Radix ophiopogonis and pericarpium citri reticulatae composition for relieving anxiety mood and improving sleep as well as preparation method and application of radix ophiopogonis and pericarpium citri reticulatae composition

Abstract

The invention discloses a radix ophiopogonis and pericarpium citri reticulatae composition for relieving anxiety mood and improving sleep. The composition comprises the following components in parts by weight: 50-80 parts of a radix ophiopogonis extract, 20-40 parts of a pericarpium citri reticulatae extract, 10-30 parts of spina date seed powder, 10-30 parts of poria cocos powder, 10-30 parts of maltodextrin and 5-10 parts of xylitol. The invention further discloses a preparation method and application of the radix ophiopogonis and pericarpium citri reticulatae composition. The composition disclosed by the invention realizes synergistic interaction through triple pathways of neuroinflammation inhibition, intestinal brain axis regulation and GABA energy system activation, and can effectively relieve anxiety mood and improve sleep disorder.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of food and health products, and particularly relates to a radix ophiopogonis and dried tangerine peel composition for relieving anxiety and improving sleep, and a preparation method and application thereof. BACKGROUND

[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 worldwide experiencing significant anxiety has continued to rise. Long-term sleep disorders affect mental and emotional states, while anxiety (such as persistent tension, worry, and irritability) is one of the factors that induce and exacerbate sleep disorders, thus forming a vicious cycle of anxiety-sleep disorders.

[0003] The pathological mechanisms of anxiety-sleep disorders include: (1) neuroendocrine imbalance: chronic anxiety activates the HPA axis, leading to abnormal elevation of cortisol, suppression of melatonin secretion, and disruption of 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] Existing intervention methods have significant defects: (1) long-term use of chemical drugs such as diazepam, estazolam, and clonazepam can induce sleep by inhibiting the excitability of the cerebral cortex, but may have adverse reactions such as daytime drowsiness and dizziness, and long-term use can lead to dependence, tolerance, and cognitive impairment; (2) traditional Chinese medicine extracts such as Suanzaoren, Baiziren, and Renshen have low bioavailability due to large molecular polysaccharides, making it difficult to achieve multi-target regulation; (3) melatonin alone is not effective in treating insomnia caused by psychological factors and environmental factors, and is only suitable for circadian rhythm disorder sleep-wake disorders, and may cause dizziness, headache, drowsiness, lack of concentration, nausea, diarrhea, and other symptoms, and may promote vasoconstriction, increase the risk of stroke, cause hormonal secretion disorders, interact with anticoagulants, antihypertensives, immunosuppressants, antidepressants, and contraceptives, increase the risk of adverse reactions or affect drug efficacy, and long-term or excessive use can inhibit the body's own melatonin secretion function, causing sleep function disorders and dependence, and may cause sleep quality to decline after discontinuation.

[0005] Therefore, there is an urgent need to develop a natural component-based composite preparation to effectively relieve anxiety and simultaneously improve sleep disorders directly caused by the emotion. SUMMARY

[0006] An object of the present application is to provide a composition that can effectively relieve anxiety and improve sleep.

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

[0008] Another object of the present application is to provide the use of the composition.

[0009] To achieve the above objects, the present application provides the following technical solutions. In a first aspect, the present application provides a Ophiopogon japonicus and Citrus reticulata Blanco composition for relieving anxiety and improving sleep, which contains Ophiopogon japonicus extract, Citrus reticulata Blanco extract, Suanzaoren powder, Fuling powder, maltodextrin and xylitol.

[0010] Preferably, the composition comprises 50-80 parts of Ophiopogon japonicus extract, 20-40 parts of Citrus reticulata Blanco extract, 10-30 parts of Suanzaoren powder, 10-30 parts of Fuling powder, 10-30 parts of maltodextrin and 5-10 parts of xylitol by weight.

[0011] More preferably, the composition comprises 80 parts of Ophiopogon japonicus extract, 20-40 parts of Citrus reticulata Blanco extract, 20-30 parts of Suanzaoren powder, 20-30 parts of Fuling powder, 20-30 parts of maltodextrin and 10 parts of xylitol by weight.

[0012] Most preferably, the composition comprises 80 parts of Ophiopogon japonicus extract, 40 parts of Citrus reticulata Blanco extract, 30 parts of Suanzaoren powder, 30 parts of Fuling powder, 30 parts of maltodextrin and 10 parts of xylitol by weight.

