Emotional health improvement composition, preparation method and application thereof

Abstract

The application discloses an emotion health improvement compound, a preparation method and application, and belongs to the technical field of medicine and functional food. Active components of the compound composition are R-carvyl ketone, yeast polysaccharide microporous carrier and lactic acid bacteria powder. The compound composition can effectively avoid the problems of R-carvyl ketone degradation by gastric acid and oxidation and volatilization in the digestive tract, and significantly improves the oral stability and bioavailability. The results of in vivo animal experiments show that the compound composition can effectively improve the irritable and irritable state, and relieve the overexcited behavior reaction induced by stress. The application provides an excellent formula basis and technical support for developing an oral product for relieving daily stress and improving emotional health.

Description

Technical Field

[0001] This invention relates to a compound for improving emotional health, its preparation method, and its application, belonging to the field of pharmaceutical and functional food technology. Background Technology

[0002] With the accelerating pace of society, increasing work and life pressures, and the rapid aging of the population, emotional health problems such as anxiety, mental stress, low mood, sleep disorders, and chronic stress-induced behavioral abnormalities are on the rise. Numerous studies have shown that emotional health is not only closely related to the balance of neurotransmitters in the central nervous system, but is also jointly regulated by multiple factors including the neuroendocrine system, the immune system, and the gut microbiota. Long-term emotional imbalance and stress not only reduce an individual's quality of life but may also further increase the risk of metabolic diseases, cardiovascular diseases, and neuropsychiatric disorders. Therefore, developing safe, effective, and suitable-for-long-term daily emotional health intervention products has become an important research direction in the pharmaceutical and functional food fields.

[0003] Currently, interventions for the aforementioned emotional health issues mainly include traditional drug therapy, psychological intervention, and functional nutritional supplements. Compared to traditional anti-anxiety chemical drugs, which are prone to side effects such as drowsiness, drug tolerance, and dependence, active ingredients derived from natural plants are highly favored due to their superior safety profile. R-carvone is a core monoterpene compound naturally found in plants such as spearmint. Modern neuropharmacological studies have confirmed that R-carvone can easily cross the blood-brain barrier, significantly relieving stress, alleviating tension and anxiety, and providing a mild sedative effect by targeting and regulating GABA receptors in the central nervous system and reducing abnormal neuronal excitability. It is a highly valuable active ingredient for emotional health interventions.

[0004] However, R-carvone faces severe physicochemical limitations in practical applications: as a volatile, lipid-soluble small molecule, it is highly susceptible to volatilization, oxidation, and degradation during processing, storage, and exposure to air, leading to the loss of its core activity. More challenging is that if ingested directly in its free form via oral administration, R-carvone is vulnerable to destruction by highly acidic gastric juices and is easily consumed by digestive enzymes and first-pass metabolism in the liver, resulting in low intestinal absorption and poor bioavailability, making it difficult to maintain long-lasting blood concentrations for relieving hypertension. Therefore, overcoming the physicochemical barriers of R-carvone and significantly improving its processing stability, resistance to gastric acid, and in vivo delivery efficiency are key technological challenges that must be overcome to achieve its large-scale industrial application.

[0005] On the other hand, recent in-depth research in the life sciences has revealed the central role of the "gut-brain axis" in the regulation of host emotions, cognition, and behavior. As an important target for intervention in the gut-brain axis, functional probiotics such as lactic acid bacteria can not only effectively reshape the intestinal microecological barrier, but also fundamentally improve the body's tolerance to long-term psychological stress by stimulating the vagus nerve, reducing systemic inflammation caused by chronic stress, and promoting the metabolism and secretion of endogenous neurotransmitters (such as serotonin precursors and GABA).

[0006] However, in practical industrialization and clinical applications, using probiotics alone to intervene in emotional health faces significant shortcomings. First, live bacteria are extremely sensitive to processing temperature, oxygen, and storage conditions, and are prone to significant inactivation during their shelf life. Second, the fragile free bacteria are highly susceptible to the corrosive effects of strong acidic gastric juices and high concentrations of bile salts, resulting in low survival and colonization rates in the gut. More importantly, the process by which probiotics regulate the gut microbiota is relatively mild and slow, typically requiring a long intervention period, making it difficult to address sudden periods of mental stress or urgent emotional needs for rapid stress relief. Therefore, single-target probiotic products often fail to comprehensively and efficiently cover the complex regulatory networks involved in emotional imbalance processes.

[0007] Based on the above analysis of mechanisms and limitations, if R-carvone, which targets the central nervous system and has a rapid onset of action, can be scientifically combined with lactic acid bacteria, which regulate the gut-brain axis and has a long-lasting effect, a natural bidirectional mood regulation system can be constructed, providing both rapid stress relief and calming from the top down and long-term ecological stability from the bottom up. However, due to the significant differences in their physicochemical properties and their shared survival crisis caused by the disruption of the acidic environment in the digestive tract, there are few existing technologies that can simultaneously consider the biological activities of both and achieve targeted and synergistic delivery. Summary of the Invention

[0008] To address the shortcomings of existing technologies, the present invention aims to provide a compound composition for improving mood health, its preparation method, and its applications. This invention, through the synergistic effect of yeast microencapsulation and gut-brain axis microecological regulation, completely solves the technical challenges of poor physicochemical stability, low oral bioavailability, and the inability of a single component to effectively reverse mood imbalance behaviors over a long period. Furthermore, the mood health improving compound can be prepared into a pharmaceutical preparation or functional food, which can be used daily to relieve stress, improve anxiety and tension, and alleviate sleep disorders related to mood imbalance. It is highly safe and suitable for long-term use.

