Composition of dendrobium huoshanense extract and lactobacillus reuteri and application of composition in preparation of medicine for treating intestinal inflammatory diseases

Abstract

The invention discloses a composition of dendrobium huoshanense extract and lactobacillus reuteri and application of the composition in preparation of medicines for treating intestinal inflammatory diseases, and belongs to the field of biological medicines. The dendrobium huoshanense is subjected to reflux extraction by using an ethanol solution to obtain a dendrobium huoshanense extract, and the dendrobium huoshanense extract is combined with lactobacillus reuteri to form the composition. Mouse model experiment results show that the composition can significantly regulate GABA metabolism of intestinal flora, reduce the level of inflammatory factors in vivo, improve depression symptoms of mice and repair intestinal barriers; compared with independent treatment of the dendrobium huoshanense extract or the lactobacillus reuteri, the dendrobium huoshanense extract has an obvious synergistic treatment effect. The preparation method of the composition is simple and easy to implement, the raw materials are wide in source, large-scale production and clinical application are facilitated, high-valued utilization of the traditional Chinese medicine-probiotic composition in the field of medicine is promoted, and a new choice is provided for treatment of intestinal inflammatory diseases and intestinal-cerebral axis related diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a composition of Dendrobium huoshanense extract and Lactobacillus reuteri and application thereof in preparation of a medicine for treating intestinal inflammatory diseases. BACKGROUND

[0002] The "gut-brain" axis, as a complex bidirectional communication system connecting the gut and the brain, plays a core role in maintaining body homeostasis and regulating disease progression through dynamic interactions among neural, immune, and endocrine networks. Previous studies have shown that once the axis system is disturbed, it is closely related to the occurrence and development of intestinal inflammatory diseases such as inflammatory bowel disease (IBD, including ulcerative colitis and Crohn's disease) and depression induced by the diseases, highlighting the key regulatory role of the axis in intestinal inflammation and related depressive disorders.

[0003] Inflammatory bowel disease belongs to chronic nonspecific intestinal inflammation with unclear etiology, and its pathogenesis is closely related to intestinal microecological imbalance and intestinal inflammatory cascade. Clinical studies have shown that the diversity of intestinal flora in IBD patients is significantly reduced, the abundance of pro-inflammatory bacteria (such as Escherichia coli and Staphylococcus) is increased, and the number of anti-inflammatory bacteria (such as Bifidobacterium and Lactobacillus) is reduced. This imbalance of intestinal flora can induce persistent inflammatory response of intestinal mucosa by activating the TLR4 / NF-κB signaling pathway. Irritable bowel syndrome, as a common functional intestinal disease, its pathological mechanism involves the multi-dimensional interaction of intestinal motility abnormalities, visceral hypersensitivity, intestinal flora imbalance, and low-grade intestinal inflammation. Studies have found that the composition of intestinal flora in IBS patients has characteristic changes, such as the decrease in the abundance of butyrate-producing bacteria (Roseburia and Faecalibacterium) and the increase in the number of gas-producing bacteria (such as Enterococcus), and this flora disorder can enhance visceral hyperalgesia through the "flora-immune-neural" pathway. Depression is a mental disorder disease with high incidence worldwide, and its pathological mechanism involves genetic susceptibility, neurobiological abnormalities (such as monoamine imbalance), and psychosocial stress. Recent studies have found that the gut-brain axis is involved in the pathogenesis of depression through the intestinal flora-brain interaction pathway. Intestinal flora imbalance can induce intestinal barrier dysfunction, trigger systemic inflammatory response (such as increasing the levels of pro-inflammatory cytokines IL-6 and TNF-α), and ultimately activate the neuro-immune interaction to cause brain functional network disorder. Current intestinal anti-inflammatory drugs (such as glucocorticoids, immunosuppressants, antibiotics, etc.) and antidepressants (such as fluoxetine, sertraline, fluvoxamine, citalopram, etc.) have the problems of large side effects, easy recurrence, etc. Therefore, it is urgent to explore safer, more effective and individualized treatment strategies for intestinal inflammatory diseases and "gut-brain" axis related diseases.

