Use of a lily saponin-lily polysaccharide composition in the preparation of an antidepressant drug

Abstract

The application discloses application of a lily saponin-lily polysaccharide composition in preparation of an antidepressant, and belongs to the technical field of biological medicines. The lily saponin (LLS) and the lily polysaccharide (LLP) are respectively extracted and purified from lily as raw materials, and are compounded to form the composition. The composition can repair the colon tight junction barrier, reduce the level of intestinal inflammatory factors, reshape the intestinal flora structure, inhibit the excessive activation of the hypothalamus-pituitary-adrenal (HPA) axis, activate the intracerebral cAMP / PKA / CREB / BDNF signal path, and play an antidepressant role through the intestinal-brain axis. The intestinal flora regulated by the LLS+LLP can independently reproduce the antidepressant effect. The application first clarifies the integrated action mechanism of the lily saponin-lily polysaccharide composition in resisting depression from the perspective of the intestinal-brain axis, and provides a scientific basis for development of the antidepressant drug based on the intestinal microecological target.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of a lily saponin-lily polysaccharide composition in the preparation of antidepressant drugs, particularly its pharmaceutical use in exerting antidepressant effects through the gut-brain axis mechanism. Background Technology

[0002] Major Depressive Disorder (MDD) is a significant public health problem worldwide, affecting approximately 3.8% of the global population, according to the World Health Organization. Currently, first-line antidepressants primarily target the monoamine neurotransmitter system, including selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine and serotonin-norepinephrine reuptake inhibitors (SRIs). However, these drugs generally suffer from slow onset of action (typically requiring 2-4 weeks), poor efficacy in approximately 30% of patients (treatment resistance), and adverse reactions such as sexual dysfunction and weight gain. Therefore, there is an urgent clinical need to develop novel antidepressants with faster onset of action, better efficacy, and fewer side effects.

[0003] In recent years, the rapid development of the gut-brain axis theory has provided a novel perspective for the study of the pathological mechanisms of depression and drug development. The gut-brain axis refers to a bidirectional communication network between the gut and the central nervous system, involving neural, endocrine, and immune pathways. Studies have found that patients with depression commonly exhibit gut microbiota dysbiosis, impaired intestinal barrier function (leaky gut), and low-grade systemic inflammation. These peripheral pathological changes can be transmitted to the central nervous system through the gut-brain axis, affecting neural plasticity and mood regulation. Specifically, intestinal barrier damage leads to increased intestinal permeability, allowing pro-inflammatory substances such as bacterial lipopolysaccharides to enter the circulation and activate peripheral and central immune inflammatory responses. Gut microbiota dysbiosis further affects the metabolism of neurotransmitters (such as 5-HT) and the synthesis of metabolites such as short-chain fatty acids, indirectly impairing neuronal function and synaptic plasticity. Therefore, drug development strategies targeting the gut-brain axis—simultaneously repairing the intestinal barrier, regulating gut microbiota, and enhancing central nervous system plasticity—are becoming an important direction in the development of new antidepressant drugs.

[0004] The cyclic adenosine monophosphate (cAMP) / protein kinase A / cAMP response element-binding protein / brain-derived neurotrophic factor (BDNF) signaling pathway is a key pathway regulating neuronal survival, synaptic plasticity, and neurogenesis. Clinical and animal studies have confirmed a close correlation between depression and the downregulation of this pathway. Activation of the cAMP / PKA / CREB / BDNF pathway can enhance neuroplasticity, promote hippocampal neuronal regeneration, and thus improve depressive-like behaviors. Furthermore, overactivation of the hypothalamus-pituitary-adrenal (HPA) axis is one of the core neuroendocrine features of depression, manifested by persistently elevated levels of corticosterone (CORT) / cortisol, adrenocorticotropic hormone (ACTH), and corticotropin-releasing hormone (CRH). There is also a close link between HPA axis dysfunction and the gut-brain axis—intestinal inflammation and dysbiosis can activate the HPA axis, while overactivation of the HPA axis, in turn, exacerbates intestinal barrier damage, creating a vicious cycle.

[0005] Lily is the plant *Tilia tigrinum* of the Liliaceae family (Lilium tigrinum). Lilium lancifolium Thunb.), lily ( Lilium brownii FEBrown var. viridulum Baker) or fine-leaved lily ( Lilium pumilum The dried, fleshy scales of lily (C. davidiana) are a representative traditional Chinese medicine with both medicinal and edible properties. As early as the Eastern Han Dynasty, Zhang Zhongjing's *Synopsis of Prescriptions of the Golden Chamber* recorded the use of lily to treat "lily disease," whose clinical manifestations included "desire to eat but inability to eat, constant silence, desire to lie down but inability to lie down, desire to walk but inability to walk… feeling neither cold nor hot, bitter taste in the mouth, red urine, incurable by various medicines, severe vomiting and diarrhea upon taking medicine, as if possessed by a spirit." This condition overlaps significantly with modern depression and its accompanying anxiety and somatization symptoms. Lily contains various active ingredients such as saponins, polysaccharides, flavonoids, and phenolic acids, among which lily saponins (LLS) and lily polysaccharides (LLP) are considered its main active material basis.

