Ophiopogon and tangerine peel composition for relieving anxiety and improving sleep, and preparation method and application thereof

Abstract

This invention discloses a composition of Ophiopogon japonicus and Citrus reticulata peel for relieving anxiety and improving sleep. The composition comprises, by weight: 50-80 parts Ophiopogon japonicus extract, 20-40 parts Citrus reticulata peel extract, 10-30 parts Ziziphus jujuba seed powder, 10-30 parts Poria cocos powder, 10-30 parts maltodextrin, and 5-10 parts xylitol. This invention also discloses a method for preparing the Ophiopogon japonicus and Citrus reticulata peel composition and its applications. The composition of this invention achieves synergistic effects through a triple pathway of neuroinflammation inhibition, gut-brain axis regulation, and GABAergic system activation, effectively relieving anxiety and improving sleep disorders.

Description

Technical Field

[0001] This invention belongs to the field of food and health product technology, specifically relating to a composition of Ophiopogon japonicus and tangerine peel that relieves anxiety and improves sleep, as well as its preparation method and application. Background Technology

[0002] The increasing prevalence of anxiety and its significant negative impact on sleep has become a major health challenge in modern society. The proportion of people experiencing significant anxiety globally continues to rise. Chronic sleep disorders affect mental and emotional states, and anxiety (such as persistent tension, worry, and irritability) is one of the factors that induce and aggravate sleep disorders, thus forming a persistent "vicious cycle of anxiety-sleep disorder."

[0003] The pathological mechanisms of anxiety-sleep disorders include: (1) neuroendocrine imbalance: chronic anxiety activates the HPA axis, leading to abnormally high cortisol levels, inhibiting melatonin secretion, and disrupting sleep rhythm; (2) inflammatory cascade: overexpression of pro-inflammatory factors TNF-α / IL-6 damages GABAergic neurons and reduces 5-HT synthesis, forming a "vicious cycle of anxiety-sleep disorders".

[0004] The existing intervention methods have significant defects: (1) Long-term use of chemical drugs, such as diazepam, estazolam, clonazepam, etc., can induce sleep by inhibiting the excitability of the cerebral cortex, but may have adverse reactions such as daytime sleepiness and dizziness. Long-term use is prone to dependence, tolerance and cognitive impairment; (2) Traditional Chinese medicine extracts, such as jujube seed, cypress seed, polygala root, etc., have low bioavailability of large molecular polysaccharides, making it difficult to achieve multi-target regulation; (3) Melatonin alone is not effective for insomnia caused by psychological factors, environmental factors, etc., and is only suitable for circadian rhythm disorders. It can regulate sleep-wake disorders and may cause symptoms such as dizziness, headache, drowsiness, poor concentration, nausea, and diarrhea. It may also promote vasoconstriction, increase the risk of stroke, and cause hormonal imbalances. It may interact with anticoagulants, antihypertensive drugs, immunosuppressants, antidepressants, and contraceptives, increasing the risk of adverse reactions or affecting efficacy. Long-term or excessive use can suppress the body's own melatonin secretion function, causing sleep disorders and dependence. After discontinuation, sleep quality may decline. Improper use may have potential harms.

[0005] Therefore, there is an urgent need to develop a compound preparation based on natural components to effectively alleviate anxiety and simultaneously improve sleep disorders directly caused by this emotion. Summary of the Invention

[0006] One object of the present invention is to provide a composition that can effectively relieve anxiety and improve sleep, in order to address the above-mentioned technical problems.

[0007] Another object of the present invention is to provide a method for preparing the composition.

[0008] Another object of the present invention is to provide applications of the composition.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a composition of Ophiopogon japonicus and tangerine peel for relieving anxiety and improving sleep, the composition containing Ophiopogon japonicus extract, tangerine peel extract, jujube seed powder, Poria cocos powder, maltodextrin, and xylitol.

[0010] Preferably, the composition comprises, by weight: 50-80 parts of Ophiopogon japonicus extract, 20-40 parts of Citrus reticulata extract, 10-30 parts of Ziziphus jujuba seed powder, 10-30 parts of Poria cocos powder, 10-30 parts of maltodextrin, and 5-10 parts of xylitol.

[0011] More preferably, the composition comprises, by weight: 80 parts of Ophiopogon japonicus extract, 20-40 parts of Citrus reticulata extract, 20-30 parts of Ziziphus jujuba seed powder, 20-30 parts of Poria cocos powder, 20-30 parts of maltodextrin, and 10 parts of xylitol.

