A ginseng mulberry leaf composition for reducing pressure, relieving anxiety and improving sleep, and a preparation method and application thereof

Abstract

The present application relates to a ginseng mulberry leaf composition for reducing pressure, relieving anxiety and improving sleep, and a preparation method and application thereof, wherein the composition is prepared by a three-stage synergistic process of ginseng and mulberry leaf through stepwise temperature control processing, enzymatic hydrolysis and fermentation. Compared with the prior art, the ginseng-mulberry leaf composition prepared by the present application is significantly superior to various control processes in terms of reducing pressure, relieving anxiety and improving sleep. The mechanism of action involves promoting 5-HT release, activating hippocampal neuron Reelin / Dab-1-GSK-3beta / beta-catenin signal axis to repair synaptic plasticity, and regulating basal forebrain adenosine signal to promote deep sleep, and can be used for preparing health care products or drugs for reducing stress, relieving anxiety and / or improving sleep disorders.

Description

Technical Field

[0001] This invention relates to the technical field of oral products, specifically to a ginseng and mulberry leaf composition for stress reduction, anxiety relief, and sleep improvement, as well as its preparation method and application. Background Technology

[0002] With the fast pace and increasing pressure of modern society, anxiety and sleep disorders have become global health problems. Chronic sleep deprivation not only affects mental state but is also significantly associated with the risk of cardiovascular disease and weakened immune function.

[0003] Current clinical interventions for stress reduction, anxiety relief, and sleep improvement have significant limitations. While benzodiazepines are fast-acting, they have side effects such as dependence, drug tolerance, daytime sleepiness, cognitive impairment, and liver and kidney damage. Melatonin alone is only suitable for circadian rhythm sleep-wake disorders, and long-term use may inhibit the body's own melatonin secretion. Although natural plant extracts have relatively high safety, their low extraction rate of large molecular active substances and limited bioavailability make it difficult to achieve efficient multi-target regulation.

[0004] Ginseng and mulberry leaves are common herbal pairs in traditional Chinese medicine, frequently used in classic prescriptions. However, existing ginseng or mulberry leaf extraction technologies have two main shortcomings: First, the extraction efficiency of active substances is low. Traditional extraction methods (such as water decoction and alcohol extraction) mainly rely on the thermodynamic leaching of solvents. Due to the physical barrier of plant cell walls, it is difficult to achieve efficient release of active ingredients. In addition, the bioavailability of macromolecular active ingredients is low, which limits their application effects in stress reduction, anxiety relief and sleep improvement.

[0005] Secondly, both ginseng and mulberry leaves are common medicinal and edible ingredients in traditional Chinese medicine, and modern research shows that they are complementary in terms of their active ingredient profiles. However, existing ginseng and mulberry leaf processing technologies rely on relatively simple extraction methods, and biological treatments such as fermentation or enzymatic hydrolysis are often used independently, lacking organic integration between processing steps. Furthermore, ginseng and mulberry leaf processing are typically carried out separately, and there is no mature solution for synergistic processing of the two within the same system to improve anxiety and sleep disorders. Therefore, developing a preparation method that enables the synergistic processing of ginseng and mulberry leaves while leveraging their complementary effects has significant application value. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a ginseng and mulberry leaf composition for stress reduction, anxiety relief, and sleep improvement, as well as its preparation method and application.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a method for preparing a ginseng and mulberry leaf composition for stress reduction, anxiety relief, and sleep improvement, comprising the following steps: S1. Grind dried ginseng and mulberry leaves into powder and sieve them separately. Mix the ginseng powder and mulberry leaf powder and perform a stepped temperature-controlled processing to obtain a processed mixture. The specific steps of the stepped temperature-controlled processing are as follows: steam the mixed ginseng powder and mulberry leaf powder at a low temperature of 40-60℃, and then raise the temperature to a high temperature of 100-121℃ for steaming. S2. Add water to the prepared mixture and stir evenly. Add phytase for enzymatic hydrolysis. After inactivation of the enzyme, the enzymatic hydrolysis product is obtained. S3. Inoculate the enzymatic hydrolysis product with Staphylococcus xylose for fermentation, sterilize and dry to obtain ginseng and mulberry leaf composition.

[0008] In the step-temperature controlled processing, the low-temperature stage is conducive to activating the endogenous enzyme system that may exist in the raw materials, promoting the adjustment of the internal state of the materials; the high-temperature stage causes cell wall rupture, releasing intracellular substances into the processing system. The step-temperature controlled processing and the subsequent exogenous addition of phytase complement each other in function: the step-temperature controlled processing focuses on the initial cell wall destruction and preliminary release, while phytase further breaks down the phytic acid barrier, providing sufficient substrate for subsequent fermentation.

[0009] The composition is prepared by step-controlled temperature processing of ginseng and mulberry leaves, phytase hydrolysis, and Staphylococcus xylosus fermentation. This invention is the first to introduce Staphylococcus xylosus into the ginseng-mulberry leaf co-fermentation system and has demonstrated its specific advantages in improving anxiety and sleep, realizing in-situ synergy of processing, hydrolysis, and fermentation in the same system.