[0004] The synergistic intervention strategy of plant-derived active ingredients and probiotics opens up a new direction for the treatment of gut-brain axis related diseases. As a traditional and precious Chinese medicinal material, Dendrobium huoshanense contains rich Dendrobium polysaccharides, alkaloids and flavonoids in its extract. Studies have shown that these components can exert multi-target pharmacological effects by resisting oxidative stress, inhibiting the NF-κB inflammatory pathway and regulating T cell immune function. Lactobacillus reuteri, as the indigenous probiotic in the intestinal tract, can inhibit the colonization of pathogenic bacteria through a competitive exclusion mechanism, enhance the expression of tight junction proteins in the intestinal epithelium to repair the mucosal barrier, and also play an anti-inflammatory role by regulating the balance of Th1 / Th2 cells. Notably, there is currently no research report on the combination of Dendrobium huoshanense extract and Lactobacillus reuteri composition in the treatment of intestinal related diseases. This combined intervention scheme is expected to break through the limitations of existing therapies by a multi-pathway synergistic mechanism of'regulating the flora-inhibiting inflammation-repairing neuro-immune interaction', and provide an innovative solution for the clinical treatment of related diseases. Therefore, it is of great practical significance to develop safe and effective drugs for the treatment of intestinal inflammatory diseases and 'gut-brain' axis related diseases. SUMMARY

[0005] The purpose of the present application is to provide a composition of Dendrobium huoshanense extract and Lactobacillus reuteri and its application in the preparation of a drug for treating intestinal inflammatory diseases, in order to solve the problems existing in the prior art. Compared with Dendrobium huoshanense extract or Lactobacillus reuteri alone, the composition provided by the present application has a significantly synergistic therapeutic effect, is conducive to large-scale production and clinical application, and promotes the high-value utilization of traditional Chinese medicine-probiotic combinations in the medical field.

[0006] To achieve the above-mentioned purpose, the present application provides the following solutions:

[0007] The present application provides a pharmaceutical composition comprising Dendrobium huoshanense extract and Lactobacillus reuteri bacterial liquid.

[0008] Further, the preparation method of the Dendrobium huoshanense extract comprises the following steps:

[0009] Take Dendrobium huoshanense, crush it, mix it with 70% ethanol solution, reflux extract for 2h, and then filter and collect the filtrate and the residue;

[0010] The residue is extracted repeatedly for 3 times, the filtrate is combined, and concentrated under reduced pressure to a paste, thereby obtaining the Dendrobium huoshanense extract.

[0011] The present application also provides the application of the above-mentioned pharmaceutical composition in the preparation of a drug for treating intestinal inflammatory diseases.

[0012] Further, the intestinal inflammatory disease is irritable bowel syndrome, Crohn's disease or inflammatory bowel disease.

[0013] The application further provides a medicine for treating intestinal inflammatory diseases, which comprises the above-mentioned medicine composition as a main effective component.

[0014] Further, the medicine further comprises a pharmaceutically acceptable excipient.

[0015] The application further provides an application of the above-mentioned medicine composition in preparing a medicine for treating intestinal-brain axis related diseases.

[0016] Further, the intestinal-brain axis related diseases are Alzheimer's disease or depression.

[0017] The application further provides a medicine for treating intestinal-brain axis related diseases, which comprises the above-mentioned medicine composition as a main effective component.

[0018] Further, the medicine further comprises a pharmaceutically acceptable excipient.

[0019] The application discloses the following technical effects:

[0020] The application uses an ethanol solution to reflux extract Dendrobium huoshanense, obtains a Dendrobium huoshanense extract, and combines the Dendrobium huoshanense extract with Lactobacillus reuteri to form a composition. Mouse model experiment results show that the composition can significantly regulate intestinal flora GABA metabolism, reduce the level of inflammatory factors in the body, improve the depression symptoms of mice, and repair the intestinal barrier; compared with Dendrobium huoshanense extract or Lactobacillus reuteri alone, the composition has an obvious synergistic treatment effect. The preparation method of the composition in the application is simple and easy to implement, raw materials are widely sourced, and the method is conducive to large-scale production and clinical application, promotes the high-value use of traditional Chinese medicine-probiotics combination in the medical field, and provides a new choice for the treatment of intestinal inflammatory diseases and intestinal-brain axis related diseases. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings described below only show some embodiments of the application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0022] Figure 1 Statistical diagram of the influence of DHEE on the culture of Lactobacillus reuteri; wherein, A is the influence of DHEE on the growth of flora; B is the determination result of the influence of different experimental groups on the GABA level; compared with the control group, *p<0.05;