[0012] Most preferably, the composition comprises, by weight: 80 parts of Ophiopogon japonicus extract, 40 parts of Citrus reticulata extract, 30 parts of Ziziphus jujuba seed powder, 30 parts of Poria cocos powder, 30 parts of maltodextrin, and 10 parts of xylitol.

[0013] Preferably, in the above composition, the Ophiopogon japonicus extract is prepared by the following steps: S1. Dry the Ophiopogon japonicus, pulverize it into powder, then add it to the fermentation liquid of Bacillus plantarum for microbial fermentation. After the fermentation is completed, sterilize it to obtain fermented Ophiopogon japonicus extract. S2. Add hemicellulase to the fermented Ophiopogon japonicus extract for enzymatic hydrolysis, inactivate the enzyme, cool down and pass through an ultrafiltration membrane to retain liquid with a molecular weight cutoff of less than 5 kDa, then spray or freeze dry to obtain Ophiopogon japonicus extract.

[0014] In step S1, the *Lactobacillus plantarum* fermentation broth includes MRS medium and *Lactobacillus plantarum* GDMCC No. 64374, and the inoculum size of *Lactobacillus plantarum* is 1×10⁻⁶. 5 -2×10 5 CFU / mL. The preferred mass ratio of Ophiopogon japonicus to Lactobacillus plantarum fermentation broth is 1:2 to 1:5. The fermentation temperature is 30-35℃, and the fermentation time is 30-36 hours.

[0015] Preferably, the formulation of the MRS culture medium is as follows: Sodium acetate 5g, peptone 10g, beef extract 10g, yeast extract 5g, dipotassium hydrogen phosphate 2g, diammonium citrate 2g, glucose 20g, magnesium sulfate heptahydrate 0.58g, manganese sulfate monohydrate 0.19g, Tween 80 1mL, distilled water 1L.

[0016] In step S2, the enzymatic hydrolysis process is as follows: First, add water with a volume of 10-20 times that of the fermented Ophiopogon japonicus extract to adjust the pH to 5.0-5.2. Then, add 5%-10% hemicellulase based on the weight of the fermented Ophiopogon japonicus extract, hydrolyze at 50-55℃ for 2-4 hours, and inactivate the enzyme at 85℃ for 20-30 minutes.

[0017] Preferably, in the above composition, the tangerine peel extract is prepared by the following steps: Step 1: Dry the dried tangerine peel, grind it into powder, then add Aspergillus niger fermentation liquid for microbial fermentation, and sterilize to obtain fermented tangerine peel extract; Step 2: Add papain to the fermented tangerine peel extract for enzymatic hydrolysis, inactivate the enzyme, cool down and pass through an ultrafiltration membrane to retain liquid with a molecular weight cutoff of less than 10 kDa, then spray or freeze dry to obtain the tangerine peel extract.

[0018] Preferably, in step 1, the Aspergillus niger fermentation broth includes a culture medium and Aspergillus niger, and the inoculum size of Aspergillus niger is 1×10⁻⁶. 5 -2×10 5 CFU / mL. The preferred mass ratio of dried tangerine peel to Aspergillus niger fermentation broth is 1:2 to 1:5. The fermentation temperature is 25-30℃, and the fermentation time is 18-24 hours.

[0019] Preferably, the culture medium contains grapefruit peel, wheat bran, soybean meal, ammonium sulfate, and dipotassium hydrogen phosphate.

[0020] More preferably, the culture medium is formulated as follows: 120g grapefruit peel, 5g wheat bran, 5g soybean meal, 4g ammonium sulfate, 0.5g dipotassium hydrogen phosphate, 1L water.

[0021] Preferably, in step 2, the enzymatic hydrolysis process is as follows: first, add 10-20 times the weight of water to the fermented tangerine peel extract, adjust the pH to 6.0-7.0, then add 1%-3% papain based on the weight of the fermented tangerine peel extract, hydrolyze at 60℃-65℃ for 2-4 hours, and inactivate the enzyme at 85℃ for 20-30 minutes.

[0022] In the above composition, the jujube seed powder and poria powder are ordinary commercially available jujube seeds and poria, which are obtained by drying, pulverizing or grinding.

[0023] In the above composition, the maltodextrin is a filler and the xylitol is a flavoring agent.

[0024] By combining the above compositions in appropriate proportions and adding other commonly used excipients such as those used in food, health products, and pharmaceuticals, the types of preparations that can be made include, but are not limited to, solid beverages, capsules, compressed candies, and beverages.

[0025] Secondly, the present invention provides a method for preparing the Ophiopogon japonicus and tangerine peel composition for relieving anxiety and improving sleep, which includes the following steps: weighing each component according to the specified amount, mixing them evenly, and thus obtaining the Ophiopogon japonicus and tangerine peel composition.

