Composite complete set of health-care product composition based on double extraction of gold and ground as well as preparation method and application thereof

Abstract

The invention provides a composite complete set of health-care product composition based on double extraction of gold and radix rehmanniae as well as a preparation method and application of the composite complete set of health-care product composition. The complete set of health-care product composition is constructed based on yin deficiency and internal heat and circadian rhythm regulation and control concepts of traditional Chinese medicine. The beverage set contains three kinds of a honeysuckle probiotic composite beverage, a Jinzhi double-extraction fermentation beverage and spina date seed composite particles. Through systematic compatibility, a specific'golden-land symbiotic fermentation 'process and a systematic time-sharing intervention scheme based on circadian rhythm, cell and animal experiments prove that the product can significantly inhibit release of inflammatory factors, improve the activity of antioxidant enzymes, relieve liver cell lipid accumulation and endoplasmic reticulum stress, improve the immunity of liver cells and improve the immunity of the liver cells. The traditional Chinese medicine composition is capable of improving anxiety-like behaviors and sleep structures, regulating intestinal flora structures, increasing abundance of beneficial bacteria such as phylum firmicalis and reducing abundance of harmful bacteria such as proteobacteria, so that an overall conditioning effect is achieved at multiple targets, and a systematic intervention scheme based on circadian rhythm is provided for improving the sub-health status of modern pressure people.

Description

Technical Field

[0001] This invention relates to the field of health food technology, and in particular to a composite set of health products based on the dual extraction of gold and earth, its preparation method and application. Background Technology

[0002] In the rapid development of contemporary society, the fast pace of life has reshaped the lifestyles of the masses. Unhealthy habits such as prolonged sleep deprivation, high stress levels, and irregular eating habits have become the norm. This has led to a significant increase in the number of people with a "Yin deficiency and internal heat" constitution, a sub-healthy condition that has become a significant area of ​​health management. This group generally suffers from a deficiency of Yin fluids and internal heat, leading to a series of complex and interconnected health problems. Elevated inflammation levels are one of its typical manifestations. A prolonged state of low-grade chronic inflammation not only causes fatigue and aches but also increases the risk of chronic diseases. Oxidative stress imbalance accelerates cell aging, resulting in poor skin condition and decreased immunity. Abnormal liver function further affects the body's metabolism and detoxification functions, creating a vicious cycle of "increasing internal heat and further depleting Yin fluids."

[0003] Meanwhile, mental and emotional distress is equally prominent. Yin deficiency and internal heat easily disturb the mind, leading to anxiety, irritability, insomnia, and excessive dreaming, severely impacting sleep quality and emotional state. Gut microbiota imbalance disrupts the body's digestive and absorptive balance, further exacerbating physical imbalances and causing multi-systemic health deterioration. From a market supply perspective, the current health supplement market is dominated by single-function products, such as plant extract preparations focused on anti-inflammation, melatonin products emphasizing sleep aid, or probiotic preparations that solely regulate gut microbiota. While these products can alleviate local symptoms, they fail to align with the core principle of Traditional Chinese Medicine's "holistic view." They neglect the core pathogenesis of "yin deficiency and internal heat" and fail to consider the influence of the body's circadian rhythm on the treatment effect, making it difficult to achieve a systematic, multi-target improvement of sub-health conditions. Therefore, the current market urgently needs a health product that not only uses a multi-target approach based on the holistic view of Traditional Chinese Medicine but also integrates with the body's circadian rhythm to form a systematic "daytime cleansing and nighttime replenishment" intervention plan, fundamentally improving the sub-health condition caused by "yin deficiency and internal heat."

[0004] It is worth noting that sub-health issues related to "yin deficiency and internal heat" are showing a trend of becoming more prevalent and affecting younger people. If these issues are not addressed scientifically and systematically over a long period, they will not only severely reduce the quality of life for individuals, but may also further induce organic diseases such as chronic liver disease, anxiety, and intestinal diseases, potentially having a lasting impact on individual quality of life and public health resources.

[0005] Modern research indicates a close correlation between "yin deficiency and internal heat" and chronic low-grade inflammation, oxidative stress, gut microbiota dysbiosis, and hypothalamic-pituitary-adrenal axis dysfunction. Therefore, developing a health product capable of systematically intervening in these physiological disorders through multiple targets is of significant practical importance.

[0006] It should be noted that the core active ingredients selected in this invention (such as honeysuckle, rehmannia root, cornus officinalis, and jujube seed) are all listed in the "List of Substances That Are Traditionally Both Food and Traditional Chinese Medicine" or the "Pharmacopoeia of the People's Republic of China" as substances with both medicinal and edible properties, possessing a long history of consumption and a recognized safety basis. The excipients and additives used (such as fructooligosaccharides, resistant dextrin, erythritol, and potassium sorbate) all comply with the provisions of GB 2760 "National Food Safety Standard for the Use of Food Additives" and GB 14880 "National Food Safety Standard for the Use of Food Fortifiers." The probiotic strains used are all derived from the "List of Microbial Strains that Can Be Used in Food." Therefore, the formula of this invention adheres to food safety regulations at the source, providing initial assurance for the long-term safety of the product.

[0007] Therefore, the present invention aims to provide a systematic intervention scheme based on the above-mentioned theory, which includes a specific product combination, a preparation process, and a method of administration designed according to the circadian rhythm theory. Summary of the Invention

[0008] Modern research indicates that the state of "yin deficiency and internal heat" is closely related to multiple systemic physiological imbalances, including chronic low-grade inflammation, oxidative stress, gut microbiota dysbiosis, and hypothalamic-pituitary-adrenal axis dysfunction. This invention systematically intervenes in these physiological imbalances through multi-target mechanisms, such as regulating the gut microbiota-brain axis and inhibiting the NF-κB inflammatory pathway. It should be clarified that this invention provides a health food combination suitable for individuals with sub-health management needs, aiming to provide auxiliary regulatory effects through dietary means; it does not have disease prevention, treatment, or diagnostic functions.

[0009] The purpose of this invention is to solve the above-mentioned problems and provide a composite health product combination based on Jindi Shuangcui, its preparation method, and its application. This composite health drink combination contains a honeysuckle probiotic compound drink for morning consumption, a Jindi Shuangcui fermented drink for bedtime consumption, and jujube seed compound granules. This combination of traditional Chinese medicine extracts, probiotics, fermentation active substances, and sleep-aiding functional factors, through a "Jindi symbiotic fermentation" process and a circadian rhythm drinking strategy, helps regulate inflammation, oxidative stress, liver function, mood, sleep, and gut microbiota.

[0010] The technical solution of this invention is: a set of health drinks based on Jindi Shuangcui compound, containing honeysuckle probiotic compound drink, Jindi Shuangcui fermented drink, and jujube seed compound granules.

[0011] The honeysuckle probiotic compound beverage contains, by weight, 100-200 parts honeysuckle extract, 300-400 parts compound probiotics, 1300-1600 parts fructooligosaccharides as a high-quality prebiotic to promote the colonization of probiotic strains, and other excipients.

[0012] The Jindi Shuangcui Fermented Beverage, by weight, contains 500-700 parts (preferably 600 parts) of Jindi symbiotic fermentation liquid, 150-300 parts (preferably 200 parts) of Cornus officinalis extract, 1400-1600 parts (preferably 1500 parts) of galactooligosaccharides, 1400-1600 parts (preferably 1500 parts) of purified water, and other excipients. The Jindi symbiotic fermentation liquid is a liquid extract of Rehmannia glutinosa and Lonicera japonica, which is a liquid obtained by bidirectional fermentation of Lactobacillus plantarum and yeast, and contains 4-6 parts of Rehmannia glutinosa and 1 part of Lonicera japonica by weight.

[0013] The jujube seed compound granules, by weight, contain 140-160 parts by weight (preferably 150 parts) of γ-aminobutyric acid (GABA), 90-110 parts by weight (preferably 100 parts) of tea theanine, and 280-320 parts by weight (preferably 300 parts) of jujube seed extract, as well as other excipients. GABA can directly inhibit neuronal activity and help promote relaxation. Tea theanine can promote the production of alpha waves in the brain, relieve anxiety, and exhibit a synergistic effect with GABA. Jujube seed extract: Jujube seed is a food and medicine homologous substance traditionally used for its calming effects.

[0014] It should be noted that the "Excipient A," "Excipient B," and "Excipient C" mentioned in this invention are conventional excipients acceptable in the food or health product formulation field, such as fillers, anti-caking agents, preservatives, and flavoring agents. Those skilled in the art can select them according to the product dosage form and general requirements.

[0015] In a preferred embodiment of the present invention, excipient A includes, but is not limited to, resistant dextrin and silicon dioxide; excipient B includes, but is not limited to, potassium sorbate; and excipient C includes, but is not limited to, erythritol and microcrystalline cellulose.

[0016] Preferably, the resistant dextrin in excipient A is water-soluble dietary fiber, used as a filler, and the silica is used as an anti-caking agent.

[0017] Honeysuckle extract is rich in active ingredients such as chlorogenic acid, with a standardized chlorogenic acid content of at least 5%. According to traditional understanding, honeysuckle is cold in nature and is classified as a heat-clearing ingredient; modern research shows that it has anti-inflammatory and antioxidant effects.