[0023] Figure 2Statistical graphs of the expression levels of inflammatory factors in the serum of mice in each group; A is the expression of IL-1β in the serum; B is the expression of TNF-α in the serum; C is the expression of IL-6 in the serum; compared with the blank group (Control) or the blank+antibiotic group (Control+Abx), *p<0.05, **p<0.01, ***p<0.001; compared with the model group (Model) or the model+antibiotic group (Model+Abx), # p<0.05, ## p<0.01; compared with the Dendrobium huoshanense ethanol extract+antibiotic group (DHEE+Abx) or the Lactobacillus reuteri+antibiotic group (L.reuteri+Abx), & p<0.05, && p<0.01;

[0024] Figure 3 Statistical graphs of the expression levels of inflammatory factors in the colon of mice in each group; A is the expression of IL-1β in the colon; B is the expression of TNF-α in the colon; C is the expression of IL-6 in the colon; compared with the blank group (Control) or the blank+antibiotic group (Control+Abx), *p<0.05, **p<0.01, ***p<0.001; compared with the model group (Model) or the model+antibiotic group (Model+Abx), # p<0.05; compared with the Dendrobium huoshanense ethanol extract+antibiotic group (DHEE+Abx) or the Lactobacillus reuteri+antibiotic group (L.reuteri+Abx), & p<0.05, && p<0.01;

[0025] Figure 4 Observation graphs of the HE section results of the colon of mice in each group; A is the HE result of the blank group, the blank+antibiotic group, the model group and the model+antibiotic group; B is the HE result of the blank+antibiotic group, the model+antibiotic group, the Lactobacillus reuteri+antibiotic group, the Dendrobium huoshanense ethanol extract+antibiotic group and the Dendrobium huoshanense ethanol extract+Lactobacillus reuteri+antibiotic group;

[0026] Figure 5 Observation graphs of the AB-APS staining results of the colon of mice in each group; A is the AB-APS staining result of the blank group, the blank+antibiotic group, the model group and the model+antibiotic group; B is the AB-APS staining result of the blank+antibiotic group, the model+antibiotic group, the Lactobacillus reuteri+antibiotic group, the Dendrobium huoshanense ethanol extract+antibiotic group and the Dendrobium huoshanense ethanol extract+Lactobacillus reuteri+antibiotic group;

[0027] Figure 6Statistical chart of the determination results of GABA expression levels of each group of mice; wherein, A is the expression of GABA in the serum of the depression mice; B is the expression of GABA in the feces of the depression mice; compared with the blank group (Control) or the blank + antibiotic group (Control + Abx), *p<0.05; compared with the model group (Model) or the model + antibiotic group (Model + Abx), ## p<0.01; compared with the Dendrobium huoshanense ethanol extract + antibiotic group (DHEE + Abx) or the Lactobacillus reuteri + antibiotic group (L. reuteri + Abx), & p<0.05. DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present application will now be described in detail, which should be considered to be illustrative of the application and not restrictive of the application. It will be appreciated that those exemplary embodiments described herein are not intended to limit the scope of the application, but rather to provide a more detailed description of certain aspects, features and embodiments of the application.

[0029] It should be understood that the terms used in the present application merely describe particular embodiments and are not intended to limit the present application. In addition, for numerical ranges in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range within any stated range or within any stated intermediate value, and any other stated value or intermediate value within the stated range is also encompassed within the present application. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict, the content of the present specification will control.

[0031] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples presented herein. The description and examples are illustrative of the application and are not intended to limit the scope of the application.

[0032] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended and do not exclude additional, unrecited elements or method steps.

[0033] The Dendrobium huoshanense of this invention was purchased from Huoshan Tianxia Zeyu Biotechnology Development Co., Ltd.; the Lactobacillus reuteri strain was purchased from DSMZ - German National Culture Collection Center.

[0034] Example 1: Preparation of Dendrobium huoshanense alcohol extract (DHEE)

[0035] 100g of dried Dendrobium huoshanense was weighed, pulverized using a pulverizer, and passed through a 24-mesh sieve to obtain coarse Dendrobium huoshanense powder. The coarse powder was placed in an extraction tank, and 70% ethanol solution was added at a material-to-liquid ratio of 1g:20mL. The mixture was refluxed at 80℃ for 2 hours, and the filtrate was collected by suction filtration. The residue was extracted three times using the same method, and the filtrates were combined. The filtrate was placed in a rotary evaporator and concentrated under reduced pressure at 60℃ to a paste-like consistency, yielding a 4g / kg Dendrobium huoshanense ethanol extract.