[0006] Existing technologies have reported on the extraction, characterization, and preliminary activity studies of lily saponins or lily polysaccharides alone, mainly focusing on in vitro antioxidant and cell protection. However, existing technologies have the following shortcomings: ① lack of systematic pharmacodynamic data in the classic chronic unpredictable mild stress (CUMS) mouse model of depression; ② limited to evaluation of a single active site, failing to elucidate the integrated mechanism of action of LLS and LLP from the perspective of the gut-brain axis; ③ failure to verify the complementary roles of LLS and LLP in different segments of the gut-brain axis (LLP focuses on intestinal repair, while LLS focuses on brain protection); ④ lack of key evidence on whether gut microbiota participates in the antidepressant effect as a causal mediator (such as fecal microbiota transplantation (FMT) verification).

[0007] Therefore, it is urgent to systematically elucidate the pharmacodynamic characteristics, synergistic mechanism, and evidence chain of the causal effect of the combined use of LLS and LLP for antidepressant treatment from the perspective of the gut-brain axis at the level of the CUMS depression model, so as to provide scientific basis and candidate solutions for the development of lily-derived antidepressant drugs based on gut-brain axis targets. Summary of the Invention

[0008] The purpose of this invention is to provide a novel use of lily saponin-lily polysaccharide composition in the preparation of antidepressant drugs, and in particular, to elucidate for the first time from the perspective of the gut-brain axis the integrated mechanism of action of LLS and LLP in antidepressant treatment, providing a scientific basis for the development of targeted drugs.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] The application of a lily saponin-lily polysaccharide composition in the preparation of antidepressant drugs, the composition comprising lily saponin LLS and lily polysaccharide LLP. LLS is the active steroidal saponin derived from lily medicinal material, containing one or more of lily saponins A, B, D, G, H, and I; LLP is an acidic pyranose polysaccharide derived from lily medicinal material, composed of seven monosaccharides: mannose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, and fucose.

[0011] Furthermore, the composition exerts its antidepressant effect through the gut-brain axis, and its peripheral gut-end mechanism includes: (1) Repairing the colonic tight junction barrier and upregulating the mRNA and protein expression levels of tight junction proteins ZO-1, Occludin, and Claudin-1. P <0.01); (2) Reduces the levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in colon tissue ( P <0.01), triggering a pullback to the abnormally high 5-HT level; (3) Remodeling the gut microbiota structure: restore the Shannon diversity index of the microbiota, increase the relative abundance of Bacteroidetes and decrease the relative abundance of Proteobacteria at the phylum level; increase the abundance of potentially beneficial bacteria (such as ParaBacteroides and Muribauliaceae) and decrease the abundance of conditionally pathogenic bacteria (such as Klebsiella and Escherichia Shigella) at the genus level.

[0012] Furthermore, the composition exerts its antidepressant effect through the gut-brain axis, with its central nervous system mechanism including: (1) Inhibit excessive activation of the HPA axis: reduce serum CORT and ACTH levels, and reduce CRH content in brain tissue (P <0.01); (2) Activation of the cAMP / PKA / CREB / BDNF signaling pathway in the brain: Increases the cAMP content in brain tissue, upregulates the expression level of PKA protein, increases the p-CREB / CREB phosphorylation ratio, and upregulates the expression level of BDNF. P <0.01), while norepinephrine (NE) levels rebounded.

[0013] Furthermore, in the composition, LLS focuses on central protection (activating cAMP / PKA / CREB / BDNF signaling and inhibiting the HPA axis), while LLP focuses on intestinal repair (restoring tight junction protein expression and reducing intestinal inflammatory factors). The two complement each other through the gut-brain axis.

[0014] Furthermore, in the CUMS mouse depression model, compared with the model group, the behavioral indicators of mice treated with the LLS+LLP combination were significantly improved ( P <0.01): The sugar water preference rate significantly rebounded, the forced swimming immobility time was significantly shortened, and the total mileage of spontaneous activities and the exploration time in the central area in the open field test both significantly increased. The improvement in the above behavioral indicators was significantly better than that of the LLS or LLP single-use group, and some indicators were close to the level of the blank control group.

[0015] Furthermore, histopathological observation showed that after the combined intervention of LLS and LLP, the colonic mucosa structure of mice tended to be intact, the crypt arrangement tended to be regular, the number of goblet cells recovered, and the inflammatory infiltration was significantly reduced; the arrangement of neurons in the CA1 area of ​​the hippocampus tended to be regular, the number of Nissl bodies was significantly restored, and neuronal damage was effectively alleviated.

[0016] Further, the mass ratio of lily saponins to lily polysaccharides in the composition is 1:(1~2), preferably 1:1. In the CUMS model, the dosage of lily saponins is 50 mg·kg⁻¹. -1 ·d -1 The dosage of lily polysaccharide is 50 mg / kg. -1 ·d -1 The total dosage was 100 mg / kg. -1 ·d -1 The administration route is by gavage.