[0018] The total live bacteria count of the compound probiotics is at least 50 billion CFU; the compound probiotics contain *Lactobacillus plantarum* LP-03, *Lactobacillus rhamnosus* LR-01, *Bifidobacterium animalis* subsp. *lactobacter* BB651, and *Bifidobacterium breve* B-3. This strain combination was selected to target the immune system and intestinal barrier. In the animal models of this study (Experiments 2 and 3), positive regulatory trends related to improved systemic inflammation, oxidative stress, and intestinal barrier function were observed; these physiological processes are known to be associated with the regulation of mood and sleep.

[0019] Preferably, the excipient B is 1-1.5 parts by weight of potassium sorbate (1.2 parts by weight is optimal), and the Cornus officinalis extract contains at least 1% mononoside. The Cornus officinalis extract, in conjunction with Rehmannia glutinosa, enhances the liver and kidney tonifying effects.

[0020] Preferably, the excipient C is 600-1000 parts by weight of erythritol (preferably 800 parts by weight) and 300-500 parts by weight of microcrystalline cellulose (preferably 400 parts by weight), used as a filler and disintegrant; the jujube seed extract contains ≥1.5% spinosin, which is the core flavonoid active ingredient in jujube seed and a key active substance for sedation, hypnosis, and anti-anxiety effects of jujube seed.

[0021] Secondly, the present invention provides a method for preparing the composite health care product combination based on Jindi Shuangcui, characterized in that the key to the preparation method is the adoption of a two-way fermentation process in the order of "yeast first, then lactobacillus" (i.e., "Jindi symbiotic fermentation process").

[0022] The described "yeast-first, lactobacillus-later" bidirectional sequential fermentation process is designed based on the principles of microbial ecology. Its advantages are as follows: the first-inoculated brewer's yeast preferentially utilizes the fermentable sugars in the extract for growth and reproduction, consuming oxygen and producing ethanol, carbon dioxide, and flavor precursors, creating a more suitable micro-anaerobic environment and flavor foundation for the subsequent growth of *Lactobacillus plantarum*; the second-inoculated *Lactobacillus plantarum* grows vigorously under suitable conditions, continuously producing acid (lactic acid, etc.), further lowering the pH value and inhibiting contaminating bacteria. Simultaneously, its rich enzyme system may transform specific glycosides and other components in *Rehmannia glutinosa* and *Lonicera japonica*, increasing the content of active small molecules. This sequential fermentation facilitates a synergistic improvement in flavor mellowness, full conversion of active ingredients, and product stability.

[0023] This process is designed based on the principles of microbial ecology. It involves first inoculating with *Saccharomyces cerevisiae* for pre-fermentation, followed by inoculation with *Lactobacillus plantarum* for primary fermentation. Fermentation parameters are set as follows: 28℃ is the optimal growth temperature for *Saccharomyces cerevisiae*, facilitating rapid oxygen consumption and flavor compound production; 37℃ is the optimal fermentation temperature for *Lactobacillus plantarum*, promoting sufficient acid production and the transformation of active ingredients in the raw materials. Fermentation durations of 48 hours and 72 hours were optimized through preliminary experiments to ensure sufficient microbial growth and component transformation, and to achieve a stable fermentation endpoint (pH ≤ 4.0). This sequence aims to optimize the fermentation process, create a unique product flavor, and maximize the accessibility and synergistic effects of functional components (such as oligosaccharides, amino acids, and catalpol) in raw materials like *Rehmannia glutinosa* and *Lonicera japonica* through microbial transformation, thereby improving the product's bioavailability and potential health benefits. The specific steps include:

[0024] S1. Preparation of Honeysuckle Probiotic Complex Drink

[0025] According to claim 1, take honeysuckle extract with ≥5% standardized chlorogenic acid, compound probiotics, and fructooligosaccharides, mix them evenly, and then package them into aluminum foil bags of 2g each after dry granulation.

[0026] S2. Preparation of Jindi Double Extract Fermented Beverage

[0027] According to claim 1, Rehmannia glutinosa and Lonicera japonica are mixed, extracted by hot water circulation, and concentrated to 1g / mL by low temperature vacuum. Then, Saccharomyces cerevisiae is first inoculated and fermented at 28℃ for 48 hours, and then Lactobacillus plantarum is inoculated and fermented at 37℃ for 72 hours. The final pH is controlled to be ≤4.0 to obtain a mellow flavor and fermented sour aroma of Rehmannia glutinosa and Lonicera japonica symbiotic fermentation liquid.

[0028] Take the above fermentation broth as the base material, add Cornus officinalis extract, galactooligosaccharides, and purified water to the total volume, stir evenly, pasteurize and fill into bottles of 15mL each.

[0029] S3. Preparation of Ziziphus jujuba seed compound granules:

[0030] According to claim 1, take γ-aminobutyric acid, tea theanine, and jujube seed extract, mix them evenly with the excipients, pass them through an 80-mesh sieve, and package them into aluminum foil bags, 2g per bag;

[0031] Steps S1, S2, and S3 are not in any particular order.

[0032] An application of the aforementioned combination of health products based on Jindi Shuangcui, or the combination of health products based on Jindi Shuangcui obtained by the aforementioned method, wherein the health drinks are used to assist in the regulation of inflammation, oxidative stress, liver function, mood and sleep, and gut microbiota in sub-healthy individuals.

[0033] Preferably, when the composite complete health care product based on Jindi Shuangcui is applied, it includes a preparation taken in the morning and a preparation taken before going to bed at night. Specifically, 1 bag (2 g) of the honeysuckle probiotic composite drink is taken on an empty stomach in the morning, and the Jindi Shuangcui fermented drink and the sour jujube kernel composite granule are taken before going to bed at night. Pour 1 bag (2 g) of the sour jujube kernel composite granule into 1 bottle (15 mL) of the Jindi Shuangcui fermented drink, shake well and drink directly. Or it can be taken with 100 mL of warm water.

[0034] Preferably, the inflammatory factors targeted by the anti-inflammatory application include at least one of IL-1β, IL-6 and TNF-α.

[0035] Preferably, the antioxidant application is to increase at least one of the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX), and / or reduce the content of malondialdehyde (MDA);

[0036] Preferably, the potential adjuvant regulation of liver health is to reduce at least one of serum glutamic pyruvic transaminase (ALT) and aspartate aminotransferase (AST), and / or reduce liver lipid accumulation;

[0037] Preferably, the application of adjuvant improvement of mood and sleep status is to reduce anxiety-like behaviors, extend sleep time and / or shorten sleep latency;

[0038] Preferably, the application of regulating intestinal flora is to reduce the abundance of harmful flora and / or increase the abundance of beneficial flora;

[0039] The harmful bacteria include Proteobacteria, and the Proteobacteria include at least one of Escherichia coli and Shigella;

[0040] The beneficial bacteria include Firmicutes, and the Firmicutes include at least one of Clostridium and Collinsella.

[0041] The three products described in the present invention are not simply superimposed, but are systematically designed based on the traditional Chinese medicine treatment principle of 'clearing - tonifying - calming' and the circadian rhythm theory. It is speculated that the synergistic mechanism may be achieved through the multi-axis linkage of the 'gut-liver axis' and the 'gut-brain axis', etc.: The morning product regulates intestinal flora and barrier function, reduces endotoxin entering the blood, and reduces the liver inflammation burden ('gut-liver axis'); while the night product improves liver function and oxidative stress, and its active ingredients (such as GABA and spinosin) and microbial metabolites (such as short-chain fatty acids) may affect the central nervous system through the circulatory system or the vagus nerve, synergistically improving mood and sleep ('gut-brain axis'). This multi-target and multi-axis systematic intervention is the key to the overall conditioning of this set of products.

[0042] Its potential pathways of action may include: regulating the gut microbiota and influencing the production of its metabolites, short-chain fatty acids (such as butyrate), which then act on the liver via blood circulation, regulating intrahepatic inflammation and lipid metabolism ('gut-liver axis'); simultaneously, gut microbiota metabolic signals or active ingredients in the product (such as GABA) may affect central nervous system function through vagal nerve conduction or the blood-brain barrier, synergistically improving mood and sleep ('gut-brain axis'). This multi-axis linkage is an important theoretical basis for the systemic conditioning achieved by this treatment.

[0043] It should be noted that the aforementioned synergistic mechanism involving multiple axes such as the 'gut-liver axis' and 'gut-brain axis' is a systemic action hypothesis based on the functions of the components of this invention, existing scientific literature, and the multi-target, simultaneous improvement trends observed in the experimental examples below (such as the simultaneous observation of improved gut microbiota, reduced liver inflammation, and reduced anxiety behavior in experimental examples 2 and 3). The focus of this invention's protection claim is on the 'cleansing-replenishing-safety' time-separated combination product system and its verified overall regulatory effects; its specific and detailed molecular pathway mechanisms are within the scope of further in-depth research.