[0013] Preferably, the Ophiopogon japonicus extract in the above composition is prepared by the following steps: S1, drying Ophiopogon japonicus, crushing it into powder, then adding Lactobacillus plantarum fermentation broth for microbial fermentation, sterilizing after fermentation to obtain fermented Ophiopogon japonicus extract; S2, adding hemicellulase to the fermented Ophiopogon japonicus extract for enzymolysis, deactivating the enzyme, passing through an ultrafiltration membrane after cooling, collecting liquid with a molecular weight of less than 5 kDa, and spray drying or freeze drying to obtain Ophiopogon japonicus extract.

[0014] In step S1, the Lactobacillus plantarum fermentation broth comprises MRS culture medium and Lactobacillus plantarum GDMCC No. 64374, and the inoculation amount of Lactobacillus plantarum is 1×10 5 -2×10 5 CFU / mL. The mass ratio of Ophiopogon japonicus to Lactobacillus plantarum fermentation broth is preferably 1:2 to 1:5. The fermentation temperature is 30-35℃, and the fermentation time is 30-36 hours.

[0015] Preferably, the formula of the MRS culture medium is as follows: Sodium acetate 5g, proteose 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 the step S2, the enzymolysis process is as follows: first, add water with a volume of 10-20 times of the fermented Ophiopogon japonicus extract, adjust pH to 5.0-5.2, then add 5%-10% hemicellulase by weight of the fermented Ophiopogon japonicus extract, and carry out enzymolysis at 50-55℃ for 2-4 hours, and then inactivate the enzyme at 85℃ for 20-30min.

[0017] Preferably, in the above composition, the Pericarpium Citri Reticulatae extract is prepared by the following steps: Step 1: dry Pericarpium Citri Reticulatae, grind into powder, then add Aspergillus niger fermentation liquor for microbial fermentation, and obtain fermented Pericarpium Citri Reticulatae extract after sterilization; Step 2: add papain to the fermented Pericarpium Citri Reticulatae extract for enzymolysis, inactivate the enzyme, pass through an ultrafiltration membrane with a molecular weight cut-off of 10kDa after cooling, and then spray dry or freeze dry to obtain the Pericarpium Citri Reticulatae extract.

[0018] Preferably, in the step 1, the Aspergillus niger fermentation liquor comprises a culture medium and Aspergillus niger, and the inoculation amount of Aspergillus niger is 1×10 5 -2×10 5 CFU / mL. The mass ratio of Pericarpium Citri Reticulatae to Aspergillus niger fermentation liquor is preferably 1:2 to 1:5. The fermentation temperature is 25-30℃, and the fermentation time is 18-24 hours.

[0019] Preferably, the culture medium comprises pomelo peel, bran, soybean cake powder, ammonium sulfate, and dipotassium hydrogen phosphate.

[0020] More preferably, the formula of the culture medium is as follows: Pomelo peel 120g, bran 5g, soybean cake powder 5g, ammonium sulfate 4g, dipotassium hydrogen phosphate 0.5g, and water 1L.

[0021] Preferably, in the step 2, the enzymolysis process is as follows: first, add water with a weight of 10-20 times of the fermented Pericarpium Citri Reticulatae extract, adjust pH to 6.0-7.0, then add 1%-3% papain by weight of the fermented Pericarpium Citri Reticulatae extract, and carry out enzymolysis at 60℃-65℃ for 2-4 hours, and then inactivate the enzyme at 85℃ for 20-30min.

[0022] In the above composition, the Semen Ziziphi Spinosae powder and the Poria cocos powder are common commercially available Semen Ziziphi Spinosae and Poria cocos, which are dried, crushed or ground.

[0023] In the above composition, the malt dextrin 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; GABA system activation: Suanzaoren saponin A and Pachymic acid can enhance the binding force of GABA_A receptor, reduce the power of β wave of electroencephalogram, and inhibit the release of CRH in amygdala, so as to realize the bidirectional regulation of anxiety and insomnia. DETAILED DESCRIPTION

[0030] The technical solutions of the present application will be further described below in combination with specific examples.

[0031] Unless otherwise specified, the instruments or reagents used in the examples are conventional instruments or reagents in the art, which are conventional products that can be purchased on the market. Unless otherwise specified, the specific experimental operations involved in the text are understood or known by those skilled in the art according to their mastery of common knowledge or conventional technical means, and will not be described one by one.

