Ophiopogon japonicus powder for relieving anxiety and improving sleep, and preparation method and application thereof

Abstract

The application discloses a powder of ophiopogon japonicus for relieving anxiety and improving sleep, a preparation method and application thereof, and belongs to the technical field of traditional Chinese medicine. The powder of ophiopogon japonicus is prepared by fermenting ophiopogon japonicus with lactobacillus plantarum GDMCC No. 64374 and then treating the ophiopogon japonicus by enzymolysis with a pullulanase. The powder of ophiopogon japonicus disclosed by the application is rich in ophiopogon saponins, small-molecule active polysaccharides and flavones and other active ingredients, and the three types of components coexist in situ and synergize under specific fermentation-enzymolysis timing process conditions, so that various forms of products can be prepared, the powder of ophiopogon japonicus has the effects of relieving anxiety and improving sleep, has high bioavailability, good safety and can be consumed for a long time. Compared with traditional ophiopogon japonicus powder, ophiopogon japonicus powder only treated by fermentation or ophiopogon japonicus powder only treated by enzymolysis, the powder of ophiopogon japonicus disclosed by the application achieves significant synergistic gains in aspects of active ingredient enrichment, bioavailability, safety and efficacy.

Description

Technical Field

[0001] This invention relates to the fields of food and health products, and in particular to a powder made from Ophiopogon japonicus used to relieve anxiety and improve sleep, as well as its preparation method and application. Background Technology

[0002] With the fast pace of modern life, sleep disorders have become a global health problem. Chronic sleep deprivation not only leads to decreased attention and weakened immunity, but also increases the risk of chronic diseases such as depression and cardiovascular disease. Currently, mainstream sleep aids (such as benzodiazepines) are fast-acting, but they have side effects such as dependence, drug tolerance, and liver and kidney damage. Therefore, developing natural and safe plant-based sleep aids has become a research hotspot in the fields of functional foods and traditional Chinese medicine.

[0003] Ophiopogon japonicus has the effects of nourishing yin and moistening the lungs, clearing the heart and relieving irritability. Traditional Ophiopogon japonicus powder preparation mainly uses water extraction, alcohol extraction or mechanical pulverization processes, which have significant drawbacks such as loss of effective ingredients, low bioavailability, high safety risks, and limited functionality. Existing technologies have reported the use of enzymatic hydrolysis to extract saponins from Ophiopogon japonicus (e.g., "Study on the Process Conditions for Extraction of Total Saponins from Ophiopogon japonicus by Compound Enzyme Method", *Food Research and Development*, 2007), as well as the use of *Lactobacillus plantarum* to ferment traditional Chinese medicine to increase the content of effective ingredients (e.g., Chinese Patent CN118217338A discloses a fermented preparation of traditional Chinese medicine probiotics and its preparation method and application), and the use of cell wall degrading enzymes and starch degrading enzymes to treat rhizomes to increase the content of effective ingredients (e.g., Korean Patent KR10-2021-0029548A discloses a method for preparing rhizome plant extracts with increased effective ingredients).

[0004] However, none of the aforementioned technical solutions discloses a specific scheme for the synergistic treatment of a particular strain of *Lactobacillus plantarum* and pullulanase at a specific time sequence, nor do they recognize that this specific combination can achieve simultaneous enrichment of saponins and active small-molecule polysaccharides, or the systemic synergistic gains in bioavailability, safety, and efficacy. In particular, the prior art does not disclose whether there is a synergistic effect between a specific strain of *Lactobacillus plantarum* and pullulanase, nor does it recognize the irreplaceable role of the specific strain in the fermentation-enzymatic hydrolysis synergistic system. Summary of the Invention

[0005] One of the objectives of this invention is to address the above-mentioned technical problems by providing Ophiopogon japonicus powder that can effectively relieve anxiety and improve sleep disorders.

[0006] The second objective of this invention is to provide a method for preparing the Ophiopogon japonicus powder.

[0007] A third objective of this invention is to provide applications for the aforementioned Ophiopogon japonicus powder.

[0008] One of the objectives of this invention is achieved through the following technical solution: A type of Ophiopogon japonicus powder for relieving anxiety and improving sleep, wherein the Ophiopogon japonicus powder is produced by fermentation of Ophiopogon japonicus with Lactiplantibacillus plantarum GDMCC No. 64374 and enzymatic hydrolysis with pullulanase.

[0009] The inventors have discovered that when Ophiopogon japonicus is first fermented with Lactobacillus plantarum GDMCC No. 64374 and then hydrolyzed with pullulanase, the resulting Ophiopogon japonicus powder exhibits a significant synergistic effect in terms of saponin content, total polysaccharide content, small molecule polysaccharide content, total flavonoid content, bioavailability, safety, and efficacy.

[0010] The synergistic mechanism is as follows: First, *Lactobacillus plantarum* GDMCC No. 64374 initially lyses the fibrous skeleton of the *Ophiopogon japonicus* cell wall during fermentation, releasing the encapsulated active ingredients and starchy substances. Second, pullulanase degrades macromolecular amylopectin into small-molecule active polysaccharides, while further promoting the dissolution of active ingredients. The two processes complement each other in terms of substrates (cell wall polysaccharides and amylopectin) and their action stages are linked (cell wall disruption and impurity removal and transformation), thus forming a sequential and synergistic two-stage directed transformation.

[0011] More importantly, in the unique material system created by this synergistic process, three types of components—Ophiopogon saponins, small molecule active polysaccharides, and flavonoids—coexist in a specific in-situ form and proportion, forming a ternary synergistic system that cannot be replicated by physical mixing.

[0012] The second objective of this invention is achieved by the following technical solution: A method for preparing Ophiopogon japonicus powder for relieving anxiety and improving sleep includes the following steps: S1: Take fresh Ophiopogon japonicus, freeze-dry it, grind it into powder, and sift it to obtain Ophiopogon japonicus powder; S2: Disperse the Ophiopogon japonicus powder evenly in water, heat and extract, then cool to obtain the fermentation substrate; S3: Inoculate the fermentation substrate with Bacillus plantarum seed liquid for fermentation culture, sterilize after the reaction is completed, and obtain Ophiopogon japonicus fermentation broth; S4: Add pullulanase to the fermentation broth of Ophiopogon japonicus for enzymatic hydrolysis. After the reaction is completed, the enzyme is inactivated to obtain Ophiopogon japonicus hydrolysate. S5: Filter the Ophiopogon japonicus enzymatic hydrolysate, discard the precipitate, collect the supernatant, dry it, and obtain the Ophiopogon japonicus powder.

[0013] As a preferred embodiment, the preparation steps of the *Lactobacillus plantarum* seed solution include: (1) Activation: Lactobacillus plantarum GDMCC No.64374 was streaked on MRS solid medium and cultured at 35℃ for 48 hours under anaerobic conditions; (2) Liquid culture: Pick a single colony from the plate, inoculate it into MRS liquid medium, and culture it under anaerobic conditions at 35°C for 48 hours to obtain the bacterial solution; (3) Subculture: Take 10% of the bacterial culture and add it to MRS liquid culture medium. Culture at 35°C for 12 hours under anaerobic conditions to obtain the seed culture of Lactobacillus plantarum.