[0044] The beneficial effects of this invention are:

[0045] The composite product combination and its time-based administration plan, designed based on the "Cleansing-Nourishing-Soothing" principle of Traditional Chinese Medicine and the theory of human circadian rhythms, provided by this invention, were validated using cell and animal models in Examples 1-4. Experimental results show that this product combination can simultaneously and multi-target regulate multiple interrelated physiological indicators. Specifically, the product combination can simultaneously exert a positive regulatory trend on multiple interrelated physiological targets, including inflammatory responses, oxidative stress, liver function indicators, mood and sleep behavior, and gut microbiota structure. This synergistic intervention capability targeting multiple aspects of complex sub-health states overcomes the shortcomings of existing single-function health products in terms of specificity, providing a systematic intervention plan based on circadian rhythms for comprehensively improving the "Yin deficiency and internal heat" type of sub-health state.

[0046] This invention provides a complete set of health drinks based on the "Golden Earth Double Extract" compound, its preparation method, and its application in improving sub-health. Based on the traditional Chinese medicine theory of "Yin deficiency and internal heat" and circadian rhythm regulation, this scheme constructs a complete product set consisting of a honeysuckle probiotic compound drink, a "Golden Earth Double Extract" fermented drink, and jujube seed compound granules. Through systematic formulation, innovative "Golden Earth Symbiotic Fermentation" technology, and based on the theoretical functions of each component during the day (clearing heat, regulating the intestines) and at night (nourishing Yin, calming the mind), combined with the human body's circadian rhythm physiological characteristics such as cortisol (high in the morning and low in the evening) and melatonin (high at night), a time-segmented administration system based on circadian rhythm is proposed and verified. Simulated feeding results in animal experiments show that the intervention using this scheme is effective. This provides new ideas and research basis for applying the human circadian rhythm theory to the system design of health products. The complete set of health drinks of this invention, through specific formulation and process, shows a positive regulatory trend on multiple targets in experimental models. Its preparation method is stable and controllable, and the resulting product is safe and free of toxic side effects, possessing broad application prospects and market value. Preliminary verification through cell and animal experiments shows that, in the experimental models, the product exhibits a positive regulatory trend in inhibiting the release of inflammatory factors, increasing the activity of antioxidant enzymes, reducing lipid accumulation in hepatocytes and endoplasmic reticulum stress, improving anxiety-like behavior and sleep structure, and regulating the gut microbiota (such as increasing the abundance of beneficial Firmicutes and decreasing the abundance of harmful Proteobacteria).

[0047] The "time-segmented administration regimen" of this invention is based on the "theoretical design" and "systematic conception" of component functions and human circadian rhythms. The "simulated time-segmented feeding" regimen in animal experiments verifies this concept. "Reverse circadian rhythm control" is a direction for future optimization research, but existing data already demonstrates the effectiveness of this regimen. The experimental data in this application provide valuable preclinical research evidence for the development of this product as a health food with potential effects such as anti-inflammatory, antioxidant, liver protection, mood and sleep improvement, and gut microbiota regulation.

[0048] In summary, the "morning cleansing and evening nourishing" time-based intervention program of this invention is not only designed based on theoretical usage methods, but also has the potential to improve overall regulatory efficiency and synergistic effects by conforming to the body's circadian rhythm (such as the secretion patterns of cortisol and melatonin) so that the two types of components, "clearing heat and regulating the intestines" and "nourishing yin and calming the mind," can exert their effects when their respective target organs (such as the intestines, liver, and nervous system) are in their optimal functional state. This provides a new research and development approach for developing systemic health care products based on chronobiology. Attached Figure Description

[0049] Figure 1 This is a graph showing the effect of each treatment group on the viability of L02 cells in Experiment Example 1;

[0050] Figure 2This is a graph showing the effects of each treatment group on the level of reactive oxygen species (ROS) in L02 cells in Experiment Example 1.

[0051] Figure 3 This is a graph showing the effect of each treatment group on the IL-6 content in the supernatant of L02 cells in Experiment Example 1;

[0052] Figure 4 This is a graph showing the effect of each treatment group on the IL-1β content in the supernatant of L02 cells in Experiment Example 1;

[0053] Figure 5 This is a graph showing the effect of each treatment group on lipid accumulation in L02 cells (quantified by Oil Red O staining) in Experiment Example 1;

[0054] Figure 6 This is a graph showing the effect of each treatment group on the apoptosis level of L02 cells in Experiment Example 1 (Annexin V-FITC / PI flow cytometry).

[0055] Figure 7 This is a graph showing the effects of each treatment group on the phosphorylation level of p65, a key protein in the NF-κB pathway of L02 cells, in Experiment Example 1 (Western Blot).

[0056] Figure 8 This is a Western Blot diagram showing the effects of each treatment group in Experiment Example 1 on the expression levels of key endoplasmic reticulum stress proteins GRP78, CHOP, p-eIF2α, and XBP-1s induced by tunicamycin in L02 cells.

[0057] Figure 9 This is a graph showing the effects of each treatment group on the serum ALT and AST levels in mice in Experiment Example 2.

[0058] Figure 10 This is a graph showing the effects of each treatment group on the serum ALP level in mice in Experiment Example 2;

[0059] Figure 11 This is a graph showing the effects of each treatment group on the levels of serum lipid metabolism indicators (TG, TC, LDL-C) in mice in Experiment Example 2.

[0060] Figure 12 This is a graph showing the effects of each treatment group on the levels of systemic inflammatory factors (TNF-α, IL-6) in mouse serum in Experiment Example 2.

[0061] Figure 13 This is a graph showing the effects of each treatment group on the serum stress hormone corticosterone (CORT) level in mice in Experiment Example 2;

[0062] Figure 14This is a graph showing the effects of each treatment group on the triglyceride (TG) content in mouse liver tissue in Experiment Example 2;

[0063] Figure 15 This is a graph showing the effects of each treatment group on the total cholesterol (TC) content in mouse liver tissue in Experiment Example 2;

[0064] Figure 16 This is a graph showing the effects of each treatment group on the levels of oxidative stress indicators (SOD, GSH, MDA) in mouse liver tissue in Experiment Example 2.

[0065] Figure 17 This is a graph showing the effects of each treatment group on the mRNA or protein expression level of adipogenesis-related genes (SREBP-1c, FASN) in mouse liver tissue in Experiment Example 2.

[0066] Figure 18 This is a graph showing the effects of each treatment group on the expression levels of fatty acid oxidation-related genes (PPARα) mRNA or protein in mouse liver tissue in Experiment Example 2.

[0067] Figure 19 This is a graph showing the effects of each treatment group on the mRNA or protein expression levels of inflammation-related genes (NF-κBp65, TNF-α) in mouse liver tissue in Experiment Example 2.

[0068] Figure 20 This is a graph showing the effects of each treatment group on the expression level of endoplasmic reticulum stress-related proteins (GRP78, CHOP) in mouse liver tissue in Experiment Example 2.

[0069] Figure 21 The figure shows the effects of each treatment group on macrophage infiltration (F4 / 80 immunohistochemistry) and oxidative metabolism (CYP2E1 immunohistochemistry) in mouse liver tissue in Experiment Example 2.

[0070] Figure 22 This is a graph showing the effect of each treatment group on the dwell time in the central region of the mouse open field experiment in Experiment Example 2;

[0071] Figure 23 This is a graph showing the effect of each treatment group on the total movement distance of mice in the open field experiment in Experiment Example 2;

[0072] Figure 24 This is a graph showing the effect of each treatment group on the percentage of time mice spend in the elevated cruciate maze with their arms open in Experiment Example 2.

[0073] Figure 25 This is a graph showing the effect of each treatment group on the sleep time induced by sodium pentobarbital in mice in Experiment Example 2;

[0074] Figure 26This is a graph showing the effects of each treatment group on the latency of sodium pentobarbital-induced sleep in mice in Experiment Example 2.

[0075] Figure 27 This is a graph showing the changes in the gut microbiota of mice at the phylum level in each treatment group in Experiment Example 3;

[0076] Figure 28 This is a graph showing the changes in the gut microbiota of mice at the genus level in each treatment group in Experiment Example 3;

[0077] Figure 29 This is a graph showing the LDA scores of the intestinal flora of mice in each treatment group in Experiment Example 3;

[0078] Figure 30 This is a graph showing the effect of each treatment group on the expression of tight junction protein ZO-1 in mouse colon tissue in Experiment Example 3 (semi-quantitative immunohistochemical analysis).

[0079] Figure 31 This is a graph showing the effect of each treatment group on the expression of the tight junction protein Occludin in mouse colon tissue in Experiment Example 3 (semi-quantitative immunohistochemical analysis).

[0080] Figure 32 This is a graph showing the changes in gut microbiota at the phylum level in fresh human fecal samples from each treatment group in Experiment Example 4.

[0081] Figure 33 This is a graph showing the changes in gut microbiota at the genus level in fresh human fecal samples from each treatment group in Experiment Example 4.

[0082] Figure 34 This is a graph showing the LDA scores of gut microbiota in fresh human fecal samples from each treatment group in Experiment Example 4.

[0083] in,

[0084] Figures 1-6 In the diagram, CT represents the control group, E1 is the honeysuckle probiotic compound drink, E2 is the Jindi Shuangcui fermented drink, E3 is the jujube seed compound granules, E4 is the "combination product" mixture (a mixture of the entire product set used in animal experiments), and there are four experimental groups.

