Nervilia fordii tissue culture rhizome proliferation culture medium and preparation method thereof

Abstract

The invention relates to the technical field of culture medium manufacturing, in particular to a ford nervilia leaf tissue culture rhizome proliferation culture medium and a preparation method of the ford nervilia leaf tissue culture rhizome proliferation culture medium. The proliferation material is prepared from the following components in parts by weight: 2 to 4 parts of 6-benayl aminopurine, 0.3 to 0.7 part of naphthylacetic acid, 0.3 to 0.6 part of indolebutyric acid, 4 to 6 parts of gibberellin, 95 to 105 parts of novel coconut juice extract, 18 to 22 parts of novel pomegranate extract, 250 to 350 parts of cane sugar, 60 to 70 parts of agar and 3 to 5 parts of activated carbon. The improved pomegranate rind extract is subjected to ultrasonic and purification composite treatment, more antioxidant components such as ellagic acid and gallic acid are released, the antioxidant components, fermentation-enhanced amino acid and a nano silicon / selenium compound in the improved coconut juice extract form a dual antioxidant barrier, free radicals in plant cells are actively removed, oxidative damage is reduced, and the anti-aging effect is achieved. The hormone combination is precisely proportioned, so that lateral bud germination and stem elongation can be promoted.

Description

Technical Field

[0001] This invention relates to the field of culture medium manufacturing technology, specifically to a tissue culture rhizome proliferation culture medium for *Gynostemma pentaphyllum* and its preparation method. Background Technology

[0002] *Cymbidium goeringii*, scientifically known as *Cymbidium goeringii*, is a perennial herbaceous plant belonging to the genus *Cymbidium* in the family Orchidaceae. Other names include *Cymbidium goeringii* var. *goeringii*, *Cymbidium pulcherrimum*, etc. It is mainly produced in Guangdong, Guangxi, Hainan, and Yunnan provinces, growing in sparse forests and shady, damp places along streams on mountain slopes at altitudes of 220 to 1000 meters. The tuber is spherical, and the leaves are basal, ovate-cordate. It flowers from April to May, producing pale green flowers with a white and purple lip. The whole plant or tuber is used medicinally. It is cool in nature and has a sweet and slightly bitter taste. It excels at clearing heat and moistening the lungs, dispersing blood stasis and detoxifying. It is mainly used to treat coughs due to lung heat, hemoptysis due to phlegm, scrofula, and traumatic injuries. It also has anti-inflammatory, antibacterial, antioxidant, and immune-enhancing effects. It is harvested in summer and autumn, dried, or granulated for later use. Those with weak spleen and stomach should use with caution.

[0003] While the tissue culture rhizome proliferation medium for *Gnaphalium affine* possesses some advantages among existing technologies, it still faces numerous challenges. Insufficient antioxidant capacity is one of the main problems leading to oxidative damage. Oxidative damage destroys cellular proteins, lipids, and DNA, severely affecting cell proliferation efficiency and growth capacity. The lack of effective antioxidant protection makes cells susceptible to free radical damage, leading to cell aging and death, thereby reducing proliferation efficiency. In addition, poor cell stress resistance during culture is also a common problem, especially when facing environmental stresses such as temperature changes and pH fluctuations, cell growth is significantly inhibited. At the same time, poor stability of the culture medium is also a key factor restricting cell culture results. Components in the culture medium are prone to oxidation, volatilization, or microbial contamination, resulting in nutrient loss or accumulation of harmful substances, ultimately affecting healthy cell growth.

[0004] Therefore, the present invention provides a tissue culture rhizome proliferation medium for *Gynostemma pentaphyllum* and its preparation method, to solve the aforementioned related technical problems. Summary of the Invention

[0005] The purpose of this invention is to provide a culture medium for the proliferation of *Gnaphalium affine* tissue culture rhizomes and its preparation method. By improving the treatment of pomegranate peel extract through ultrasound and purification, more antioxidant components such as ellagic acid and gallic acid are released. These, along with fermentation-enhanced amino acids and nano-silicon / selenium complexes in improved coconut juice extract, form a dual antioxidant barrier, actively scavenging free radicals within plant cells and reducing oxidative damage. The precise ratio of 6-BA, NAA, and IBA in the hormone combination can activate the signaling pathways for cell division and differentiation in rhizome meristems, promoting lateral bud germination and stem elongation, while avoiding abnormal growth caused by high concentrations of hormones. Vitamins C and E and tea polyphenols in the stress-resistance enhance cell membrane stability through a synergistic antioxidant network, while proline and betaine maintain cell turgor pressure through osmotic regulation. ABA and SA, as stress signaling molecules, can induce the expression of drought- and salt-resistant genes in plants, forming a stress-resistance mechanism that allows rhizomes to maintain stable proliferation capacity under adverse conditions.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A culture medium for the proliferation of tissue culture rhizomes of *Gynostemma pentaphyllum*, wherein the culture medium is composed of MS medium and proliferation material at a mass ratio of 1:26-28, and the proliferation material comprises the following components: 2-4 parts 6-benzylaminopurine, 0.3-0.7 parts naphthaleneacetic acid, 0.3-0.6 parts indolebutyric acid, 4-6 parts gibberellin, 95-105 parts novel coconut juice extract, 18-22 parts novel pomegranate extract, 250-350 parts sucrose, 60-70 parts agar, and 3-5 parts activated carbon; The novel pomegranate extract is prepared by mixing chitosan solution, gum arabic solution and pomegranate extract, and then by ultrasound and chelation. The novel coconut juice extract is obtained by preparing fermented coconut juice, nano-coconut juice containing nano-silicon / selenium complex, and fortified coconut juice containing fortifiers, and then mixing the three types of coconut juice with seaweed extract and humic acid.

[0007] MS culture medium was purchased from OMAS Biotechnology Co., Ltd., sucrose from Langfang Qianyao Technology Co., Ltd., agar was selected from Sigma-Aldrich (Shanghai) Trading Co., Ltd., and activated carbon was purchased from Zhengzhou Kelin Water Purification Materials Co., Ltd.