[0010] Preferably, in step S1, the weight ratio of ginseng powder to mulberry leaf powder is (1-5):(1-5), more preferably 1:1; the low-temperature steaming time is 2-4 hours, the high-temperature steaming time is 0.5-2 hours, and after being taken out, it is dried until the moisture content is less than 10%.

[0011] Preferably, in step S2, the mass ratio of the prepared mixture to the water is 1:(10-50); the amount of phytase used is 0.5-2% of the weight of the prepared mixture; the enzymatic hydrolysis temperature is 45-55℃, the pH is 4.5-5.5, the stirring speed is 50-100 r / min, and the time is 4-6 h; the enzyme inactivation temperature is 90-100℃, and the time is 10-20 min.

[0012] Preferably, the preparation steps of the Staphylococcus xylose seed culture include: streaking Staphylococcus xylose onto a nutrient agar solid medium and culturing it at 30-37℃ for 24-48 hours; picking a single colony and inoculating it into a nutrient broth liquid medium and culturing it at 30-37℃ on a shaker for 12-24 hours to obtain the Staphylococcus xylose seed culture.

[0013] Preferably, in step S3, the inoculum amount of Staphylococcus xylose is 1-5% of the volume of the enzymatic hydrolysis product; the fermentation temperature is 30-37℃, the pH is 6.5-7.5, and the time is 12-24h; the sterilization temperature is 95-100℃, and the time is 15-20min.

[0014] Secondly, the present invention provides a ginseng and mulberry leaf composition for stress reduction, anxiety relief, and improved sleep, which is prepared by the method described in the first aspect for preparing the ginseng and mulberry leaf composition for stress reduction, anxiety relief, and improved sleep.

[0015] Thirdly, the present invention provides the use of the ginseng and mulberry leaf composition of the second aspect in the preparation of health products or pharmaceuticals for stress reduction, anxiety relief, and / or sleep disorder improvement. The ginseng and mulberry leaf composition reduces stress, relieves anxiety, and / or improves sleep disorders through at least one of the following three mechanisms: (I) Promotes 5-HT release and regulates neurotransmitter balance; (II) Activate the Reelin / Dab-1-GSK-3β / β-catenin signaling axis of hippocampal neurons, promote Dab1 phosphorylation, inhibit GSK-3β activity, stabilize β-catenin protein levels, repair stress-damaged synaptic plasticity, and restore synaptic homeostasis. (III) Regulates adenosine signaling in the basal forebrain, promotes deep sleep drive, and improves sleep quality.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention is the first to combine stepped temperature-controlled processing, phytase hydrolysis, and Staphylococcus xylose fermentation, achieving "in-situ synergistic" enhancement of medicinal materials through a three-stage synergistic process. Compared with existing technologies, the ginseng-mulberry leaf composition prepared by this invention is significantly more effective than various control processes in improving anxiety-like behaviors and prolonging sleep time. Its mechanism of action involves promoting 5-HT release, activating the Reelin / Dab-1-GSK-3β / β-catenin signaling axis in hippocampal neurons to repair synaptic plasticity, and regulating adenosine signaling in the basal forebrain to promote deep sleep. It can be used to prepare health products or medicines for stress reduction, relieving anxiety, and / or improving sleep disorders. Detailed Implementation

[0017] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0018] The raw materials used in the following examples and comparative examples are as follows: Staphylococcus xylosus: purchased from China Industrial Microbial Culture Collection Center, product code 0605K; Lactobacillus plantarum: purchased from China General Microbiological Culture Collection Center, CGMCC number 1.16089; Phytase: Purchased from Hebei Runbu Biotechnology Co., Ltd., CAS No. 37288-11-2; 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. Example 1

[0019] The preparation method of the ginseng and mulberry leaf composition includes the following steps: S1. Take dried ginseng and dried mulberry leaves, crush them separately and pass them through a 20-mesh sieve to obtain ginseng powder and mulberry leaf powder. Mix the ginseng powder and mulberry leaf powder evenly in a weight ratio of 1:1. Steam the mixed powder at 50℃ for 3 hours, then raise the temperature to 115℃ and steam for 1 hour. After taking it out, dry it until the moisture content is less than 10% to obtain the processed mixture.

[0020] S2. Add water at a mass ratio of 1:25 between the prepared mixture and water and stir evenly. Add 1.0% (w / w) phytase based on the weight of the prepared mixture, adjust the pH to 5, and enzymatically hydrolyze for 5 hours at 50°C and a stirring speed of 75 r / min. Then, heat to 95°C to inactivate the enzyme for 15 minutes to obtain the enzymatic hydrolysis product.

[0021] S3. Adjust the pH of the enzymatic hydrolysis product to 7 with food-grade alkaline solution, inoculate with Staphylococcus xylose seed liquid at a volume ratio of 3% (cell concentration OD600 is 1.5), ferment at 35℃ for 18h, heat to 100℃ for 15min for sterilization, and spray dry to obtain ginseng and mulberry leaf composition. Example 2

[0022] The preparation method of the ginseng and mulberry leaf composition includes the following steps: S1. Take dried ginseng and dried mulberry leaves, grind them separately and pass them through a 20-mesh sieve to obtain ginseng powder and mulberry leaf powder. Mix the ginseng powder and mulberry leaf powder evenly at a weight ratio of 5:1. Steam the mixed powder at 40℃ for 4 hours, then raise the temperature to 100℃ and steam for 2 hours. After taking it out, dry it until the moisture content is less than 10% to obtain the processed mixture.