[0026] Thirdly, the present invention also provides the application of the Ophiopogon japonicus and Citrus reticulata peel composition in the preparation of foods, health products, and pharmaceuticals for relieving anxiety and improving sleep disorders.

[0027] Compared with the prior art, the present invention has the following advantages: (1) High bioconversion process of Ophiopogon japonicus extract Directional fermentation of Ophiopogon japonicus was performed using *Lactobacillus plantarum*, utilizing its extracellular enzyme system (β-glucosidase, xylanase, etc.) to degrade large polysaccharides (molecular weight >20 kDa) into small oligosaccharides (molecular weight <5 kDa), thereby improving bioavailability. Simultaneously, Ophiopogon japonicus saponin D and free amino acids were released. Subsequent enzymatic hydrolysis using hemicellulase further controlled the molecular weight of the active ingredients to 1-3 kDa, and ultrafiltration (with a 5 kDa cutoff) was used to enrich the active ingredients. The final product was metabolized by intestinal flora to generate short-chain fatty acids, which regulate neurotransmitter secretion via the gut-brain axis pathway.

[0028] (2) Two-stage bioactivation process of tangerine peel extract Fermentation with Aspergillus niger hydrolyzes the flavonoid glycoside bonds in tangerine peel, converting hesperidin into free hesperidin and enhancing its antioxidant activity. After fermentation, papain is used to target and enzymatically hydrolyze the tangerine peel protein, eliminating bitter and pungent substances while increasing the flavonoid dissolution rate to 89.7±2.4%. Ultrafiltration (10 kDa) removes large molecular impurities, allowing small molecule active ingredients to directly regulate anxiety-related neural pathways.

[0029] (3) Multi-target synergistic regulation of anxiety-insomnia comorbidity mechanism The composition achieves synergistic effects through a triple pathway: Inhibition of neuroinflammation: Ophiopogon japonicus saponin D and hesperidin synergistically downregulate TNF-α / IL-6 expression, block NF-κB signal transduction, and reduce hippocampal neuroinflammation; Gut-brain axis regulation: Ophiopogon japonicus oligosaccharide and Poria cocos polysaccharide promote the proliferation of Bifidobacteria in the gut, accelerate the conversion of tryptophan to 5-hydroxytryptamine, and simultaneously increase serum melatonin levels; GABAergic system activation: Jujube seed saponin A and pachymic acid jointly enhance the binding force of GABA_A receptors, reduce the power of β waves on electroencephalogram, and inhibit the release of CRH from the amygdala, thereby achieving bidirectional regulation of anxiety and insomnia. Detailed Implementation

[0030] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0031] 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.

[0032] Sour jujube seed powder and poria powder are ordinary commercially available sour jujube seeds and poria, which are dried, pulverized or ground.

[0033] The fermentation broth of *Lactobacillus plantarum* includes MRS medium and *Lactobacillus plantarum* GDMCC No. 64374. *Lactobacillus plantarum* ( Lactiplantibacillus plantarum GDMCC No. 64374 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 formula for MRS medium is as follows: 5g sodium acetate, 10g peptone, 10g beef extract, 5g yeast extract, 2g dipotassium hydrogen phosphate, 2g diammonium citrate, 20g glucose, 0.58g magnesium sulfate heptahydrate, 0.19g manganese sulfate monohydrate, 1mL Tween 80, and 1L distilled water.

[0035] The Aspergillus niger fermentation broth includes the culture medium and Aspergillus niger. The Aspergillus niger strain used is CICC 40273, purchased from the China Industrial Microbial Culture Collection Center.

[0036] The culture medium formula is as follows: 120g grapefruit peel, 5g wheat bran, 5g soybean meal powder, 4g ammonium sulfate, 0.5g dipotassium hydrogen phosphate, and 1L water.

[0037] Example 1 A composition of Ophiopogon japonicus and tangerine peel for relieving anxiety and improving sleep, comprising the following components in parts by weight: 80 parts Ophiopogon japonicus extract, 40 parts tangerine peel extract, 30 parts Ziziphus jujuba seed powder, 30 parts Poria cocos powder, 30 parts maltodextrin, and 10 parts xylitol.

[0038] The preparation method of Ophiopogon japonicus extract is as follows: Ophiopogon japonicus is dried, pulverized into powder, passed through a 20-mesh sieve, and then 3 times its weight of Lactobacillus plantarum fermentation broth is added (inoculum amount is 2×10). 5 Ferment at 35°C for 36 hours (CFU / mL), sterilize at 100°C for 10 minutes, then cool to room temperature, add 10 times the volume of water, adjust the pH to 5.0, then add 10% (based on the weight of the fermented Ophiopogon japonicus extract) of hemicellulase, enzymatically hydrolyze at 50°C for 4 hours, inactivate the enzyme at 85°C for 20 minutes, cool down, filter, retain liquid below 5 kDa, and spray dry to obtain Ophiopogon japonicus extract.