[0008] Furthermore, the preparation method of the novel pomegranate extract includes the following steps: S1: Mix chitosan with a 1% acetic acid solution at a solid-liquid ratio of 0.01-0.03 g / mL and stir for 25-35 min to form a chitosan solution; S2: Mix gum arabic with deionized water at a mass ratio of 1:99-101, and treat with ultrasound for 8-12 minutes to obtain a gum arabic solution. The gum arabic was purchased from Shanghai Kaisheng Chemical Co., Ltd. S3: Mix pomegranate peel extract with anhydrous ethanol at a mass ratio of 1:99-101, and treat with ultrasound for 4-6 minutes to obtain pomegranate extract; S4: Chitosan solution, gum arabic solution and pomegranate extract are mixed at a volume ratio of 1.8-2.2:1:1 and ultrasonically emulsified for 4-6 minutes to obtain a mixed emulsion. Copper sulfate solution with a mass ratio of 120-125:1 and a concentration of 0.08-0.12 mol / L is added dropwise to the mixed emulsion and stirred for 25-35 minutes to obtain a chelated emulsion. The chelated emulsion is then processed to obtain a novel pomegranate extract.

[0009] The chelated emulsion is processed by a spray dryer with an inlet air temperature of 180-185℃ and an outlet air temperature of 85-95℃, and then passed through a 150-200 mesh sieve to obtain a novel pomegranate extract.

[0010] Furthermore, the preparation process of the pomegranate peel extract in step S3 is as follows: Peel the pomegranate peel, rinse it with clean water 2-4 times, air dry it for 23-25 ​​hours, and then crush it through a pulverizer and pass it through a 35-45 mesh sieve to obtain pomegranate peel powder. The pomegranates are produced from seedlings sold by Taian Hengyue Agricultural Technology Co., Ltd. Pomegranate peel powder was mixed with 35% edible ethanol at a solid-liquid ratio of 0.08–0.12 g / mL and extracted by reflux in a water bath at 75–85°C for 2–3 times, with an interval of 1.5–2.5 h each time, to obtain a crude extract. The crude extract was then concentrated and purified to obtain pomegranate peel extract.

[0011] It should be noted that after obtaining the pomegranate peel extract, HPLC and infrared spectroscopy can be used for verification to ensure the quality of the pomegranate peel extract.

[0012] Furthermore, the step of concentrating and purifying the crude extract is as follows: The water bath temperature was set to 45–55℃ and the vacuum degree to 0.08–0.09 MPa. The crude extract was concentrated under reduced pressure to obtain pomegranate extract. The pomegranate extract was mixed with deionized water at a mass ratio of 1:3.5–4.5 and stirred until completely dissolved to form an extract solution. The macroporous resin was soaked in ethanol and loaded into a chromatography column to form a resin column. The resin column was rinsed with deionized water until no ethanol residue was left. The extract solution was then passed into the resin column to obtain a resin column that was saturated with adsorption. Among them, macroporous resins are a class of synthetic resin materials with large pore structures. Their pore sizes are usually between tens and hundreds of nanometers. Due to their large pore space, they can accommodate larger molecules and have high adsorption capacity and selectivity.

[0013] The saturated resin column was washed with a 35% (v / v) ethanol solution, and the eluent was collected. The eluent was concentrated at 45–55°C and 0.08–0.09 MPa to obtain a concentrate. The concentrate was then dried at 55–65°C and 0.08–0.09 MPa for 11–13 hours to obtain pomegranate peel extract.

[0014] Furthermore, the macroporous resin can be selected from any one of the following types: D101, D101-I, HPD-100, HPD300, and SIP-1100. All of the above macroporous resin types are purchased from Tianjin Yunkai Resin Technology Co., Ltd.

[0015] Furthermore, the preparation method of the novel coconut milk extract is as follows: Fresh coconuts were selected for pretreatment to obtain raw coconut juice. The raw coconut juice was ultrasonically treated at 35-45℃ for 20-30 minutes to form optimized coconut juice. Lactic acid bacteria accounting for 4%-6% of the volume of optimized coconut juice were inoculated and fermented at 35-39℃ for 35-37 hours to obtain fermented coconut juice, which was then divided into two equal portions. The fresh coconuts were purchased from Wangfa Fruit Seedling Plantation in Huicheng District, Huizhou City. It should be added that the pretreatment of fresh coconuts includes: placing the raw coconut juice in an ultrasonic processor and ultrasonically treating it at 40-45°C for 25-30 minutes (power 200W, frequency 40kHz) to break down macromolecular polysaccharides into soluble sugars and release bound amino acids and minerals.

[0016] Add 30% to 35% of the weight of the enhanced coconut juice to one portion of the fermented coconut juice, stir until completely dissolved, and then divide the enhanced coconut juice into two equal portions. Nano-silicon and nano-selenium were added to one portion of the fortified coconut juice at solid-liquid ratios of 75–85 mg / L and 0.04–0.06 mg / L, respectively, and then ultrasonically dispersed to obtain nano-coconut juice; Fermented coconut juice, fortified coconut juice, seaweed extract, humic acid, and nano coconut juice were mixed in a mass ratio of 9–11:5–7:2–4:1:1 and stirred for 10–15 minutes. The pH was adjusted to 6.0–6.5 and refrigerated at 3–5°C for 20–30 days to obtain a novel coconut juice extract.

[0017] Seaweed extract can be purchased from Shanxi Yirun Biotechnology Co., Ltd., while humic acid can be purchased from Shandong Binhai Biotechnology Co., Ltd.

[0018] Furthermore, the lactic acid bacteria can be any one of Lactobacillus plantarum, Lactobacillus acidophilus, Bifidobacterium lactis, and Lactococcus lactis, all of which were purchased from the Henan Provincial Engineering Technology Research Center for Industrial Microbial Strains.