[0014] As a preferred embodiment, the concentration (OD) of *Lactobacillus plantarum* in the *Lactobacillus plantarum* seed solution is... 600 The value is 1.0-2.0, more preferably 2.0.

[0015] As a preferred embodiment, in step S2, the mass ratio of the Ophiopogon japonicus powder to the water is 1:100-5:100; in step S3, the volume ratio of the Bacillus plantarum seed liquid to the fermentation substrate is 1:100-5:100, more preferably 5:100.

[0016] As a preferred embodiment, the fermentation culture temperature is 33-37℃, more preferably 35℃, and the time is 10-15 hours, more preferably 12 hours; the heating extraction temperature is 70-90℃, more preferably 80℃, and the time is 1-3 hours, more preferably 2 hours.

[0017] As a preferred embodiment, the sterilization process is carried out at a temperature of 100°C for 20-40 minutes, more preferably 30 minutes.

[0018] As a preferred embodiment, in step S4, the amount of pullulanase used is 0.1-0.5% w / w of the Ophiopogon japonicus fermentation broth, more preferably 0.2-0.5% w / w.

[0019] As a preferred embodiment, the enzymatic hydrolysis treatment temperature is 55-65℃, more preferably 60℃, the pH is 4.5-6, more preferably 5, the stirring speed is 150-250rpm, more preferably 200rpm, and the time is 5-7 hours, more preferably 6 hours.

[0020] As a preferred embodiment, the temperature of the enzyme inactivation treatment is 90-100℃, more preferably 90℃; and the time is 10-20 minutes, more preferably 15 minutes.

[0021] The third objective of this invention is achieved by the following technical solution: The application of Ophiopogon japonicus powder, which is used to relieve anxiety and improve sleep, in the preparation of food, health products or medicines for relieving anxiety and improving sleep.

[0022] The Ophiopogon japonicus powder can be combined with commonly used excipients in food, health products, or pharmaceuticals to prepare various forms of products.

[0023] The beneficial effects of this invention are: (1) Synergistic enrichment of active ingredients: The present invention obtains Ophiopogon japonicus powder through the synergistic effect of Lactobacillus plantarum GDMCC No.64374 and pullulanase. The enrichment of Ophiopogon japonicus powder has been significantly improved in three types of active ingredients: saponins, small molecule active polysaccharides and flavonoids.

[0024] The content of small molecule polysaccharides in Comparative Example 1 (fermentation only) and Comparative Example 2 (enzymatic hydrolysis only) was significantly lower than that in Example 1. The saponin content in Example 1 was significantly higher than the simple sum of Comparative Example 1 (fermentation only) and Comparative Example 2 (enzymatic hydrolysis only), with a synergistic effect coefficient of 3.25 times. The total flavonoid content in Example 1 was significantly higher than the simple sum of Comparative Example 1 (fermentation only) and Comparative Example 2 (enzymatic hydrolysis only), with a synergistic effect coefficient of 1.29 times. This invention achieves the simultaneous enrichment of three types of components: saponins, small molecule active polysaccharides, and flavonoids.

[0025] (2) Significantly improved bioavailability: The bioavailability of the Ophiopogon japonicus powder of the present invention is 9.78%-19.54%. The bioavailability of Example 1 is 11.6 times that of the conventional water extraction of Comparative Example 3, 2.4 times that of the fermentation of Comparative Example 1, and 9.2 times that of the enzymatic hydrolysis of Comparative Example 2.

[0026] (3) Significantly improved safety: The maximum safe concentration of Ophiopogon japonicus powder of the present invention for RIN-14B cells is 4 mg / mL, which is 4 times that of conventional water extraction in Comparative Example 3.

[0027] (4) Excellent effects in relieving anxiety and improving sleep: The 5-HT promotion rate of the Ophiopogon japonicus powder of the present invention is as high as 41.20%-56.80%, and the 5-HT promotion rate of Example 1 is 6.6 times that of the traditional water extract of Comparative Example 3; The zebrafish sedative-hypnotic effect of the Ophiopogon japonicus powder of the present invention is 29.49%-42.5%, and the zebrafish sedative-hypnotic effect of Example 1 is 6.25 times that of the traditional water extract of Comparative Example 3.

[0028] (5) Irreplaceability of strains: Under completely identical conditions, the saponin yield of the strains of the present invention is 5.2 times that of common Lactobacillus plantarum, 7.3 times that of Lactobacillus acidophilus, 6.0 times that of Lactobacillus rhamnosus, 9.4 times that of Lactobacillus casei, and 12.4 times that of Candida utilis.

[0029] (6) Complete verification of the in-situ synergistic system of the three components: In Comparative Example 12, the purified total saponins of Ophiopogon japonicus, small molecule active polysaccharides of Ophiopogon japonicus, and total flavonoids of Ophiopogon japonicus were physically mixed in the same proportion as in Example 1, and the 5-HT promoting rate was much lower than that in Example 1. In Comparative Example 13, pure Rusco saponins were used instead of total saponins of Ophiopogon japonicus for the same physical mixing, and the results were also lower than those in Example 1. In Comparative Examples 14-15, commercially available Ganoderma lucidum polysaccharides or Tremella fuciformis polysaccharides were used instead of the small molecule active polysaccharides of Ophiopogon japonicus unique to this invention, and the effect was even worse.

[0030] The above results consistently demonstrate that the efficacy of this invention does not stem from a simple combination of the three components, but rather from a ternary in-situ synergistic system created by a specific fermentation-enzymatic hydrolysis process that cannot be replicated by physical mixing. Detailed Implementation

[0031] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0032] Unless otherwise specified, all instruments and reagents used in the examples are conventional instruments or reagents in the art and are commercially available products. Unless otherwise specified, all specific experimental operations involved in the text are understandable or known to those skilled in the art based on their common knowledge or conventional technical means, and will not be described in detail here.

[0033] The following Lactiplantibacillus plantarum GDMCC No. 64374 was independently screened and has been deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) with accession number GDMCC No. 64374, deposit date of February 5, 2024, and deposit address of Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province.

[0034] The following are the steps for preparing *Lactobacillus plantarum* seed culture: (1) Activation: Lactobacillus plantarum GDMCC No.64374 was streaked on MRS solid medium and cultured at 35℃ for 48 hours under anaerobic conditions; (2) Liquid culture: Pick a single colony from a plate, inoculate it into MRS liquid medium, and culture it under anaerobic conditions at 35°C for 48 hours to obtain bacterial solution; (3) Subculture: Take 10% of the bacterial solution and add it to MRS liquid culture medium. Culture at 35°C for 12 hours under anaerobic conditions to obtain the seed culture of Lactobacillus plantarum.

[0035] The formula for MRS solid culture medium is as follows: Dissolve the following components in 1L of distilled water: 10g peptone, 10g beef extract, 5g yeast extract, 2g dipotassium hydrogen phosphate, 2g diammonium citrate, 5g sodium acetate, 20g glucose, 1mL Tween 80, 0.5g magnesium sulfate, 0.25g manganese sulfate, and 15g agar powder. Adjust the pH to 6.2-6.4 and autoclave (101 kPa, 121℃) for 15 minutes.