[0039] The preparation method of the tangerine peel extract is as follows: tangerine peel is dried, ground into powder, passed through a 20-mesh sieve, and then three times its weight of Aspergillus niger fermentation broth is added (inoculation amount is 2×10⁻⁶). 5 Ferment at 33℃ for 24 hours (CFU / mL), sterilize at 100℃ for 20 minutes, then cool to room temperature, add 10 times the volume of water, adjust pH to 7.0, then add 3% (by weight of fermented tangerine peel extract) papain, hydrolyze at 60℃ for 4 hours, inactivate enzyme at 85℃ for 20 minutes, cool, filter, retain liquid below 10 kDa, and spray dry to obtain tangerine peel extract.

[0040] According to the above-mentioned mass proportions, Ophiopogon japonicus extract, tangerine peel extract, jujube seed powder, Poria cocos powder, maltodextrin and xylitol are mixed evenly to obtain an Ophiopogon japonicus and tangerine peel composition that relieves anxiety and improves sleep.

[0041] Example 2 A composition of Ophiopogon japonicus and tangerine peel for relieving anxiety and improving sleep, comprising the following components in parts by weight: 80 parts Ophiopogon japonicus extract, 20 parts tangerine peel extract, 20 parts Ziziphus jujuba seed powder, 20 parts Poria cocos powder, 20 parts maltodextrin, and 10 parts xylitol.

[0042] The preparation method of Ophiopogon japonicus extract is as follows: Ophiopogon japonicus is dried, pulverized into powder, passed through a 20-mesh sieve, and then 5 times its weight of Bacillus plantarum fermentation broth is added (inoculum amount is 1×10). 5 Ferment at 30°C for 30 hours (CFU / mL), sterilize at 100°C for 10 minutes, then cool to room temperature, add 10 times the volume of water, adjust the pH to 5.2, then add 8% (based on the weight of the fermented Ophiopogon japonicus extract) of hemicellulase, enzymatically hydrolyze at 55°C for 2 hours, inactivate the enzyme at 85°C for 30 minutes, cool down, filter, retain liquid below 5 kDa, and spray dry to obtain Ophiopogon japonicus extract.

[0043] The preparation method of tangerine peel extract is as follows: tangerine peel is dried, ground into powder, passed through a 20-mesh sieve, and then 5 times its weight of Aspergillus niger fermentation broth is added (inoculum size is 1×10⁻⁶). 5Ferment at 37°C for 18 hours (CFU / mL), sterilize at 100°C for 20 minutes, then cool to room temperature, add 15 times the volume of water, adjust pH to 6.5, then add 2% (by weight of fermented tangerine peel extract) papain, hydrolyze at 65°C for 2 hours, inactivate enzyme at 85°C for 20 minutes, cool, filter, retain liquid below 10 kDa, and spray dry to obtain tangerine peel extract.

[0044] According to the above-mentioned mass proportions, Ophiopogon japonicus extract, tangerine peel extract, jujube seed powder, Poria cocos powder, maltodextrin and xylitol are mixed evenly to obtain an Ophiopogon japonicus and tangerine peel composition that relieves anxiety and improves sleep.

[0045] Example 3 A composition of Ophiopogon japonicus and tangerine peel for relieving anxiety and improving sleep, comprising the following components in parts by weight: 50 parts Ophiopogon japonicus extract, 40 parts tangerine peel extract, 10 parts Ziziphus jujuba seed powder, 10 parts Poria cocos powder, 20 parts maltodextrin, and 8 parts xylitol.

[0046] The preparation method of Ophiopogon japonicus extract is as follows: Ophiopogon japonicus is dried, pulverized into powder, passed through a 20-mesh sieve, and then 2 times the mass of Bacterium plantarum fermentation broth is added (inoculum amount is 2×10). 5 Ferment at 32℃ for 34 hours (CFU / mL), sterilize at 100℃ for 10 minutes, then cool to room temperature, add 20 times the volume of water, adjust the pH to 5.0, then add 6% (based on the weight of the fermented Ophiopogon japonicus extract) of hemicellulase, enzymatically hydrolyze at 52℃ for 4 hours, inactivate the enzyme at 85℃ for 20 minutes, cool down, filter, retain liquid below 5 kDa, and spray dry to obtain Ophiopogon japonicus extract.