[0085] Figures 7-8 In the diagram, CT represents the control group, PA+S represents palmitic acid plus silymarin (a classic liver-protecting agent), and E represents the experimental group.

[0086] Figures 9-31 In the diagram, M represents the model group, CT represents the control group, and LD, MD, and HD represent the low, medium, and high consumption groups based on the Jindi Shuangcui compound health drink, respectively.

[0087] Figures 32-34In the figures, C represents the blank control group, M represents the model group, and E represents the experimental group including intestinal phase samples; E0 represents the product of sample example one or two after mixing Jindi Shuangcui compound health drink, bacterial suspension, and GAM culture medium for 0 hours; E12 represents the product of sample example one or two after mixing Jindi Shuangcui compound health drink, bacterial suspension, and GAM culture medium for 12 hours; E24 represents the product of sample example one or two after mixing Jindi Shuangcui compound health drink, bacterial suspension, and GAM culture medium for 24 hours; E48 represents the product of sample example one or two after mixing Jindi Shuangcui compound health drink, bacterial suspension, and GAM culture medium for 48 hours; and E72 represents the product of sample example one or two after mixing Jindi Shuangcui compound health drink, bacterial suspension, and GAM culture medium for 72 hours.

[0088] Figure 1-6 In 9-16 and 19-26, one-way ANOVA and Tukey post-hoc test were used for statistical analysis. In the figure, the presence of the same character indicates no significant difference (p>0.05), and the absence of the same character indicates a significant difference between groups (p<0.05).

[0089] Based on the positive regulatory trends observed in the aforementioned cell experiments (Example 1) and animal experiments (Examples 2 and 3), this invention also claims protection for the application of the compound health drink in specific aspects (as described in claims 6-12). It should be noted that the application to 'sub-healthy individuals' involved in the claims is based on the anticipated use and potential application direction of the aforementioned preclinical experimental results. This invention has provided preliminary support for its functional feasibility through in vitro and in vivo experiments, which constitutes the basis for claiming protection for this use. The actual human application effects of this product fall within the scope of extended development and verification of the patented technical solution. Detailed Implementation

[0090] Example 1:

[0091] A composite set of health care products based on Jindi Shuangcui, characterized in that the combination comprises the following three independent preparations: a. honeysuckle probiotic compound beverage, b. Jindi Shuangcui fermented beverage, c. jujube seed compound granules;

[0092] The honeysuckle probiotic compound beverage contains, by weight, 150 parts honeysuckle extract, 350 parts compound probiotics, 1500 parts fructooligosaccharides, and other excipient A, of which fructooligosaccharides are high-quality prebiotics that promote the colonization of probiotic strains.

[0093] The excipient A is resistant dextrin (resistant dextrin is a water-soluble dietary fiber used as a filler) and silica (anti-caking effect); the honeysuckle extract contains at least 5% standardized chlorogenic acid, which is the core heat-clearing component, anti-inflammatory, antioxidant and antiseptic, and improves the intestinal environment.

[0094] The total live bacteria count of the compound probiotics is at least 50 billion CFU; the compound probiotics contain *Lactobacillus plantarum* LP-03, *Lactobacillus rhamnosus* LR-01, *Bifidobacterium animalis* subsp. *lactobacter* BB651, and *Bifidobacterium breve* B-3. This strain combination was selected to target the immune system and intestinal barrier. In the animal models of this study (Experiments 2 and 3), positive regulatory trends related to improved systemic inflammation, oxidative stress, and intestinal barrier function were observed; these physiological processes are known to be associated with the regulation of mood and sleep.

[0095] The aforementioned Jindi Shuangcui fermented beverage, by weight, contains 600 parts by weight of Jindi symbiotic fermentation liquid, 200 parts by weight of Cornus officinalis extract, 1500 parts by weight of galactooligosaccharides, 1500 parts by weight of purified water, and other excipients. The Jindi symbiotic fermentation liquid is a liquid extract of Rehmannia glutinosa and Lonicera japonica, fermented by both Lactobacillus plantarum and yeast, containing 5 parts by weight of Rehmannia glutinosa and 1 part by weight of Lonicera japonica. Excipient B is 1.2 parts by weight of potassium sorbate (preservative). The Cornus officinalis extract contains at least 1% monosodium glutamate. The two main raw materials, Rehmannia glutinosa and Lonicera japonica, are processed together (symbiotically), simultaneously extracted, concentrated, and fermented, thereby simplifying the process, reducing costs, and creating synergistic value. The fermented beverage is rich in small molecule active substances such as Rehmannia glutinosa oligosaccharides, amino acids, and catalpol. Fermentation transforms large molecules into smaller molecules, which helps improve bioavailability and facilitates absorption by the user. Rehmannia glutinosa enhances its yin-nourishing effect, while Lonicera japonica enhances its heat-clearing effect. Cornus officinalis extract, in synergy with Rehmannia glutinosa, enhances the liver and kidney tonifying effects, while galactooligosaccharides act as prebiotics, continuously nourishing the gut microbiota.

[0096] The jujube seed compound granules, by weight, contain 150 parts by weight of γ-aminobutyric acid (GABA), 100 parts by weight of tea theanine, and 300 parts by weight of jujube seed extract, as well as other excipients. GABA can directly inhibit neuronal activity, promote relaxation, and has a sedative effect. Tea theanine can promote the production of alpha waves in the brain, relieve anxiety, and exhibits a synergistic effect with GABA. The excipients C are 800 parts by weight of erythritol and 500 parts by weight of microcrystalline cellulose, used as fillers and disintegrants. Jujube seed extract is a traditional food and medicine substance used for sedation. Preferably, the jujube seed extract contains ≥1.5% spinosin, a core flavonoid active ingredient in jujube seed and a key active substance for its sedative, hypnotic, and anti-anxiety effects.

[0097] This embodiment is based on the Jindi Shuangcui compound health drink, which combines Chinese herbal extracts, probiotics, fermentation active substances and sleep-aiding functional factors. Through the "Jindi symbiotic fermentation" process and the circadian rhythm drinking strategy, it achieves multi-target synergistic conditioning effects such as anti-inflammation, anti-oxidation, liver protection, assistance in improving mood and sleep, and regulation of intestinal flora.

[0098] Example 2:

[0099] A method for preparing a complete set of health drinks based on the dual-extract formula of Jindi is as follows:

[0100] S1. Preparation of Honeysuckle Probiotic Complex Drink

[0101] According to the proportions in Example 1, take honeysuckle extract with ≥5% standardized chlorogenic acid, compound probiotics, and fructooligosaccharides, mix them evenly, and then package them into aluminum foil bags, 2g per bag after dry granulation.

[0102] S2. Preparation of Jindi Double Extract Fermented Beverage

[0103] According to the ratio in Example 1, Rehmannia glutinosa and honeysuckle were mixed and extracted using hot water circulation and concentrated to 1g / mL under low temperature and reduced pressure. Then, Saccharomyces cerevisiae was first inoculated and fermented at 28°C for 48 hours, followed by Lactobacillus plantarum inoculation and fermentation at 37°C for 72 hours. The final pH was controlled to be ≤4.0, resulting in a mellow flavor and fermented sour aroma of Rehmannia glutinosa and honeysuckle symbiotic fermentation liquid. This process helps to form a mellow flavor and may improve the taste of the product.

[0104] Take the above fermentation broth as the base material, add Cornus officinalis extract, galactooligosaccharides, and purified water to the total volume, stir evenly, pasteurize and fill into bottles of 15mL each.

[0105] S3. Jujube seed compound granules

[0106] According to the proportions in Example 1, take γ-aminobutyric acid, tea theanine, and jujube seed extract, mix them evenly with the excipients, pass them through an 80-mesh sieve, and package them into aluminum foil bags, 2g per bag;

[0107] The steps S1, S2, and S3 described above are not in any particular order.

[0108] The specific steps of S1 are as follows:

[0109] S1.1 Raw material pretreatment:

[0110] Honeysuckle extract: pass through an 80-mesh sieve and collect the sieve residue; fructooligosaccharides and resistant dextrin: pass through a 60-mesh sieve and collect the sieve residue; sieving is to prevent clumping in the powder.

[0111] Compound probiotic powder: Remove from refrigeration and place in a dry environment until room temperature;

[0112] S1.2 premix

[0113] According to the weight proportions of Example 1, take the honeysuckle extract, fructooligosaccharides, and excipients after step S1.1.

[0114] S1.2.1 First, honeysuckle extract and resistant dextrin of equal increment are premixed by equal increment method for a mixing time of t1 to obtain premix A. The equal increment method of premixing is to ensure that honeysuckle extract and resistant dextrin are mixed evenly.

[0115] S1.2.2 Then, the fructooligosaccharides are premixed with the remaining resistant dextrin and silicon dioxide to obtain premix B.

[0116] S1.3 Total Mixture

[0117] S1.3.1 First, mix premix A and premix B for time t2 until they are evenly mixed.

[0118] S1.3.2 Then, according to the weight proportions of Example 1, take the compound probiotic powder completed in S1.1 and gently mix for time t3 until uniform. The probiotics are added after the other raw materials are mixed evenly, and the mixture is gently mixed to minimize the risk of damage to the live bacteria caused by mechanical shearing force during the operation of the mixer.