[0017] FMT causality verification: Furthermore, fecal microbiota transplantation experiments have demonstrated for the first time that the gut microbiota remodeled after gavage administration of the LLS+LLP combination can independently reproduce the following antidepressant effects after transplantation into antibiotic-pretreated pseudo-germ-free recipient mice: (1) Behavioral level—Mice in the FMTL group (transplanted with LLS+LLP gut microbiota) showed significant improvement in depressive-like behavior; (2) Histopathological level—the integrity of the colonic mucosa structure was restored, and the damage to neurons in the CA1 area of ​​the hippocampus was relieved; (3) At the molecular level, the expression of colonic tight junction protein was restored, the level of pro-inflammatory factors was reduced, the cAMP / PKA / CREB / BDNF pathway in the brain was activated, and the HPA axis function was restored to normal. (4) Microbiome level—The Shannon diversity index of the gut microbiota in the FMTL group was restored, the microbiome structure converged with that of the healthy control group, and the microbiome characteristics reproduced the donor state.

[0018] The FMT causal validation results directly demonstrate that gut microbiota remodeling is an important mediating link in the antidepressant effect of LLS+LLP combination therapy, rather than an accompanying phenomenon.

[0019] Furthermore, the lily saponin-lily polysaccharide composition is used in the preparation of functional foods, health foods, special medical purpose formula foods, or dietary supplements that improve depression-related intestinal barrier damage and neuroinflammation through the gut-brain axis.

[0020] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention is the first to systematically elucidate the pharmacodynamic characteristics and integrated mechanism of action of the lily saponin-lily polysaccharide composition on the level of CUMS mouse depression model from the perspective of gut-brain axis, providing sufficient in vivo pharmacodynamic evidence for the development of this composition as an antidepressant drug; (2) This invention reveals for the first time the complementary division of labor between LLS and LLP in different links of the gut-brain axis—LLP focuses on the gut end (repairing the intestinal barrier and reducing intestinal inflammation), while LLS focuses on the brain end (activating cAMP / PKA / CREB / BDNF signals and inhibiting the HPA axis). This discovery provides a scientific basis for natural multi-component synergistic antidepressant strategies. (3) This invention provides causal evidence for the first time through fecal microbiota transplantation experiments, confirming that the gut microbiota regulated by LLS+LLP can independently transmit antidepressant effects, providing key methodological reference for the development of traditional Chinese medicine antidepressant drugs based on gut microbiota targets; (4) The composition of the present invention is derived from lily, a medicinal and edible herb, and has the natural advantages of high safety and low toxicity and side effects, which is in line with the direction of modern antidepressant drug development. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments are briefly described below. Obviously, the drawings described below are only some embodiments of the present invention, and those skilled in the art can obtain other drawings based on the content disclosed in the present invention without creative effort. The following drawings are used to further illustrate the technical solutions of the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0022] Figure 1 Total ion chromatograms of lily saponin A, lily saponin B, and lily saponin D identified by UHPLC-MS / MS; Figure 2 Total ion chromatograms of lily saponin G, lily saponin H, and lily saponin I identified by UHPLC-MS / MS; Figure 3 Infrared spectrum of lily polysaccharide; Figure 4 This is a schematic diagram of the CUMS mouse experimental procedure; Figure 5 Effects of the LLS+LLP combination on the behavior of CUMS mice: Sugar water preference rate (SPT); Figure 6 Effects of the LLS+LLP combination on the behavior of CUMS mice: Forced swimming immobility time (FST); Figure 7 Effects of the LLS+LLP combination on the behavior of CUMS mice: Figure 7 a is a schematic diagram of the motion trajectory. Figure 7 b is a heatmap of activity distribution. Figure 7 c is the number of squares traversed. Figure 7 d represents the percentage of cells traversed in the central area; Figure 8 HE staining results showing the effects of the LLS+LLP combination on the histopathology of colon tissue in CUMS mice; Figure 9 Nissl staining results (400×) showing the effect of the LLS+LLP combination on neurons in the CA1 region of the hippocampus of CUMS mice. Figure 10 This is a schematic diagram illustrating the integrated mechanism by which the lily saponin-lily polysaccharide composition of the present invention exerts its antidepressant effect through the gut-brain axis; Figure 11 The figure shows the RT-qPCR results of the effect of the LLS+LLP combination on the mRNA expression of the colonic tight junction proteins ZO-1, Occludin, and Claudin-1 in CUMS mice. Figure 12The Western blot results show the effect of the LLS+LLP combination on the expression of tight junction proteins ZO-1, Occludin, and Claudin-1 in the colon of CUMS mice. Figure 13 Bar chart showing the effect of LLS+LLP composition on serum CORT, ACTH and brain tissue BDNF, CRH, NE and cAMP levels in CUMS mice; Figure 14 The Western blot results show the effects of the LLS+LLP combination on the expression of PKA, BDNF, and p-CREB / CREB proteins in the brain tissue of CUMS mice. Figure 15 16S rRNA sequencing analysis of the effects of the LLS+LLP combination on the gut microbiota of CUMS mice: ( Figure 15 a) Shannon's diversity index; Figure 15 b) PCoA analysis; Figure 15 c) Phylum-level bacterial community composition; Figure 15 d) Belongs to the category of horizontal heatmaps; Figure 16 Figure showing the results of a fecal microbiota transplantation (FMT) experiment validating the antidepressant effect mediated by gut microbiota: ( Figure 16 (Top row) FMTL group colon HE staining; Figure 16 Bottom row) FMTL group hippocampal Nielsen staining; Figure 17 The figure shows the results of Shannon diversity index and PCoA analysis of the gut microbiota in FMT receptor mice. Figure 17 'a' represents the Shannon diversity index. Figure 17 b is a graph showing the PCoA analysis results. Detailed Implementation

[0023] The present invention will be further described below with reference to embodiments and comparative examples. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. All equivalent substitutions, conventional adjustments or improvements made based on the technical concept of the present invention should fall within the scope of protection of the present invention.