[0023] S2. Add water at a mass ratio of 1:10 to the prepared mixture and stir evenly. Add 0.5% (w / w) phytase based on the weight of the prepared mixture, adjust the pH to 4.5, stir at 50 r / min, and enzymatically hydrolyze at 45℃ for 6 h. Then, heat to 90℃ to inactivate the enzyme for 20 min to obtain the enzymatic hydrolysis product.

[0024] S3. Adjust the pH of the enzymatic hydrolysis product to 7.5 with food-grade alkaline solution, inoculate with Staphylococcus xylose seed liquid at a volume ratio of 1% (cell concentration OD600 is 1.0), ferment at 30℃ for 24h, heat to 95℃ for 20min for sterilization, and spray dry to obtain ginseng and mulberry leaf composition. Example 3

[0025] The preparation method of the ginseng and mulberry leaf composition includes the following steps: S1. Take dried ginseng and dried mulberry leaves, grind them separately and pass them through a 20-mesh sieve to obtain ginseng powder and mulberry leaf powder. Mix the ginseng powder and mulberry leaf powder evenly at a weight ratio of 1:5. Steam the mixed powder at 60℃ for 2 hours, then raise the temperature to 121℃ and steam for 0.5 hours. After taking it out, dry it until the moisture content is less than 10% to obtain the processed mixture.

[0026] S2. Add water at a mass ratio of 1:50 for the prepared mixture and stir evenly. Add 2% (w / w) phytase based on the weight of the prepared mixture to the prepared mixture, adjust the pH to 5.5, stir at 100 r / min, and enzymatically hydrolyze at 55℃ for 4 hours. Then, heat to 100℃ to inactivate the enzyme for 10 minutes to obtain the enzymatic hydrolysis product.

[0027] S3. Adjust the pH of the enzymatic hydrolysis product to 6.5 with food-grade alkaline solution, inoculate the enzymatic hydrolysis product with 5% by volume of Staphylococcus xylose seed liquid (cell concentration OD600 is 2.0), ferment at 37℃ for 12h, heat to 100℃ for 20min for sterilization, and spray dry to obtain ginseng and mulberry leaf composition. Example 4

[0028] The only difference between Example 4 and Example 1 is that in step S1, the weight ratio of ginseng powder to mulberry leaf powder is 10:1. Comparative Example 1

[0029] The only difference between Comparative Example 1 and Example 1 is that the ginseng and mulberry leaf composition of Comparative Example 1 is not processed, enzymatically hydrolyzed, or fermented; it is simply prepared by decoction. The specific preparation steps are as follows: Dried ginseng and dried mulberry leaves were pulverized separately and passed through a 20-mesh sieve to obtain ginseng powder and mulberry leaf powder. The ginseng powder and mulberry leaf powder were mixed evenly at a weight ratio of 1:1. Water was added at a mass ratio of 1:25 between the mixed powder and water, and the mixture was heated and extracted in an 80℃ water bath for 3 hours. The liquid was then filtered. The residue was added to water again at the same material-liquid ratio and extracted at 80℃ for 1 hour. The liquid was then filtered. The two filtrates were combined and spray-dried to obtain the ginseng and mulberry leaf composition. Comparative Example 2

[0030] The only difference between Comparative Example 2 and Example 1 is that the ginseng and mulberry leaf composition of Comparative Example 2 only underwent processing and fermentation, without enzymatic hydrolysis. The specific steps are as follows: (1) After processing according to step S1 of Example 1, a processed mixture is obtained.

[0031] (2) Add water at a mass ratio of 1:25 to the prepared mixture and stir evenly. Adjust the pH to 7 with food-grade alkali solution. Inoculate with Staphylococcus xylose seed liquid at a volume ratio of 3% (the bacterial concentration OD600 is 1.5). Ferment at 35℃ for 18 hours, heat to 100℃ for 15 minutes for sterilization, and spray dry to obtain the ginseng and mulberry leaf composition. Comparative Example 3

[0032] The only difference between Comparative Example 3 and Example 1 is that the ginseng and mulberry leaf composition of Comparative Example 3 only underwent processing and enzymatic hydrolysis, without fermentation. The specific steps are as follows: (1) After processing according to step S1 of Example 1, a processed mixture is obtained.

[0033] (2) Add water at a mass ratio of 1:25 to the prepared mixture and stir evenly. Add 1.0% (w / w) phytase based on the weight of the prepared mixture, adjust the pH to 5, and enzymatically hydrolyze for 5 hours at 50°C and a stirring speed of 75 r / min. Then, heat to 95°C to inactivate the enzyme for 15 minutes and spray dry to obtain the ginseng and mulberry leaf composition. Comparative Example 4

[0034] The only difference between Comparative Example 4 and Example 1 is that the ginseng and mulberry leaf composition of Comparative Example 4 only underwent enzymatic hydrolysis and fermentation, without any processing. The specific steps are as follows: (1) Take dried ginseng and dried mulberry leaves, crush them separately and pass them through a 20-mesh sieve to obtain ginseng powder and mulberry leaf powder. Mix the ginseng powder and mulberry leaf powder evenly in a weight ratio of 1:1.