[0036] The formula for MRS liquid culture medium is as follows: Dissolve the following components in 1L of distilled water: 10g peptone, 10g beef extract, 5g yeast extract, 2g dipotassium hydrogen phosphate, 2g diammonium citrate, 5g sodium acetate, 20g glucose, 1mL Tween 80, 0.5g magnesium sulfate, and 0.25g manganese sulfate. Adjust the pH to 6.2-6.4 and autoclave (101 kPa, 121°C) for 15 minutes.

[0037] Example 1 Prepare Ophiopogon japonicus powder according to the following steps: (1) Take fresh Ophiopogon japonicus, freeze dry, grind into powder, and sieve (20 mesh) to obtain Ophiopogon japonicus powder.

[0038] (2) Take 50g of Ophiopogon japonicus powder, add it to 1000g of water, heat water extraction (80℃, 2h), cool down to 37℃, and obtain fermentation substrate.

[0039] (3) Take 50 mL of Lactobacillus plantarum seed solution (concentration of Lactobacillus plantarum OD) 600 2.0) was inoculated into 1000 mL of fermentation substrate to obtain a fermentation system. The fermentation system was fermented in a shaker at 35°C for 12 hours to obtain the fermentation product.

[0040] (4) Sterilize the fermentation product at 100°C for 30 minutes to inactivate the bacteria.

[0041] (5) Add 0.2% (w / w) pullulanase (Sigma CAS#: 9075-68-7), and incubate at pH=5, 60℃ with constant stirring (200rpm) for 6 hours, followed by enzyme inactivation at 90℃ for 15 minutes.

[0042] (6) Filter, discard the precipitate, collect the supernatant, and spray dry to obtain Ophiopogon japonicus powder.

[0043] Example 2 Prepare Ophiopogon japonicus powder according to the following steps: (1) Take fresh Ophiopogon japonicus, freeze dry, grind into powder, and sieve (20 mesh) to obtain Ophiopogon japonicus powder.

[0044] (2) Take 30g of Ophiopogon japonicus powder, add it to 1000g of water, heat water extraction (70℃, 3h), cool down to 37℃, and obtain fermentation substrate.

[0045] (3) Take 30 mL of Lactobacillus plantarum seed solution (concentration of Lactobacillus plantarum OD) 600 2.0) was inoculated into 1000 mL of fermentation substrate to obtain a fermentation system. The fermentation system was fermented in a shaker at 37°C for 10 hours to obtain the fermentation product.

[0046] (4) Sterilize the fermentation product at 100°C for 40 minutes to inactivate the bacteria.

[0047] (5) Add 0.5% (w / w) pullulanase, and incubate at pH=4.5 at 65℃ with stirring (150 rpm) for 7 hours, followed by enzyme inactivation at 90℃ for 20 minutes.

[0048] (6) Filter, discard the precipitate, collect the supernatant, and spray dry to obtain Ophiopogon japonicus powder.

[0049] Example 3 Prepare Ophiopogon japonicus powder according to the following steps: (1) Take fresh Ophiopogon japonicus, freeze dry, grind into powder, and sieve (20 mesh) to obtain Ophiopogon japonicus powder.

[0050] (2) Take 10g of Ophiopogon japonicus powder, add it to 1000g of water, heat water extraction (90℃, 1h), cool down to 37℃, and obtain fermentation substrate.

[0051] (3) Take 10 mL of Lactobacillus plantarum seed solution (concentration of Lactobacillus plantarum OD) 600 1.0) was inoculated into 1000 mL of fermentation substrate to obtain a fermentation system. The fermentation system was fermented in a shaker at 33°C for 15 hours to obtain the fermentation product.

[0052] (4) Sterilize the fermentation product at 100°C for 20 minutes to inactivate the bacteria.

[0053] (5) Add 0.1% (w / w) pullulanase, and incubate at pH=6, 55℃ with constant stirring (250 rpm) for 5 hours, followed by enzyme inactivation at 100℃ for 10 minutes.

[0054] (6) Filter, discard the precipitate, collect the supernatant, and spray dry to obtain Ophiopogon japonicus powder.

[0055] Comparative Example 1 Prepare Ophiopogon japonicus powder according to the following steps: (1) Take fresh Ophiopogon japonicus, freeze dry, grind into powder, and sieve (20 mesh) to obtain Ophiopogon japonicus powder.

[0056] (2) Take 50g of Ophiopogon japonicus powder, add it to 1000g of water, heat water extraction (80℃, 2h), cool down to 37℃, and obtain fermentation substrate.

[0057] (3) Take 50 mL of Bacillus plantarum seed liquid and inoculate it into 1000 mL of fermentation substrate to obtain a fermentation system. Ferment the fermentation system in a shaker at 35°C for 12 hours to obtain the fermentation product.

[0058] (4) Sterilize the fermentation product at 100°C for 30 minutes to inactivate the bacteria.

[0059] (5) Filter, discard the precipitate, collect the supernatant, and spray dry to obtain Ophiopogon japonicus powder.

[0060] Comparative Example 2 Prepare Ophiopogon japonicus powder according to the following steps: (1) Take fresh Ophiopogon japonicus, freeze dry, grind into powder, and sieve (20 mesh) to obtain Ophiopogon japonicus powder.

[0061] (2) Take 50g of Ophiopogon japonicus powder, add it to 1000g of water, heat it with hot water (80℃, 2h), and cool it down to 37℃.

[0062] (3) Add 0.2% (w / w) pullulanase (Sigma CAS#: 9075-68-7), and incubate at pH=5, 60℃ with constant stirring (200rpm) for 6 hours, followed by enzyme inactivation at 90℃ for 15 minutes.

[0063] (4) Filter, discard the precipitate, collect the supernatant, and spray dry to obtain Ophiopogon japonicus powder.

[0064] Comparative Example 3 Prepare Ophiopogon japonicus powder according to the following steps: (1) Take fresh Ophiopogon japonicus, freeze dry, grind into powder, and sieve (20 mesh) to obtain Ophiopogon japonicus powder.

[0065] (2) Take 50g of Ophiopogon japonicus powder, add it to 1000g of water, and extract with hot water (80℃, 2h).

[0066] (3) Filter, discard the precipitate, collect the supernatant, and spray dry to obtain Ophiopogon japonicus powder.

[0067] Comparative Example 4 The difference between Comparative Example 4 and Example 1 is that commercially available common Lactobacillus plantarum (purchased from China General Microbiological Culture Collection Center, CGMCC No. 1.16089) was used instead of Lactobacillus plantarum GDMCC No. 64374 for fermentation. Apart from the different strains, the fermentation and enzymatic hydrolysis conditions were the same.

[0068] Comparative Example 5 The difference between Comparative Example 5 and Example 1 is that commercially available Lactobacillus acidophilus (purchased from China General Microbiological Culture Collection Center, CGMCC No. 1.1854) was used instead of Lactobacillus plantarum GDMCC No. 64374 for fermentation. Apart from the different strains, the other fermentation and enzymatic hydrolysis conditions were the same.