[0024] Example 1: Preparation of lily saponins (LLS) and lily polysaccharides (LLP) (1) Preparation of lily saponins Dried scales and leaves of lily were pulverized and passed through a 40-mesh sieve. 80% ethanol solution was added at a material-to-liquid ratio of 1:10 (g / mL), and the mixture was extracted using ultrasound-assisted extraction at 40 ℃ and 100 W for 30 min. This extraction was repeated three times. The extracts were combined, filtered, and concentrated under reduced pressure to recover the ethanol, yielding a crude extract of lily saponins. The crude extract was dispersed in distilled water and loaded onto a pre-treated AB-8 macroporous adsorption resin column. The column was first eluted with distilled water until the eluent was clear, then eluted with 80% ethanol. The ethanol eluent was collected, concentrated under reduced pressure, and freeze-dried to obtain lily saponins LLS. The total saponin content in LLS was determined to be 76.5% by the vanillin-perchloric acid colorimetric method. UHPLC-MS / MS analysis identified six steroidal saponin components (lily saponins A, B, D, G, H, and I) in LLS. The total ion chromatogram is shown below. Figure 1 and Figure 2 As shown.

[0025] (2) Preparation of lily polysaccharides Dried scales and leaves of lily were pulverized and passed through a 40-mesh sieve. Distilled water was added at a material-to-liquid ratio of 1:10 (g / mL), and the mixture was extracted with ultrasound at 70℃ and 100 W for 40 min. This extraction was repeated twice. The extracts were combined, filtered, and concentrated under reduced pressure to 1 / 5 of the original volume. Trichloroacetic acid was added to a final concentration of 5%, and the mixture was allowed to stand overnight at 4℃ to remove proteins. The supernatant was then centrifuged. The supernatant was dialyzed against a dialysis bag with a molecular weight cutoff of 3500 Da at 4℃ for 48 h to remove small molecule impurities. After concentration under reduced pressure, the mixture was freeze-dried to obtain lily polysaccharide (LLP). The polysaccharide content in LLP was determined to be 70.53% by the phenol-sulfuric acid method, 7.22% by the Coomassie brilliant blue method, and 8.78% by the sulfuric acid-carbazole method. Infrared spectroscopy analysis (…) Figure 3 LLP exhibits typical polysaccharide characteristic absorption peaks, indicating it is an acidic pyranose polysaccharide. High-performance gel permeation chromatography (HPLC) determined the molecular weight distribution of LLP to be 4.45 × 10⁻⁶. 6 Da and 2.14×10 3 Between Da. Analysis by PMP pre-column derivatization HPLC method revealed that LLP is composed of seven monosaccharides: mannose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, and fucose, with a molar ratio of 1:0.34:0.30:0.29:0.39:0.19:0.02.

[0026] Example 2: Antidepressant effect of LLS+LLP composition on CUMS depression model mice This embodiment establishes a chronic unpredictable mild stress (CUMS) mouse model of depression and systematically evaluates the in vivo antidepressant efficacy of the LLS+LLP combination.

[0027] (1) Laboratory animals and grouping Sixty SPF-grade male C57BL / 6J mice, weighing (20±2) g, were randomly divided into 6 groups (n=10) according to body weight after 7 days of acclimatization: blank control group (CON group), model group (MOD group), fluoxetine positive control group (FLX group, 8 mg·kg). -1 ·d -1 LLS monotherapy group (100 mg·kg) -1 ·d -1 LLP monotherapy group (100 mg·kg) -1 ·d -1 ), LLS+LLP combination group (lily saponins 50 mg·kg) -1 ·d -1 +Lily polysaccharide 50 mg·kg -1 ·d -1 Total dosage: 100 mg / kg -1 ·d -1 ).

[0028] (2) CUMS modeling and drug administration regimen Except for the CON group, mice in the other groups received a 56-day CUMS program. Stressors included restraint, wet cages, tilted cages, cold / heat stimulation, reversed day / night cycles, and fasting / water restriction. One to two stressors were administered daily, and mice were randomly assigned to avoid developing adaptation (experimental procedure as follows). Figure 4 (As shown). Administration of medication by gavage began on day 28: the CON and MOD groups were administered an equal volume of distilled water (20 mL / kg) by gavage. -1 ·d -1 The following groups were administered medications by gavage: FLX group received fluoxetine solution, LLS group received lily saponin solution, LLP group received lily polysaccharide solution, and LLS+LLP group received a mixed solution of lily saponin and lily polysaccharide. Administration continued for 28 days.

[0029] (3) Behavioral testing The following behavioral evaluations were conducted sequentially after the administration of the medication: Sugar water preference test (SPT): Mice were housed individually and given one bottle each of 1% sucrose solution and pure water. After 24 hours of fasting with unlimited water, the consumption of both liquids was recorded. Sugar water preference rate (%) = (sucrose solution consumption / (sucrose solution consumption + pure water consumption)) × 100%.