[0035] (2) Add water at a mass ratio of 1:25 between the mixed powder and water and stir evenly. Add 1.0% (w / w) phytase based on the weight of the mixed powder, adjust the pH to 5, and enzymatically hydrolyze for 5 hours at 50°C and a stirring speed of 75 r / min. Then, raise the temperature to 95°C to inactivate the enzyme for 15 minutes to obtain the enzymatic hydrolysis product.

[0036] (3) The pH of the enzymatic hydrolysis product was adjusted to 7 with food-grade alkaline solution, and Staphylococcus xylose seed liquid was inoculated at a volume ratio of 3% (the bacterial concentration OD600 was 1.5). The mixture was fermented at 35℃ for 18 hours, then heated to 100℃ for 15 minutes for sterilization, and spray-dried to obtain the ginseng and mulberry leaf composition. Comparative Example 5

[0037] The only difference between Comparative Example 5 and Example 1 is that the ginseng and mulberry leaf composition of Comparative Example 5 is processed using conventional constant temperature processing, without step-controlled temperature processing. The specific steps are as follows: (1) Take dried ginseng and dried mulberry leaves, crush them separately and pass them through a 20-mesh sieve, and mix them evenly at a weight ratio of 1:1. Steam the mixed powder at 100℃ for 4 hours (one-time high-temperature steaming). After taking it out, dry it until the moisture content is less than 10% to obtain the processed mixture.

[0038] (2) The remaining steps are the same as steps S2 and S3 in Example 1. Comparative Example 6

[0039] The only difference between Comparative Example 6 and Example 1 is that Lactobacillus plantarum is used instead of Staphylococcus xylose in step S3. Comparative Example 7

[0040] The only difference between Comparative Example 7 and Example 1 is that mulberry leaves are not added to the raw materials in the composition; only ginseng is processed. The specific steps are as follows: (1) Take dried ginseng, crush it and pass it through a 20-mesh sieve. Then, carry out step-by-step temperature-controlled processing under the same conditions as step S1 in Example 1 (steam at 50°C for 3 hours, then raise the temperature to 115°C and steam for 1 hour), and dry it until the moisture content is less than 10%.

[0041] (2) Add water at a mass ratio of 1:25 between the processed ginseng powder and water and stir evenly. Add 1.0% (w / w) phytase based on the weight of the processed powder, adjust the pH to 5, and enzymatically hydrolyze for 5 hours at 50℃ and a stirring speed of 75r / min. Then, raise the temperature to 95℃ to inactivate the enzyme for 15 minutes.

[0042] (3) The pH of the enzymatic hydrolysis product was adjusted to 7 with food-grade alkaline solution, and Staphylococcus xylose seed liquid was inoculated at a volume ratio of 3% (the bacterial concentration OD600 was 1.5). The mixture was fermented at 35℃ for 18 hours, then heated to 100℃ for 15 minutes for sterilization, and spray-dried to obtain the ginseng composition (excluding mulberry leaves). Comparative Example 8

[0043] The only difference between Comparative Example 8 and Example 1 is that ginseng is not added to the raw materials in the composition; only mulberry leaves are processed. The specific steps are as follows: (1) Take dried mulberry leaves, crush them and pass them through a 20-mesh sieve. Then, process them under the same conditions as step S1 in Example 1 (steam at 50°C for 3 hours, then raise the temperature to 115°C and steam for 1 hour) and dry them until the moisture content is less than 10%.

[0044] (2) Add water at a mass ratio of 1:25 between processed mulberry leaf powder and water and stir evenly. Add 1.0% (w / w) phytase based on the weight of the processed powder, adjust the pH to 5, and enzymatically hydrolyze for 5 hours at 50℃ and stirring speed of 75r / min. Then, raise the temperature to 95℃ to inactivate the enzyme for 15 minutes.

[0045] (3) The pH of the enzymatic hydrolysis product was adjusted to 7 with food-grade alkaline solution, and Staphylococcus xylose seed liquid was inoculated at a volume ratio of 3% (the bacterial concentration OD600 was 1.5). The mixture was fermented at 35℃ for 18 hours, then heated to 100℃ for 15 minutes for sterilization, and spray-dried to obtain the mulberry leaf composition (excluding ginseng). Comparative Example 9

[0046] The only difference between Comparative Example 9 and Example 1 is that the same three-stage synergistic process as in Example 1 is used, but the ginseng and mulberry leaves are processed separately and then mixed in a 1:1 ratio. The specific steps are as follows: (1) Take dried ginseng and obtain ginseng powder by following the complete three-stage synergistic process of Example 1 (step-controlled temperature processing → phytase hydrolysis → Staphylococcus xylose fermentation).

[0047] (2) Take dried mulberry leaves and follow the complete three-stage synergistic process of Example 1 (step-controlled temperature processing → phytase hydrolysis → Staphylococcus xylose fermentation) to obtain mulberry leaf powder.

[0048] (3) The two are physically mixed at a weight ratio of 1:1 to obtain the composition of Comparative Example 9. Comparative Example 10

[0049] The only difference between Comparative Example 10 and Example 1 is that mulberry leaves are replaced with chrysanthemums. Comparative Example 11

[0050] The only difference between Comparative Example 11 and Example 1 is that ginseng is replaced with Panax notoginseng.