[0009] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a compound composition for improving emotional health, wherein the active ingredients of the compound composition include, by weight, 5-30 parts of R-carvone, 30-60 parts of yeast polysaccharide microporous carrier, and 10-50 parts of lactic acid bacteria powder.

[0010] In one embodiment of the present invention, the improvement of emotional health includes at least one of relieving anxiety, improving depression, improving irritability, relieving mood swings, stabilizing emotions, and reducing aggressive behavior.

[0011] In one embodiment of the present invention, the yeast polysaccharide microporous carrier comprises one or more of inactivated whole cells of Saccharomyces cerevisiae, purified yeast cell wall extract, or yeast β-glucan.

[0012] The yeast polysaccharide microporous carrier has a microporous multi-network structure, serving as a physical encapsulation carrier. R-carvone is loaded and embedded within its internal micropores or lumens, forming a stable microencapsulated structure. This microencapsulated structure remains highly dense in the strongly acidic gastric juice environment, while swelling in the near-neutral intestinal environment, enabling the targeted and long-lasting release of active monoterpenoid compounds from lactic acid bacteria, thereby significantly improving bioavailability after oral ingestion.

[0013] In one embodiment of the present invention, the lactic acid bacteria powder includes *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum Lactobacillus gasseri ( Lactobacillus gasseri Lactobacillus rhamnosus ( Lactobacillus rhamnosus ) or fermenting lactobacillus ( Limosilactobacillus fermentum At least one of the following.

[0014] In one embodiment of the present invention, the lactic acid bacteria powder is a lyophilized powder or a microencapsulated lyophilized powder. Preferably, the number of viable bacteria in the lyophilized powder is not less than 1 × 10⁻⁶. 10 CFU / g.

[0015] A second aspect of the present invention provides a method for preparing the compound composition, comprising the following steps: S1. Mixing: Disperse the yeast polysaccharide microporous carrier in water to form a uniform suspension, and then add R-carvone dropwise to obtain a mixed solution; S2, Ultrasonic treatment: The mixture of S1 is ultrasonically treated to form an R-carvone-yeast complex suspension; S3. Drying: The R-carvone-yeast complex suspension of S2 is dried, pulverized, and sieved to obtain R-carvone-yeast microcapsule powder; S4. Dry mixing and compounding: The R-carvone-yeast microcapsule powder of S3 is uniformly mixed with the lactic acid bacteria powder to obtain the compound composition.

[0016] In one embodiment of the present invention, the yeast polysaccharide microporous carrier comprises one or more of inactivated whole cells of Saccharomyces cerevisiae, purified yeast cell wall extract, or yeast β-glucan.

[0017] In one embodiment of the present invention, the ultrasound conditions are 0~4℃, 300~500 W ultrasound for 20~40 minutes.

[0018] In one embodiment of the present invention, in S3, the drying is vacuum drying, spray drying, or freeze drying.

[0019] In one embodiment of the present invention, in S4, the lactic acid bacteria include *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum Lactobacillus gasseri ( Lactobacillus gasseri Lactobacillus rhamnosus ( Lactobacillus rhamnosus ) or fermenting lactobacillus ( Limosilactobacillus fermentum At least one of the following.

[0020] In one embodiment of the present invention, the lactic acid bacteria powder is a lyophilized powder or a microencapsulated lyophilized powder. Preferably, the number of viable bacteria in the lyophilized powder is not less than 1 × 10⁻⁶. 10 CFU / g.

[0021] A third aspect of the present invention provides a medicament comprising the compound composition.

[0022] In one embodiment of the present invention, the drug is an oral dosage form, and specific dosage forms include, but are not limited to: hard capsules, tablets, microemulsions, dry suspensions, solid beverages, or gel candies.

[0023] A fourth aspect of the invention provides the use of the compound composition in the preparation of a medicament for improving mood or in the preparation of a medicament for the prevention and / or adjunctive treatment of neuropsychiatric disorders accompanied by irritability and aggressive behavior.

[0024] In one embodiment of the present invention, the application includes: (1) Relieve anxiety; (2) Improves depression; (3) Improve irritability; (4) Alleviate emotional fluctuations and stabilize emotions; (5) Reduce aggressive behavior; In one embodiment of the present invention, the neuropsychiatric abnormalities accompanying irritability and aggressive behavior include: persistent mental stress, sudden irritability, spatial exploration anxiety disorder, and stress-induced gastrointestinal discomfort caused by abnormally elevated levels of monoamine neurotransmitters (such as 5-HT and GABA) and peripheral corticosterone (CORT) in the central nervous system.