[0019] Furthermore, the fortifying agent is prepared by mixing antioxidants, osmotic regulators and hormone regulators in a mass ratio of 140-160:1-1.5:0.7-0.8.

[0020] Furthermore, the antioxidant is prepared by mixing vitamin C, vitamin E, and tea polyphenols in a mass ratio of 4.5–5.5:1.5–2.5:7–9. The osmotic regulator is prepared by mixing proline, betaine, and trehalose in a mass ratio of 0.7–0.9: 0.25–0.35: 0.15–0.25. The hormone regulator is prepared by mixing abscisic acid, salicylic acid and indolebutyric acid in a mass ratio of 0.25-0.35:0.07-0.09:0.35-0.45.

[0021] A method for preparing a culture medium for the proliferation of tissue culture rhizomes of *Gynostemma pentaphyllum* includes the following steps: Step 1: Prepare MS base solution, novel pomegranate extract and coconut extract separately. Add 6-benzylaminopurine, naphthaleneacetic acid, indolebutyric acid and gibberellin to MS base solution in proportion and stir until completely dissolved. Then add novel pomegranate extract and coconut extract and continue stirring until homogeneous to obtain premixed culture medium solution. Step 2: Add agar and sucrose to the premixed culture medium solution, heat to boiling and stir continuously until completely dissolved; when the solution cools to 45-55℃, add activated carbon and stir for 10-15 minutes to obtain a solidified culture medium solution. Step 3: Pour the solidified culture medium solution into the tissue culture bottle, seal it, and place it in an autoclave. Sterilize it at 120-125℃ for 20-25 minutes to obtain the tissue culture rhizome proliferation medium for *Gynostemma pentaphyllum*.

[0022] Compared with the prior art, the beneficial effects of the present invention are: This invention achieves efficient rhizome proliferation and enhanced stress resistance through multi-dimensional component synergy. An improved pomegranate peel extract, after ultrasonic and purification treatment, releases more antioxidants such as ellagic acid and gallic acid. These, along with fermentation-enhanced amino acids and nano-silicon / selenium complexes in the improved coconut juice extract, form a dual antioxidant barrier, actively scavenging free radicals within plant cells and reducing oxidative damage. The precise ratio of 6-BA, NAA, and IBA in the hormone combination activates the cell division and differentiation signaling pathways in rhizome meristems, promoting lateral bud germination and stem elongation while avoiding abnormal growth caused by high hormone concentrations. Vitamins C and E, along with tea polyphenols in the stress-resistance enhancer, strengthen cell membrane stability through a synergistic antioxidant network. Proline and betaine maintain cell turgor pressure through osmotic regulation. ABA and SA, as stress signaling molecules, induce the expression of drought- and salt-tolerant genes, forming a stress-resistance mechanism that allows rhizomes to maintain stable proliferation capacity under adverse conditions. The basic MS medium provides comprehensive nutritional support, while the improved extract and stress-resistance enhancer achieve stress resistance enhancement through complementary components, avoiding the limitations of single components. The three-dimensional network structure formed by the agar solidifier not only provides physical support for the rhizomes but also allows for the slow release of water, nutrients, and gases through its porous structure, simulating the natural soil microenvironment. The adsorption effect of activated carbon reduces the accumulation of metabolic waste, avoiding the risk of browning and contamination. The sterilization step in the preparation process ensures the sterility of the medium, enabling it to have both high-efficiency proliferation and long-term stability, significantly improving the tissue culture efficiency and stress resistance of *Gynostemma pentaphyllum* rhizomes. 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 process of a novel pomegranate extract in the tissue culture rhizome proliferation medium of *Gnaphalium affine* according to the present invention. Figure 2 This is a flowchart illustrating the preparation process of pomegranate peel extract in the tissue culture rhizome proliferation medium of *Gnaphalium affine* according to the present invention. Figure 3 This is a flowchart of the process for concentrating and purifying the crude extract according to the present invention. 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 the proliferation of tissue culture rhizomes of *Gynostemma pentaphyllum*, wherein the culture medium is composed of MS medium and proliferation material at a mass ratio of 1:26, and the proliferation material comprises the following components: 2 parts 6-benzylaminopurine, 0.3 parts naphthaleneacetic acid, 0.3 parts indolebutyric acid, 4 parts gibberellin, 95 parts novel coconut juice extract, 18 parts novel pomegranate extract, 250 parts sucrose, 60 parts agar, and 3 parts activated carbon; The novel pomegranate extract is prepared by mixing chitosan solution, gum arabic solution and pomegranate extract, and then by ultrasound and chelation. The novel coconut juice extract is obtained by preparing fermented coconut juice, nano-coconut juice containing nano-silicon / selenium complex, and fortified coconut juice containing fortifiers, and then mixing the three types of coconut juice with seaweed extract and humic acid.

[0027] MS culture medium was purchased from OMAS Biotechnology Co., Ltd., sucrose from Langfang Qianyao Technology Co., Ltd., agar was selected from Sigma-Aldrich (Shanghai) Trading Co., Ltd., and activated carbon was purchased from Zhengzhou Kelin Water Purification Materials Co., Ltd.

[0028] Furthermore, such as Figure 1 As shown, the preparation method of the novel pomegranate extract includes the following steps: S1: Mix chitosan with a 1% acetic acid solution at a solid-liquid ratio of 0.01 g / mL and stir for 25 min to form a chitosan solution; S2: Gum arabic and deionized water were mixed at a mass ratio of 1:99 and ultrasonically treated for 8 minutes to obtain a gum arabic solution. The gum arabic was purchased from Shanghai Kaisheng Chemical Co., Ltd. S3: Mix pomegranate peel extract with anhydrous ethanol at a mass ratio of 1:99, and sonicate for 4 minutes to obtain pomegranate extract; S4: Chitosan solution, gum arabic solution and pomegranate extract were mixed at a volume ratio of 1.8:1:1 and ultrasonically emulsified for 4 min to obtain a mixed emulsion. Copper sulfate solution with a mass ratio of 120:1 and a concentration of 0.08 mol / L was added dropwise to the mixed emulsion and stirred for 25 min to obtain a chelated emulsion. The chelated emulsion was then processed to obtain a novel pomegranate extract.