[0051] Test Example 1: Effect of the sample on the release of serotonin (5-HT) in RIN-14B cells 5-HT is an important monoamine neurotransmitter in the central nervous system, known as the "happy hormone," and is closely related to stress and anxiety, while also participating in the regulation of the sleep-wake rhythm. RIN-14B cells have the function of synthesizing, storing, and secreting 5-HT, and their secretion mechanism is similar to that of 5-HTergic neurons in vivo, which can be used to evaluate the effects of samples on reducing stress, alleviating anxiety, and improving sleep.

[0052] The effect of each composition on serotonin (5-HT) release was detected using the RIN-14B (rat insulinoma cells, ATCC) cell model. Cells were digested and counted when they reached 80-90% confluence, and then analyzed at 30 × 10⁻⁶ cells / cells. 4 Cells were seeded at a density of [number] cells / well in 24-well plates and allowed to adhere overnight. The experiment included a blank control group and a sample group. Cells in each group were washed with a wash buffer containing fluoxetine (10 μM) and BSA to inhibit 5-HT reuptake. Subsequently, the sample group was treated with the compositions of Examples 1-4 and the comparative examples (prepared with PBS) at a final concentration of 1 mg / mL. After 4 hours of treatment, the supernatant was collected, and the 5-HT content was detected using an ELISA kit. The results are shown in Table 1.

[0053] Table 1. Cellular serotonin promotion rate

[0054] As shown in Table 1, all three groups of Examples 1-3 significantly promoted the release of 5-HT from RIN-14B cells (p<0.05), with Example 1 showing the highest 5-HT promotion rate. The 5-HT promotion rates of Comparative Example 7 (single ginseng) and Comparative Example 8 (single mulberry leaf) were significantly lower than those of Example 1, while the promotion rate of Example 1 was significantly higher than the theoretical sum of the two, indicating that the combination of ginseng and mulberry leaf also produced a significant synergistic effect in promoting 5-HT release.

[0055] Test Example 2: In vitro stress reduction and anxiety relief experiments of various compositions 1. Mouse elevated cruciate maze test To verify the stress and anxiety-relieving effect of the composition of this invention, SPF-grade Kunming mice (half male and half female, 18-22g), 10 mice per group, were randomly divided into groups. A blank control group (physiological saline), a positive control group (diazepam, 0.5mg / kg), groups of Examples 1-3, and comparative examples 1-11 were established. Each group was administered the medication by gavage once daily at a volume of 0.1mL / 10g body weight. The concentration of the gavage solution for each composition group was 250mg / mL (prepared with physiological saline), for 7 consecutive days.

[0056] Thirty minutes after the last administration, the mice were placed in an elevated cruciate maze (open arm / closed arm) to induce anxiety and stress responses. The time spent in the open arm and the number of times the mice entered the open arm were recorded within 5 minutes, and the percentage of time spent in the open arm was calculated. The results are shown in Table 2.

[0057] Table 2 Results of the elevated cross maze experiment

[0058] As shown in Table 2, the open arm residence time in Example 1 reached 51.5%, close to the 53.8% of the positive control group (diazepam). Comparative Example 7 (ginseng) had 22.2%, Comparative Example 8 (mulberry leaf) had 26.5%, and Comparative Example 9 (treated separately and then mixed) had 35.5%, all significantly lower than the 51.5% of Example 1. Based on simple summation, if ginseng and mulberry leaf only had an additive effect, the expected value would be (22.2% + 26.5%) / 2 ≈ 24.35%, while the actual value of 51.5% in Example 1 far exceeded this expected value, proving that the combination of ginseng and mulberry leaf produced a significant "1+1>2" synergistic effect. In Example 4, after changing the mixing ratio to 10:1, the open arm residence time decreased to 38.2%, further demonstrating the specificity of the optimal ratio formed by co-processing. The effects of Comparative Examples 10 and 11 were significantly lower than those of Example 1, indicating that replacing mulberry leaves with chrysanthemum, which has similar efficacy, or replacing ginseng with Panax notoginseng, a closely related herb, could not achieve the synergistic effect of the composition of the present invention, demonstrating the specificity of the selection of raw materials in the present invention. The above results indicate that the ginseng and mulberry leaf composition of the present invention can significantly reduce stress avoidance behavior in mice in a suspended open environment, confirming its clear stress-reducing and anxiety-relieving effects from a behavioral perspective.

[0059] 2. Sodium pentobarbital synergistic sleep experiment Thirty minutes after the last administration, sodium pentobarbital (35 mg / kg, suprathreshold dose) was administered intraperitoneally, and the total sleep duration (time from the disappearance to the recovery of the righting reflex) was recorded. The results are shown in Table 3.

[0060] Table 3 Results of the sodium pentobarbital-assisted sleep experiment

[0061] As shown in Table 3, the sleep time of Example 1 reached 97 minutes, which was significantly improved compared with the blank control group. The sleep time of Comparative Example 9 (treated separately and then mixed) was 77 minutes, and the simple summation average of Comparative Examples 7 and 8 was 61 minutes, which was much lower than the 97 minutes of Example 1, further demonstrating that ginseng and mulberry leaf compound produced a significant synergistic effect.