[0025] Beneficial effects: (1) This invention innovatively utilizes the microporous structure of yeast polysaccharides to physically encapsulate R-carvone through microencapsulation. This structure is resistant to gastric acid degradation, can target and swell in intestinal fluid, and provide long-lasting sustained release, perfectly solving the technical problem that volatile monoterpenoids are easily destroyed orally and have low bioavailability.

[0026] (2) This composition breaks through the bottleneck of single-component efficacy and achieves bidirectional regulation: R-carvone efficiently crosses the blood-brain barrier and activates central GABA receptors to quickly suppress nerve hyperactivity; lactic acid bacteria reshape the intestinal microecology through the "gut-brain axis" and long-term downregulate the stress response of the HPA axis (hypothalamus-pituitary-adrenal axis).

[0027] (3) The compound of this invention can completely suppress abnormally elevated central nervous system excitatory neurotransmitters (such as NE and octopamine) and peripheral stress hormones (CORT) to a healthy baseline, significantly improving mental anxiety and emotional imbalance. Its efficacy is significantly better than that of single-ingredient preparations, and it can even restore the emotional stability of animals to a level better than that of the normal control group, which represents a substantial technological advancement. Attached Figure Description

[0028] Figure 1 Example 1 of this invention: Two-stage accumulation and release kinetic curves of free R-carvone and formulation A in in vitro simulated gastric juice (SGF) and simulated intestinal juice (SIF).

[0029] Figure 2 The effect of the compound in Example 1 of this invention on the baseline movement distance and anxiety index (central zone dwell time) of chronically socially isolated mice in the open field test (OFT).

[0030] Figure 3 The effect of the compound in Example 1 of this invention on the number of overreactions in the test tube brush test of mice with chronic social isolation.

[0031] Figure 4 The figure shows the effect of the compound in Example 1 of this invention on the total duration and frequency of territorial attacks in resident-intruder experiments in mice with chronic social isolation.

[0032] Figure 5 The diagram shows the effect of the compound in Example 1 of this invention on the levels of central norepinephrine (NE) and peripheral serum corticosterone (CORT) in mice with chronic social isolation.

[0033] Biological Preservation Materials A strain of Lactobacillus plantarum ( Lactiplantibacillus plantarum Y24, taxonomically named Lactiplantibacillus plantarumIt was deposited on February 4, 2026 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO:67804, located at the Guangdong Institute of Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Detailed Implementation

[0034] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0035] Test method: Determination of embedding rate: The encapsulation efficiency of the microcapsule powder was determined by gas chromatography (GC). First, the surface of the microcapsule powder was rapidly washed with petroleum ether to remove free R-carvone from the surface (recorded as M). 游离 ); In addition, the microcapsule structure was completely destroyed by Soxhlet extraction using anhydrous ethanol / n-hexane mixed solvent, and the total weight of R-carvone inside was extracted and measured (recorded as M). 总 ).

[0036] The formula for calculating the embedding rate is: EE% = [(M 总 - M 游离 ) / M 总 × 100%. Each formulation was tested in parallel for 3 independent batches (n=3).

[0037] Raw materials used in the examples: R-carvone: ee value ≥ 99.5% Purified Saccharomyces cerevisiae cell wall extract: purchased from Angel Yeast Co., Ltd., mainly composed of β-glucan (29%–64%), mannan (31%), protein (13%), lipids (9%) and chitin (1%–2%).

[0038] Yeast β-glucan: purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number G1501903.

[0039] Whole-cell brewer's yeast: purchased from Angel Yeast Co., Ltd.

[0040] Lactobacillus plantarum ( Lactiplantibacillus plantarum ): GDMCC No: 67804.

[0041] Lactobacillus gasseri ( Lactobacillus gasseri ): GDMCC No: 64320.

[0042] Fermented Lactobacillus mucinus ( Limosilactobacillus fermentum ): GDMCC No:65071.

[0043] Lactobacillus rhamnosus ( Lactobacillus rhamnosus ): GDMCC No:65131.

[0044] The preparation method of the lactic acid bacteria freeze-dried powder is as follows: After activation and expansion culture, the bacterial cells were collected by centrifugation, resuspended in a freeze-drying protectant (composed of 10 wt% skim milk powder and 5 wt% trehalose), pre-frozen at -40℃ for 4 h, and then freeze-dried at a vacuum of <10 Pa and a cold trap temperature of -50℃ for 48 h. The resulting product was then pulverized and passed through a 100-mesh sieve to obtain the freeze-dried lactic acid bacteria powder (viable count ≥1×10¹). 0 CFU / g).