[0047] The preparation method of tangerine peel extract is as follows: tangerine peel is dried, ground into powder, passed through a 20-mesh sieve, and then twice the mass of Aspergillus niger fermentation broth is added (inoculum size is 2×10⁻⁶). 5 Ferment at 36℃ for 22 hours (CFU / mL), sterilize at 100℃ for 20 minutes, then cool to room temperature, add 20 times the volume of water, adjust pH to 7.0, then add 1% (by weight of fermented tangerine peel extract) papain, hydrolyze at 65℃ for 2 hours, inactivate enzyme at 85℃ for 30 minutes, cool, filter, retain liquid below 10 kDa, and spray dry to obtain tangerine peel extract.

[0048] According to the above-mentioned mass proportions, Ophiopogon japonicus extract, tangerine peel extract, jujube seed powder, Poria cocos powder, maltodextrin and xylitol are mixed evenly to obtain an Ophiopogon japonicus and tangerine peel composition that relieves anxiety and improves sleep.

[0049] Example 4 A composition of Ophiopogon japonicus and tangerine peel for relieving anxiety and improving sleep, comprising the following components in parts by weight: 50 parts Ophiopogon japonicus extract, 20 parts tangerine peel extract, 30 parts Ziziphus jujuba seed powder, 30 parts Poria cocos powder, 10 parts maltodextrin, and 5 parts xylitol.

[0050] The preparation method of Ophiopogon japonicus extract is as follows: Ophiopogon japonicus is dried, pulverized into powder, passed through a 20-mesh sieve, and then 4 times its weight of Lactobacillus plantarum fermentation broth is added (inoculum amount is 1×10). 5 Ferment at 33°C for 32 hours (CFU / mL), sterilize at 100°C for 10 minutes, then cool to room temperature, add 18 times the volume of water, adjust the pH to 5.0, then add 5% (based on the weight of the fermented Ophiopogon japonicus extract) of hemicellulase, enzymatically hydrolyze at 53°C for 3 hours, inactivate the enzyme at 85°C for 20 minutes, cool down, filter, retain liquid below 5 kDa, and spray dry to obtain Ophiopogon japonicus extract.

[0051] The preparation method of tangerine peel extract is as follows: tangerine peel is dried, ground into powder, passed through a 20-mesh sieve, and then four times its weight of Aspergillus niger fermentation broth is added (inoculum size is 1×10⁻⁶). 5 Ferment at 35°C for 20 hours (CFU / mL), sterilize at 100°C for 20 minutes, then cool to room temperature. Add 16 times the volume of water to adjust the pH to 6.8. Then add 2% (by weight of fermented tangerine peel extract) of papain. Incubate at 63°C for 3 hours, then inactivate the enzyme at 85°C for 20 minutes. After cooling, filter, retain the liquid below 10 kDa, and spray dry to obtain the tangerine peel extract.

[0052] According to the above-mentioned mass proportions, Ophiopogon japonicus extract, tangerine peel extract, jujube seed powder, Poria cocos powder, maltodextrin and xylitol are mixed evenly to obtain an Ophiopogon japonicus and tangerine peel composition that relieves anxiety and improves sleep.

[0053] Comparative Example 1 In the composition, Ophiopogon japonicus extract is replaced by water-extracted Ophiopogon japonicus powder, and Citrus reticulata extract is replaced by water-extracted Citrus reticulata powder. The remaining components and process parameters are the same as in Example 1.

[0054] The preparation methods of water-extracted Ophiopogon japonicus powder and water-extracted tangerine peel powder are as follows: After drying and pulverizing Ophiopogon japonicus or tangerine peel, it is soaked in 10 times its weight of 80℃ hot water for 2 hours, filtered, and the filtrate is spray-dried.

[0055] Comparative Example 2 The composition does not contain Ophiopogon japonicus extract, and the other components and process parameters are the same as in Example 1.

[0056] Comparative Example 3 The composition does not contain tangerine peel extract, and the other components and process parameters are the same as in Example 1.

[0057] Comparative Example 4 The composition does not contain jujube seed powder or poria cocos powder, and the other components and process parameters are the same as in Example 1.

[0058] Effect verification 1. Animal experiments SPF-grade Kunming mice (half male and half female, 18-22g), 10 mice per group, were randomly assigned to groups. A blank control group (physiological saline), a positive control group, and experimental groups (Examples 1-4, Comparative Examples 1-4) were established. Each group was administered 0.1mL / 10g via gavage once daily for 7 consecutive days.

[0059] The positive control group was given 0.5 mg / kg of diazepam by gavage.