[0036] Example 2: Preparation of Lactobacillus reuteri bacterial culture

[0037] Lactobacillus reuteri was inoculated into MRS liquid medium and activated in a 37°C incubator for 24 h. The activated bacterial culture was then transferred to fresh MRS liquid medium at a 5% inoculation rate and cultured at 37°C and 200 rpm for 12 h. The cultured bacterial culture was centrifuged at 4°C and 3000 rpm for 10 min, and the bacterial pellet was collected. The bacterial cells were washed twice with sterile physiological saline, and then resuspended in sterile PBS to achieve a bacterial concentration of 1 × 10⁻⁶. 9 CFU / mL.

[0038] Example 3

[0039] With the aim of investigating the synergistic effect of Dendrobium huoshanense ethanol extract and Lactobacillus reuteri in increasing GABA levels, activated Lactobacillus reuteri from Example 2 was used, and the concentration was adjusted to 1×10⁻⁶. 9 Four groups were established using CFU / mL: Lactobacillus reuteri group (inoculation only), 7 mg / mL, 14 mg / mL, and 28 mg / mL Dendrobium huoshanense alcohol extract group (inoculation + corresponding concentration of Dendrobium huoshanense alcohol extract). All groups were cultured on MRS medium at 37℃ for 24 h. After the culture was completed, the absorbance of the bacterial solution was measured. The supernatant was collected by centrifugation at 3000 rpm for 10 min according to the GABA kit instructions. A standard curve was plotted and the absorbance of the samples was detected to calculate the GABA content.

[0040] The results are as follows Figure 1 As shown, by Figure 1 As shown in section A, there was no significant difference in the absorbance (ABS) of *Lactobacillus reuteri* after treatment with different concentrations of DHEE extract, indicating that different concentrations of extract had no significant effect on the growth of *Lactobacillus reuteri*; however, from...Figure 1 As shown in FIG. 10B, the GABA level of L. reuteri was changed after the treatment of DHEE extract. Compared with the L. reuteri group without DHEE, the GABA content of each treatment group increased with the increase of DHEE concentration (7-28 mg / mL), and there was a significant difference between the 28 mg / mL DHEE extract group and the L. reuteri group (P < 0.05), indicating that different concentrations of DHEE extract can significantly promote the GABA metabolism and synthesis of L. reuteri, i.e., have a significant effect on the GABA metabolism and synthesis of L. reuteri.

[0041] Example 4 Animal experiment for treating intestinal inflammation and depression in combination

[0042] The D. altissima ethanol extract prepared in Example 1 and the L. reuteri bacterial solution prepared in Example 2 were used for animal experiments.

[0043] 1. Establishment of experimental model

[0044] Seventy SPF male C57BL / 6 mice (weight 18 g ± 2 g, 6-8 weeks old) were randomly divided into 7 groups, 10 mice in each group, and each group was treated as follows:

[0045] Control group (Control): no antibiotic treatment + no modeling + physiological saline gavage;

[0046] Control + antibiotic group (Control + Abx): antibiotic treatment + no modeling + physiological saline gavage;

[0047] Model group (Model): no antibiotic treatment + modeling + physiological saline gavage;

[0048] Model + antibiotic group (Model + Abx): antibiotic treatment + modeling + physiological saline gavage;

[0049] L. reuteri + antibiotic group (L. reuteri + Abx): antibiotic treatment + modeling + 200 μL of L. reuteri bacterial solution (concentration 1 × 10 9 CFU / mL per mouse gavage;

[0050] D. altissima ethanol extract + antibiotic group (DHEE + Abx): antibiotic treatment + modeling + D. altissima ethanol extract gavage at a dose of 4 g / kg;

[0051] D. altissima ethanol extract + L. reuteri + antibiotic group (DHEE + L. reuteri + Abx): antibiotic treatment + modeling + D. altissima ethanol extract gavage at a dose of 2 g / kg + 200 μL of L. reuteri bacterial solution (concentration 5 × 10 8 CFU / mL);

[0052] Antibiotic treatment: The mice were treated with a quadruple antibiotic (vancomycin 0.5 g / L, metronidazole 1 g / L, ampicillin 1 g / L, neomycin 1 g / L) for 14 days, i.e. the mice were treated with "germ-free".

[0053] Modeling: The mice were modeled by intraperitoneal injection of LPS at a dose of 1 mg / mL to establish an intestinal inflammation and depression model.

[0054] Drug administration: After 7 days of modeling, the mice were administered drugs once a day by gavage, continuously for 14 days.