[0069] Comparative Example 6 The difference between Comparative Example 6 and Example 1 is that commercially available Lactobacillus rhamnosus (purchased from China General Microbiological Culture Collection Center, CGMCC No. 1.8882) was used instead of Lactobacillus plantarum GDMCC No. 64374 for fermentation. Apart from the different strains, the fermentation and enzymatic hydrolysis conditions were the same.

[0070] Comparative Example 7 The difference between Comparative Example 7 and Example 1 is that commercially available Lactobacillus casei (purchased from China General Microbiological Culture Collection Center, CGMCC No. 1.8727) was used instead of Lactobacillus plantarum GDMCC No. 64374 for fermentation. Apart from the different strains, the other fermentation and enzymatic hydrolysis conditions were the same.

[0071] Comparative Example 8 The difference between Comparative Example 8 and Example 1 is that Commercially available common Candida utilis (purchased from China General Microbiological Culture Collection Center, CGMCC No. 2.3047) was used instead of Bacillus plantarum GDMCC No. 64374 for fermentation. Apart from the different strains, the other fermentation and enzymatic hydrolysis conditions were the same.

[0072] The preparation method for purified component A (pure total saponins from Ophiopogon japonicus) includes the following steps: (1) Take 100g of Ophiopogon japonicus powder prepared in Example 1, add 800mL of 70% ethanol, and extract by ultrasonication for 30min (power 400W, temperature 60℃). Filter, and extract the residue once more. Combine the filtrates.

[0073] (2) The filtrate was concentrated under reduced pressure until there was no alcohol odor, and water was added to disperse it to 500 mL. It was then extracted and defatted with petroleum ether (500 mL × 2) and ethyl acetate (500 mL × 2) in sequence, and the aqueous layer was collected.

[0074] (3) The aqueous layer was loaded onto a D101 macroporous adsorption resin column (column volume 1L), and eluted sequentially with distilled water, 30% ethanol, 70% ethanol and 95% ethanol. The eluted portion of 70% ethanol was collected, concentrated under reduced pressure, and freeze-dried to obtain pure total saponins of Ophiopogon japonicus.

[0075] Test results showed that the total saponin content was ≥85% (calculated as rusco saponin), the total polysaccharide content was <2%, and the total flavonoid content was <1%.

[0076] The preparation method for purified component B (pure small molecule active polysaccharide from Ophiopogon japonicus) includes the following steps: (1) Take 100g of Ophiopogon japonicus powder prepared in Example 1, add 1000mL of distilled water, stir and disperse, heat at 80℃ for 1 hour, centrifuge (8000rpm, 15min), collect the supernatant, repeat the extraction of the residue once, and combine the supernatants.

[0077] (2) Concentrate the supernatant under reduced pressure to 200 mL, add 50 mL of Sevage reagent (chloroform: n-butanol = 4:1), shake vigorously for 30 minutes, centrifuge (4000 rpm, 10 minutes) to remove the denatured protein layer. Repeat the operation 5 times until no protein layer appears.

[0078] (3) Slowly add anhydrous ethanol to the supernatant until the ethanol concentration reaches 80%, let stand overnight at 4°C, collect the precipitate by centrifugation, wash twice with anhydrous ethanol, freeze dry to obtain crude polysaccharide of Ophiopogon japonicus.

[0079] (4) Dissolve the crude polysaccharide of Ophiopogon japonicus in water to prepare a 5% solution, centrifuge (10000 rpm, 10 minutes), take the supernatant and pass it through a 10 kDa ultrafiltration membrane and a 1 kDa nanofiltration membrane in sequence, collect the retentate with a molecular weight between 1.5-10 kDa, concentrate under reduced pressure, freeze dry, and obtain pure Ophiopogon japonicus small molecule active polysaccharide.

[0080] Tests showed that the total polysaccharide content was 86.5%, the small molecule polysaccharide content was 76.1%, saponins were not detected (<0.01%), and total flavonoids were not detected.

[0081] The preparation method for purified component C (pure total flavonoids from Ophiopogon japonicus) includes the following steps: (1) Take 100g of Ophiopogon japonicus powder prepared in Example 1, add 800mL of 70% ethanol, and extract by ultrasonication for 30min (power 400W, temperature 60℃). Filter, and extract the residue once more. Combine the filtrates.

[0082] (2) The filtrate was concentrated under reduced pressure until there was no alcohol odor, and water was added to disperse it to 500 mL. It was then extracted and defatted with petroleum ether (500 mL × 2) in sequence, and the aqueous layer was collected.

[0083] (3) The aqueous layer was extracted with ethyl acetate (500mL×3), the ethyl acetate layers were combined, and the mixture was evaporated under reduced pressure to obtain pure total flavonoids from Ophiopogon japonicus.

[0084] Tests showed that the total flavonoid content was ≥85% (calculated as rutin), the total saponins were <0.5%, and the total polysaccharides were <1%.

[0085] Comparative Example 9 Prepare mixture one according to the following steps: (1) Take purified component A (pure total saponins of Ophiopogon japonicus) and purified component B (pure small molecule active polysaccharides of Ophiopogon japonicus) and physically mix them according to the ratio of saponin content and small molecule polysaccharide content in Example 1.

[0086] (2) Preparation method: Weigh 0.233g of pure total saponins of Ophiopogon japonicus (calculated based on 85% purity, the actual saponins are 0.198g), 1.858g of pure small molecule active polysaccharides of Ophiopogon japonicus (calculated based on 76.1% purity, the actual small molecule polysaccharides are 1.414g), add maltodextrin to make up to 10g, grind and mix thoroughly in a mortar for 30 minutes, and pass through a 100-mesh sieve three times to obtain mixture one.

[0087] This mixture does not contain total flavonoids from Ophiopogon japonicus.

[0088] Comparative Example 10 Prepare mixture two according to the following steps: (1) Take purified component A (pure total saponins of Ophiopogon japonicus) and purified component C (pure total flavonoids of Ophiopogon japonicus) and mix them physically according to the ratio of saponin content and flavonoid content in Example 1.

[0089] (2) Preparation method: Weigh 0.233g of pure total saponins of Ophiopogon japonicus (calculated based on 85% purity, the actual saponins are 0.198g) and 0.100g of pure total flavonoids of Ophiopogon japonicus (calculated based on 85% purity, the actual flavonoids are 0.085g). Add maltodextrin to make up to 10g. Grind and mix thoroughly in a mortar for 30 minutes. Pass through a 100-mesh sieve three times to obtain mixture two.

[0090] This mixture does not contain small molecule active polysaccharides from Ophiopogon japonicus.

[0091] Comparative Example 11 Prepare mixture three according to the following steps: (1) Take purified component B (pure small molecule active polysaccharide of Ophiopogon japonicus) and purified component C (pure total flavonoids of Ophiopogon japonicus) and physically mix them according to the ratio of small molecule polysaccharide content and flavonoid content in Example 1.

