was randomly measured and the average value (cm) was taken.

[0070] Table 3: Table of Asexual Reproduction In summary, the *Scutellaria barbata* culture medium provided by this invention, through the synergistic effect of functional additives and novel nano-silica, can significantly improve the accumulation of flavonoids, effectively reduce the risk of browning in tissue culture, and greatly promote the induction and growth of clustered shoots. It is a highly efficient and stable culture medium specifically for *Scutellaria barbata* tissue culture. Comparative experiments have verified that both the functional additives and the novel nano-silica are indispensable key innovations for achieving the above-mentioned excellent effects.

[0071] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0072] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A culture medium for Scutellaria barbata, characterized in that: The culture medium for Scutellaria barbata is prepared by MS medium and basic raw materials at a mass ratio of 6-7:

1. The basic raw materials are made from the following ingredients in parts by weight: 2-4 parts of novel nano-silica, 0.05-0.08 parts of cerium nitrate, 1-2 parts of organic bacterial agent, 0.7-0.8 parts of 6-benzyladenine, 0.2-0.4 parts of α-naphthaleneacetic acid, 0.1-0.3 parts of methyl jasmonate, and 2-4 parts of functional additives.

2. The culture medium for Scutellaria barbata according to claim 1, characterized in that, The preparation method of the functional additive includes the following steps: S1: Wash Scutellaria barbata with deionized water, drain and dry in a hot air drying oven at 35-40℃ until the moisture content is ≤5%, then pulverize and pass through a 50-60 mesh sieve to obtain Scutellaria barbata powder. S2: Prepare the extraction solvent. Mix the Scutellaria barbata powder with the extraction solvent at a mass ratio of 1:18-22. Place the mixture in an ultrasonic extractor and set the temperature to 45-50℃ for 30-35 minutes to obtain a crude extract. Centrifuge the crude extract, collect the supernatant, and filter it to obtain the extract. S3: Mix the extract, cysteine, and disodium EDTA at a mass ratio of 1:0.0008-0.012:0.0002-0.0004 and stir at 23-27℃ for 25-35 minutes to form an inhibitory extract. Then mix the inhibitory extract, ascorbic acid, glutathione, and citric acid at a mass ratio of 1:0.0002-0.0004:0.0008-0.0012:0.0004-0.0006 and add sodium hydroxide solution to adjust the pH to 5.0-5.2 to obtain the antioxidant treatment solution. S4: The antioxidant treatment solution is filtered and purified to obtain a clear solution, which is then treated to prepare the functional additive.

3. The culture medium for Scutellaria barbata according to claim 2, characterized in that: The method for preparing the extraction solvent in step S2 is as follows: Prepare citric acid solution and disodium hydrogen phosphate solution separately. Mix the citric acid solution and disodium hydrogen phosphate solution at a volume ratio of 1:1.2-1.4 to obtain a buffer solution. Dissolve ascorbic acid in the buffer solution at a solid-liquid ratio of 0.0015-0.002 g / mL and stir for 10-15 min. Nitrogen gas was introduced into the buffer solution containing ascorbic acid and stirred for 25–35 minutes to obtain the extraction solvent.

4. The culture medium for Scutellaria barbata according to claim 2, characterized in that: The antioxidant treatment solution is filtered and purified to obtain a clarified solution. The specific steps are as follows: The antioxidant treatment solution was filtered using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa. The ultrafiltrate was collected and adsorbed using a macroporous adsorption resin column. After adsorption saturation, it was eluted with 70% ethanol to form an eluent. The eluent was evaporated to one-fifth of its original volume to obtain a concentrated solution. Add activated carbon to the concentrate at a solid-liquid ratio of 0.0015–0.002 g / mL, stir in a water bath at 50–55°C for 40–50 min, and filter after stirring to obtain a clear liquid.

5. The culture medium for Scutellaria barbata according to claim 2, characterized in that: The specific steps for further processing the clarified liquid are as follows: The clarified liquid was mixed with β-cyclodextrin at a mass ratio of 4.5–5.5:1 and stirred in a water bath at 40–45°C for 1.5–2.5 h to form an inclusion complex solution. The inclusion complex solution was then transferred to a spray dryer to obtain microcapsule powder. Gelatin and gum arabic were mixed at a mass ratio of 1:1 and dissolved in deionized water to prepare an 8%–10% wall material solution. Microcapsule powder was mixed with the wall material solution at a mass ratio of 1:4.8–5.2 and stirred at 55–65°C to form an emulsion. The pH of the emulsion was adjusted to 4.4–4.

6. After adjustment, 0.4%–0.6% (by mass) of transglutaminase was added to the wall material solution, and the reaction was carried out at 35–45°C for 0.8–1 h. After the reaction was completed, a 5% (by mass) calcium chloride solution was added dropwise to prepare microcapsules. The microcapsules were washed 2–4 times with deionized water to obtain the functional additive.

6. The culture medium for Scutellaria barbata according to claim 3, characterized in that: The citric acid solution is prepared by mixing citric acid and deionized water at a mass ratio of 1:35-40; The disodium hydrogen phosphate solution is prepared by mixing disodium hydrogen phosphate and deionized water at a mass ratio of 1:20-24.

7. The culture medium for Scutellaria barbata according to claim 1, characterized in that, The preparation steps of the novel nano-silica are as follows: Nano-sized silica with a particle size of 20 nm, anhydrous ethanol, and 3-aminopropyltriethoxysilane were mixed at a mass ratio of 6–7:13–15:1 and stirred under reflux at 65–75 °C for 5.5–6.5 h to obtain a reaction solution. After the reaction solution was cooled to 25 °C, it was washed 2–4 times by centrifugation with ethanol and dried under vacuum at 55–65 °C for 10–12 h to obtain modified silica. Modified silica was grafted to obtain grafted silica. The grafted silica, phosphate buffer, and 6-benzyladenine were mixed at a mass ratio of 23-27:80-85:1 and stirred at 23-27°C for 20-25 hours to obtain a loading solution. The loading solution was dialyzed, the dialysate was collected, and then freeze-dried to obtain novel nano-silica.

8. The culture medium for Scutellaria barbata according to claim 7, characterized in that: The specific steps for grafting modified silica are as follows: Modified silica, deionized water, and polyethylene glycol are mixed in a mass ratio of 1:2.5-3.5:1.5-2.5 to obtain a polymer solution. Ammonium persulfate at a mass of 0.4%-0.6% of polyethylene glycol is added to the polymer solution, and the mixture is stirred at 55-65°C for 7.5-8.5 hours under nitrogen protection to form a grafting solution. The grafting solution was dialyzed with deionized water, and after dialyzing, it was freeze-dried to obtain grafted silica.

9. The culture medium for Scutellaria barbata according to claim 1, characterized in that: The organic microbial agent is selected from any one of Bacillus subtilis, Bacillus belye, and Bacillus amyloliquefaciens.

10. A method for preparing a culture medium for Scutellaria barbata according to any one of claims 1 to 9, characterized in that, Includes the following steps: Step 1: Weigh each salt according to the MS medium standard formula, dissolve in deionized water, heat to dissolve, add sucrose and agar, adjust the pH to 5.8-6, and sterilize at 121-125℃ for 15-20 minutes. Step 2: Activate the organic bacteria to obtain organic bacterial agent, and prepare functional adjuvants and novel nano silica according to the steps. After the MS medium is cooled to 45°C, add novel silica, cerium nitrate, organic bacterial agent, 6-benzyladenine, α-naphthaleneacetic acid, methyl jasmonate and functional adjuvant respectively. After adding, stir evenly and let it cool and solidify to obtain a culture medium for Scutellaria barbata.

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