[0119] S1.4 Sterilization Step: After the final mixing in S1.3, the powder is sterilized by "cobalt-60 gamma ray irradiation". Through process validation, it has been determined that precisely controlling the irradiation dose within the range of 5-8 kGy can ensure that the product meets commercial sterility requirements (total bacterial count, mold and yeast, and pathogenic bacteria indicators are qualified) while minimizing the loss of activity of the compound probiotics. The survival rate of live bacteria after sterilization can be maintained at over 90% according to process validation.

[0120] S1.5 Packaging: In a clean, dry environment with humidity <30%, the powder after S1.4 sterilization should be individually packaged in moisture-proof, light-proof, and high-barrier packaging materials (such as aluminum foil composite bags) and sealed immediately. After packaging, the finished product must be stored in a cool, dry place. To ensure that the viable bacterial count remains stable at or above the nominal value (≥50 billion CFU / unit dose) throughout the shelf life, it is recommended and preferred to store and transport the product under a cold chain condition of 2-8°C throughout the entire process.

[0121] The following steps are preferred.

[0122] The dry environment in steps S1.1 and S1.4 refers to a clean environment with humidity <30%.

[0123] The mixing equipment used in steps S1.2 and S1.3 is a three-dimensional motion mixer.

[0124] The mixing time is at least 15 minutes for t1, at least 20 minutes for t2, and 15 minutes for t3.

[0125] This honeysuckle probiotic compound beverage is used to clear internal heat and regulate intestinal flora. The honeysuckle extract plays a key role in clearing heat and detoxifying, and has anti-inflammatory effects. Targeting the common sub-health state of "yin deficiency and internal heat" and its accompanying chronic low-grade inflammation in modern people, this product helps regulate related physiological imbalances and assists in improving inflammatory responses. The compound probiotics can regulate the intestines, improve the intestinal microecology, promote intestinal mucosal repair, reduce intestinal permeability, and improve intestinal problems such as diarrhea, constipation, and irritable bowel syndrome, while also reducing the entry of inflammatory factors into the bloodstream via the intestines. Fructooligosaccharides are high-quality prebiotics that promote the colonization of compound probiotic strains, while resistant dextrin is a water-soluble dietary fiber that can absorb water and metabolic waste in the intestines, helping to expel "damp heat" from the body. Its preparation method uses multiple batches of premixing, secondary mixing, and flexible mixing of probiotics to ensure uniform mixing of all raw materials and the activity of probiotics, facilitating the effective delivery of this solid beverage. It is highly suitable for people currently seeking to clear internal heat and regulate intestinal flora.

[0126] The specific steps of S2 are as follows:

[0127] S2.1 Feeding and Extraction

[0128] Take 5 parts of raw Rehmannia root and 1 part of honeysuckle according to the weight ratio, add 10 times the total weight of purified water, and use a hot water circulation extraction unit to extract twice at a temperature T1, with each extraction time being t1.

[0129] S2.2 Concentration:

[0130] After mixing the two extracts, the mixture was placed in a double-effect concentrator and concentrated into a clear extract under reduced pressure at a temperature T2≤60℃.

[0131] S2.3 fermentation

[0132] S2.3.1 Yeast fermentation: First, transfer the product from step S2.2 to the fermentation tank, cool it to T3, inoculate it with 1% yeast inoculum, and ferment for t2.

[0133] S2.3.2 Fermentation of Lactobacillus plantarum: After completing step S2.3.1, adjust the temperature to T4, then inoculate with 2% Lactobacillus plantarum inoculum, and ferment for t3;

[0134] In the described sequential fermentation process of 'yeast first, then lactobacillus', the yeast fermentation stage (28℃, 48 hours) aims to consume oxygen, produce flavor compounds, and create a micro-anaerobic environment for the system; the lactobacillus fermentation stage (37℃, 72 hours) aims to fully produce acid, transform components, and inhibit contaminating microorganisms. The fermentation endpoint is marked by a stable pH value ≤4.0 and the production of typical alcohol and acid aromas. These process parameters are an optimized preferred scheme that can stably obtain fermentation broth with excellent flavor, activity, and stability.

[0135] The selection of fermentation temperature and time is based on the optimal growth conditions of microorganisms and the results of preliminary experiments, aiming to balance fermentation efficiency, conversion of active ingredients and product flavor formation.

[0136] S2.4 blending

[0137] According to the weight proportions of Example 1, the fermentation broth after S2.3 was transferred into a mixing tank; under stirring, Cornus officinalis extract and galactooligosaccharides were added sequentially according to the weight proportions of Example 1, and stirred until completely dissolved; then purified water was added according to the formula; finally, potassium sorbate was added and stirred evenly.

[0138] S2.5 Pasteurization and Filling;

[0139] The pasteurization and filling process is as follows: the prepared liquid in step S2.4 is pasteurized through a plate heat exchanger; the preferred sterilization temperature is 85°C and the sterilization time is 15 minutes, and then it is rapidly cooled to below 25°C;

[0140] An automatic liquid filling machine is used to fill brown oral liquid glass bottles in a clean area of ​​at least Class C. The bottles are then filled with nitrogen and sealed to protect them from light, prevent oxidation, and ensure airtightness.

[0141] The following steps are preferred.

[0142] In step S2.1, the extraction temperature T1 is 90℃, and the extraction time t1 is 1 hour each time.

[0143] The concentration temperature T2 in step S2.2 shall not exceed 60°C.

[0144] In step S2.3.1, the yeast fermentation temperature T3 is 28℃ and the fermentation time t2 is 48 hours.

[0145] In step S2.3.2, the fermentation temperature T4 of Lactobacillus plantarum is 37°C, and the fermentation time t3 is 72 hours.

[0146] The density of the concentrated extract in step S2.2 is 1.10-1.15 g / cm³, which is equivalent to 1 g / mL.

[0147] The fermentation endpoint in step S3.2 is as follows: the pH value of the fermentation broth stabilizes at ≤4.0, and the fermentation broth has an mellow fermented sour aroma. This mellow aroma can reduce bitterness, help improve palatability, make it easier to accept, and reduce the difficulty of consumption.

[0148] The Jindi Shuangcui fermented beverage prepared in step S2 is based on the traditional efficacy theory of raw materials such as Rehmannia glutinosa and Cornus officinalis, aiming to exert the conditioning effects of nourishing yin and clearing heat. The first-inoculated brewer's yeast consumes oxygen and monosaccharides, lowers the system's redox potential, and produces ethanol, creating a selectively advantageous environment for the subsequent anaerobic fermentation and acid production of Lactobacillus plantarum. Simultaneously, yeast metabolism may convert some glycosides into more easily absorbed aglycones. Step S2 combines the two main raw materials (symbiotic processing), thereby simplifying the process, reducing costs, and creating synergistic value. In particular, the co-fermentation of the two ingredients transforms large molecules in the extract into smaller molecules, helping to improve bioavailability and facilitate absorption by the user. Rehmannia glutinosa enhances the yin-nourishing effect, while honeysuckle enhances the heat-clearing effect. It can be used for sub-healthy individuals with yin deficiency and excessive fire, achieving the purpose of nourishing yin and blood, clearing heat, and calming the mind.

[0149] The specific steps of S3 are as follows:

[0150] S3.1 Raw Material Pretreatment

[0151] Gamma-aminobutyric acid, tea theanine, and jujube seed extract were each passed through an 80-mesh sieve, and the sieve-passing material was collected for later use.

[0152] Erythritol and microcrystalline cellulose were passed through a 60-mesh sieve, and the sieve-passing material was collected for use as a filler and disintegrant.

[0153] S3.2 Mixing - Soft Material

[0154] The undersize material processed in step S3.1 is simultaneously fed into a trough mixer according to the formula ratio of Example 1. The mixing time is t1. Then, a wetting agent is added, and the mixture is continued to form a soft material. The goal is to achieve uniform mixing between the various raw materials. The addition of the wetting agent is beneficial for the bonding between the various powders, which facilitates the formation of granules in the subsequent granulation process.

[0155] S3.3 Granulation

[0156] The soft material obtained in step S3.2 is fed into a gyratory pellet mill for granulation, using a primary screen for granulation.

[0157] S3.4 Drying

[0158] The wet granules are evenly spread on a drying tray and placed in a hot air circulating oven for drying. This process removes some of the wetting agent and helps the granules to solidify.

[0159] S3.5 whole grains

[0160] After step S3.4, the granules are fed back into the pendulum pellet mill and granulated using a secondary screen that is slightly finer than the primary screen to break up clumps and obtain uniform granules. This process achieves a second extrusion and shaping of the granule surface formed in the granulation step, ensuring that the granule surface has a regular shape and that the particle size is more uniform, thus avoiding clumping.

[0161] S3.6 total mix

[0162] The granulated particles are fed into a three-dimensional motion mixer and mixed for a total time of t2 until homogeneous. The purpose of total mixing is to solve the problem of uneven mixing of raw materials before granulation. After total mixing, the particles can be mixed evenly, avoiding excessive errors in the composition of products in different bags after subsequent packaging.

[0163] S3.7 Packaging

[0164] Packed in food-grade aluminum foil bags.