[0029] The chelated emulsion is processed by a spray dryer with an inlet air temperature of 180°C and an outlet air temperature of 85°C, and then passed through a 150-mesh sieve to obtain a novel pomegranate extract.

[0030] Furthermore, such as Figure 2 As shown, the preparation process of the pomegranate peel extract in step S3 is as follows: The pomegranate peel was removed, rinsed twice with clean water, air-dried for 23 hours, and then crushed in a pulverizer and passed through a 35-mesh sieve to obtain pomegranate peel powder. The pomegranates were produced from seedlings sold by Taian Hengyue Agricultural Technology Co., Ltd. Pomegranate peel powder was mixed with 35% edible ethanol at a solid-liquid ratio of 0.08 g / mL and extracted twice by reflux in a water bath at 75°C, with an interval of 1.5 h between each extraction to obtain a crude extract. The crude extract was then concentrated and purified to obtain pomegranate peel extract.

[0031] It should be noted that after obtaining the pomegranate peel extract, HPLC and infrared spectroscopy can be used for verification to ensure the quality of the pomegranate peel extract.

[0032] Furthermore, please refer to Figure 3 The steps for concentrating and purifying the crude extract are as follows: The water bath temperature was set at 45℃ and the vacuum degree was 0.08MPa. The crude extract was concentrated under reduced pressure to obtain pomegranate extract. The pomegranate extract was mixed with deionized water at a mass ratio of 1:3.5 and stirred until completely dissolved to form an extract solution. The macroporous resin was soaked in ethanol and loaded into a chromatography column to form a resin column. The resin column was rinsed with deionized water until no ethanol residue was left. The extract solution was then passed into the resin column to obtain a resin column that was saturated with adsorption. Among them, macroporous resins are a class of synthetic resin materials with large pore structures. Their pore sizes are usually between tens and hundreds of nanometers. Due to their large pore space, they can accommodate larger molecules and have high adsorption capacity and selectivity.

[0033] The saturated resin column was washed with a 35% (v / v) ethanol solution, and the eluent was collected. The eluent was concentrated at 45°C and 0.08 MPa to obtain a concentrate, which was then dried at 55°C and 0.08 MPa for 11 h to obtain pomegranate peel extract.

[0034] Furthermore, the macroporous resin can be of type D101.

[0035] Furthermore, the preparation method of the novel coconut milk extract is as follows: Fresh coconuts were selected for pretreatment to obtain raw coconut juice. The raw coconut juice was ultrasonically treated at 35℃ for 20 minutes to form optimized coconut juice. Lactic acid bacteria accounting for 4% of the volume of the optimized coconut juice were inoculated and fermented at 35℃ for 35 hours to obtain fermented coconut juice, which was then divided into two equal portions. The fresh coconuts were purchased from Wangfa Fruit Seedling Plantation in Huicheng District, Huizhou City. It should be added that the pretreatment of fresh coconuts includes: placing the raw coconut juice in an ultrasonic processor and ultrasonically treating it at 40°C for 25 minutes (power 200W, frequency 40kHz) to break down macromolecular polysaccharides into soluble sugars and release bound amino acids and minerals.

[0036] Add 30% to 35% of the weight of the enhanced coconut juice to one portion of the fermented coconut juice, stir until completely dissolved, and then divide the enhanced coconut juice into two equal portions. Nano-silicon and nano-selenium were added to one portion of the fortified coconut juice at solid-liquid ratios of 75 mg / L and 0.04 mg / L, respectively, and then ultrasonically dispersed to obtain nano-coconut juice; Fermented coconut juice, fortified coconut juice, seaweed extract, humic acid, and nano coconut juice were mixed in a mass ratio of 9:5:2:1:1 and stirred for 10 minutes. The pH was adjusted to 6.0 and the mixture was refrigerated at 3°C ​​for 20 days to obtain a novel coconut juice extract.

[0037] Furthermore, the lactic acid bacteria may be selected from Lactobacillus plantarum.

[0038] Furthermore, the fortifying agent is prepared by mixing antioxidants, osmotic regulators and hormone regulators in a mass ratio of 140:1:0.7.

[0039] Furthermore, the antioxidant is prepared by mixing vitamin C, vitamin E, and tea polyphenols in a mass ratio of 4.5:1.5:7; The osmotic regulator is prepared by mixing proline, betaine, and trehalose in a mass ratio of 0.7:0.25:0.15. The hormone regulator is prepared by mixing abscisic acid, salicylic acid and indolebutyric acid in a mass ratio of 0.25:0.07:0.35.

[0040] A method for preparing a culture medium for the proliferation of tissue culture rhizomes of *Gynostemma pentaphyllum* includes the following steps: Step 1: Prepare MS base solution, novel pomegranate extract and coconut extract separately. Add 6-benzylaminopurine, naphthaleneacetic acid, indolebutyric acid and gibberellin to MS base solution in proportion and stir until completely dissolved. Then add novel pomegranate extract and coconut extract and continue stirring until homogeneous to obtain premixed culture medium solution. Step 2: Add agar and sucrose to the premixed culture medium solution, heat to boiling and stir continuously until completely dissolved; when the solution cools to 45°C, add activated carbon and stir for 10 minutes to obtain a solidified culture medium solution. Step 3: Pour the solidified culture medium solution into the tissue culture bottle, seal it, and place it in an autoclave. Sterilize it at 120℃ for 20 minutes to obtain the tissue culture rhizome proliferation medium for *Gynostemma pentaphyllum*.

[0041] Example 2 The preparation method of the tissue culture rhizome proliferation medium 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 proliferation medium is composed of MS medium and proliferation material at a mass ratio of 1:27. The proliferation material includes the following components: 3 parts 6-benzylaminopurine, 0.5 parts naphthaleneacetic acid, 0.5 parts indolebutyric acid, 5 parts gibberellin, 100 parts novel coconut juice extract, 20 parts novel pomegranate extract, 300 parts sucrose, 65 parts agar, and 4 parts activated carbon.