[0092] (2) Preparation method: Weigh 1.858g of pure small molecule active polysaccharide of Ophiopogon japonicus (calculated based on 76.1% purity, the actual small molecule polysaccharide is 1.414g), and 0.100g of pure total flavonoids of Ophiopogon japonicus (calculated based on 85% purity, the actual flavonoids are 0.085g). Add maltodextrin to make up to 10g. Grind and mix thoroughly in a mortar for 30 minutes, and pass through a 100-mesh sieve three times to obtain mixture three. This mixture does not contain Ophiopogon japonicus saponins.

[0093] Comparative Example 12 Prepare mixture four according to the following steps: (1) Take purified component A (pure total saponins of Ophiopogon japonicus), purified component B (pure small molecule active polysaccharide of Ophiopogon japonicus), and purified component C (pure total flavonoids of Ophiopogon japonicus) and mix them physically according to the ratio of saponin content, small molecule polysaccharide content and flavonoid content in Example 1.

[0094] (2) Preparation method: Weigh 0.233g of pure total saponins of Ophiopogon japonicus (calculated based on 85% purity, actual saponins 0.198g), 1.858g of pure small molecule active polysaccharides of Ophiopogon japonicus (calculated based on 76.1% purity, actual small molecule polysaccharides 1.414g), and 0.100g of pure total flavonoids of Ophiopogon japonicus (calculated based on 85% purity, actual flavonoids 0.085g). Add maltodextrin to make up to 10g. Grind and mix thoroughly in a mortar for 30 minutes. Pass through a 100-mesh sieve three times to obtain mixture four.

[0095] Comparative Example 13 Prepare mixture five according to the following steps: (1) Rusco saponin standard (purity ≥98%, purchased from China National Institutes for Food and Drug Control, CAS#472-11-7); pure small molecule active polysaccharide of Ophiopogon japonicus (purified component B); pure total flavonoids of Ophiopogon japonicus (purified component C).

[0096] (2) Physically mix according to the ratio of saponin content, small molecule polysaccharide content and flavonoid content in Example 1.

[0097] (3) Preparation method: Weigh 0.202g of Rusco saponin (calculated based on 98% purity, actual saponin 0.198g), 1.858g of pure small molecule active polysaccharide of Ophiopogon japonicus (calculated based on 76.1% purity, actual small molecule polysaccharide 1.414g), and 0.100g of pure total flavonoids of Ophiopogon japonicus (calculated based on 85% purity, actual flavonoids 0.085g). Add maltodextrin to make up to 10g. Grind and mix thoroughly in a mortar for 30 minutes. Pass through a 100-mesh sieve three times to obtain mixture five.

[0098] In this comparative example, commercially available Ruscosaponin standard was used instead of the Ophiopogon japonicus saponin unique to this invention.

[0099] Comparative Example 14 Prepare mixture six according to the following steps: (1) Ganoderma lucidum polysaccharide (commercially available, purity ≥50%); purified component A (pure total saponins of Ophiopogon japonicus); purified component C (pure total flavonoids of Ophiopogon japonicus).

[0100] (2) Physically mix according to the ratio of saponin content, small molecule polysaccharide content and flavonoid content in Example 1.

[0101] (3) Preparation method: Weigh 0.233g of pure total saponins of Ophiopogon japonicus (calculated based on 85% purity, the actual saponins are 0.198g), 2.828g of Ganoderma lucidum polysaccharide (calculated based on 50% purity, the actual Ganoderma lucidum polysaccharide is 1.414g), and 0.100g of pure total flavonoids of Ophiopogon japonicus (calculated based on 85% purity, the actual flavonoids are 0.085g). Add maltodextrin to make up to 10g. Grind and mix thoroughly in a mortar for 30 minutes. Pass through a 100-mesh sieve three times to obtain mixture six.

[0102] In this comparative example, commercially available Ganoderma lucidum polysaccharides were used to replace the Ophiopogon japonicus small molecule active polysaccharides unique to this invention.

[0103] Comparative Example 15 Prepare mixture seven according to the following steps: (1) Tremella polysaccharide (commercially available, purity ≥50%); purified component A (pure total saponins of Ophiopogon japonicus); purified component C (pure total flavonoids of Ophiopogon japonicus).

[0104] (2) Physically mix according to the ratio of saponin content, small molecule polysaccharide content and flavonoid content in Example 1.

[0105] (3) Preparation method: Weigh 0.233g of pure total saponins of Ophiopogon japonicus (calculated based on 85% purity, the actual saponins are 0.198g), 2.828g of polysaccharide of Tremella fuciformis (calculated based on 50% purity, the actual polysaccharide of Tremella fuciformis is 1.414g), and 0.100g of pure total flavonoids of Ophiopogon japonicus (calculated based on 85% purity, the actual flavonoids are 0.085g). Add maltodextrin to make up to 10g. Grind and mix thoroughly in a mortar for 30 minutes. Pass through a 100-mesh sieve three times to obtain mixture seven.

[0106] In this comparative example, commercially available Tremella fuciformis polysaccharide was used to replace the small-molecule active polysaccharide of Ophiopogon japonicus unique to this invention.

[0107] Efficacy Experiment 1. Saponin content detection The saponin content of the Ophiopogon japonicus powder obtained in Examples 1-3 and Comparative Examples 1-8 was determined by HPLC. The results are shown in Table 1.

[0108] Table 1. Results of Saponin Content Detection

[0109] As shown in Table 1, the saponin content of Example 1 (1.98%) was significantly higher than the simple sum of Comparative Example 1 (fermentation only, 0.47%) and Comparative Example 2 (enzymatic hydrolysis only, 0.14%) (0.61%), with a synergistic effect coefficient of 3.25 times. The saponin yield of the strain of this invention was 5.2 times that of Comparative Example 4 (fermentation by *Lactobacillus plantarum*), 7.3 times that of Comparative Example 5 (fermentation by *Lactobacillus acidophilus*), 6.0 times that of Comparative Example 6 (fermentation by *Lactobacillus rhamnosus*), 9.4 times that of Comparative Example 7 (fermentation by *Lactobacillus casei*), and 12.4 times that of Comparative Example 8 (fermentation by *Candida utilis*).

[0110] Comparative Example 1 showed that fermentation alone released a certain amount of saponins from Ophiopogon japonicus, while Comparative Example 2 showed extremely poor saponin extraction with only enzymatic hydrolysis. Compared to Example 1, when fermentation and enzymatic hydrolysis worked synergistically, the saponin content increased significantly beyond the sum of their individual contributions, indicating a clear synergistic effect between fermentation and enzymatic hydrolysis. The mechanism is that fermentation lyses the cell walls of Ophiopogon japonicus, releasing the encapsulated saponins, while enzymatic hydrolysis further reduces mass transfer resistance and promotes saponin dissolution. The two processes are functionally complementary and temporally sequential, forming a complete extraction chain.

[0111] Under identical fermentation and enzymatic hydrolysis conditions, simply replacing the strain of this invention with other commercially available strains resulted in a significant decrease in saponin content, indicating that the strain of this invention makes an irreplaceable and specific contribution to this synergistic system.