[0030] Forced swimming test (FST): Mice were placed in a cylindrical container with a water depth of 15 cm and a water temperature of (25±1) ℃. After acclimatization for 2 minutes, the cumulative immobility time over the following 4 minutes was recorded. Immobility was defined as the minimum movement required for the mouse to stop struggling and keep only its head above water.

[0031] Open field test (OFT): Mice were placed in the center of an open field box measuring 40×40×30 cm, and the total distance of spontaneous activity and the exploration time in the central area (20×20 cm) were recorded within 5 minutes.

[0032] Behavioral test results such as Figure 5 , Figure 6 , Figure 7 As shown. Compared with the MOD group, the sucrose preference rate of mice in the LLS+LLP combination group was significantly increased (P<0.01). Figure 5 The time spent immobile during forced swimming was significantly shortened. P <0.01, Figure 6 ), total distance of autonomous activity and exploration time in the central area during the open field experiment ( Figure 7 All significantly increased ( P <0.01). The improvement in the above behavioral indicators was significantly better than that in the LLS or LLP single-use group, and some indicators were close to the level of the CON group.

[0033] (4) Histopathological observation After the behavioral tests were completed, colon and brain tissues from each group of mice were collected for histopathological examination. Colon tissues were fixed in 10% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE). Brain tissues were fixed, embedded in paraffin, sectioned coronally, and stained with Nissl stain, with particular attention paid to the CA1 region of the hippocampus.

[0034] Colon HE staining results as follows Figure 8 As shown, the CON group exhibited intact colonic mucosal structure, regular crypt arrangement, abundant goblet cells, and no submucosal edema or inflammatory infiltration. The MOD group showed significant damage to the colonic mucosal structure, manifested as disordered crypt arrangement, reduced goblet cells, submucosal edema, and extensive inflammatory cell infiltration. After LLS+LLP combined intervention, the colonic mucosal structure tended to be more intact, the crypt arrangement more regular, goblet cells showed significant recovery, and inflammatory infiltration was significantly reduced.

[0035] The results of hippocampal Nielsen staining are as follows: Figure 9 As shown, in the CON group, neurons in the CA1 region of the hippocampus were densely packed, with full cell bodies and abundant Nissl bodies. In the MOD group, neurons were sparsely packed and fewer in number, with significantly reduced or absent Nissl bodies. After LLS+LLP combined intervention, the neuronal arrangement became more regular, cell body morphology was restored, the number of Nissl bodies increased significantly, and neuronal damage was effectively alleviated.

[0036] Example 3: The regulatory effect of LLS+LLP composition on intestinal barrier function and intestinal inflammation This embodiment examines the regulatory effect of the LLS+LLP composition on the expression of tight junction protein in the colon and the level of intestinal inflammatory factors in CUMS mice.

[0037] (1) Detection of tight junction proteins in the colon Colon tissues were collected from mice in each group, and the mRNA and protein expression levels of tight junction proteins ZO-1, Occludin, and Claudin-1 were detected by RT-qPCR and Western blot, respectively.

[0038] The RT-qPCR primer sequences are as follows: ZO-1-F: 5'-GCCGCTAAGAGCACAGCAA-3' (SEQ ID NO.1) ZO-1-R: 5'-TCCCCACTCTGAAAATGAGGA-3' (SEQ ID NO.2) Occludin-F: 5'-TTGAAAGTCCACCTCCTTACAGA-3' (SEQ ID NO.3) Occludin-R: 5'-CCGGATAAAAAGAGTACGCTGG-3' (SEQ ID NO.4) Claudin-1-F: 5'-GGGGACAACATCGTGACCG-3' (SEQ ID NO.5) Claudin-1-R: 5'-AGGAGTCGAAGACTTTGCACT-3' (SEQ ID NO.6) GAPDH-F: 5'-AGGTCGGTGTGAACGGATTTG-3' (SEQ ID NO.7) GAPDH-R: 5'-GGGGTCGTTGATGGCAACA-3' (SEQ ID NO.8) The results are as follows Figure 11 and Figure 12 As shown in the figure, compared with the MOD group, the mRNA and protein expression levels of ZO-1, Occludin, and Claudin-1 in the colon tissue of the LLS+LLP combination group were significantly upregulated (P<0.01), and the degree of recovery was better than that of the LLS or LLP single-use groups. The comparison of single-use treatments showed that LLP restored the three tight junction proteins to a greater extent than LLS, indicating that LLP plays a more prominent role in intestinal barrier repair.

[0039] (2) Detection of intestinal inflammatory factors and 5-HT levels The levels of pro-inflammatory cytokines IL-1β, IL-6, TNF-α and neurotransmitter 5-HT in the colon tissue of mice in each group were detected using an ELISA kit.

[0040] The results showed that, compared with the MOD group, the levels of IL-1β, IL-6, and TNF-α in the colon tissue of the LLS+LLP combination group were significantly reduced. P (<0.01), and the abnormally elevated 5-HT levels were effectively reduced. Single-use comparisons showed that LLP was superior to LLS in improving intestinal inflammatory markers, further supporting the advantages of LLP in intestinal protection.