[0060] The gavage solution for each experimental group was a combination of the corresponding group, prepared at a concentration of 250 mg / mL (the solvent was physiological saline).

[0061] (1) Mouse elevated cruciate maze test Thirty minutes after the last administration, mice were placed in an elevated cruciate maze (open arm / closed arm). The time spent in the open arm within 5 minutes was recorded, and the percentage of time spent in the open arm was calculated (open arm time / total time). The number of times mice entered the open arm was also recorded.

[0062] (2) Mouse light-dark box experiment Thirty minutes after the last administration, mice were placed in the dark area of ​​a dark-light box, and their behavior was recorded for 5 minutes. This included the duration of time spent in the light area and the number of times the mice traversed between light and dark zones.

[0063] (3) Sodium pentobarbital-induced sleep experiment in mice Sleep onset rate (subthreshold dose) (30 mg / kg): 30 minutes after the last administration, sodium pentobarbital (30 mg / kg) was injected intraperitoneally, and the number of babies falling asleep within 15 minutes was recorded (the righting reflex was absent for more than 1 minute).

[0064] Sleep duration (supreme dose): 30 minutes after the last administration, sodium pentobarbital (35 mg / kg) was injected intraperitoneally, and the total sleep duration (time from the disappearance of the righting reflex to its recovery) was recorded.

[0065] (4) Mouse spontaneous activity inhibition experiment Twenty minutes after the last administration, the mice were placed on a self-movement device, and the number of squares walked and the number of forelimbs raised were recorded within 10 minutes.

[0066] Table 1 shows the results of the mouse anxiety behavior experiment.

[0067] Table 1. Results of mouse anxiety behavior experiments

[0068] In the elevated cross maze experiment, anxious mice tended to avoid open arms, and a higher value indicated a better anti-anxiety effect. As shown in Table 1, the open arm dwell time in Example 1 group reached 45.3%, close to the 49.8% of the positive control group, representing a 198% increase compared to the blank control group, and significantly higher than Comparative Examples 1-4. The number of times the mice entered the open arm was 6, similar to the 6.5 times in the positive control group, both significantly higher than Comparative Examples 1-4. Comparing the results of Examples 1-4, changes in different components or processes affected the results, but under appropriate ratios, all showed significantly better effects than the blank control group and Comparative Examples 1-4.

[0069] In the light-dark box experiment, anxious mice reduced their exploration of the light area; a higher value indicated a better anti-anxiety effect. As shown in Table 1, the dwell time in the light area in Example 1 group was 185 seconds, which was 155% higher than the blank control group and significantly higher than Comparative Examples 1-4. The number of light area shuttles was 11.9 times, which was 129% higher than the blank control group and significantly higher than Comparative Examples 1-4. This indicates that the composition of the present invention has a good anxiety-relieving effect.

[0070] Table 2 shows the results of the mouse sleep experiment.

[0071] Table 2. Results of mouse sleep experiment

[0072] As shown in Table 2 above, the sleep onset rate and sleep duration of Examples 1-4 were similar to those of the positive control group. However, the sleep onset rate of Comparative Example 1 was only 30%, and the sleep duration was 34% shorter than that of Example 1. This indicates that fermentation / enzymatic hydrolysis of Ophiopogon japonicus and Citrus reticulata significantly increases the content of their active ingredients and enhances bioavailability. In Comparative Examples 2-4, where no Ophiopogon japonicus extract, Citrus reticulata extract, Ziziphus jujuba seed powder, or Poria cocos powder were added, the sleep onset rate and sleep duration were lower than those of Example 1, highlighting the necessity of the synergistic effect of Ophiopogon japonicus extract, Citrus reticulata extract, Ziziphus jujuba seed powder, and Poria cocos powder. Similarly, regarding the spontaneous activity inhibition rate, a higher value indicates a better sedative effect. From the data of each group, Examples 1-4 showed effects similar to the positive control group, while Comparative Examples 1-4 were not as effective as Examples 1-4. Therefore, this demonstrates that the compositions of the present invention have a good sleep-improving effect.

[0073] (5) Neurotransmitter level experiment One hour after the last administration, blood was collected from the orbital cavity, and serum was separated by centrifugation at 3000 rpm. The levels of 5-HT, MT, and COR were detected by enzyme-linked immunosorbent assay (ELISA). In addition, the whole brain was removed after decapitation, and the hypothalamus region was separated. The levels of GABA and CRH in the hypothalamus were detected by chemiluminescent immunoassay (CLIA).