[0045] Example 1: A method for preparing a compound composition for improving emotional health (Formula A, standard type) This embodiment provides a method for preparing a compound composition for improving mood health. R-carvone is encapsulated in purified Saccharomyces cerevisiae cell wall extract and then compounded with lyophilized lactic acid bacteria powder (Lactobacillus plantarum) to obtain finished product Formula A. Details are as follows: S1. Hydration and Mixing: Weigh 40.0 g of purified Saccharomyces cerevisiae cell wall extract and suspend it evenly in 1000 mL of ultrapure water. Stir at 500 rpm for 30 min to fully hydrate and expand the polysaccharide network. Then, slowly add R-carvone (15.0 g) dropwise at 0.5 g / min while stirring continuously at 800 rpm. S2, Ultrasonic Treatment: The mixture from S1 was placed in an ice-water bath at 0°C and ultrasonicated at 400 W for 30 min. Utilizing the strong acoustic cavitation effect generated by ultrasound, the free essential oil was sheared into nano-sized droplets and forcibly squeezed into the inner cavity of the polysaccharide microporous network, forming a highly stable oil-in-water (O / W) microemulsion; S3, vacuum freeze-drying: The microemulsion of S2 was pre-frozen at -80℃ for 12 h, then transferred to a vacuum dryer (vacuum degree <10Pa, cold trap temperature -50℃) and dried for 48 h. It was then pulverized through an 80-mesh sieve and collected to obtain R-carvone-yeast microcapsule powder. S4, Dry Mixing: In a Class 100,000 cleanroom with relative humidity ≤20% and temperature 20℃, mix the R-carvone-yeast microcapsule powder of S3 with *Lactobacillus plantarum* (… L . plants 20.0 g of freeze-dried powder was placed in a V-type mixer and dry-mixed for 20 min until uniform to obtain the finished product of formulation A, with an effective loading of 17.5% R-carvone.

[0046] Example 2: Preparation of a compound composition for improving mood health (Formula B, immediate release type) This embodiment provides a method for preparing a compound composition for improving mood health. R-carvone is encapsulated with yeast β-glucan and then compounded with lyophilized lactic acid bacteria powder (Lactobacillus paracasei) to obtain the finished product, Formulation B. Details are as follows: S1. Hydration and Mixing: Weigh out 30.0 g of yeast β-glucan and suspend it evenly in 1000 mL of ultrapure water. Stir at 500 rpm for 30 min to fully hydrate and expand the polysaccharide network. Then, slowly add R-carvone (20.0 g) dropwise at 1.0 g / min while stirring continuously at 800 rpm.

[0047] S2. Ultrasonic treatment: Same as in Example 1; S3. Spray drying: The microemulsion of S2 is rapidly dried using a spray drying process (with inlet air temperature set at 120℃, outlet air temperature at 60℃, and feed rate at 15 mL / min), and the microcapsule powder is collected. S4. Dry Mixing: In a Class 100,000 cleanroom with relative humidity ≤20% and temperature 20℃, mix the microcapsule powder of S3 with lyophilized Lactobacillus paracasei powder (40.0 g) evenly to obtain the finished product of Formula B, with an effective loading of 17.6% R-carvone.

[0048] Example 3: Preparation of a compound composition for improving emotional health (Formula C, compound type) This embodiment provides a method for preparing a compound composition for improving mood health. R-carvone is encapsulated in whole cells of *Saccharomyces cerevisiae* and then compounded with lyophilized lactic acid bacteria powder (*Lactobacillus rhamnosus* and *Lactobacillus fermentum*) to obtain the finished product, formulation C. Details are as follows: S1. Hydration and Mixing: Weigh out 60.0 g of whole cells of Saccharomyces cerevisiae and suspend them evenly in 1000 mL of ultrapure water. Stir at 500 rpm for 30 min to fully hydrate and expand the polysaccharide network. Then, slowly add R-carvone (10.0 g) dropwise at 0.5 g / min while stirring continuously at 800 rpm. S2. Ultrasonic treatment: Same as in Example 1; S3, Vacuum freeze drying: Same as in Example 1; S4. Dry Mixing: In a Class 100,000 cleanroom with relative humidity ≤20% and temperature 20℃, mix the microcapsule powder of S3 with lyophilized Lactobacillus rhamnosus (10.0 g) and lyophilized Lactobacillus fermentum (10.0 g) evenly to obtain the finished product of Formula C, with an effective loading of 9.6% R-carvone.

[0049] The encapsulation efficiency of the finished products from Examples 1-3 was determined, and the results are shown in Table 1. The effects of different encapsulation carriers and drying processes on the encapsulation efficiency of R-carvone showed that the encapsulation efficiency of formulation A in Example 1 was (82.3±2.6)%, that of formulation B in Example 2 was (78.5±3.1)%, and that of formulation C in Example 3 was (85.1±1.9)%. It can be seen that all three preparation processes can achieve high encapsulation efficiency. Among them, formulation C, which uses whole cells of Saccharomyces cerevisiae as a carrier, has the highest encapsulation efficiency, while formulation B, which uses spray drying, has a slightly lower encapsulation efficiency, but it still meets the development needs of functional products. Moreover, spray drying is more suitable for large-scale industrial production, with lower production costs and higher production efficiency, and is more in line with the industrialization requirements of immediate-release products.

[0050] Table 1: Results of R-carvone microencapsulation and embedding efficiency under different carrier formulations and processes (n=3)

[0051] The results in Table 1 show that the yeast physical microencapsulation process in Examples 1-3 all exhibited extremely high loading efficiency (>85%), indicating that the yeast polysaccharide micropores have a strong hydrophobic locking ability for highly lipid-soluble monoterpenoid molecules, which can ensure the stable loading of R-carvone in this invention and provide a basis for targeted release via oral administration.