[0032] Suanzaoren powder and Pachyman powder are ordinary commercially available Suanzaoren and Pachyman, which are obtained by drying, crushing or grinding.

[0033] The Lactobacillus plantarum fermentation broth includes MRS culture medium and Lactobacillus plantarum GDMCC No. 64374. Lactiplantibacillus plantarum Lactobacillus plantarum (Lactobacillus plantarum) GDMCC No. 64374 has been preserved in Guangdong Microbial Culture Collection Center (GDMCC), with the preservation number GDMCC No. 64374, the preservation date February 5, 2024, and the preservation address 5th Floor, Building 59, 100 Middle Field Road, Guangzhou, Guangdong Province.

[0034] The formula of the MRS culture medium is as follows: sodium acetate 5g, proteose peptone 10g, beef extract 10g, yeast extract 5g, potassium phosphate dibasic 2g, diammonium citrate 2g, glucose 20g, magnesium sulfate heptahydrate 0.58g, manganese sulfate monohydrate 0.19g, Tween 80 1mL, distilled water 1L.

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

[0036] The formula of the culture medium is as follows: pomelo peel 120g, bran 5g, soybean cake powder 5g, ammonium sulfate 4g, potassium phosphate dibasic 0.5g, water 1L.

[0037] Example 1 A Ophiopogon japonicus and dried tangerine peel composition for relieving anxiety and improving sleep, the component ingredient mass fraction is: 80 parts of Ophiopogon japonicus extract, 40 parts of dried tangerine peel extract, 30 parts of Suanzaoren powder, 30 parts of Pachyman powder, 30 parts of malt dextrin, and 10 parts of 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). 5The olibanum extract, the pericarpium citri reticulatae extract, the semen ziziphi spinosae powder, the poria cocos powder, the malt dextrin and the xylitol are mixed according to the above mass fractions to obtain the olibanum pericarpium citri reticulatae composition for relieving anxiety and improving sleep.

[0044] The olibanum extract, the pericarpium citri reticulatae extract, the semen ziziphi spinosae powder, the poria cocos powder, the malt dextrin and the xylitol are mixed according to the above mass fractions to obtain the olibanum pericarpium citri reticulatae composition for relieving anxiety and improving sleep.

[0045] Example 3 The olibanum extract, the pericarpium citri reticulatae extract, the semen ziziphi spinosae powder, the poria cocos powder, the malt dextrin and the xylitol are mixed according to the above mass fractions to obtain the olibanum pericarpium citri reticulatae composition for relieving anxiety and improving sleep.

[0046] The preparation method of the olibanum extract is as follows: the olibanum is dried, crushed into powder, and then sieved through a 20-mesh screen, then 2 times of mass of the bacillus lactimorbus fermentation liquor (inoculation amount is 2x10 5 CFU / mL) is added, fermentation is carried out at 32 DEG C for 34 hours, after sterilization at 100 DEG C for 10 min and cooling to room temperature, 20 times of volume of water is added, pH is adjusted to 5.0, then 6% (according to the weight of the fermented olibanum extract) of hemicellulase is added, enzymolysis is carried out at 52 DEG C for 4 hours, after enzyme inactivation at 85 DEG C for 20 min, cooling and filtering, liquid below 5 kDa is intercepted, and the olibanum extract is obtained by spray drying.

[0047] The preparation method of the pericarpium citri reticulatae extract is as follows: the pericarpium citri reticulatae is dried, ground into powder, and then sieved through a 20-mesh screen, then 2 times of mass of the aspergillus niger fermentation liquor (inoculation amount is 2x10 5 CFU / mL) is added, fermentation is carried out at 36 DEG C for 22 hours, after sterilization at 100 DEG C for 20 min and cooling to room temperature, 20 times of volume of water is added, pH is adjusted to 7.0, then 1% (according to the weight of the fermented pericarpium citri reticulatae extract) of papain is added, enzymolysis is carried out at 65 DEG C for 2 hours, enzyme inactivation is carried out at 85 DEG C for 30 min, after cooling and filtering, liquid below 10 kDa is intercepted, and the pericarpium citri reticulatae extract is obtained by spray drying.

[0048] The olibanum extract, the pericarpium citri reticulatae extract, the semen ziziphi spinosae powder, the poria cocos powder, the malt dextrin and the xylitol are mixed according to the above mass fractions to obtain the olibanum pericarpium citri reticulatae composition for relieving anxiety and improving sleep.