[0165] The following steps are preferred.

[0166] In step S2.3.2, the wetting agent is an 85% ethanol solution. This concentration of ethanol solution can achieve wetting and granulation while also enabling low-temperature rapid drying and volatilization during subsequent drying, without affecting the activity and efficacy of the raw materials. In step S3.2, the soft material is prepared until it can be formed into a ball by hand and crumbles easily when lightly pressed.

[0167] The mixing time t1 in step S3.2 is 20 minutes, and the total mixing time t2 in step S3.6 is 30 minutes.

[0168] In step S3.4, the drying temperature in the hot air circulating oven is 60-65℃, and the drying continues until the moisture content of the particles is 3.0-5.0%.

[0169] The primary sieve in step S3.3 is 14 mesh, and the secondary sieve in step S5 is 16 mesh.

[0170] Example 3:

[0171] An application of the Jindi Shuangcui complete set of health drinks in Example 1, or the Jindi Shuangcui compound complete set of health drinks obtained by the preparation method in Example 2, to improve sub-health. The complete set of health drinks is applied to the auxiliary regulation of sub-healthy people or to oxidative stress, or to liver function, or to mood and sleep, or to gut microbiota.

[0172] The aforementioned combination of health products based on Jindi Shuangcui includes a morning dose and a bedtime dose. Specifically, one sachet (2g) of honeysuckle probiotic compound drink is taken on an empty stomach in the morning, and the Jindi Shuangcui fermented drink and jujube seed compound granules are taken before bedtime. Pour one sachet (2g) of jujube seed compound granules into one bottle (15mL) of Jindi Shuangcui fermented drink, shake well, and drink directly. Alternatively, it can be mixed with 100mL of warm water.

[0173] The anti-inflammatory application targets at least one of the inflammatory factors including IL-1β, IL-6, and TNF-α.

[0174] The antioxidant application is to increase the activity of at least one of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX), and / or reduce the content of malondialdehyde (MDA).

[0175] The potential auxiliary regulatory effect on liver health is to reduce at least one of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and / or reduce lipid accumulation in the liver;

[0176] The aforementioned applications to assist in improving mood and sleep include reducing anxiety-like behaviors, prolonging sleep time and / or shortening sleep latency;

[0177] The application of regulating intestinal flora includes at least one of reducing the abundance of harmful bacteria and / or increasing the abundance of beneficial bacteria; the harmful bacteria include Proteobacteria, which includes at least one of Escherichia coli and Shigella; the beneficial bacteria include Firmicutes, which includes at least one of Clostridium spp. and Collins spp.

[0178] It should be noted that the experimental data in this application are derived from in vitro cell models and animal models, primarily used to elucidate the effects and potential mechanisms of the product under experimental conditions, providing preclinical research evidence for its functional feasibility. Based on the experimental results from the cell and animal models in this application, it can be inferred that the product is used to regulate sub-health conditions in humans. The specific regulatory effects of this product on sub-health conditions and the optimal usage plan need to be verified and confirmed through further human clinical trials in accordance with relevant national laws and regulations.

[0179] To verify the above application effect, the inventors conducted the following verification experiments.

[0180] Experiment Example 1: In vitro cell experiments to verify the anti-inflammatory, antioxidant, and cell-protective effects of the product.

[0181] [This experimental example aims to provide in vitro experimental evidence for the use of the methods described in claims 6, 8, and 9 in assisting the regulation of inflammation, oxidative stress, and liver function.]

[0182] 1.1 Experimental System and Model Establishment

[0183] Cell line: Human normal liver cell line L02 (purchased from ATCC), cultured strictly according to ATCC standards.

[0184] Model 1:

[0185] A model of hepatocyte injury induced by lipid peroxidation and inflammation. Modeling agent: palmitic acid (PA, 400 μM).

[0186] Model 2:

[0187] Endoplasmic reticulum stress model. Modeling agent: Tunicamycin (2 μg / mL).

[0188] Grouping:

[0189] The study included a normal control group (CT), a PA model group (M), a positive control group (PC), and the experimental group containing the product of this invention. The experimental group included:

[0190] Single group: test the water extract of "Honeysuckle Probiotic Compound Drink" (E1), the fermentation liquid of "Jindi Double Extract Fermented Drink" (E2), and the water extract of "Sour Jujube Seed Compound Granules" (E3) respectively. The water extract refers to the aqueous solution of the product in the previous examples.

[0191] Combination Product Group: The "combination product" mixture (E4) is made by mixing one of each of the actual products produced by substances E1, E2, and E3 according to the previous examples.

[0192] Four concentration gradients of 100, 200, 400, and 800 μg / mL were set for all experimental groups (E1 to E4). These concentrations represent the final concentrations of each sample in the cell culture system.

[0193] deal with:

[0194] Cells were incubated with each sample for 4 hours before being co-cultured with the modeling agent for 24 hours.

[0195] 1.2 Detection Indicators and Results

[0196] Cell viability (CCK-8 assay):

[0197] like Figure 1 As shown, compared with the normal control group (100.0±3.5%), the cell viability of the PA model group was significantly reduced to (55.2±4.1%) (p<0.01).

[0198] Based on the fact that each dose group of the Jindi Shuangcui compound health drink can increase cell viability in a dose-dependent manner, the viability of the high-dose group recovered to (88.7±5.2%) (p<0.01 vs. model group), which was not statistically different from the positive control group (p>0.05).

[0199] Intracellular reactive oxygen species (ROS, DCFH-DA probe):

[0200] like Figure 2 As shown, the ROS level in the model group was significantly higher than that in the normal group by approximately 2.5 times. All experimental groups effectively reduced ROS levels, with the high-dose honeysuckle probiotic compound beverage group showing a ROS level that decreased to (62.3±6.8)% of the model group (p<0.01). This result suggests that the antioxidant effect of honeysuckle extract in this product is one of the contributing factors to its antioxidant effect.

[0201] Inflammatory factors (ELISA method):

[0202] like Figure 3 As shown, the IL-6 content in the cell supernatant of the model group was significantly increased to (285.4±22.5) pg / mL. All experimental groups could significantly inhibit the release of IL-6, and the IL-6 level in the high-dose group of Jindi Shuangcui fermented beverage decreased to (132.7±15.8) pg / mL (p<0.01).

[0203] Lipid accumulation (quantitative analysis using Oil Red O staining):

[0204] like Figure 5 As shown, all experimental groups were able to effectively reduce fat deposition in hepatocytes and alleviate fatty degeneration.

[0205] Hepatocellular stress (Western Blot analysis):

[0206] like Figure 7-8 As shown, each experimental group, especially the Jindi Shuangcui fermented beverage, significantly downregulated the phosphorylation level of NF-κBp65 and the expression of GRP78 and CHOP, key endoplasmic reticulum stress proteins (p<0.05 vs. model group).

[0207] In this experimental example, the combined product group (E4) and each individual group (E1, E2, E3) showed a positive regulatory trend in most indicators. Although the numerical advantage of the combined group in some indicators did not always reach statistical significance at the current experimental concentration, it simultaneously covers multiple key pathways such as inflammation, oxidation, lipid accumulation, and cellular stress. This suggests that the value of the product of this invention lies in its 'multi-target coverage' capability, rather than pursuing the ultimate inhibition of a single pathway. The synergistic effect among the components, especially the systemic effect based on time-sharing administration verified by subsequent animal experiments, constitutes the main innovation of this invention.

[0208] It should be noted that the data in this experiment are derived from in vitro cell models and are primarily used to elucidate the trends and potential mechanisms of action of the product at the cellular level, providing preliminary evidence for the feasibility of its auxiliary regulatory functions such as anti-inflammatory, antioxidant, and cell protection. Based on the experimental results from the cell and animal models described in this application, it can be inferred that this product can be used to regulate sub-health conditions in humans. Its specific effects on humans require further research and verification.

[0209] Experiment Example 2: In vivo animal experiments verified the product's anti-inflammatory, antioxidant, and sleep-inducing effects.

[0210] This experimental example aims to verify the overall regulatory effect of the complete product combination of the present invention in a complex sub-health animal model, and to 'simulate' its designed 'morning cleansing and evening supplementation' timed administration regimen. This experimental design verifies the 'systematic concept' of the present invention. The results show that intervention according to this concept can effectively improve multiple abnormal indicators in the model animals. This provides preclinical evidence for the effectiveness of this systematic approach.

[0211] [This experimental example aims to provide in vivo animal evidence for the use of the auxiliary regulation of inflammation, oxidative stress, liver function, and mood and sleep as described in claims 6, 8, 9, 10, and 11.]

[0212] The treatment regimen in this experiment was designed based on the traditional Chinese medicine principle of "clearing in the morning and nourishing at night" and the principles of modern chronobiology. Human cortisol secretion follows a circadian rhythm, with mornings being suitable for "clearing heat and regulating the intestines," and nights for "nourishing yin and calming the mind." This experiment simulated this rhythm, administering "honeysuckle probiotic compound drink" at 9:00 AM and "Jindi Shuangcui fermented drink" and "jujube seed compound granules" at 7:00 PM to verify the overall regulatory effect of this time-segmented intervention regimen in an animal model.