[0041] Example 4: The regulatory effect of the LLS+LLP composition on the central cAMP / PKA / CREB / BDNF pathway and HPA axis This embodiment examines the regulatory effect of the LLS+LLP composition on the cAMP / PKA / CREB / BDNF signaling pathway and HPA axis-related indices in the brains of CUMS mice.

[0042] (1) HPA axis index detection The levels of CORT and ACTH in the serum of mice in each group and the content of CRH in brain tissue were detected using an ELISA kit.

[0043] The results are as follows Figure 13 As shown. Compared with the MOD group, the LLS+LLP combination group showed a highly significant decrease in serum CORT, ACTH levels, and brain tissue CRH content. P <0.01), the overactivation state of the HPA axis was effectively suppressed.

[0044] (2) Detection of the cAMP / PKA / CREB / BDNF pathway The levels of cAMP, BDNF, and NE in the brain tissue of mice in each group were detected using an ELISA kit. The protein expression levels of PKA, p-CREB, CREB, and BDNF were detected using Western blot.

[0045] The results are as follows Figure 13 and Figure 14 As shown. Compared with the MOD group, the LLS+LLP combination group showed a highly significant increase in cAMP content, PKA protein expression level, p-CREB / CREB phosphorylation ratio, and BDNF expression level in brain tissue. P <0.01), NE levels rebounded significantly. Single-use comparisons showed that LLS restored the above central nervous system indicators to a greater extent than LLP, indicating that LLS has a more prominent effect in protecting the central nervous system at the brain level.

[0046] Based on the results of Examples 3 and 4, this invention reveals for the first time the complementary and synergistic antidepressant mechanism of the combined use of LLS and LLP: LLP primarily acts on the gut, repairing the colonic tight junction barrier and reducing intestinal inflammation; LLS primarily acts on the brain, activating the cAMP / PKA / CREB / BDNF pathway and inhibiting excessive activation of the HPA axis. The two complement each other through the gut-brain axis, producing a synergistic antidepressant effect. A schematic diagram of the mechanism is shown below. Figure 10 As shown.

[0047] Example 5: The regulatory effect of LLS+LLP composition on gut microbiota This embodiment uses 16S rRNA gene sequencing technology to analyze the effect of the LLS+LLP combination on the composition of the gut microbiota in CUMS mice.

[0048] Colon contents were collected from mice in each group, total bacterial DNA was extracted, and the V3-V4 variable region of the 16S rRNA gene was amplified by PCR and sequenced using Illumina high-throughput sequencing. Bioinformatics analysis was performed using QIIME2 and R.

[0049] The results are as follows Figure 15 As shown, CUMS stress significantly reduced the Shannon diversity index of the gut microbiota in mice (P<0.01) and disrupted the microbiota structure. After intervention with LLS+LLP, the microbiota diversity was significantly restored (…). Figure 15 a) The abundance curve tends to flatten. PCoA analysis showed that the microbial community structure of the LLS+LLP group was similar to that of the CON group ( Figure 15 b). At the phylum level, the relative abundance of Bacteroidetes significantly increased in the LLS+LLP group, while the abundance of Proteobacteria significantly decreased. Figure 15 c). At the genus level, the abundance of potentially beneficial bacteria such as ParaBacteroides and Muribauraceae increased, while the abundance of opportunistic pathogens such as Klebsiella and Escherichia Shigella decreased. Figure 15 d).

[0050] LEfSe differential analysis showed that the LLS+LLP group had the fewest characteristic differentially expressed bacteria, and its bacterial community structure was closest to that of the healthy control group. Spearman correlation analysis revealed that the restored beneficial bacteria were positively correlated with neuroprotective indicators such as BDNF and cAMP, and negatively correlated with inflammatory factors and HPA axis-related indicators.

[0051] Example 6: Fecal microbiota transplantation (FMT) to verify the causal mediating role of gut microbiota This embodiment uses an FMT experiment to verify whether the gut microbiota regulated by the LLS+LLP composition is sufficient to independently reproduce the antidepressant effect.

[0052] (1) Establishment of a pseudo-germless mouse model SPF-grade male C57BL / 6J mice were administered a mixture of antibiotics (ampicillin 1 g / L, neomycin 1 g / L, metronidazole 1 g / L, vancomycin 0.5 g / L) by gavage daily for 7 consecutive days to establish a pseudo-germ-free mouse model.

[0053] (2) FMT Experiment Grouping Pseudo-germ-free mice were randomly divided into three groups (n=8): FMTC group (transplanted with fecal microbiota suspension from CON group mice), FMTM group (transplanted with fecal microbiota suspension from MOD group mice), and FMTL group (transplanted with fecal microbiota suspension from LLS+LLP group mice). Each mouse was administered 0.2 mL of fecal microbiota suspension by gavage daily for 14 consecutive days.

[0054] (3) FMT histopathological examination Colon HE staining results ( Figure 16 (Top row) shows that the FMTL group exhibited significantly restored colonic mucosal structural integrity and reduced inflammatory infiltration. Hippocampal Nielsen staining results ( Figure 16 (Bottom row) shows that the arrangement of neurons in the CA1 region of the FMTL group tends to be more regular, and the number of Nissl bodies has been significantly restored.