[0074] Table 3 Serum 5-HT, MT, and hypothalamic GABA levels in mice

[0075] 5-Hydroxytryptamine (5-HT) is an important monoamine neurotransmitter in the central nervous system, involved in sleep-wake regulation. RIN-14B is a rat islet tumor-derived cell line with the functions of synthesizing, storing, and secreting 5-HT, and its secretion mechanism is similar to that of 5-HTergic neurons in vivo. It can be used to evaluate the anti-anxiety and sleep-aiding effects of samples; a higher value indicates a better effect.

[0076] Melatonin (MT) is an indole heterocyclic hormone secreted by the pineal gland. Its core function is to regulate circadian rhythms and sleep-wake cycles. It promotes deep sleep and shortens sleep latency by activating MT1 / MT2 receptors and inhibiting the hypothalamic-pituitary-gonadal axis. Rat or mouse animal models are widely used to evaluate the sleep-aiding effects of melatonin. By measuring parameters such as serum melatonin levels, prolonged sleep duration, and shortened sleep latency, the neuroendocrine regulatory process in vivo can be simulated, making it suitable for verifying the improvement effect of samples on sleep disorders. Higher values ​​indicate better effects.

[0077] Cortisol (COR) is a steroid hormone secreted by the adrenal cortex, which dominates the body's stress response and metabolic regulation. Its core functions include: (1) stress regulation: rapid increase in blood sugar under stress, promotion of fat breakdown, and activation of the "fight or flight" response; (2) circadian rhythm: secretion is high in the morning and low at night, and imbalance can lead to insomnia, anxiety, and central obesity; (3) metabolic interference: long-term excessive levels lead to increased hunger and appetite, inducing a preference for high-fat and high-sugar diets and abnormal fat accumulation. Clinically, serum time-segmented detection is used to assess HPA axis function and assist in the diagnosis and treatment of anxiety-sleep comorbidity. Its value fluctuates, and an increase in the afternoon or at night indicates an abnormality in the body.

[0078] Gamma-aminobutyric acid (GABA) is an important inhibitory neurotransmitter in the mammalian central nervous system, mediating over 40% of inhibitory neurotransmission and participating in physiological functions such as calming anxiety, improving sleep, and regulating blood pressure. Its mechanism of action includes reducing neuronal excitability through postsynaptic hyperpolarization and inhibiting glutamate release, thereby promoting deep sleep and shortening sleep onset time. It can also be used to evaluate the sleep-aiding effect of samples; a higher value indicates a better effect.

[0079] Hypothalamic CRH is a peptide neurohormone synthesized by the paraventricular nucleus, serving as the initiation signal for the HPA axis. Its core functions include: (1) ACTH-driven: activating the anterior pituitary gland to release ACTH via the pituitary portal system, thereby stimulating cortisol synthesis; (2) stress integration: converging physiological / psychological stress signals and enhancing anxiety behavior through the amygdala (such as reducing open-arm exploration in elevated mazes); (3) multi-system regulation: inhibiting gastric acid secretion, delaying gastrointestinal motility, and participating in blood pressure and blood glucose regulation. Hypothalamic CRH detection is a key indicator for studying anxiety neural pathways and the mechanisms of anti-anxiety drugs. When there is a lesion in the hypothalamic-pituitary regulatory link or a state of stress, the value will increase, that is, the higher the value, the higher the degree of anxiety.

[0080] As shown in Table 3, compared with the blank control group, in Example 1, 5-HT increased by 154%, MT increased by 324%, GABA increased by 146%, COR decreased by 52%, and CRH decreased by 56%. Examples 2-4, due to variations in components or processes, yielded results different from Example 1, but their trends and magnitudes were the same as the positive control group. This indicates that in the composition of the present invention, each component, under appropriate proportions, has a synergistic effect. In Comparative Examples 1-4, no Ophiopogon japonicus extract, Citrus reticulata extract, Ziziphus jujuba seed powder, or Poria cocos powder were added to the composition. Although the levels of 5-HT, MT, and GABA were all increased to varying degrees compared with the blank control group, and the levels of COR and CRH were decreased to varying degrees compared with the blank control group, the improvement magnitudes were significantly different from those of Examples 1-4, further confirming the synergistic effect of each component in the composition of the present invention.

[0081] In summary, the composition of the present invention has a synergistic effect: Ophiopogon japonicus reduces internal heat, Citrus reticulata peel regulates qi, and Ziziphus jujuba var. spinosa and Poria cocos calm the mind. By regulating multiple pathways such as 5-HT, MT, COR, GABA and CRH, it achieves the effect of relieving anxiety and improving sleep at multiple targets.