[0052] Example 4: In vitro simulated gastrointestinal release and stability evaluation of the compound composition This embodiment describes the release pattern of the composition (formulation A) prepared in Example 1 in a simulated human gastrointestinal environment.

[0053] Artificial gastric juice (SGF: pH 1.2, containing 10 g / L pepsin) and artificial intestinal juice (SIF: pH 6.8, containing 10 g / L pancreatic enzyme) were prepared.

[0054] Powder from Formulation A and free R-carvone (control group) containing an equivalent dose of R-carvone were weighed separately and added to a dissolution vessel containing 900 mL of SGF medium (37±0.5℃, 100 rpm), and incubated for 2 h. At the end of the 2h, the powder was centrifuged to remove impurities and then rapidly transferred to SIF medium for further incubation until the 6th h. Samples were taken periodically, and the concentration of R-carvone was quantitatively determined by high-performance liquid chromatography (HPLC). Each sample was measured in triplicate.

[0055] The results are as follows Figure 1 As shown in Table 2, after incubation in simulated gastric juice (pH 1.2, containing pepsin) for 2 h, the retention rate of unencapsulated free R-carvone reached 35.93 ± 2.63%, while the cumulative release rate of R-carvone in the powder of formulation A in Example 1 was 11.52 ± 1.32%.

[0056] After the gastric-incubated sample was transferred to simulated intestinal fluid (pH 6.8, containing pancreatic enzymes), R-carvone in Formula A powder showed a continuous release trend, with a cumulative release rate of 87.63 ± 1.69% over 6 hours.

[0057] The above results demonstrate that the powder of formulation A in Example 1 has good gastric stability and enteric release characteristics: it can effectively protect the active ingredients in the gastric environment, and achieve sustained release in the intestinal environment as the polysaccharide pores swell and open, confirming that the composition of Example 1 can achieve intestinal-targeted sustained-release delivery of active essential oils.

[0058] Table 2: Comparison of stability and release rate of free R-carvone and formulation A in two simulated gastrointestinal fluid stages (n=3)

[0059] Example 5: Effects of the compound composition on central neurotransmitters and irritability in cold-treated bumblebees This embodiment uses invertebrates (bumblebees) as experimental subjects to investigate the effects of Formula A from Example 1 on central neurotransmitters and irritable behavior in bumblebees, as detailed below: Animal grouping: 100 healthy 7-day-old worker bees (bumblebees) of the same batch with consistent physiological state were selected and randomly divided into blank control group, model group, treatment group 1 (formula A group), treatment group 2 (R-carvone single formula group) and treatment group 3 (Lactobacillus plantarum single formula group), with 20 animals in each group.

[0060] Cold treatment molding: Each group underwent cold treatment molding for 7 consecutive days: each day, the molding process was carried out at 4℃ for 20 min, followed by recovery at 25℃ for 10 min, and the cycle was repeated 5 times.

[0061] Blank control group: fed at room temperature at 25℃ without cold treatment for modeling, and provided with a basal 40% (w / v, g / mL) sugar solution.

[0062] Model group: Models were cold-treated and provided with a basal 40% (w / v, g / mL) sugar solution during modeling.

[0063] Treatment Group 1 (Formula A): The compound composition (Formula A) prepared in Example 1 was mixed with 40% sucrose solution to prepare a sugar solution containing 1.0 mg / mL of the compound composition. Throughout the experiment, the sugar solution was placed in a feeder for bumblebees to freely consume.

[0064] Treatment group 2 (R-carvone monotherapy group): Microcapsule powder containing only R-carvone was mixed with 40% sucrose water to prepare a sugar solution containing 1.0 mg / mL of microcapsule powder. Throughout the experiment, bumblebees were allowed to freely ingest the solution via a feeder.

[0065] Treatment group 3 (Lactobacillus plantarum single-ingredient group): Lactobacillus plantarum lyophilized powder was mixed with 40% sucrose solution to prepare a sugar solution containing 1.0 mg / mL of Lactobacillus plantarum lyophilized powder. Throughout the experiment, the sugar solution was placed in a feeder for bumblebees to freely consume.

[0066] Indicator testing: After 7 consecutive days of intervention, standardized mechanical probing was performed on the abdomen of bumblebees using soft silicone. The number of individuals in each group exhibiting obvious defensive aggressive behavior (such as raising forelegs and extending the stinger) within 5 seconds of stimulation was recorded. The formula for calculating the incidence of irritable aggression is as follows: The incidence of irritable aggression (%) = (N1 / N2) × 100%; In the formula, N1 represents the number of individuals in the experimental group who exhibited obvious defensive aggressive behavior; N2 represents the total number of surviving individuals in the experimental group who underwent standardized mechanical testing (N2 = 20).

[0067] Immediately after the behavioral tests, bumblebee brain ganglia were dissected and isolated on ice. The supernatant was extracted by ultrasonic homogenization and centrifugation, and the absolute concentration of the excitatory neurotransmitter octopamine was quantitatively determined using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). The results are shown below. Figure 2 And Table 3.