[0055] (4) FMT receptor microbiota analysis 16S rRNA sequencing results showed that the Shannon diversity index of the gut microbiota in the FMTL group recipient mice was significantly restored, and the microbiota structure clustered with that of the FMTC group in PCoA analysis. Figure 17 The abundance of beneficial bacteria such as Bacteroides has rebounded, and the characteristics of the bacterial community have been successfully reproduced from the donor state.

[0056] (5) Detection of FMT molecular index ELISA and Western blot results showed that in the FMTL group of recipient mice, the expression of tight junction protein in the colon was restored, and the levels of IL-1β, IL-6, and TNF-α were reduced; the cAMP / PKA / CREB / BDNF pathway in the brain was activated, and serum CORT, ACTH, and brain tissue CRH levels returned to normal. The simultaneous restoration of these molecular indicators directly demonstrates that gut microbiota remodeling is an important causal mediator of the antidepressant effect of the combined use of LLS and LLP.

[0057] Comparative Example 1 LLS and LLP were prepared according to the method in Example 1. The antidepressant efficacy was evaluated according to the method in Example 2, except that only LLS was used alone (100 mg / kg). -1 ·d -1 ) or LLP alone (100 mg·kg -1 ·d -1The administration was performed as described in Examples 2 to 5, with other steps identical. Results showed that both LLS and LLP alone improved behavioral and molecular indicators in CUMS mice to some extent, but the improvement was significantly lower than that of the LLS+LLP combination group (P<0.05 or P<0.01). Mechanistically, LLS alone showed better regulation of the central cAMP / PKA / CREB / BDNF pathway and HPA axis, but weaker effects on intestinal barrier repair and gut microbiota regulation; LLP alone showed better effects on intestinal barrier repair, intestinal inflammation suppression, and gut microbiota regulation, but weaker effects on central pathways and the HPA axis. This comparative study directly demonstrates the synergistic effect of LLS and LLP combination in achieving complementary advantages in the gut-brain axis.

[0058] Comparative Example 2 The efficacy and mechanism of the antidepressant were evaluated according to the methods of Examples 2 to 5, the difference being that fluoxetine hydrochloride (FLX, 8 mg·kg) was used. -1 ·d -1 (Administered by gavage) as a positive control drug, in combination with LLS+LLP (lily saponins 50 mg·kg) -1 ·d -1 +Lily polysaccharide 50 mg·kg -1 ·d -1 Parallel comparisons were conducted. Results showed that the LLS+LLP combination group achieved comparable efficacy to the fluoxetine group in terms of restoration of sucrose preference rate, shortened immobility time during forced swimming, improved open field spontaneous activity, and enhanced exploratory behavior (no statistically significant difference, P>0.05), with some behavioral indicators even slightly superior to the fluoxetine group. Mechanistically, fluoxetine primarily acts on the central 5-HT system, and its effects on intestinal barrier repair, intestinal inflammation suppression, and gut microbiota diversity restoration were significantly weaker than those of the LLS+LLP combination group. However, the LLS+LLP combination group exhibited a more comprehensive bidirectional regulatory effect on both the gut (restoration of tight junction proteins, reduction of IL-1β / IL-6 / TNF-α, restoration of Shannon's index in gut microbiota) and the brain (activation of cAMP / PKA / CREB / BDNF pathways, inhibition of the HPA axis). This comparison indicates that the antidepressant efficacy of the LLS+LLP combination is comparable to that of the first-line clinical drug fluoxetine, but it has unique advantages in multi-target comprehensive regulation of the gut-brain axis, making it particularly suitable for patients with depression accompanied by intestinal dysfunction.

[0059] Comparative Example 3 LLS and LLP were prepared according to the method in Example 1. The antidepressant efficacy was evaluated according to the methods in Examples 2 to 5, the difference being the setting of combination groups with different LLS:LLP mass ratios: LLS:LLP = 1:2 group (LLS 33.3 mg·kg⁻¹). -1 ·d -1+ LLP 66.7 mg·kg -1 ·d -1 Total dose 100 mg·kg -1 ·d -1 The LLS:LLP=2:1 group (LLS 66.7 mg·kg) and the LLS:LLP=2:1 group (LLS 66.7 mg·kg) -1 ·d -1 + LLP 33.3 mg·kg -1 ·d -1 Total dose 100 mg·kg -1 ·d -1 The results were compared with those of the LLS:LLP=1:1 combination group in Example 2. The results showed that all three ratios could improve depressive-like behavior and related molecular markers in CUMS mice to some extent. The LLS:LLP=1:1 combination group showed the best performance in behavioral aspects (SPT, FST, and OFT), intestinal barrier repair (upregulation of ZO-1, Occludin, and Claudin-1), intestinal inflammation suppression (reduction of IL-1β, IL-6, and TNF-α), restoration of gut microbiota diversity (Shannon index), and activation of central pathways (inhibition of cAMP / PKA / CREB / BDNF and HPA axis) (P<0.05 vs 1:2 and 2:1 groups). The LLS:LLP=2:1 group showed the second best performance in central pathway activation but a weaker effect on intestinal barrier repair; the LLS:LLP=1:2 group showed the second best performance in intestinal barrier repair but a weaker effect on central pathway activation. This comparison indicates that an optimal gut-brain axis synergistic effect can be achieved when the mass ratio of LLS to LLP is 1:1, and bias towards either side will lead to a decrease in the overall antidepressant efficacy.