[0049] Example 4 The olibanum extract, the pericarpium citri reticulatae extract, the semen ziziphi spinosae powder, the poria cocos powder, the malt dextrin and the xylitol are mixed according to the above mass fractions to obtain the olibanum pericarpium citri reticulatae composition for relieving anxiety and improving sleep.

[0050] The preparation method of the Ophiopogon japonicus extract is as follows: Ophiopogon japonicus is dried, crushed into powder, and then passed through a 20-mesh sieve. Then, 4 times the mass of Lactobacillus plantarum fermentation liquor (inoculum amount: 1 x 10 5 CFU / mL) is added, and fermentation is carried out at 33°C for 32 hours. After sterilization at 100°C for 10 min and cooling to room temperature, 18 times the volume of water is added, and the pH is adjusted to 5.0. Then, 5% (based on the weight of the fermented Ophiopogon japonicus extract) of hemicellulase is added, and enzymatic hydrolysis is carried out at 53°C for 3 hours. Enzyme inactivation is carried out at 85°C for 20 min. After cooling, filtration is carried out, and liquid below 5 kDa is collected. The Ophiopogon japonicus extract is obtained by spray drying.

[0051] The preparation method of the Pericarpium Citri Reticulatae extract is as follows: Pericarpium Citri Reticulatae is dried, ground into powder, and then passed through a 20-mesh sieve. Then, 4 times the mass of Aspergillus niger fermentation liquor (inoculum amount: 1 x 10 5 CFU / mL) is added, and fermentation is carried out at 35°C for 20 hours. After sterilization at 100°C for 20 min and cooling to room temperature, 16 times the volume of water is added, and the pH is adjusted to 6.8. Then, 2% (based on the weight of the fermented Pericarpium Citri Reticulatae extract) of papain is added, and enzymatic hydrolysis is carried out at 63°C for 3 hours. Enzyme inactivation is carried out at 85°C for 20 min. After cooling, filtration is carried out, and liquid below 10 kDa is collected. The Pericarpium Citri Reticulatae extract is obtained by spray drying.

[0052] The Ophiopogon japonicus extract, the Pericarpium Citri Reticulatae extract, the Spondias dulcis powder, the Poria cocos powder, the malt dextrin, and the xylitol are mixed in the above-mentioned mass fractions to obtain the Ophiopogon japonicus and Pericarpium Citri Reticulatae composition for relieving anxiety and improving sleep.

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

[0054] The preparation method of the water-extracted Ophiopogon japonicus powder and the water-extracted Pericarpium Citri Reticulatae powder is as follows: Ophiopogon japonicus or Pericarpium Citri Reticulatae is dried and crushed. Then, 10 times the mass of 80°C hot water is added for extraction for 2 hours. Filtration is carried out, and the filtrate is spray dried to obtain the water-extracted Ophiopogon japonicus powder or the water-extracted Pericarpium Citri Reticulatae powder.

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

[0056] Comparative Example 3 In the composition, the Pericarpium Citri Reticulatae extract is not added. The other components and process parameters are the same as in Example 1.

[0057] Comparative Example 4 In the composition, the Spondias dulcis powder and the Poria cocos powder are not added. The other components and process parameters are the same as in Example 1.

[0058] Effect verification 1. Animal experiment SPF grade Kunming mice (half male and half female, 18-22 g), 10 in each group, were randomly divided into groups. Set up a blank control group (normal saline), a positive control group, and an experimental group (Examples 1-4, Comparative Examples 1-4). Each group was given intragastric administration once a day, with a volume of 0.1 mL / 10 g, for 7 consecutive days.

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

[0060] The intragastric administration solution of each experimental group was the corresponding composition, prepared at a concentration of 250 mg / mL (solvent: normal saline).

[0061] (1) Elevated plus maze test in mice Thirty minutes after the last administration, the mice were placed in the elevated plus maze (open arm / closed arm), and the time spent in the open arm was recorded for 5 minutes. The proportion of open arm residence time (open arm residence time / total time) was calculated. The number of entries into the open arm was also recorded.

[0062] (2) Light-dark box test in mice Thirty minutes after the last administration, the mice were placed in the dark zone of the light-dark box, and the behavior was recorded for 5 minutes. The time spent in the light zone and the number of light-dark transitions were recorded.

[0063] (3) Pentobarbital sodium synergistic sleep test in mice Sleep rate (subthreshold dose) (30 mg / kg): 30 minutes after the last administration, pentobarbital sodium (30 mg / kg) was injected intraperitoneally, and the number of mice that fell asleep within 15 minutes was recorded. (The righting reflex disappeared for more than 1 minute).