[0068] The experimental results in Table 3 show that after low-temperature treatment, the excitatory neurotransmitter octopamine in the bumblebee brain increased, and the frequency of violent defensive aggression behavior significantly increased. While the R-carvone single-agent group (treatment group 2) and the Lactobacillus plantarum single-agent group (treatment group 3) showed some intervention effects, the compound composition of this invention (treatment group 1) exhibited a more significant sedative effect (P < 0.01). This data confirms that the compound composition of this invention successfully overcomes the effect of R-carvone alone, demonstrating a strong synergistic effect in improving the emotional health of bumblebees.

[0069] Table 3: Bumblebee Behavior and Changes in Central Octopamine Levels

[0070] Note: P < 0.05 P < 0.01, compared with the blank control; # P < 0.05 ## P < 0.01, compared with the model group; n = 20.

[0071] Example 6: Validation of the effect of the compound composition on improving the emotional health of chronically socially isolated mice This embodiment uses an animal model of chronic social isolation to investigate the effects of Formula A from Example 1 on improving anxiety, sudden irritability, and social impairment induced by long-term psychological stress. Details are as follows: 1. Construction of an Irritable Isolation Model One hundred healthy male C57BL / 6J mice were randomly divided into a blank control group, a model group, a treatment group 1, a treatment group 2, and a treatment group 3 (n=20 / group).

[0072] Control group: The animals were housed in a standard group setting (5 animals per cage) for 6 consecutive weeks, with plenty of social interaction provided.

[0073] Model group and treatment group: Mice were housed alone in opaque cages (1 mouse per cage), strictly deprived of visual and physical contact with other mice, and kept in isolation for 6 consecutive weeks.

[0074] Treatment Group 1: Mice were acclimatized for one week and then subjected to isolation stress for six weeks. Subsequently, they were administered a corn oil solution of Formula A (Formula A was dissolved in corn oil to prepare a 5 mg / mL solution, with an administration volume of 10 mL / kg body weight) by gavage daily for 7 consecutive days at a dose of 50 mg / kg body weight (based on the total mass of Formula A).

[0075] Treatment Group 2: Mice were acclimatized for one week and then subjected to isolation stress for six weeks. Subsequently, they were administered a corn oil solution of R-carvone (prepared by dissolving R-carvone in corn oil to a concentration of 5 mg / mL, with an administration volume of 10 mL / kg body weight) by gavage daily for 7 consecutive days at a dose of 50 mg / kg body weight (based on the total mass of R-carvone).

[0076] Treatment Group 3: Mice were acclimatized for one week and then subjected to isolation stress for six weeks. Subsequently, they were administered a corn oil suspension of Lactobacillus plantarum lyophilized powder (prepared by suspending Lactobacillus plantarum lyophilized powder in corn oil to a concentration of 5 mg / mL, with a dosage of 10 mL / kg body weight) by gavage daily for 7 consecutive days at a dose of 50 mg / kg body weight.

[0077] The blank control group and the model group were given the same amount of pure corn oil by gavage.

[0078] 2. Behavioral testing and data analysis The day after 7 consecutive days of intervention, the following tests were conducted using the EthoVision XT animal behavior video tracking system: 2.1 Open Field Test (OFT): Assessing basic motor skills and anxiety levels Mice were placed in the center of a standardized open field box (50 cm × 50 cm × 50 cm, divided into a central area and a peripheral area). The system automatically tracked and recorded the total distance the mice traveled and the time spent in the central area over 5 minutes.

[0079] Table 4: Effects of the compound on basal motor function and anxiety indices in mice (n=20)

[0080] Note: P < 0.05 P < 0.01, compared with the blank control group; # P < 0.05 ## P < 0.01, compared with the model group; n = 20.

[0081] Figure 2 Table 4 shows the behavioral test results of each group of subjects in the open field experiment. Compared with the normal control group, long-term isolation modeling resulted in a significantly shorter central zone dwell time in the model group in the open field (P < 0.01) and a significantly increased total movement distance (P < 0.01), exhibiting obvious anxiety and tension behaviors. After intervention, the use of R-carvone (treatment group 2) and Lactobacillus plantarum (treatment group 3) alone could improve the above indicators to some extent. Furthermore, the central zone dwell time of the compound composition group of the present invention (treatment group 1) showed a highly significant recovery (P < 0.01), and the total movement distance steadily decreased to near the level of the normal control group. The above data indicate that the compound composition of the present invention can produce a highly significant synergistic effect, thereby more effectively relieving mental stress and improving emotional health.

[0082] 2.2 Test tube brush test The experimenter quickly brought a standard test tube brush close to and touched the mouse's face and whiskers from above, repeating the test five times and recording the total number of times the mouse exhibited excessive agitation. (1) Criteria for judging an overreaction include: biting the brush bristles, stiffening the body or emitting a threatening scream.

[0083] (2) Recording rules: If a mouse exhibits any one or more of the above behaviors, it is counted as one positive reaction, and no duplicates are accumulated.

[0084] Table 5: Effects of the compound on sudden irritability in isolated mice (n=20)

[0085] Note: P < 0.05 P < 0.01, compared with the blank control group; # P < 0.05 ## P < 0.01, ### P < 0.001 compared with the model group; n = 20.