[0060] In summary, this invention, through a CUMS mouse depression model, 16S rRNA gut microbiota analysis, and FMT causal verification experiments, systematically elucidates for the first time from the perspective of the gut-brain axis the integrated mechanism of action of the lily saponin-lily polysaccharide combination in antidepressant effects. The combination of LLS and LLP can reshape the gut microbiota, repair the colonic tight junction barrier, alleviate intestinal inflammation (peripheral end), and further inhibit excessive activation of the HPA axis and activate the cAMP / PKA / CREB / BDNF signaling pathway in the brain (central end), thereby producing a synergistic antidepressant effect. Crucially, the FMT experiment directly demonstrates that the gut microbiota regulated by LLS+LLP can independently transmit antidepressant effects, providing key causal evidence and candidate solutions for the development of natural antidepressant drugs based on gut microbiota targets. This highly aligns with the millennia-old clinical experience of using lily to treat "lily disease" in the *Jinkui Yaolue* (Essential Prescriptions of the Golden Chamber), providing a systematic scientific explanation of this traditional efficacy from the perspective of modern gut-brain axis theory.

[0061] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of a lily saponin-lily polysaccharide composition in the preparation of antidepressant drugs, characterized in that, The composition includes lily saponins and lily polysaccharides, wherein the lily saponins are steroidal saponin active fractions derived from lily medicinal materials, and the lily polysaccharides are acidic pyranoid polysaccharides derived from lily medicinal materials.

2. The application according to claim 1, characterized in that, The lily saponins are one or more of lily saponins A, B, D, G, H, and I; the lily polysaccharides are composed of mannose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, and fucose.

3. The application according to claim 1, characterized in that, The preparation method of the lily saponins includes the following steps: Dried scales and leaves of lily were pulverized and passed through a 40-mesh sieve. 80% ethanol solution was added at a material-to-liquid ratio of 1:10 (g / mL), and the mixture was extracted with ultrasound at 40℃ and 100 W for 30 min. This extraction was repeated three times. The extracts were combined, filtered, and concentrated under reduced pressure to recover the ethanol, yielding a crude extract of lily saponins. The crude extract was dispersed in distilled water and loaded onto a pre-treated AB-8 macroporous adsorption resin column. The column was first eluted with distilled water until the eluent was clear, then eluted with 80% ethanol. The ethanol-eluted fraction was collected, concentrated under reduced pressure, and freeze-dried to obtain lily saponins.

4. The application according to claim 1, characterized in that, The preparation method of the lily polysaccharide includes the following steps: Dried scales and leaves of lily were pulverized and passed through a 40-mesh sieve. Distilled water was added at a material-to-liquid ratio of 1:10 (g / mL), and the mixture was extracted with ultrasound at 70 ℃ and 100W for 40 min. The extraction was repeated twice. The extracts were combined, filtered, and concentrated under reduced pressure to 1 / 5 of the original volume. Trichloroacetic acid was added to a final concentration of 5%, and the mixture was allowed to stand overnight at 4 ℃ to remove proteins. The supernatant was then centrifuged. The supernatant was dialyzed at 4 ℃ for 48 h using a dialysis bag with a molecular weight cutoff of 3500 Da to remove small molecule impurities. After concentration under reduced pressure, the mixture was freeze-dried to obtain lily polysaccharide.

5. The application according to claim 1, characterized in that, The lily saponin-lily polysaccharide composition significantly improved the sucrose preference rate, shortened the immobility time during forced swimming, and increased the total mileage of spontaneous activity and the exploration time in the central region during the open field test in a mouse model of chronic unpredictable mild stress (CUMS). P <0.01), and the combined effect is significantly better than that of lily saponins or lily polysaccharides alone.

6. The application according to any one of claims 1 to 4, characterized in that, The mass ratio of lily saponins to lily polysaccharides is 1:1~2.

7. The application according to claim 6, characterized in that, The dosage of lily saponins and lily polysaccharides in this composition is 50 mg / kg each. -1 ·d -1 The total dosage was 100 mg / kg. -1 ·d -1 The administration route is by gavage.

8. The application according to claim 1, characterized in that, Lily polysaccharides focus on repairing intestinal barrier function and reducing intestinal inflammation, while lily saponins focus on activating the central cAMP / PKA / CREB / BDNF signaling pathway and inhibiting excessive activation of the HPA axis. The two complement each other through the gut-brain axis.

9. The application according to claim 1, characterized in that, The gut microbiota remodeled after oral administration of the lily saponin-lily polysaccharide composition can independently reproduce antidepressant effects after transplantation into recipient animals, including improving depressive-like behaviors, repairing the colonic barrier, reducing inflammation levels, inhibiting excessive activation of the HPA axis, and activating the cAMP / PKA / CREB / BDNF pathway in the brain.

10. The use of a lily saponin-lily polysaccharide composition in the preparation of a product that improves depression-related intestinal barrier damage and neuroinflammation via the gut-brain axis, characterized in that, The product is a functional food, health food, special medical purpose formula food, or dietary supplement.

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