[0213] 2.1 Laboratory Animals and Models

[0214] animal:

[0215] SPF-grade C57BL / 6J mice, 8 weeks old, half male and half female, were acclimatized for one week before the experiment began.

[0216] Model:

[0217] A complex sub-health model was successfully established by combining chronic sleep deprivation (CSD, 20 hours per day) with a high-fat diet (HFD, 45% fat for energy) for 6 weeks.

[0218] Grouping:

[0219] Normal control group (normal feed, no sleep deprivation, gavage with solvent), model control group (HFD+CSD, gavage with solvent), and low (1×human equivalent dose HED), medium (2.5×HED), and high (5×HED) dose groups based on the Jindi Shuangcui compound health drink combination, n=12 / sex / group.

[0220] Dosage instructions:

[0221] Simultaneous with model establishment, the treatment lasted for 6 weeks. Strictly mimicking human circadian rhythms: "Honeysuckle Probiotic Complex Drink" solution was administered by gavage at 9:00 AM, and "Jindi Double Extract Fermented Drink" and "Ziziphus Jujuba Compound Granules" were mixed and administered by gavage at 7:00 PM. The dosage was calculated at 0.1 mL / 10g body weight.

[0222] 2.2 Detection Indicators and Results

[0223] Liver function and lipid metabolism:

[0224] like Figures 9-11 , Figures 14-15 As shown, serum ALT, AST, ALP, and liver TG and TC levels were significantly elevated in the model group (p<0.01). The Jindi Shuangcui compound health drink at various dosage levels reduced these indicators in a dose-dependent manner. For example, the ALT level in the high-dose group was reduced by approximately 45% compared to the model group (p<0.01).

[0225] Stress hormones:

[0226] like Figure 13 As shown, serum corticosterone (CORT) levels were significantly elevated in the model group (p<0.01), indicating that the model animals were in a state of high stress. After administration of Jindi Shuangcui compound health drink, CORT levels in each dose group decreased in a dose-dependent manner, with significant differences observed in the high-dose group (p<0.05 vs. model group).

[0227] Inflammation and oxidative stress:

[0228] like Figure 12 As shown, ELISA detection revealed significantly elevated serum TNF-α and IL-6 levels in the model group, while these levels decreased after administration of the Jindi Shuangcui Compound Health Drink (p<0.05 vs. model group). Figure 16 As shown, after taking the Jindi Shuangcui compound health drink, the MDA content in the liver tissue decreased (p<0.05) and the SOD and GSH activities increased (p<0.05) compared with the model group.

[0229] Behavioral Science and Sleep:

[0230] like Figure 24As shown, the time spent in the central region of the open field experiment was significantly shortened in the model group mice (p<0.01), indicating an increase in anxiety-like behavior. The medium and high dose groups of the Jindi Shuangcui compound health drink significantly prolonged the time spent in the central region (p<0.05).

[0231] like Figure 25 As shown, the sleep time in the model group was significantly shortened, while the high-dose group of Jindi Shuangcui compound health drink significantly prolonged the sleep time induced by sodium pentobarbital (p<0.05 vs. model group).

[0232] It should be noted that the results of this experimental case were obtained based on a composite sub-healthy animal model, simulating the "morning cleansing and evening supplementation" time-sharing intervention program. The aim was to verify the overall regulatory trend of this product combination in animals, providing preclinical experimental evidence for its use in assisting the regulation of inflammation, oxidative stress, liver function, and mood and sleep. Its human application and effects need to be confirmed through clinical trials.

[0233] Experiment Example 3: In vivo animal experiments to verify the product's effect on the intestines

[0234] [This experimental example aims to provide supplementary in vitro fermentation model experiments for the use of the gut microbiota-assisted regulation described in claims 6 and 12.]

[0235] 3.1 Experimental animals, models and grouped animals: SPF grade C57BL / 6J mice (same as Experimental Example 2).

[0236] Model: A composite sub-health model was established by combining chronic sleep deprivation (CSD) with a high-fat diet (HFD) for 6 weeks (same as Experiment 2).

[0237] Grouping: Same as in Experiment 2, divided into: normal control group (CT), model control group (M), and low (LD), medium (MD), and high (HD) dose groups based on Jindi Shuangcui compound health drink (n=12 / sex / group). Feeding regimen was the same as in Experiment 2.

[0238] 3.2 Sample Collection

[0239] After the experiment, fresh fecal samples and colon tissue were collected from each group of mice.

[0240] 3.3 Detection Indicators and Results

[0241] 3.3.1 Gut microbiota analysis (16S rRNA gene sequencing)

[0242] Mouse fecal samples were collected, and genomic DNA was extracted using the OMEGA Mag-bind soil DNA kit and sent to Shanghai Zhongke New Life Biotechnology Co., Ltd. for sequencing analysis.

[0243] Result: As Figure 27-29 As shown,

[0244] Phylum level changes: In the model group (M), the relative abundance of Proteobacteria in the gut microbiota was significantly increased, while the relative abundance of Firmicutes was decreased. After administration of the complete set of health drinks of the present invention, each dosage group (LD, MD, HD) was able to reverse this trend in a dose-dependent manner, significantly increasing the Firmicutes / Proteobacteria ratio (p<0.05 vs. model group).

[0245] At the genus level, this product can significantly increase the relative abundance of beneficial bacteria such as Clostridium sensustricto 1 and Collinsella, while decreasing the relative abundance of potentially harmful bacteria such as Escherichia-Shigella.

[0246] Differential species analysis: Linear discriminant analysis (LDA) showed that there were multiple differential species (biomarkers) with LDA scores >4 between the treatment group and the model group.

[0247] 3.3.2 Intestinal barrier function assessment (immunohistochemistry)

[0248] Immunohistochemical staining was performed on mouse colon tissue to detect the expression of tight junction proteins ZO-1 and Occludin.

[0249] Intestinal barrier (immunohistochemistry):

[0250] like Figures 30-31 As shown, the expression of tight junction proteins ZO-1 and Occludin in the colon tissue of the model group was significantly decreased (p<0.01), while the medium and high dose groups of Jindi Shuangcui compound health drink significantly increased the expression levels of ZO-1 and Occludin (p<0.05 vs. model group), indicating that it can effectively improve intestinal barrier damage caused by HFD+CSD.

[0251] Based on the results of Experiment 3, the aforementioned gut microbiota structure (increased Firmicutes / Proteobacteria ratio and increased abundance of specific beneficial bacterial genera) and the upregulation of intestinal barrier protein (ZO-1, Occludin) expression suggest that this product may regulate the gut microbiota and thus affect its metabolic functions (such as the synthesis of short-chain fatty acids), thereby synergistically improving the intestinal barrier and regulating systemic inflammation. It should be further noted that the product of this invention showed a trend of regulating gut microbiota structure in experimental animal models, for example, changes in the Firmicutes / Proteobacteria ratio and an increase in the abundance of specific bacterial genera (such as Clostridium). It should be pointed out that the relationship between gut microbiota composition and health status is complex. The above changes are correlational phenomena observed in a specific model in this study, and the conclusion that they serve as universal beneficial indicators needs to be confirmed by further research. This invention focuses on the observation that the product 'has the potential to regulate gut microbiota' itself, and considers it as a component of its multi-target effects. In-depth exploration of the metabolic functions of the microbiota will be an important direction for subsequent research to elucidate the overall mechanism of action of this product.

[0252] It should be noted that the improved gut microbiota and barrier function observed in the animal model in this experiment provide in vivo experimental evidence for the product's potential to regulate the gut microbiota. Based on the effects of the cell and animal models presented in this application, it can be inferred that this product is used to regulate sub-health conditions in humans, but its specific effects on the human gut environment require further research and verification.

[0253] Experiment Example 4: Direct Effects of In Vitro Fermentation Products on Gut Microbiota Metabolic Activity

[0254] [This experimental example aims to provide in vitro experimental evidence for the use of the gut microbiota-adjusting agent as described in claim 12.]

[0255] 4.1 Experimental Objective

[0256] Using an in vitro batch fermentation model, we conducted a preliminary investigation into the direct impact of the product of this invention on the metabolic activities of the intestinal flora.

[0257] 4.2 Experimental Methods

[0258] Inoculum preparation: Fecal samples were selected from healthy volunteers aged 25-40 years with no history of gastrointestinal diseases, and homogenized bacterial suspensions were prepared.

[0259] Fermentation system: In GAM medium, the following were established:

[0260] Blank control group (C): Contains only culture medium and bacterial suspension.

[0261] Experimental group (E): Contains culture medium, bacterial suspension and a certain concentration of the homogenized health drink of the present invention (the product prepared in Example 1 or 2, the three are mixed together and used together, the dosage is the same as in Experiment 1).

[0262] Fermentation and sampling: Fermentation was carried out under anaerobic conditions at 37℃. Samples were taken at 0h, 12h, 24h, 48h, and 72h of fermentation and labeled as C0 / C12 / C24 / C48 / C72 and E0 / E12 / E24 / E48 / E72, respectively.

[0263] Detection and analysis: 16S rRNA gene sequencing was performed on the samples to analyze the dynamic changes of the bacterial community.