[0086] Figure 3 Table 5 presents the behavioral results of the mouse tube brush test. Compared with the normal control group, the modeling resulted in a significant increase in the number of overreactions when faced with sudden contact. After intervention, the number of overreactions in treatment groups 2 and 3 decreased (P < 0.05 compared with the model group); while the number of overreactions in treatment group 1 was significantly reduced to 2.4 times (P < 0.001 compared with the model group).

[0087] The data above indicate that the compound composition of Example 1 can effectively alleviate the irritability response of test subjects when faced with sudden stimuli. Furthermore, it is confirmed that the compound composition is significantly more effective than the single component in stabilizing behavior and improving emotional health (reducing anxiety and aggressive behavior stemming from irritability).

[0088] 2.3 Inhabitant-Intruder Experiment On the day following 7 days of continuous drug intervention, the territorial aggression phenotype of mice in each group was assessed using a video tracking system. Mice in the model group and treatment group, kept in isolated individual cages, resided directly in their original cages. In the group-housed control group, all other mice in the same cage were removed 30 minutes before the test, leaving only one test mouse as the resident. Subsequently, a smaller (approximately 10-15% lighter) syngeneic normal male mouse in a group-housed configuration was introduced into the cage of each resident as an intruder. The total duration and number of explicit physical attacks (biting, aggressive chasing, lateral threatening) initiated by the resident within 10 minutes were recorded.

[0089] Table 6: Effects of the compound on resident-intruder territorial aggression behavior in isolated mice (n=20)

[0090] Note: P < 0.05 P < 0.01, compared with the blank control group; # P < 0.05 ## P < 0.01, compared with the model group; n = 20.

[0091] Figure 4Table 6 presents the behavioral results of each group of mice in the resident-intruder experiment. Compared with the blank control group, long-term isolation modeling led to a significant increase in the total duration and number of attacks in the model group (P < 0.01). After equal-volume gavage intervention, the duration and number of attacks in treatment groups 2 and 3 decreased; while the duration and number of attacks in treatment group 1 decreased significantly (compared with the model group, P < 0.01), and both indicators had basically returned to the level of the normal control group.

[0092] The data above indicate that the compound composition of Example 1 can effectively suppress aggressive behavior induced by long-term isolation stress. Furthermore, it is confirmed that the compound composition is significantly more effective than the single-component composition in stabilizing mood and improving emotional health.

[0093] 2.4 Physiological Indicator Detection Peripheral blood was collected and serum was separated by centrifugation. The absolute concentration of serum corticosterone (CORT) was detected using a high-sensitivity mouse enzyme-linked immunosorbent assay (ELISA) kit.

[0094] Mice were euthanized, and their brains were rapidly dissected. Hypothalamic tissue blocks were precisely excised and flash-frozen in liquid nitrogen. The abundance of norepinephrine (NE) per milligram of tissue protein was quantitatively determined using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS).

[0095] Table 7: Effects of intervention on central NE and peripheral corticosterone physiological indicators in isolated mice (n=20)

[0096] Note: P < 0.05 P < 0.01, compared with the blank control group; # P < 0.05 ## P < 0.01, compared to the model group; n = 20.

[0097] Figure 5 Table 7 shows the changes in neuroendocrine indicators in each group of subjects.

[0098] Compared with the blank control group, long-term isolation modeling led to a significant increase in the levels of the central neurotransmitter norepinephrine (NE) and the peripheral stress hormone corticosterone (CORT) in the model group (P < 0.01). After equal-volume gavage intervention, the NE and CORT levels in treatment groups 2 and 3 decreased; while the NE and CORT levels in treatment group 1 decreased significantly (compared with the model group, P < 0.01), and both indicators had basically returned to the levels of the normal control group.

[0099] The data above indicate that the compound composition of Example 1 can effectively downregulate central NE and peripheral CORT levels under stress. By correcting this neuroendocrine imbalance, the compound composition is significantly more effective than the single component in relieving stress and improving emotional health.

[0100] In summary, the compound composition of this invention successfully achieves a synergistic effect of "1+1>2" while remaining safe and reliable. Through a dual action of "brain soothing + intestinal regulation," it fundamentally alleviates anxiety, tension, and mood swings induced by long-term psychological stress. These experimental data fully demonstrate the excellent stress-relieving effect of this formula, laying a solid foundation for its future development into oral products (such as functional foods or health supplements) for improving emotional health and relieving daily stress.

[0101] Comparative Example 1: Referring to Example 1, the difference is that in S1, R-carvone is not encapsulated, and the free R-carvone is directly mixed with the purified Saccharomyces cerevisiae cell wall extract. In S2, ultrasonic treatment is omitted, and the product is directly obtained by vacuum freeze drying in S3 and dry mixing in S4.

[0102] The results showed that, due to the lack of ultrasonic embedding, free R-carvone underwent significant volatilization and degradation during vacuum freeze-drying and was unable to withstand gastric acid damage. Specific comparative results are shown in Table 8. Table 8: Comparison of core physicochemical and behavioral indicators between Comparative Example 1 and Example 1

[0103] Comparative Example 2: Refer to Example 1, except that no lactic acid bacteria freeze-dried powder is added, otherwise it is the same as Example 1.