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

[0065] (4) Inhibition of spontaneous activity in mice Twenty minutes after the last administration, the mice were placed in the spontaneous activity instrument, and the number of walking grids and the number of forelimb lifts within 10 minutes were recorded.

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

[0067] Table 1. Results of the anxiety behavior experiment in mice

[0068] In the elevated plus maze experiment, the more the anxious mice tend to avoid the open arms, the better the anti-anxiety effect. From the results in Table 1, the open arm residence time ratio of Example 1 group reached 45.3%, close to 49.8% of the positive control group, increased by 198% compared with the blank control group, and was significantly higher than that of Comparative Examples 1-4. The open arm entry times were 6, close to 6.5 of the positive control group, and were significantly higher than those of Comparative Examples 1-4. By comparing the results of Comparative Examples 1-4, it was found that the change of different components or processes had some influence on the results, but under the appropriate ratio, they were all significantly better than the blank control group and Comparative Examples 1-4.

[0069] In the light-dark box experiment, the anxious mice will reduce the exploration of the light area, and the larger the value, the better the anti-anxiety effect. From the results in Table 1, the light area residence time of Example 1 group was 185 s, which increased by 155% compared with the blank control group, and was significantly higher than that of Comparative Examples 1-4. The light area shuttle times were 11.9 times, which increased by 129% compared with the blank control group, and were significantly higher than those of Comparative Examples 1-4. It shows that the composition of the application has a good effect of relieving anxiety.

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

[0071] Table 2. Results of the mouse sleep experiment

[0072] From the results in Table 2 above, the sleep rate and sleep time of Examples 1-4 were close to those of the positive control group. The sleep rate of Comparative Example 1 was only 30%, and the sleep time was 34% shorter than that of Example 1, which shows that the content of active ingredients of Ophiopogon japonicus and dried tangerine or orange peel can be greatly improved by fermentation / enzymatic hydrolysis, and the bioavailability is enhanced. In Comparative Examples 2-4, Ophiopogon japonicus extract, dried tangerine or orange peel extract, or acid jujube powder and tuckahoe powder are not added in the composition, and the sleep rate and sleep time are not as good as Example 1, which shows the necessity of the synergistic effect of Ophiopogon japonicus extract, dried tangerine or orange peel extract, acid jujube powder and tuckahoe powder. Similarly, in terms of the spontaneous activity inhibition rate, the larger the value, the better the sedative effect. From the data of each group, Examples 1-4 have an effect close to that of the positive control group, and Comparative Examples 1-4 are not as good as Examples 1-4. Therefore, it shows that the composition of the application has a good effect of improving sleep.

[0073] (5) Neurotransmitter level experiment One hour after the last administration, blood was taken from the orbit, 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 taken by decapitation, and the hypothalamic region was separated. The levels of hypothalamic GABA and CRH were detected by chemiluminescence immunoassay (CLIA).

[0074] Table 3 shows the levels of mouse serum 5-HT, MT and hypothalamic GABA

[0075] 5-hydroxytryptamine (5-HT) is an important monoamine neurotransmitter in the central nervous system, which is involved in sleep-wake regulation. RIN-14B is a rat pancreatic tumor-derived cell line with the function of synthesizing, storing and secreting 5-HT, and its secretion mechanism is similar to that of 5-HT neurons in vivo. It can be used to evaluate the anxiolytic and sleep-promoting effects of samples, and the larger the value is, the better the effect is.

[0076] Melatonin (MT) is an indole heterocyclic hormone secreted by the pineal gland, which regulates circadian rhythm and sleep-wake cycle, and inhibits the hypothalamic-pituitary-gonadal axis by activating MT1 / MT2 receptors, promotes deep sleep and shortens sleep latency. Rat or mouse animal models are widely used to evaluate the sleep-promoting effect of melatonin, by measuring serum melatonin levels, sleep time extension and sleep latency reduction, etc. It can simulate the process of neuroendocrine regulation in vivo, and is suitable for verifying the improvement effect of samples on sleep disorders. The larger the value is, the better the effect is.