[0112] 2. Determination of polysaccharide content and molecular weight distribution The total polysaccharide content and molecular weight distribution of the Ophiopogon japonicus powder obtained in Examples 1-3 and Comparative Examples 1-8 were determined using the following methods. The results are shown in Table 2.

[0113] The total polysaccharide content was determined by the phenol-sulfuric acid method: 10 mg of sample was accurately weighed, dissolved in water and diluted to 10 mL. 1 mL of the solution was added to 1 mL of 5% phenol solution, followed by 5 mL of concentrated sulfuric acid. The mixture was shaken well, heated in a boiling water bath for 15 min, cooled to room temperature, and the absorbance was measured at 490 nm. The total polysaccharide content was calculated using glucose as a standard.

[0114] The molecular weight distribution of polysaccharides was determined by gel permeation chromatography (GPC): the column was a TSKgel GMPWXL (7.8 × 300 mm), the mobile phase was 0.1 M NaNO3 solution, the flow rate was 0.6 mL / min, the column temperature was 35 ℃, and a differential refractive index detector was used. Calibration curves were plotted using dextran standards of different molecular weights (1 kDa, 5 kDa, 10 kDa, 40 kDa, 70 kDa, and 500 kDa). The proportion of small molecule polysaccharides (molecular weight 1.5-10 kDa) in the samples was calculated, and the absolute content of small molecule active polysaccharides was calculated accordingly (small molecule polysaccharide content = total polysaccharide content × small molecule polysaccharide proportion).

[0115] Table 2. Results of polysaccharide content and molecular weight distribution detection

[0116] The total polysaccharide content of Examples 1-3 of this invention is 15.1%-20.2%, the small molecule polysaccharide content is 8.31%-14.14%, and the proportion of small molecule polysaccharides is 55%-70%. In Comparative Example 3, the total polysaccharide content of conventional water extraction is 10.2%, the proportion of small molecule polysaccharides is extremely low (only 8%), and the small molecule polysaccharide content is only 0.82%. In Comparative Example 2, the total polysaccharide content of enzymatic hydrolysis alone is 14.3%, but the proportion of small molecule polysaccharides is only 16%, the small molecule polysaccharide content is only 2.29%, and the saponin content is extremely low (0.14%). In Comparative Example 1, the small molecule polysaccharide content of fermentation alone is 5.37%, far lower than the 14.14% of Example 1. These results indicate that only this invention, through the synergistic treatment of fermentation and enzymatic hydrolysis, achieves the simultaneous enrichment of high saponin and high small molecule polysaccharide content.

[0117] When fermentation and enzymatic hydrolysis work synergistically, the content of active small-molecule polysaccharides is significantly increased, far exceeding the simple sum of the effects of either enzyme alone. The mechanism lies in the fact that fermentation not only releases a massive amount of polysaccharide substrate, but more importantly, it alters the ultrastructure of the cell wall, allowing pullulanase to obtain entirely new sites of action that are inaccessible under conditions of enzymatic hydrolysis alone, thereby achieving a leap in enzymatic hydrolysis efficiency.

[0118] Comparing Comparative Examples 4-8 with Example 1, the yield of active small molecule polysaccharides was significantly reduced after replacing them with other strains, further demonstrating the irreplaceable role of the strains of the present invention in the active transformation of Ophiopogon japonicus polysaccharides.

[0119] 3. Flavonoid content determination The total flavonoid content was determined using the aluminum nitrate-sodium nitrite colorimetric method. An appropriate amount of sample was accurately weighed, dissolved in 70% ethanol by sonication, and diluted to volume to prepare the test solution. 1.0 mL of the test solution was accurately measured, and 0.3 mL of 5% sodium nitrite solution was added, shaken well, and allowed to stand for 6 min. Then, 0.3 mL of 10% aluminum nitrate solution was added, shaken well, and allowed to stand for 6 min. Finally, 4.0 mL of 4% sodium hydroxide solution was added, shaken well, and allowed to stand for 15 min. The absorbance was measured at 510 nm. A standard curve was plotted using rutin as a standard, and the total flavonoid content (as rutin, %) in the sample was calculated. The total flavonoid content of the *Ophiopogon japonicus* powder obtained in Examples 1-3 and Comparative Examples 1-8 was determined using the above method. The results are shown in Table 3.

[0120] Table 3. Results of Flavonoid Content Detection

[0121] As shown in Table 3, the total flavonoid content of Example 1 (0.85%) was significantly higher than the simple sum of Comparative Example 1 (fermentation only, 0.48%) and Comparative Example 2 (enzymatic hydrolysis only, 0.18%) (0.66%), with a synergistic effect coefficient of 1.29 times. Example 2 (0.66%) and Example 3 (0.52%) were also higher than their respective comparative examples. This indicates that the synergistic process of the present invention not only promotes the release and transformation of saponins and polysaccharides, but also promotes the dissolution and enrichment of flavonoid components, and the enrichment patterns of the three types of components are consistent.

[0122] The strain of this invention also showed the best performance in flavonoid enrichment, with the flavonoid content of the strain being 2.2-4.5 times that of other strains. The specific contribution of the strain was further verified in the enrichment of the third type of active ingredients.

[0123] 4. Bioavailability test The bioavailability of the Ophiopogon japonicus powder obtained in Examples 1-3 and Comparative Examples 1-8 was determined using the following methods. The results are shown in Table 4.

[0124] (1) Prepare fresh culture medium: DMEM cell culture medium (Gibco, USA): fetal bovine serum (Gibco, USA): penicillin antibiotic (Guangzhou Ruishu Biotechnology Co., Ltd.) = 9:1:0.1. When CaCo-2 (ATCC cell bank) cells have grown and fused to 80-90%, digest the cells, gently pipette to mix, and count them. Seed 0.5 mL of cell suspension (2×10⁵) onto the wells of a 12-well Transwell plate and add 1.5 mL of complete culture medium.

[0125] (2) The culture medium was replaced daily with fresh culture medium to obtain differentiated monolayers. On days 11, 13, 15, 17, 19 and 21, the integrity of the Caco-2 cell monolayer was measured by epithelial resistance (TEER) using a Millicell-ERS electrode.

[0126] (3) When the TEER value reaches 400Ω / cm on the 21st day 2 At this point, further transport experiments were conducted using a single-layer membrane.

[0127] (4) Wash the filter holder twice with Hanks balanced salt solution (HBSS, pH 6.8, 37°C) to remove residual culture medium. Add 1 mL of substrate (2 mg / mL) dissolved in HBSS to the upper chamber and 1 mL of HBSS (37°C) to the outer chamber of the substrate.

[0128] (5) At 30, 60, 90 and 120 min, 0.6 mL of sample was collected from the lower chamber, and the same volume (0.6 mL) of HBSS was added to the receiving chamber at the same time.

[0129] (6) Liquid phase detection of the content of the sample permeating into the lower chamber at different time periods, and use permeability to evaluate bioavailability.