[0062] 3. Biomarker detection and analysis in mice. One hour after the last administration, the mice in each group were tested for the following biomarkers to verify the mechanism of action of the composition of the present invention in repairing synaptic plasticity through the Reelin / Dab-1-GSK-3β / β-catenin signaling axis and promoting deep sleep through basal forebrain adenosine signaling.

[0063] (1) Reelin / Dab-1-GSK-3β / β-catenin signal axis related indicators (synaptic plasticity repair) The entire brain was harvested after decapitation, and the hippocampus was isolated. Western blot was used to detect Dab1 phosphorylation levels (p-Dab1 / Dab1 ratio), GSK-3β phosphorylation levels (p-GSK-3β(Ser9) / GSK-3β ratio, with Ser9 phosphorylation in an inactivated form), and β-catenin protein expression levels in the hippocampus. Golgi staining was used to detect dendritic spine density in pyramidal neurons in the CA1 region of the hippocampus to assess synaptic structure and plasticity changes. The results are shown in Table 4.

[0064] Table 4. Results of Reelin / Dab-1-GSK-3β / β-catenin signal axis correlation indicators.

[0065] Table 4 shows that, compared with the blank control group, the p-Dab1 / Dab1 ratio, p-GSK-3β(Ser9) / GSK-3β ratio, and β-catenin protein expression levels in the hippocampus of the Example 1 group were significantly increased, and the dendritic spine density in the CA1 region of the hippocampus was significantly increased. The positive control group (diazepam) showed no significant difference from the blank control group in the above indicators, indicating that although benzodiazepines can provide rapid sedation, they do not directly repair hippocampal neuronal synaptic structural damage. The composition of this invention can effectively activate the Reelin / Dab-1-GSK-3β / β-catenin signaling axis—by promoting Dab1 phosphorylation to initiate upstream signals, thereby inducing GSK-3β inactivation (increased Ser9 phosphorylation), reducing its degradation of β-catenin, and ultimately stabilizing and upregulating β-catenin protein levels, promoting the expression of genes related to synaptic plasticity.

[0066] Although the indicators of Comparative Example 7 (ginseng treated alone) and Comparative Example 8 (mulberry leaf treated alone) were improved compared with the blank control group, they were still significantly lower than those of Example 1. Similarly, the indicators of Comparative Example 9 (treated separately and then mixed) were also lower than those of Example 1, further demonstrating that ginseng and mulberry leaf must be processed together in the same system to achieve the best activation effect on this signal axis. These results indicate that the ginseng and mulberry leaf composition of the present invention effectively promotes the repair of synaptic plasticity in stress-induced damage and restores synaptic homeostasis by activating the Reelin / Dab-1-GSK-3β / β-catenin signal axis of hippocampal neurons. This is highly consistent at the mechanistic level with its anti-anxiety effect shown in the elevated cross maze experiment.

[0067] (2) Basal forebrain adenosine signaling related indicators (driven by deep sleep) The entire brain was harvested after decapitation, and the basal forebrain tissue was separated. The adenosine content in the basal forebrain tissue was detected using ELISA. The results are shown in Table 5.

[0068] Table 5. Detection results of adenosine signal-related indicators in the basal forebrain.

[0069] As shown in Table 5, compared with the blank control group, the adenosine content in the basal forebrain tissue of Example 1 group was significantly increased. However, the adenosine content in the positive control group (diazepam) was not significantly different from that in the blank control group. This explains why benzodiazepines, while prolonging total sleep time, are less effective in truly improving deep sleep quality. Adenosine is a key endogenous molecule driving deep non-rapid eye movement (NREM) sleep, and increased adenosine levels in the basal forebrain can directly promote the initiation and maintenance of deep sleep. The composition of this invention, by specifically regulating basal forebrain adenosine signaling, acts at the source of driving deep sleep, achieving a sleep-aiding mechanism completely different from that of diazepam.

[0070] Although the adenosine content of Comparative Example 7 (ginseng treated alone) and Comparative Example 8 (mulberry leaf treated alone) was higher than that of the blank control group, it was significantly lower than that of Example 1. The indicators of Comparative Example 9 (treated separately and then mixed) were also lower than those of Example 1, indicating that ginseng and mulberry leaf must be processed together in the same system to achieve the best regulatory effect on adenosine signal in the basal forebrain.

[0071] The above results indicate that the ginseng and mulberry leaf composition of the present invention effectively promotes deep sleep drive by regulating basal probrain adenosine signaling, which is highly consistent with its significant effect on prolonging sleep time in the sodium pentobarbital synergistic sleep experiment at the mechanistic level.

[0072] 4. Comprehensive analysis of the experimental results of each group (1) Originality of the three-stage synergistic process: Comparative Example 5 (conventional constant temperature processing) was inferior to Example 1 in all efficacy indicators, which shows the unique value of step-by-step temperature-controlled processing in achieving synergistic effects. The indicators of Comparative Example 2 (no enzymatic hydrolysis), Comparative Example 3 (no fermentation) and Comparative Example 4 (no processing) were significantly lower than those of Example 1, proving that processing, enzymatic hydrolysis and fermentation are indispensable and form a complete synergistic effect chain.

[0073] (2) Specificity of strain selection: Comparative Example 6 used Lactobacillus plantarum instead of Staphylococcus xylose, and all efficacy indicators were significantly lower than those in Example 1, proving that Staphylococcus xylose has unique and irreplaceable advantages in this system.