[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: rapidly increasing blood glucose under stress, promoting fat decomposition, and activating the "fight or flight" response; (2) circadian rhythm: secretion is high in the morning and low at night, imbalance can cause insomnia, anxiety and central obesity; (3) metabolic interference: long-term over-high leads to increased hunger, appetite, high-fat and high-sugar diet preference and abnormal fat accumulation. Clinically, serum fraction detection is used to evaluate HPA axis function and assist in the diagnosis and treatment of anxiety-sleep comorbidity. The value is fluctuating, and the afternoon or night is still elevated, indicating abnormal body.

[0078] Gamma-aminobutyric acid (GABA) is an important inhibitory neurotransmitter in the central nervous system of mammals, which mediates more than 40% of inhibitory neural transmission and is involved in physiological functions such as calming, anti-anxiety, improving sleep and blood pressure regulation. Its mechanism of action includes reducing neuronal excitability through postsynaptic membrane hyperpolarization and inhibiting glutamate release, thereby promoting deep sleep and shortening sleep time. It can also be used to evaluate the sleep-promoting effect of samples, and the larger the value is, the better the effect is.

[0079] Hypothalamic CRH is a peptide neurohormone synthesized by paraventricular nucleus, as the starting signal of HPA axis. Its core functions include: (1) ACTH drive: activate pituitary to release ACTH through the pituitary portal system, and then stimulate cortisol synthesis; (2) stress integration: converge physiological / psychological stress signals, enhance anxiety behavior (such as reducing open arm exploration in elevated maze) through amygdala; (3) multisystem regulation: inhibit gastric acid secretion, delay gastrointestinal peristalsis, and participate in blood pressure and blood glucose regulation. Hypothalamic CRH detection is a key indicator for studying anxiety neural pathways and the mechanism of anti-anxiety drugs. When the hypothalamic-pituitary regulation link is diseased or under stress, the value will increase, that is, the higher the value, the higher the anxiety level.

[0080] From the results of Table 3, compared with the blank control group, 5-HT in Example 1 increased by 154%, MT increased by 324%, GABA increased by 146%, COR decreased by 52%, and CRH decreased by 56%. Examples 2-4 have different results due to changes in different components or processes, but the change trend and amplitude are the same as the positive control group. It shows that in the composition of the present application, each component has a synergistic gain effect under the appropriate ratio. In Comparative Examples 1-4, the composition does not add Ophiopogon japonicus extract, Pericarpium Citri Reticulatae extract or Suanzaoren powder and Tuckahoe powder, although the levels of 5-HT, MT and GABA are improved to different degrees compared with the blank control group, and the levels of COR and CRH are decreased to different degrees compared with the blank control group, but the improvement amplitude is greatly different from that of Examples 1-4, which also confirms the synergistic gain effect of each component in the composition of the present application.

[0081] In summary, the composition of the present application has synergistic effects of components: Ophiopogon japonicus reduces fire, Pericarpium Citri Reticulatae regulates qi, Suanzaoren and Tuckahoe calm the nerves, and achieves the effect of relieving anxiety and improving sleep through regulating multiple pathways of 5-HT, MT, COR, GABA and CRH.

[0082] The above description is only the preferred embodiment of the present application, and does not limit the present application in any form, so any simple modification, equivalent change and modification of the above embodiment according to the technical essence of the present application, which does not deviate from the technical solution of the present application, still belongs to the scope of the technical solution of the present application.

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: 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.

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 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 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.

4. The Ophiopogon japonicus and dried tangerine peel composition according to claim 3, characterized in that, 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 was 1:2 to 1:

5. The fermentation temperature was 30-35℃, and the fermentation time was 30-36 hours.

5. The Ophiopogon japonicus and dried tangerine peel composition according to claim 3, characterized in that, 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.

6. The Ophiopogon japonicus and dried tangerine peel composition according to claim 1 or 2, characterized in that, 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.

7. The Ophiopogon japonicus and dried tangerine peel composition according to claim 6, characterized in that, 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 The optimal mass ratio of tangerine peel to Aspergillus niger fermentation broth is 1:2 to 1:5, with a fermentation temperature of 25-30℃ and a fermentation time of 18-24 hours.

8. The Ophiopogon japonicus and dried tangerine peel composition according to claim 6, characterized in that, 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.

9. A method for preparing the Ophiopogon japonicus and dried tangerine peel composition according to any one of claims 1 to 8, 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.

10. The use of the Ophiopogon japonicus and Citrus reticulata peel composition according to any one of claims 1 to 8 in the preparation of food, health products, and pharmaceuticals for relieving anxiety and improving sleep disorders.

Images