[0130] Table 4. Bioavailability Test Results

[0131] The results showed that the bioavailability of the Ophiopogon japonicus powder described in this invention was 9.78%-19.54%, which was significantly higher than that of other comparative examples. This indicates that the Ophiopogon japonicus powder described in this invention has higher bioavailability than Ophiopogon japonicus powder fermented by other bacteria.

[0132] 5. RIN-14B Cytotoxicity Assay The RIN-14B cytotoxicity of the Ophiopogon japonicus powders obtained in Examples 1-3 and Comparative Examples 1-15 was determined using the following methods. The results are shown in Table 5.

[0133] Prepare fresh culture medium: 1640 basal medium (Gibco, USA): fetal bovine serum (Gibco, USA): double antibiotics (Guangzhou Ruishu Biotechnology Co., Ltd.) = 9:1:0.1.

[0134] When RIN-14B cells (rat insulinoma cells, ATCC cell bank) reached 80-90% confluence, the cells were digested, gently pipetted to mix, and then counted. Cells were seeded at a density of 1 × 10⁴ cells / well in 96-well plates, with 0.1 mL of cell suspension per well. After 4 hours of cell attachment, the culture supernatant was discarded, and complete culture medium containing different concentrations of Ophiopogon japonicus powder (0.5, 1, 2, 4, 6 mg / mL) was added, followed by incubation for another 24 hours. Cell viability was then assessed according to the CCK-8 assay kit instructions.

[0135] Table 5. Results of RIN-14B cytotoxicity assay

[0136] As shown in Table 5, the maximum safe concentration of the Ophiopogon japonicus powder of this invention is 4 mg / mL, which is 4 times that of the traditional water extraction (1 mg / mL). Although the saponin content of the product of Comparative Example 2, which was only enzymatically hydrolyzed, was low, its cytotoxicity (maximum safe concentration 2 mg / mL) was better than that of the traditional water extraction (1 mg / mL), indicating that enzymatic hydrolysis itself has a certain detoxification effect. The saponin content of the product of Comparative Example 1, which was only fermented, was not only higher than that of Comparative Example 2, which was only enzymatically hydrolyzed, but its maximum safe concentration also reached 4 mg / mL. This indicates that fermentation, while breaking down cell walls and releasing effective components, also has a more significant detoxification and efficacy-enhancing effect.

[0137] Although the contents of saponins, small molecule polysaccharides and flavonoids in Comparative Example 12 are almost the same as those in Example 1, its maximum safe concentration is only half that of Example 1. This shows that the safety improvement of the present invention also comes from the unique material form formed by the specific process, rather than a simple superposition of components.

[0138] 6. Effect of the sample on serotonin (5-HT) release in RIN-14B cells 5-HT is an important monoamine neurotransmitter in the central nervous system, often referred to as the "happy hormone," and also participates in sleep-wake regulation. RIN-14B is a rat islet tumor-derived cell line that synthesizes, stores, and secretes 5-HT, with a secretion mechanism similar to that of 5-HTergic neurons in vivo. It can be used to evaluate the anxiety-relieving and sleep-improving effects of samples.

[0139] The effects of the Ophiopogon japonicus powder obtained in Examples 1-3 and Comparative Examples 1-15 on the release of serotonin (5-HT) from RIN-14B cells were determined using the following methods. The results are shown in Table 6.

[0140] Experimental methods: When RIN-14B (rat insulinoma cells, ATCC cell bank) cells reached 80-90% confluence, the cells were digested, gently pipetted to mix, and then counted. Cells were seeded in 24-well plates at a density of 30 × 10⁴ cells / well and allowed to adhere overnight.

[0141] The experiment was divided into a normal group and a sample group. Cells in each group were washed with a washing solution containing fluoxetine and BSA to inhibit the reuptake of 5-HT. After the sample group was treated with the sample solution for 24 hours, the supernatant was taken and the 5-HT content was tested using an ELISA kit.

[0142] Table 6. Serotonin promotion rate in RIN-14B cells

[0143] As shown in Table 6, the 5-HT promotion rate of Example 1 (56.8%) is 6.6 times that of Comparative Example 3 (8.6%) using traditional water extraction, 1.54 times that of Comparative Example 1 (36.9%) using only fermentation, and 6.06 times that of Comparative Example 2 (9.38%) using only enzymatic hydrolysis. The 5-HT promotion rate of Comparative Example 9 (flavonoid deficiency) is 22.50%, that of Comparative Example 10 (polysaccharide deficiency) is 14.5%, and that of Comparative Example 11 (saponin deficiency) is 19.91%, all significantly lower than that of Example 1. This indicates that in the in-situ synergistic system of the present invention, saponins, polysaccharides, and flavonoids are all indispensable; the absence of any one of them will lead to the collapse of the synergistic effect.

[0144] The results of Comparative Example 12 (38.51%) and Comparative Example 13 (25.60%) of the three-type physical compound group were much lower than those of Example 1. This shows that the simple physical mixing of the three types of components cannot produce a synergistic effect. Even if the types and proportions of components are exactly the same, only through the specific fermentation-enzymatic hydrolysis in-situ process of the present invention can a substance with synergistic effects be formed.

[0145] The exogenous polysaccharide substitution group (Comparative Example 14-15) showed the worst effect, indicating that known anti-anxiety polysaccharides from other sources cannot form a synergistic system with the saponins and flavonoids of this invention, further proving that the material basis of the polysaccharides of this invention is unique.

[0146] 7. Sedative and hypnotic effects of zebrafish on insomnia The sedative-hypnotic effects of Ophiopogon japonicus powder obtained in Examples 1-3 and Comparative Examples 1-15 on zebrafish insomnia were determined using the following methods. The results are shown in Table 7.

[0147] 120 hpf embryos were selected and set up as blank control group, model control group, positive control group (melatonin, 1 μM) and sample group (2 g / L). The blank control group and model control group were given embryo buffer water, while the positive control group and sample group were given the corresponding concentration of drug solution. The embryos were incubated in a constant temperature incubator at (28.5±0.5)℃ for 1 day.

[0148] Embryos cultured with the drug were selected into 96-well plates according to their groups, and the drug solution was replaced (100 μL per well). The model control group was given the modeling agent (PTZ, 5 mM), while the positive control group and the sample group were given the corresponding concentrations of the modeling agent mixture. The samples were then transferred to a zebrafish behavioral analysis system, and the movement trajectory of zebrafish under dark conditions was recorded using the device's built-in infrared tracking software for 1 hour. The reduction rate of the total movement distance of the zebrafish was used to indicate the sedative-hypnotic effect of the samples.

[0149] Table 7. Sedative-hypnotic effects of zebrafish on insomnia

[0150] As shown in Table 7, the percentages of comparative examples 9-11 (10.82%-17.2%), comparative example 12 (27.22%), comparative example 13 (19.53%), and comparative example 14-15 (13.01%-14.1%) were all significantly lower than those of example 1 (42.5%).