[0042] MS culture medium was purchased from OMAS Biotechnology Co., Ltd., sucrose from Langfang Qianyao Technology Co., Ltd., agar was selected from Sigma-Aldrich (Shanghai) Trading Co., Ltd., and activated carbon was purchased from Zhengzhou Kelin Water Purification Materials Co., Ltd.

[0043] Furthermore, the preparation method of the novel pomegranate extract includes the following steps: S1: Mix chitosan with a 1% acetic acid solution at a solid-liquid ratio of 0.02 g / mL and stir for 30 min to form a chitosan solution; S2: Gum arabic and deionized water were mixed at a mass ratio of 1:100 and ultrasonically treated for 10 minutes to obtain a gum arabic solution. The gum arabic was purchased from Shanghai Kaisheng Chemical Co., Ltd. S3: Mix pomegranate peel extract with anhydrous ethanol at a mass ratio of 1:100, and sonicate for 5 minutes to obtain pomegranate extract. S4: Chitosan solution, gum arabic solution and pomegranate extract were mixed at a volume ratio of 2:1:1 and ultrasonically emulsified for 5 minutes to obtain a mixed emulsion. Copper sulfate solution with a mass ratio of 123:1 and a concentration of 0.1 mol / L was added dropwise to the mixed emulsion and stirred for 30 minutes to obtain a chelated emulsion. The chelated emulsion was then processed to obtain a novel pomegranate extract.

[0044] The chelated emulsion is processed by a spray dryer with an inlet air temperature of 183°C and an outlet air temperature of 90°C, and then passed through a 180-mesh sieve to obtain a novel pomegranate extract.

[0045] Furthermore, the preparation process of the pomegranate peel extract in step S3 is as follows: The pomegranate peel was removed, rinsed three times with clean water, air-dried for 24 hours, and then crushed in a pulverizer and passed through a 40-mesh sieve to obtain pomegranate peel powder. The pomegranates were produced from seedlings sold by Taian Hengyue Agricultural Technology Co., Ltd. Pomegranate peel powder was mixed with 35% edible ethanol at a solid-liquid ratio of 0.1 g / mL and extracted twice by reflux in a water bath at 80°C, with an interval of 2 h between each extraction, to obtain a crude extract. The crude extract was then concentrated and purified to obtain pomegranate peel extract.

[0046] It should be noted that after obtaining the pomegranate peel extract, HPLC and infrared spectroscopy can be used for verification to ensure the quality of the pomegranate peel extract.

[0047] Furthermore, the step of concentrating and purifying the crude extract is as follows: The water bath temperature was set to 50℃ and the vacuum degree to 0.09MPa. The crude extract was concentrated under reduced pressure to obtain pomegranate extract. The pomegranate extract was mixed with deionized water at a mass ratio of 1:4 and stirred until completely dissolved to form an extract solution. The macroporous resin was soaked in ethanol and loaded into a chromatography column to form a resin column. The resin column was rinsed with deionized water until no ethanol residue was left. The extract solution was then passed into the resin column to obtain a resin column that was saturated with adsorption. Among them, macroporous resins are a class of synthetic resin materials with large pore structures. Their pore sizes are usually between tens and hundreds of nanometers. Due to their large pore space, they can accommodate larger molecules and have high adsorption capacity and selectivity.

[0048] The saturated resin column was washed with a 35% (v / v) ethanol solution, and the eluent was collected. The eluent was concentrated at 50°C and 0.09 MPa to obtain a concentrate, which was then dried at 60°C and 0.09 MPa for 12 hours to obtain pomegranate peel extract.

[0049] Furthermore, the macroporous resin can be of type D101-I.

[0050] Furthermore, the preparation method of the novel coconut milk extract is as follows: Fresh coconuts were selected for pretreatment to obtain raw coconut juice. The raw coconut juice was ultrasonically treated at 40℃ for 25 minutes to form optimized coconut juice. Lactic acid bacteria accounting for 5% of the volume of optimized coconut juice were inoculated and fermented at 37℃ for 36 hours to obtain fermented coconut juice, which was then divided into two equal portions. The fresh coconuts were purchased from Wangfa Fruit Seedling Plantation in Huicheng District, Huizhou City. It should be added that the pretreatment of fresh coconuts includes: placing the raw coconut juice in an ultrasonic processor and ultrasonically treating it at 43°C for 28 minutes (power 200W, frequency 40kHz) to break down macromolecular polysaccharides into soluble sugars and release bound amino acids and minerals.

[0051] Add a fortifier at a mass of 32% of the optimized coconut juice to one portion of the fermented coconut juice, stir until completely dissolved, and obtain fortified coconut juice, which is then divided into two equal portions. Nano-silicon and nano-selenium were added to one portion of the fortified coconut juice at solid-liquid ratios of 80 mg / L and 0.05 mg / L, respectively, and then ultrasonically dispersed to obtain nano-coconut juice; Fermented coconut juice, fortified coconut juice, seaweed extract, humic acid, and nano coconut juice were mixed in a mass ratio of 10:6:3:1:1 and stirred for 12 minutes. The pH was adjusted to 6.3 and the mixture was refrigerated at 4°C for 25 days to obtain a novel coconut juice extract.

[0052] Furthermore, the lactic acid bacteria may be selected from Lactobacillus acidophilus.

[0053] Furthermore, the fortifier is prepared by mixing antioxidants, osmotic regulators and hormone regulators in a mass ratio of 150:1.2:0.75.

[0054] Furthermore, the antioxidant is prepared by mixing vitamin C, vitamin E, and tea polyphenols in a mass ratio of 5:2:8; The osmotic regulator is prepared by mixing proline, betaine, and trehalose in a mass ratio of 0.8:0.3:0.2. The hormone regulator is prepared by mixing abscisic acid, salicylic acid and indolebutyric acid in a mass ratio of 0.3:0.08:0.4.