[0074] (3) Synergistic effect of ginseng and mulberry leaf compound: A comparison of data from Comparative Example 7 (ginseng), Comparative Example 8 (mulberry leaf), and Comparative Example 9 (treated separately and then mixed) shows that Example 1 is significantly superior to the other three in behavioral indicators. For example, sleep time in Example 1 is 26% longer than in Comparative Example 9; and the percentage of open arm dwell time is 45.1% higher in Example 1 than in Comparative Example 9. These data fully demonstrate that ginseng and mulberry leaf must be processed together in the same system to achieve the synergistic effect that cannot be achieved by treating and mixing individual herbs separately. Comparative Examples 10 and 11, after replacing mulberry leaf with chrysanthemum and ginseng with Panax notoginseng, showed significantly lower effects than Example 1, proving that replacing herbs with similar efficacy or closely related species cannot achieve the synergistic effect of this invention, indicating the specificity of the raw material selection.

[0075] (4) Specificity of the co-processing ratio: In Example 4, after adjusting the ratio of ginseng to mulberry leaf powder from 1:1 to 10:1, the percentage of open arm dwell time decreased from 51.5% in Example 1 to 38.2%, a reduction of 26%. This indicates that the optimal anti-anxiety effect can be achieved when the weight ratio of ginseng powder to mulberry leaf powder is within the range of (1-5):(1-5). This further proves the non-arbitrary nature of the process parameters of the present invention.

[0076] (5) Overall synergy of the three mechanisms: The composition of Example 1 simultaneously and significantly promoted 5-HT release, activated the Reelin / Dab-1-GSK-3β / β-catenin signaling axis, and regulated basal forebrain adenosine signaling. These three mechanisms work synergistically at three levels: neurotransmitter regulation, synaptic plasticity repair, and deep sleep drive, achieving a complete closed-loop intervention from "repairing stress-damaged neural circuits" to "promoting restorative deep sleep," effectively breaking the vicious cycle of anxiety and insomnia.

[0077] Test Example 3: Toxicity Tests of Each Composition (1) Acute toxicity test Twenty SPF-grade Kunming mice (half male and half female, 18-22g) were randomly divided into a treatment group and a control group, with 10 mice in each group. The treatment group received a single gavage administration of the composition from Example 1 at the maximum administered concentration (500mg / mL) and maximum gavage volume (0.2mL / 10g body weight), while the control group received an equal volume of physiological saline. The mice were observed for 14 consecutive days after administration, and their general condition, behavior, food and water intake, weight changes, and mortality were recorded. After the final weighing, serum alanine aminotransferase (ALT) and blood urea nitrogen (BUN) were measured to assess the acute effects of a single high-dose administration on liver and kidney function. The results are shown in Table 6.

[0078] Table 6 Results of Acute Toxicity Tests

[0079] As shown in Table 6, none of the mice in the treatment group died during the 14-day observation period. No abnormalities were observed in appearance, behavior, food and water intake, or weight gain. ALT and BUN levels showed no significant difference compared to the blank control group. The maximum tolerated dose (MTD) of the composition of this invention, administered orally, is >10 g / kg (based on crude drug weight), classifying it as practically non-toxic.

[0080] (2) 28-day repeated-dose toxicity test Thirty SPF-grade SD rats (half male and half female, 180-220g) were randomly divided into a low-dose group (1250mg / kg), a high-dose group (2500mg / kg), and a blank control group, with 10 rats in each group. The rats were administered the drug by gavage for 28 consecutive days, while the blank control group received an equal volume of physiological saline. Twenty-four hours after the last administration, blood was collected from the abdominal aorta to measure serum alanine aminotransferase (ALT) and blood urea nitrogen (BUN). The gross morphology of major organs (heart, liver, spleen, lungs, and kidneys) was also observed by dissection. The results are shown in Table 7.

[0081] Table 7 Results of the 28-day repeated-dose toxicity study

[0082] As shown in Table 7, rats in each dose group had normal weight gain during the 28-day administration period. The levels of ALT and BUN were not significantly different from those in the blank control group. No pathological changes were observed in the major organs, and no drug-related toxic reactions were observed.

[0083] The results of the above-mentioned acute and 28-day repeated-dose toxicity tests show that the ginseng and mulberry leaf composition of the present invention did not show obvious toxic reactions even at doses far higher than those used in humans, indicating good safety and long-term use.

[0084] Test Example 4: Human Efficacy Experiments of Various Compositions Eighty adult volunteers (aged 25-55 years, half male and half female) who reported experiencing anxiety and sleep disturbances were randomly divided into four groups of 20 each. Each group received either the composition of Example 1, the composition of Comparative Example 7 (single participant in the tertiary process), the composition of Comparative Example 8 (mulberry leaf alone with tertiary process), or a placebo (equal amount of maltodextrin, with appearance and taste identical to the test substance), once daily at a dose of 2g for 28 consecutive days. A randomized, double-blind design was used, and neither the participants nor the outcome assessors were aware of their group assignments.