[0264] 4.3 Experimental Results

[0265] like Figures 32-34 As shown in the figure, during in vitro fermentation, the experimental group (E) exhibited a different microbial succession pattern from the control group (C) in the mid-to-late stages of fermentation (e.g., E24, E48, E72). For example, at time point E24, Firmicutes became the dominant phylum; at the genus level, the relative abundance of Clostridium sensustricto and Collinsella increased at specific time points in the experimental group. These in vitro results are consistent with the trends observed in in vivo animal experiments, providing supplementary evidence for the direct regulation of the intestinal microecology by this product.

[0266] 4.3.1 Extraction of intestinal flora

[0267] Genomic DNA was extracted from the samples using the OMEGA Mag-bindsoil DNA kit according to the manufacturer's instructions. The samples were then sent to Shanghai Zhongke New Life Biotechnology Co., Ltd. for DNA purity and concentration testing.

[0268] Among them, the samples are

[0269] The experimental groups were: 0h (E0), 12h (E12), 24h (E24), 48h (E48), and 72h (E72).

[0270] The samples were formed from the 0h blank group (C0), the 12h blank group (C12), the 24h blank group (C24), the 48h blank group (C48), and the 72h blank group (C72).

[0271] 4.3.2 Analysis of Intestinal Microbiota Experiment Results

[0272] 16S rRNA gene sequencing analysis showed that, in the in vitro fermentation model, the gut microbiota structure of the experimental group (E) and the blank control group (C) exhibited different dynamic changes.

[0273] Analysis based on amplicon sequence variants (ASVs) revealed that 75 shared ASVs existed between the experimental group and the blank control group during fermentation (0-72 h). Figures 32-33 As shown, with the extension of fermentation time, the differences in the microbial community composition between the experimental group (E12, E24, E48, E72) and the blank control group (C12, C24, C48, C72) gradually became apparent.

[0274] Impact factor analysis (LEfSe) and linear discriminant analysis (LDA) results showed that there were statistically significant differences in the number of species between the experimental and control groups at different fermentation time points (LDA score > 4). For example, at time points E12, E24, and E72, the experimental group had 10, 5, and 6 ASVs that were significantly different from the control group, respectively. Notably, the relative abundance of beneficial bacteria such as *Clostridium sensustricto* and *Collinsella* showed an increasing trend at specific fermentation time points in the experimental group (e.g., E24, E48), while Firmicutes became the dominant phylum in the middle and late stages of fermentation.

[0275] These in vitro fermentation results indicate that the product of this invention can directly affect the metabolic activities of intestinal microorganisms and promote the growth of beneficial bacteria. This trend is consistent with the results of in vivo animal experiments (Experimental Example 3), providing in vitro experimental evidence for further elucidating the mechanism of action of this product in regulating intestinal microecology.

[0276] It should be noted that this experimental example simulated the interaction between the product and gut microbiota using an in vitro fermentation model. The results complement the trends observed in in vivo experiments, jointly supporting the conclusion that the product has the potential to regulate gut microbiota. However, the results of this in vitro model cannot be directly equated with actual effects in the human body.

[0277] Based on Experiments 1 to 4, in the cell and animal models described herein, the health drink based on the Jindi Shuangcui compound provided by this invention showed potential positive regulatory effects in anti-inflammation, anti-oxidation, liver protection, mood and sleep improvement, and intestinal flora regulation.

[0278] It should be noted that the experimental data in this application are derived from in vitro cell models and animal models, primarily used to elucidate the effects and potential mechanisms of the product under experimental conditions, providing preclinical research evidence for its feasibility in assisting regulation. The specific regulatory effects and optimal usage of this product on human sub-health states can be inferred from the experimental results in the cell and animal models presented in this application. Further verification and confirmation through human clinical trials are required, in accordance with relevant national laws and regulations.

[0279] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A composite set of health care products based on the dual extraction of gold and earth essences, characterized in that, The combination comprises the following three independent formulations: a. honeysuckle probiotic compound beverage, b. Jindi double-extract fermented beverage, and c. jujube seed compound granules; the honeysuckle probiotic compound beverage includes, but is not limited to, honeysuckle extract and compound probiotics; the Jindi double-extract fermented beverage includes, but is not limited to, Jindi symbiotic fermentation liquid; and the jujube seed compound granules include, but are not limited to, γ-aminobutyric acid, tea theanine, and jujube seed extract.

2. The composite health product combination based on Jindi Dual Extraction as described in claim 1, characterized in that: The honeysuckle probiotic compound beverage contains, by weight, 100-200 parts honeysuckle extract, 300-400 parts compound probiotics, 1300-1600 parts fructooligosaccharides, and other excipients. The Jindi Shuangcui fermented beverage, by weight, contains 500-700 parts of Jindi symbiotic fermentation liquid, 150-300 parts of Cornus officinalis extract, 1400-1600 parts of galactooligosaccharides, 1400-1600 parts of purified water, and other excipients. The Jindi symbiotic fermentation liquid is a liquid extract of Rehmannia glutinosa and Lonicera japonica, which is a liquid obtained by bidirectional fermentation of Lactobacillus plantarum and yeast, and contains 4-6 parts of Rehmannia glutinosa and 1 part of Lonicera japonica by weight. The jujube seed compound granules, by weight, contain 140-160 parts by weight of γ-aminobutyric acid, 90-110 parts by weight of tea theanine, 280-320 parts by weight of jujube seed extract, and other excipients.

3. The composite health product combination based on Jindi Dual Extraction as described in claim 2, characterized in that: The excipient A includes, but is not limited to, resistant dextrin and silicon dioxide; the honeysuckle extract contains at least 5% standardized chlorogenic acid; the total live bacteria count of the compound probiotics is at least 50 billion CFU; the compound probiotics contain Lactobacillus plantarum LP-03, Lactobacillus rhamnosus LR-01, Bifidobacterium animalis subsp. lactis BB651, and Bifidobacterium breve B-3.

4. The health product combination according to claim 2, characterized in that: The excipient B includes, but is not limited to, potassium sorbate, which is 1-1.5 parts by weight, and the extract of Cornus officinalis contains at least 1% mononoside.

5. The composite health product combination based on Jindi Dual Extraction as described in claim 2, characterized in that: The excipients include, but are not limited to, erythritol and microcrystalline cellulose, with 600-1000 parts by weight of erythritol and 300-500 parts by weight of microcrystalline cellulose; the jujube seed extract contains ≥1.5% spinosin.

6. A method for preparing a composite health product combination based on the dual-extracting formula of gold and earth as described in any one of claims 1-5, characterized in that, Includes the following steps: S1. Preparation of honeysuckle probiotic compound beverage: According to claim 1, take honeysuckle extract with ≥5% standardized chlorogenic acid, compound probiotics, and fructooligosaccharides, mix them evenly, and then package them into aluminum foil bags using a dry granulation process. S2. Preparation of Jindi Double Extract Fermented Beverage: According to claim 1, Rehmannia glutinosa and Lonicera japonica are mixed, extracted by hot water circulation, and concentrated to 1g / mL by low temperature vacuum. Then, Saccharomyces cerevisiae is first inoculated and fermented at 28℃ for 48 hours, and then Lactobacillus plantarum is inoculated and fermented at 37℃ for 72 hours. The final pH is controlled to be ≤4.0 to obtain a mellow flavor and fermented sour aroma of Rehmannia glutinosa and Lonicera japonica symbiotic fermentation liquid. Take the above fermentation broth as the base material, add Cornus officinalis extract, galactooligosaccharides, and purified water to the total volume, stir evenly, and then fill after pasteurization. S3. Preparation of Ziziphus jujuba seed compound granules: According to claim 1, take γ-aminobutyric acid, tea theanine, and jujube seed extract, mix them evenly with the excipients, pass them through an 80-mesh sieve, and package them into aluminum foil bags. Steps S1, S2, and S3 are not in any particular order.

7. The application of a composite health product set based on Jin Di Shuang Cui as described in any one of claims 1-5, or a composite health product set based on Jin Di Shuang Cui obtained by the method of claim 6, characterized in that: This complete set of health drinks is used to assist in the regulation of inflammation, oxidative stress, liver function, mood and sleep, and gut microbiota in sub-healthy individuals; the combined use of the Jindi Shuangcui compound health products includes a preparation taken in the morning and a preparation taken before bedtime.

8. The application according to claim 7, characterized in that, The auxiliary regulation of inflammation includes reducing the level of at least one of IL-1β, IL-6, and TNF-α.

9. The application according to claim 7, characterized in that, The auxiliary regulation of oxidative stress includes: increasing the activity of at least one of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX), and / or decreasing the content of malondialdehyde (MDA).

10. The application according to claim 7, characterized in that, The auxiliary regulation of liver function includes: assisting in reducing the level of at least one of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and / or assisting in reducing lipid accumulation in the liver.

11. The application according to claim 7, characterized in that, The auxiliary regulation of mood and sleep includes: reducing anxiety-like behaviors, prolonging sleep time and / or shortening sleep latency.

12. The application according to claim 7, characterized in that, The auxiliary regulation of gut microbiota includes at least one of: reducing the abundance of harmful bacteria and / or increasing the abundance of beneficial bacteria; The beneficial bacteria include Firmicutes, which include at least one of Clostridium and Collins.

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