[0055] Example 3 The preparation method of the tissue culture rhizome proliferation medium 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 proliferation medium is composed of MS medium and proliferation material at a mass ratio of 1:28. The proliferation material includes the following components: 4 parts 6-benzylaminopurine, 0.7 parts naphthaleneacetic acid, 0.6 parts indolebutyric acid, 6 parts gibberellin, 105 parts novel coconut juice extract, 22 parts novel pomegranate extract, 350 parts sucrose, 70 parts agar, and 5 parts activated carbon.

[0056] MS culture medium was purchased from OMAS Biotechnology Co., Ltd., sucrose from Langfang Qianyao Technology Co., Ltd., agar was selected from Sigma-Aldrich (Shanghai) Trading Co., Ltd., and activated carbon was purchased from Zhengzhou Kelin Water Purification Materials Co., Ltd.

[0057] Furthermore, the preparation method of the novel pomegranate extract includes the following steps: S1: Mix chitosan with a 1% acetic acid solution at a solid-liquid ratio of 0.03 g / mL and stir for 35 min to form a chitosan solution; S2: Gum arabic and deionized water were mixed at a mass ratio of 1:101 and ultrasonically treated for 12 minutes to obtain a gum arabic solution. The gum arabic was purchased from Shanghai Kaisheng Chemical Co., Ltd. S3: Mix pomegranate peel extract with anhydrous ethanol at a mass ratio of 1:101, and sonicate for 6 minutes to obtain pomegranate extract; S4: Chitosan solution, gum arabic solution and pomegranate extract were mixed at a volume ratio of 2.2:1:1 and ultrasonically emulsified for 6 minutes to obtain a mixed emulsion. Copper sulfate solution with a mass ratio of 125:1 and a concentration of 0.12 mol / L was added dropwise to the mixed emulsion and stirred for 35 minutes to obtain a chelated emulsion. The chelated emulsion was then processed to obtain a novel pomegranate extract.

[0058] The chelated emulsion is processed by a spray dryer with an inlet air temperature of 185°C and an outlet air temperature of 95°C, and then passed through a 200-mesh sieve to obtain a novel pomegranate extract.

[0059] Furthermore, the preparation process of the pomegranate peel extract in step S3 is as follows: The pomegranate peel was removed, rinsed four times with clean water, air-dried for 25 hours, and then crushed in a pulverizer and passed through a 45-mesh sieve to obtain pomegranate peel powder. The pomegranates were produced from seedlings sold by Taian Hengyue Agricultural Technology Co., Ltd. Pomegranate peel powder was mixed with 35% edible ethanol at a solid-liquid ratio of 0.12 g / mL and extracted by reflux in a water bath at 85°C three times, with an interval of 2.5 h each time, to obtain a crude extract. The crude extract was then concentrated and purified to obtain pomegranate peel extract.

[0060] It should be noted that after obtaining the pomegranate peel extract, HPLC and infrared spectroscopy can be used for verification to ensure the quality of the pomegranate peel extract.

[0061] Furthermore, the step of concentrating and purifying the crude extract is as follows: The water bath temperature was set to 55℃ and the vacuum degree to 0.09MPa. The crude extract was concentrated under reduced pressure to obtain pomegranate extract. The pomegranate extract was mixed with deionized water at a mass ratio of 1:4.5 and stirred until completely dissolved to form an extract solution. The macroporous resin was soaked in ethanol and loaded into a chromatography column to form a resin column. The resin column was rinsed with deionized water until no ethanol residue was left. The extract solution was then passed into the resin column to obtain a resin column that was saturated with adsorption. Among them, macroporous resins are a class of synthetic resin materials with large pore structures. Their pore sizes are usually between tens and hundreds of nanometers. Due to their large pore space, they can accommodate larger molecules and have high adsorption capacity and selectivity.

[0062] The saturated resin column was washed with a 35% (v / v) ethanol solution, and the eluent was collected. The eluent was concentrated at 55°C and 0.09 MPa to obtain a concentrate, which was then dried at 65°C and 0.09 MPa for 13 hours to obtain pomegranate peel extract.

[0063] Furthermore, the macroporous resin can be of type HPD-100.

[0064] Furthermore, the preparation method of the novel coconut milk extract is as follows: Fresh coconuts were selected for pretreatment to obtain raw coconut juice. The raw coconut juice was ultrasonically treated at 45℃ for 30 minutes to form optimized coconut juice. Lactic acid bacteria accounting for 6% of the volume of optimized coconut juice were inoculated and fermented at 39℃ for 37 hours to obtain fermented coconut juice, which was then divided into two equal portions. The fresh coconuts were purchased from Wangfa Fruit Seedling Plantation in Huicheng District, Huizhou City. It should be added that the pretreatment of fresh coconuts includes: placing the raw coconut juice in an ultrasonic processor and ultrasonically treating it at 45°C for 30 minutes (power 200W, frequency 40kHz) to break down macromolecular polysaccharides into soluble sugars and release bound amino acids and minerals.

[0065] Add a fortifier of 35% of the weight of the optimized coconut juice to one portion of the fermented coconut juice, stir until completely dissolved, and obtain fortified coconut juice and divide it into two equal portions; Nano-silicon and nano-selenium were added to one portion of the fortified coconut juice at solid-liquid ratios of 85 mg / L and 0.06 mg / L, respectively, and then ultrasonically dispersed to obtain nano-coconut juice; Fermented coconut juice, fortified coconut juice, seaweed extract, humic acid, and nano coconut juice were mixed in a mass ratio of 11:7:4:1:1 and stirred for 15 minutes. The pH was adjusted to 6.5 and the mixture was refrigerated at 5°C for 30 days to obtain a novel coconut juice extract.

[0066] Furthermore, the lactic acid bacteria may be Bifidobacterium.

[0067] Furthermore, the fortifying agent is prepared by mixing antioxidants, osmotic regulators and hormone regulators in a mass ratio of 160:1.5:0.8.