[0085] The Hamilton Anxiety Rating Scale (HAMA) was used to assess changes in anxiety, and the Pittsburgh Sleep Quality Index (PSQI) was used to assess changes in sleep quality. Saliva samples were collected at three time points: upon waking (0 min), 30 min after waking, and 45 min after waking, before treatment and on the same day 28 days after treatment. The cortisol wakefulness response (CAR, i.e., the net change in cortisol concentration at 30 min or 45 min relative to 0 min, reflecting HPA axis stress sensitivity) was calculated. The results are shown in Table 8.

[0086] Table 8 Results of Human Trial Experiments

[0087] Table 8 shows that there were no significant changes in any indicators in the placebo group before and after treatment. In Example 1 group, the HAMA score decreased by 36.9%, the PSQI score decreased by 36.8%, and the CAR decreased by 21.7% compared to before treatment, all of which were significantly better than the single ginseng group, the single mulberry leaf group, and the placebo group. P <0.01). The cortisol arousal response (CAR) is an internationally recognized objective indicator of HPA axis function. Its decrease reflects a reduction in HPA axis stress sensitivity, indicating that the composition of this invention can effectively regulate stress system function. The above results demonstrate that the composition of this invention has significant stress-reduction, anxiety-relieving, and sleep-improving effects in humans, and the effect of ginseng and mulberry leaf combination is significantly better than that of single herbs.

[0088] No significant adverse reactions were observed in any of the volunteers during the course of treatment, and there was no significant difference in the incidence of adverse reactions among the groups, further demonstrating that the composition of the present invention has good safety.

[0089] In summary, the ginseng and mulberry leaf composition disclosed in this invention is prepared by a three-stage synergistic process of ginseng and mulberry leaves through stepwise temperature-controlled processing, phytase hydrolysis, and Staphylococcus xylose fermentation. This invention also discloses the preparation method of the ginseng and mulberry leaf composition and its application. The preparation method of this invention is based on a three-stage synergistic process of stepwise temperature-controlled processing, enzymatic hydrolysis, and fermentation. The ginseng-mulberry leaf composition obtained can (1) promote the release of 5-HT and regulate neurotransmitter balance; (2) activate the Reelin / Dab-1-GSK-3β / β-catenin signal axis of hippocampal neurons, repair synaptic plasticity damaged by stress, and restore synaptic homeostasis, thereby reducing stress and relieving anxiety; (3) regulate the adenosine signal of the basal forebrain and promote deep sleep drive, thereby improving sleep quality and achieving multi-target closed-loop intervention against the vicious cycle of anxiety and insomnia. The composition of this invention can significantly reduce anxiety-like behavior, prolong sleep time, and has no toxic side effects, and can be taken safely for a long time.

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a ginseng and mulberry leaf composition for stress reduction, anxiety relief, and sleep improvement, characterized in that, Includes the following steps: S1. Grind dried ginseng and mulberry leaves into powder and sieve them separately. Mix the ginseng powder and mulberry leaf powder and perform a stepped temperature-controlled processing to obtain a processed mixture. The specific steps of the stepped temperature-controlled processing are as follows: steam the mixed ginseng powder and mulberry leaf powder at a low temperature of 40-60℃, and then raise the temperature to a high temperature of 100-121℃ for steaming. S2. Add water to the prepared mixture and stir evenly. Add phytase for enzymatic hydrolysis. After inactivation of the enzyme, the enzymatic hydrolysis product is obtained. S3. Inoculate the enzymatic hydrolysis product with Staphylococcus xylose for fermentation, sterilize and dry to obtain ginseng and mulberry leaf composition.

2. The method for preparing the ginseng and mulberry leaf composition for stress reduction, anxiety relief, and sleep improvement as described in claim 1, characterized in that, In step S1, the weight ratio of ginseng powder to mulberry leaf powder is (1-5):(1-5); the low-temperature steaming time is 2-4 hours, the high-temperature steaming time is 0.5-2 hours, and after being taken out, it is dried until the moisture content is less than 10%.

3. The method for preparing the ginseng and mulberry leaf composition for stress reduction, anxiety relief, and sleep improvement as described in claim 1, characterized in that, In step S2, the mass ratio of the prepared mixture to the water is 1:(10-50); the amount of phytase used is 0.5-2% of the weight of the prepared mixture; the enzymatic hydrolysis temperature is 45-55℃, the pH is 4.5-5.5, the stirring speed is 50-100 r / min, and the time is 4-6 h; the enzyme inactivation temperature is 90-100℃, and the time is 10-20 min.

4. The method for preparing the ginseng and mulberry leaf composition for stress reduction, anxiety relief, and sleep improvement as described in claim 1, characterized in that, In step S3, the inoculum size of Staphylococcus xylose is 1-5% of the volume of the enzymatic hydrolysis product; the fermentation temperature is 30-37℃, the pH is 6.5-7.5, and the time is 12-24h; the sterilization temperature is 95-100℃, and the time is 15-20min.

5. A ginseng and mulberry leaf composition for stress reduction, anxiety relief, and improved sleep, characterized in that, The ginseng and mulberry leaf composition for stress reduction, anxiety relief, and sleep improvement, as described in any one of claims 1-4, is prepared by the method described in claims 1-4.

6. The use of the ginseng and mulberry leaf composition according to claim 5 in the preparation of health products or pharmaceuticals for stress reduction, relieving anxiety and / or improving sleep disorders.

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