[0082] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Therefore, any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A composition of Ophiopogon japonicus and dried tangerine peel for relieving anxiety and improving sleep, characterized in that, The composition comprises, by weight, the following components: 50-80 parts of Ophiopogon japonicus extract, 20-40 parts of Citrus reticulata extract, 10-30 parts of Ziziphus jujuba seed powder, 10-30 parts of Poria cocos powder, 10-30 parts of maltodextrin, and 5-10 parts of xylitol. The Ophiopogon japonicus extract was prepared by the following steps: S1. Dry the Ophiopogon japonicus, pulverize it into powder, then add it to the fermentation liquid of Lactobacillus plantarum for microbial fermentation. After the fermentation is completed, sterilize it to obtain fermented Ophiopogon japonicus extract. S2. Add hemicellulase to fermented Ophiopogon japonicus extract for enzymatic hydrolysis, inactivate enzyme, cool down and pass through ultrafiltration membrane, retain liquid with molecular weight below 5kDa, spray or freeze dry to obtain Ophiopogon japonicus extract. In step S1, the *Lactobacillus plantarum* fermentation broth includes MRS medium and *Lactobacillus plantarum* GDMCC No. 64374, and the inoculum size of *Lactobacillus plantarum* is 1×10⁻⁶. 5 -2×10 5 CFU / mL; the mass ratio of Ophiopogon japonicus to Lactobacillus plantarum fermentation broth is 1:2 to 1:5; the fermentation temperature is 30-35℃ and the fermentation time is 30-36 hours. In step S2, the enzymatic hydrolysis process is as follows: First, add water with a volume of 10-20 times to the fermented Ophiopogon japonicus extract, adjust the pH to 5.0-5.2, then add 5%-10% hemicellulase based on the weight of the fermented Ophiopogon japonicus extract, hydrolyze at 50-55℃ for 2-4 hours, and inactivate the enzyme at 85℃ for 20-30 minutes. The tangerine peel extract was prepared through the following steps: Step 1: Dry the dried tangerine peel, grind it into powder, then add Aspergillus niger fermentation liquid for microbial fermentation, and sterilize to obtain fermented tangerine peel extract; Step 2: Add papain to the fermented tangerine peel extract for enzymatic hydrolysis, inactivate the enzyme, cool down and pass through an ultrafiltration membrane to retain liquid with a molecular weight cutoff of less than 10 kDa, then spray or freeze dry to obtain the tangerine peel extract. In step 1, the Aspergillus niger fermentation broth includes a culture medium and Aspergillus niger, with an inoculum size of 1×10⁻⁶. 5 -2×10 5 CFU / mL, the mass ratio of dried tangerine peel to Aspergillus niger fermentation broth is 1:2 to 1:5, the fermentation temperature is 25-30℃, and the fermentation time is 18-24 hours; In step 2, the enzymatic hydrolysis process is as follows: First, add 10-20 times the weight of water to the fermented tangerine peel extract, adjust the pH to 6.0-7.0, then add 1%-3% papain based on the weight of the fermented tangerine peel extract, hydrolyze at 60℃-65℃ for 2-4 hours, and inactivate the enzyme at 85℃ for 20-30 minutes.

2. The Ophiopogon japonicus and dried tangerine peel composition according to claim 1, characterized in that, The composition comprises, by weight: 80 parts of Ophiopogon japonicus extract, 20-40 parts of Citrus reticulata extract, 20-30 parts of Ziziphus jujuba seed powder, 20-30 parts of Poria cocos powder, 20-30 parts of maltodextrin, and 10 parts of xylitol.

3. The Ophiopogon japonicus and dried tangerine peel composition according to claim 1 or 2, characterized in that, The formula for the MRS medium is as follows: 5g sodium acetate, 10g peptone, 10g beef extract, 5g yeast extract, 2g dipotassium hydrogen phosphate, 2g diammonium citrate, 20g glucose, 0.58g magnesium sulfate heptahydrate, 0.19g manganese sulfate monohydrate, 1mL Tween 80, and 1L distilled water.

4. The Ophiopogon japonicus and dried tangerine peel composition according to claim 1 or 2, characterized in that, The culture medium formula of the Aspergillus niger fermentation broth is as follows: 120g grapefruit peel, 5g wheat bran, 5g soybean meal, 4g ammonium sulfate, 0.5g dipotassium hydrogen phosphate, and 1L water.

5. A method for preparing the Ophiopogon japonicus and dried tangerine peel composition according to any one of claims 1 to 4, comprising the following steps: Weigh out each component according to the specified amount, mix them evenly, and you will get the Ophiopogon japonicus and dried tangerine peel composition.

6. The use of the Ophiopogon japonicus and Citrus reticulata composition according to any one of claims 1 to 4 in the preparation of a medicament for relieving anxiety and improving sleep disorders.

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