[0068] Furthermore, the antioxidant is prepared by mixing vitamin C, vitamin E, and tea polyphenols in a mass ratio of 5.5:2.5:9; The osmotic regulator is prepared by mixing proline, betaine, and trehalose in a mass ratio of 0.9:0.35:0.25. The hormone regulator is prepared by mixing abscisic acid, salicylic acid and indolebutyric acid in a mass ratio of 0.35:0.09:0.45.

[0069] Comparative Example 1: The preparation method and specific ratio of raw materials of the tissue culture rhizome proliferation medium provided in this example are roughly the same as those in Example 1. The main difference is that this example does not contain the novel pomegranate extract.

[0070] Comparative Example 2: The preparation method and specific ratio of raw materials of the tissue culture rhizome proliferation medium provided in this example are roughly the same as those in Example 1. The main difference is that ordinary pomegranate extract is used instead of novel pomegranate extract, and the ordinary pomegranate extract is purchased from Xi'an Feida Biotechnology Co., Ltd.

[0071] Comparative Example 3: The preparation method and specific ratio of raw materials of the tissue culture rhizome proliferation medium provided in this example are roughly the same as those in Example 1. The main difference is that in this example, ordinary coconut juice extract is used instead of novel coconut juice extract, and the ordinary coconut juice extract is selected from Shaanxi Shengmi Biotechnology Co., Ltd. Comparative Example 4: The preparation method and specific ratio of raw materials of the tissue culture rhizome proliferation medium provided in this example are roughly the same as those in Example 1. The main difference is that the novel coconut juice extract in this example does not contain nano coconut juice.

[0072] Effect test The tissue culture rhizome proliferation culture media prepared by Examples 1-3 of the present invention are respectively designated as Experimental Examples 1-3; the tissue culture rhizome proliferation culture media prepared by Comparative Examples 1-4 are respectively designated as Comparative Examples 1-4; and then they are tested respectively.

[0073] Experimental setup: Stress resistance test: Stress resistance was assessed using the survival rate of *Gnaphalium affine* tissue-cultured rhizomes under low temperature (4℃) and high salt (0.3 mol / L NaCl) stress as the core indicator, combined with the evaluation of antioxidant enzyme (SOD, POD) activity. Examples 1-3 exhibited the best stress resistance due to the nano-components and fortifiers of the novel coconut milk extract, and the chelate structure of the novel pomegranate extract. Comparative Example 1 lacked the novel pomegranate extract, and Comparative Examples 3-4 had incomplete coconut milk extracts, resulting in significantly reduced stress resistance, as detailed in Table 1. Table 1: Stress Resistance Test Table Quality testing Quality was assessed according to the Chinese Pharmacopoeia, testing for the content of active ingredients (flavonoids, polysaccharides) and pesticide residues (heavy metals, pesticide residues) in *Gynostemma pentaphyllum* to ensure compliance with standards. The novel extracts in Examples 1-3 improved the accumulation of active ingredients, and the purification process reduced pesticide residues; the comparative examples, due to extract defects, had lower active ingredient content, as detailed in Table 2. Table 2: Quality Test Table Germination rate test Germination rate was determined according to the national occupational standards for plant tissue culture workers, and the germination rate and uniformity within 7 days after inoculation were statistically analyzed. The hormone ratios in Examples 1-3, combined with the novel extract, synergistically promoted germination, resulting in high and uniform germination rates. The comparative examples, due to the lack of extract function, exhibited low germination rates and poor uniformity. Detailed experimental results are shown in Table 3. Table 3: Germination Rate Test Table Rapid proliferation test Rapid proliferation was assessed using the rhizome proliferation coefficient (number of rhizomes after proliferation / initial number) and fresh weight growth rate within 30 days, according to the Good Manufacturing Practices (GMP) for Chinese medicinal materials. The novel extracts in Examples 1-3 provided sufficient nutrition and hormone signals, resulting in significant proliferation effects; the comparative examples, due to insufficient extracts, showed a significant decrease in proliferation coefficient and fresh weight growth rate (see Table 4). Table 4: Rapid Proliferation Test Table In summary, based on the above four types of data, we can obtain Table 5: Table 5: Comprehensive Performance Test Table 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.

[0074] 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 the proliferation of tissue-cultured rhizomes of *Gynostemma pentaphyllum*, characterized in that: The proliferation medium is composed of MS medium and proliferation material at a mass ratio of 1:26-28. The proliferation material includes the following components: 2-4 parts 6-benzylaminopurine, 0.3-0.7 parts naphthaleneacetic acid, 0.3-0.6 parts indolebutyric acid, 4-6 parts gibberellin, 95-105 parts novel coconut juice extract, 18-22 parts novel pomegranate extract, 250-350 parts sucrose, 60-70 parts agar, and 3-5 parts activated carbon. The novel pomegranate extract is prepared by mixing chitosan solution, gum arabic solution and pomegranate extract, and then by ultrasound and chelation. The novel coconut juice extract is obtained by preparing fermented coconut juice, nano-coconut juice containing nano-silicon / selenium complex, and fortified coconut juice containing fortifiers, and then mixing the three types of coconut juice with seaweed extract and humic acid.

2. The culture medium for the proliferation of tissue culture rhizomes of *Gynostemma pentaphyllum* according to claim 1, characterized in that, The preparation method of the novel pomegranate extract includes the following steps: S1: Mix chitosan with a 1% acetic acid solution at a solid-liquid ratio of 0.01-0.03 g / mL and stir for 25-35 min to form a chitosan solution; S2: Mix gum arabic with deionized water at a mass ratio of 1:99-101, and treat with ultrasound for 8-12 minutes to obtain a gum arabic solution; S3: Mix pomegranate peel extract with anhydrous ethanol at a mass ratio of 1:99-101, and treat with ultrasound for 4-6 minutes to obtain pomegranate extract; S4: Chitosan solution, gum arabic solution and pomegranate extract are mixed at a volume ratio of 1.8-2.2:1:1 and ultrasonically emulsified for 4-6 minutes to obtain a mixed emulsion. Copper sulfate solution with a mass ratio of 120-125:1 and a concentration of 0.08-1.2 mol / L is added dropwise to the mixed emulsion and stirred for 25-35 minutes to obtain a chelated emulsion. The chelated emulsion is then processed to obtain a novel pomegranate extract.