[0151] This demonstrates that the material basis of the efficacy of this invention exhibits a stable and consistent effect in both cell models (5-HT) and whole animal models (zebrafish), verifying the reliability of the results. Furthermore, from the material basis (saponins + polysaccharides + flavonoids) to the absorption pathway (bioavailability), to safety (cytotoxicity), to the target of action (5-HT), to the biological effect (sedation), seven experiments form a complete chain of evidence, consistently pointing to the same conclusion: the inventiveness and irreplaceability of this invention stem from the complete synergistic system created by the specific process, rather than any single component or simple combination.

[0152] Based on the efficacy experiment results above, it can be seen that the content of small molecule polysaccharides in Comparative Example 1 (fermentation only) was 5.37%, and the proportion of small molecule polysaccharides was 37%. The content of small molecule polysaccharides in Comparative Example 2 (enzymatic hydrolysis only) was only 2.29%, and the proportion of small molecule polysaccharides was only 16%, both significantly lower than the content (14.14%) and proportion (70%) of small molecule polysaccharides in Example 1. The saponin content (1.98%) in Example 1 was significantly higher than the simple sum (0.61%) of Comparative Example 1 (fermentation only, 0.47%) and Comparative Example 2 (enzymatic hydrolysis only, 0.14%), with a synergistic effect coefficient of 3.25 times. The total flavonoid content (0.85%) in Example 1 was significantly higher than the simple sum (0.66%) of Comparative Example 1 (fermentation only, 0.48%) and Comparative Example 2 (enzymatic hydrolysis only, 0.18%), with a synergistic effect coefficient of 1.29 times. This invention achieves the simultaneous enrichment of three types of components: saponins, small molecule active polysaccharides, and flavonoids.

[0153] Comparing Example 1 with Comparative Examples 9-11 reveals that the efficacy of Example 1 is significantly lower when any of the saponins, polysaccharides, or flavonoids are absent. This indicates that all three types of components are indispensable in the in-situ synergistic system of this invention. Furthermore, even when all three types of components are physically mixed in the same proportions, the efficacy of Comparative Example 12 is still significantly lower than that of Example 1. This demonstrates that the efficacy of this invention does not derive from the "presence" of the three types of components, but rather from their "in-situ coexistence" under a specific fermentation-enzymatic hydrolysis process. This coexistence cannot be replicated by physical mixing; rather, it is an unreplicable multi-component synergistic system produced by a specific fermentation-enzymatic hydrolysis process.

[0154] Comparing Example 1 with Comparative Examples 13-15, it can be seen that even if pure Rusco saponin, Ganoderma lucidum polysaccharide, or Tremella fuciformis polysaccharide are used to replace the Ophiopogon japonicus saponin or Ophiopogon japonicus small molecule active polysaccharide unique to this invention, physical mixing is still ineffective. This shows that known anti-anxiety polysaccharides from other sources cannot replace the Ophiopogon japonicus small molecule active polysaccharide generated in situ in this invention, and the material basis of the polysaccharide of this invention is unique.

[0155] Under the same conditions, the strain of this invention has a saponin yield 5-12 times higher than other strains, a small molecule polysaccharide content 2.8-4.7 times higher than other strains, and a flavonoid content 2.2-4.5 times higher than other strains. It has achieved synchronous advantages in all three dimensions, and the strain of this invention is irreplaceable in the fermentation system of Ophiopogon japonicus.

[0156] This invention generates systematic synergistic gains from five dimensions: material basis (the ternary synergy of saponins, small molecule polysaccharides, and flavonoids), absorption pathway (bioavailability), safety (cytotoxicity), target of action (5-HT), and biological effects (sedation).

[0157] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention shall fall within the protection scope of the present invention.

Claims

1. A type of Ophiopogon japonicus powder for relieving anxiety and improving sleep, characterized in that, The Ophiopogon japonicus powder is produced by fermentation of Ophiopogon japonicus with Lactobacillus plantarum GDMCC No. 64374 and enzymatic hydrolysis with pullulanase.

2. A method for preparing Ophiopogon japonicus powder for relieving anxiety and improving sleep as described in claim 1, characterized in that, Includes the following steps: S1: Take fresh Ophiopogon japonicus, freeze-dry it, grind it into powder, and sift it to obtain Ophiopogon japonicus powder; S2: Disperse the Ophiopogon japonicus powder evenly in water, heat and extract, then cool to obtain the fermentation substrate; S3: Inoculate the fermentation substrate with Bacillus plantarum seed liquid for fermentation culture, sterilize after the reaction is completed, and obtain Ophiopogon japonicus fermentation broth; S4: Add pullulanase to the fermentation broth of Ophiopogon japonicus for enzymatic hydrolysis. After the reaction is completed, the enzyme is inactivated to obtain Ophiopogon japonicus hydrolysate. S5: Filter the Ophiopogon japonicus enzymatic hydrolysate, discard the precipitate, collect the supernatant, dry it, and obtain the Ophiopogon japonicus powder.

3. The method for preparing Ophiopogon japonicus powder for relieving anxiety and improving sleep according to claim 2, characterized in that, The preparation steps of the *Lactobacillus plantarum* seed solution include: (1) Activation: Lactobacillus plantarum GDMCC No.64374 was streaked on MRS solid medium and cultured at 35℃ for 48 hours under anaerobic conditions; (2) Liquid culture: Pick a single colony from the plate, inoculate it into MRS liquid medium, and culture it under anaerobic conditions at 35°C for 48 hours to obtain the bacterial solution; (3) Subculture: Take 10% of the bacterial culture and add it to MRS liquid culture medium. Culture at 35°C for 12 hours under anaerobic conditions to obtain the seed culture of Lactobacillus plantarum.

4. The method for preparing Ophiopogon japonicus powder for relieving anxiety and improving sleep according to claim 2, characterized in that, In step S2, the mass ratio of the Ophiopogon japonicus powder to the water is 1:100-5:

100.

5. The method for preparing Ophiopogon japonicus powder for relieving anxiety and improving sleep according to claim 2, characterized in that, In step S3, the volume ratio of the *Lactobacillus plantarum* seed culture to the fermentation substrate is 1:100-5:

100.

6. The method for preparing Ophiopogon japonicus powder for relieving anxiety and improving sleep according to claim 2, characterized in that, The fermentation culture temperature is 33-37℃, and the time is 10-15 hours.

7. The method for preparing Ophiopogon japonicus powder for relieving anxiety and improving sleep according to claim 2, characterized in that, The extraction temperature is 70-90℃, and the time is 1-3 hours.

8. The method for preparing Ophiopogon japonicus powder for relieving anxiety and improving sleep according to claim 2, characterized in that, In step S4, the amount of pullulanase used is 0.1-0.5% w / w of the Ophiopogon japonicus fermentation broth.

9. The method for preparing Ophiopogon japonicus powder for relieving anxiety and improving sleep according to claim 2, characterized in that, The enzymatic hydrolysis treatment was carried out at a temperature of 55-65℃, a pH of 4.5-6, a stirring speed of 150-250 rpm, and a time of 5-7 hours.

10. The use of Ophiopogon japonicus powder as described in claim 1 for relieving anxiety and improving sleep in the preparation of food, health products or medicines for relieving anxiety and improving sleep.

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