[0104] The results showed that, due to the lack of microecological regulation by lactic acid bacteria, the composition could not effectively downregulate peripheral stress hormones in model animals for a long period. Specific comparative results are shown in Table 9. Table 9: Comparison of core physiological and behavioral indicators between Comparative Example 2 and Example 1

[0105] Comparative Example 3: Refer to Example 1, except that R-carvone microcapsules are not added, otherwise the same as in Example 1.

[0106] The results showed that, due to the lack of R-carvone microcapsules, which target the central nervous system and have a rapid onset of action, the composition was less effective in responding to sudden psychological stimuli. Specific comparative results are shown in Table 10. Table 10: Comparison of core biochemical and behavioral indicators between Comparative Example 3 and Example 1

[0107] Comparative Example 4: Referring to Example 1, the difference is that the purified Saccharomyces cerevisiae cell wall extract was replaced with sodium alginate (40.0g), and the following steps were added: a 2% calcium chloride solution was prepared as the curing solution, the mixture after S2 ultrasonic treatment was dripped into the calcium chloride solution and cured for 30 min, and the cured microcapsules were collected and freeze-dried. The rest is the same as in Example 1.

[0108] The results showed that after replacing the *Saccharomyces cerevisiae* cell wall extract of this invention with conventional wall material sodium alginate, the product could not achieve the unique anti-gastric acid degradation properties. Specific comparative results are shown in Table 11: Table 11: Comparison of core physicochemical and behavioral indicators between Comparative Example 4 and Example 1

[0109] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A compound composition for improving emotional health, characterized in that, The active ingredients of the compound composition, by weight, include: 5-30 parts of R-carvone, 30-60 parts of yeast polysaccharide microporous carrier, and 10-50 parts of lactic acid bacteria powder. The yeast polysaccharide microporous carrier includes one or more of the following: inactivated whole cells of Saccharomyces cerevisiae, purified yeast cell wall extract, or yeast β-glucan. The lactic acid bacteria powder includes *Lactobacillus plantarum* (… Lactiplantibacillus plantarum Lactobacillus gasseri ( Lactobacillus gasseri Lactobacillus rhamnosus ( Lactobacillus rhamnosus ) or fermenting lactobacillus ( Limosilactobacillus fermentum At least one of the following; The improvement of emotional health includes at least one of the following: relieving anxiety, improving depression, improving irritability, relieving mood swings, stabilizing emotions, and reducing aggressive behavior.

2. A method for preparing the compound composition of claim 1, characterized in that, Includes the following steps: S1. Mixing: Disperse the yeast polysaccharide microporous carrier in water to form a uniform suspension, and then add R-carvone dropwise to obtain a mixed solution; S2, Ultrasonic treatment: The mixture of S1 is ultrasonically treated to form an R-carvone-yeast complex suspension; S3. Drying: The R-carvone-yeast complex suspension of S2 is dried, pulverized, and sieved to obtain R-carvone-yeast microcapsule powder; S4. Dry mixing and compounding: The R-carvone-yeast microcapsule powder of S3 is uniformly mixed with the lactic acid bacteria powder to obtain the compound composition.

3. The method according to claim 2, characterized in that, In S1, the yeast polysaccharide microporous carrier includes one or more of the following: inactivated whole cells of Saccharomyces cerevisiae, purified yeast cell wall extract, or yeast β-glucan.

4. The method according to claim 2, characterized in that, In S3, the drying process is either vacuum drying, spray drying, or freeze drying.

5. The method according to claim 2, characterized in that, In S4, the lactic acid bacteria include *Lactobacillus plantarum* (… Lactiplantibacillus plantarum Lactobacillus gasseri ( Lactobacillus gasseri Lactobacillus rhamnosus ( Lactobacillus rhamnosus ) or fermenting lactobacillus ( Limosilactobacillus fermentum At least one of the following.

6. The method according to claim 2, characterized in that, In S4, the lactic acid bacteria powder is lyophilized powder or microencapsulated lyophilized powder; the number of live bacteria in the lyophilized powder is not less than 1×10⁻⁶. 10 CFU / g.

7. A drug, characterized in that, The drug contains the compound composition of claim 1.

8. The medicament according to claim 7, characterized in that, The drug is an oral dosage form, and specific dosage forms include, but are not limited to: hard capsules, tablets, microemulsions, dry suspensions, solid beverages, or gel candies.

9. The use of the compound composition of claim 1 or the compound composition prepared by any of the methods of claims 2 to 6 in the preparation of a medicament for improving mood or in the preparation of a medicament for the prevention and / or adjunctive treatment of neuropsychiatric disorders accompanied by irritability and aggressive behavior.

10. The application according to claim 9, characterized in that, The application includes at least one of the following (1) to (5): (1) Relieve anxiety; (2) Improves depression; (3) Improve irritability; (4) Alleviate emotional fluctuations and stabilize emotions; (5) Reduce aggressive behavior.

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