3. The culture medium for the proliferation of tissue culture rhizomes of *Gynostemma pentaphyllum* according to claim 2, characterized in that: The preparation process of the pomegranate peel extract in step S3 is as follows: Peel the pomegranate peel, rinse it with water 2-4 times, air dry it for 23-25 ​​hours, and then grind it into powder by crushing it through a 35-45 mesh sieve. Pomegranate peel powder was mixed with 35% edible ethanol at a solid-liquid ratio of 0.08–0.12 g / mL and extracted by reflux in a water bath at 75–85°C for 2–3 times, with an interval of 1.5–2.5 h each time, to obtain a crude extract. The crude extract was then concentrated and purified to obtain pomegranate peel extract.

4. The culture medium for the proliferation of tissue culture rhizomes of *Gynostemma pentaphyllum* according to claim 3, characterized in that: The steps for concentrating and purifying the crude extract are as follows: The water bath temperature was set to 45–55℃ and the vacuum degree to 0.08–0.09 MPa. The crude extract was concentrated under reduced pressure to obtain pomegranate extract. The pomegranate extract was mixed with deionized water at a mass ratio of 1:3.5–4.5 and stirred until completely dissolved to form an extract solution. The macroporous resin was soaked in ethanol and loaded into a chromatography column to form a resin column. The resin column was rinsed with deionized water until no ethanol residue was left. The extract solution was then passed into the resin column to obtain a resin column that was saturated with adsorption. The saturated resin column was washed with a 35% (v / v) ethanol solution, and the eluent was collected. The eluent was concentrated at 45–55°C and 0.08–0.09 MPa to obtain a concentrate. The concentrate was then dried at 55–65°C and 0.08–0.09 MPa for 11–13 hours to obtain pomegranate peel extract.

5. The culture medium for the proliferation of tissue culture rhizomes of *Gynostemma pentaphyllum* according to claim 4, characterized in that: The macroporous resin can be any one of the following types: D101, D101-I, HPD-100, HPD300, and SIP-1100.

6. The culture medium for the proliferation of tissue culture rhizomes of *Gynostemma pentaphyllum* according to claim 1, characterized in that: The preparation method of the novel coconut milk extract is as follows: Fresh coconuts were pretreated to obtain raw coconut juice. The raw coconut juice was ultrasonically treated at 35-45℃ for 20-30 minutes to form optimized coconut juice. Lactic acid bacteria accounting for 4%-6% of the volume of the optimized coconut juice were inoculated and fermented at 35-39℃ for 35-37 hours to obtain fermented coconut juice, which was then divided into two equal portions. Add 30% to 35% of the weight of the enhanced coconut juice to one portion of the fermented coconut juice, stir until completely dissolved, and then divide the enhanced coconut juice into two equal portions. Nano-silicon and nano-selenium were added to one portion of the fortified coconut juice at solid-liquid ratios of 75–85 mg / L and 0.04–0.06 mg / L, respectively, and then ultrasonically dispersed to obtain nano-coconut juice; Fermented coconut juice, fortified coconut juice, seaweed extract, humic acid, and nano coconut juice were mixed in a mass ratio of 9–11:5–7:2–4:1:1 and stirred for 10–15 minutes. The pH was adjusted to 6.0–6.5 and refrigerated at 3–5°C for 20–30 days to obtain a novel coconut juice extract.

7. The culture medium for the proliferation of tissue culture rhizomes of *Gynostemma pentaphyllum* according to claim 6, characterized in that, The lactic acid bacteria can be any one of Lactobacillus plantarum, Lactobacillus acidophilus, Bifidobacterium lactis, and Lactococcus lactis.

8. The culture medium for the proliferation of tissue culture rhizomes of *Gynostemma pentaphyllum* according to claim 6, characterized in that, The fortifier is prepared by mixing antioxidants, osmotic regulators and hormone regulators in a mass ratio of 140-160:1-1.5:0.7-0.

8.

9. The culture medium for the proliferation of tissue culture rhizomes of *Gynostemma pentaphyllum* according to claim 8, characterized in that, The antioxidant is prepared by mixing vitamin C, vitamin E, and tea polyphenols in a mass ratio of 4.5–5.5:1.5–2.5:7–9. The osmotic regulator is prepared by mixing proline, betaine, and trehalose in a mass ratio of 0.7–0.9: 0.25–0.35: 0.15–0.

25. The hormone regulator is prepared by mixing abscisic acid, salicylic acid and indolebutyric acid in a mass ratio of 0.25-0.35:0.07-0.09:0.35-0.

45.

10. A method for preparing a culture medium for the proliferation of tissue-cultured rhizomes of *Gnaphalium affine* according to any one of claims 1 to 9, characterized in that, Includes the following steps: Step 1: Prepare MS base solution, novel pomegranate extract and coconut extract separately. Add 6-benzylaminopurine, naphthaleneacetic acid, indolebutyric acid and gibberellin to MS base solution in proportion and stir until completely dissolved. Then add novel pomegranate extract and coconut extract and continue stirring until homogeneous to obtain premixed culture medium solution. Step 2: Add agar and sucrose to the premixed culture medium solution, heat to boiling and stir continuously until completely dissolved; when the solution cools to 45-55℃, add activated carbon and stir for 10-15 minutes to obtain a solidified culture medium solution. Step 3: Pour the solidified culture medium solution into the tissue culture bottle, seal it, and place it in an autoclave. Sterilize it at 120-125℃ for 20-25 minutes to obtain the tissue culture rhizome proliferation medium for *Gynostemma pentaphyllum*.

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