[0055] 2. Intestinal inflammation factor detection and experimental results

[0056] After the last gavage, the mice were sacrificed, and serum and colon tissues were taken. The expression levels of IL-1β, TNF-α and IL-6 were detected by enzyme-linked immunosorbent assay (ELISA).

[0057] Figure 2 Table A-C presents the detection results of the expression levels of inflammatory factors (IL-1β, TNF-α and IL-6) in the serum of mice under different treatment methods, to explore the regulation effect of DHEE, L. reuteri and their combined use on the serum inflammatory factors of LPS-induced intestinal inflammation model mice. By comparing the expression differences of inflammatory factors in the blank group, the model group and the different intervention groups, the anti-inflammatory effects of each administration method were discussed. It can be seen that overall, LPS modeling can induce the up-regulation of pro-inflammatory factors such as IL-1β, TNF-α and IL-6 in the serum of mice (P<0.05), simulating the intestinal inflammation state; the combined use of DHEE and L. reuteri shows better effect in down-regulating the expression of serum pro-inflammatory factors (IL-1β, TNF-α and IL-6) compared with single drug intervention (P<0.05), indicating that the synergistic effect of the two may regulate the inflammatory factor pathway to improve LPS-induced intestinal inflammation, providing potential microbial-traditional Chinese medicine combined strategy basis for the intervention of inflammation-related diseases.

[0058] Figure 3 The detection results of the expression levels of inflammatory factors in the colon tissues of mice in different experimental groups are shown in Table A-C, covering IL-1β, TNF-α and IL-6, which are used to explore the influence of experimental intervention on the inflammatory microenvironment of colon tissues. The results show that LPS modeling can induce the abnormal increase of pro-inflammatory factors such as IL-1β, TNF-α and IL-6 in the colon tissues of mice (P<0.05), successfully constructing an intestinal inflammation model; the combined intervention of DHEE and L. reuteri shows better effect in down-regulating the expression of pro-inflammatory factors in the colon tissues compared with single drug (P<0.05), indicating that the synergistic effect of the two can regulate the local inflammatory factors to improve the intestinal inflammation microenvironment.

[0059] In summary, compared with the model group, the DHEE+L.reuteri group significantly reduced the expression levels of IL-1β, TNF-α and IL-6 in the serum and colon tissue of mice (P<0.05), and the effect was significantly better than that of the DHEE and L.reuteri groups alone.

[0060] 3. Depression-related index detection and experimental results

[0061] After the last gavage, the following tests were performed:

[0062] Behavioral test: Open field test, elevated plus maze test, forced swimming test, and tail suspension test were used to verify the depression of mice. The results of the behavioral test are shown in Tables 1-4.

[0063] The open field test was used to detect the effect of different treatments on the activity and anxiety state of LPS-induced depression model mice, and the results are shown in Table 1. The results show that the total distance of the blank group mice is 13072.86±2585.10 mm, the central area distance is 2424.53±774.63 mm, and the central area time is 22.48±7.31 s, indicating that the normal mice have strong activity and high willingness to explore the central open area; the total distance of the blank+antibiotic group is 8980.39±857.90 mm, the central area distance is 2174.47±2170.51 mm, and the central area time is 14.56±2.94 s, which are lower than those of the blank group (P<0.01), indicating that antibiotic treatment may inhibit the activity of mice and increase their anxiety tendency. After LPS modeling, the total distance of the model group decreased to 6801.86±1432.37 mm, the central area distance was 687.81±133.86 mm, and the central area time was only 6.56±3.94 s, which was significantly lower than that of the blank group (P<0.001), confirming that the activity of the depression model mice was weakened and they avoided the central open area; the above-mentioned indexes of the model+antibiotic group were further reduced, and the central area time of some mice was less than 2 s, indicating that the combination of the two exacerbated the depression-like behavior of mice. After intervention, the total distance, central area distance, and central area time of the L.reuteri and DHEE groups were significantly increased compared with the model+antibiotic group (P<0.05), and the combined intervention of DHEE and L.reuteri had the best effect (P<0.001), with a total distance of 12308.70±446.24 mm, a central area distance of 2427.12±643.37 mm, and a central area time of 25.70±6.38 s, which was close to the blank group level and significantly better than the single drug intervention, indicating that the combination of the two can effectively improve the activity inhibition and anxiety state of LPS+antibiotic-induced mice, and the mechanism may be related to the regulation of the gut microbiota metabolism-neuroimmune axis.

[0064] Table 1 Values of total distance, central area distance, and central area time of mice in the open field test

[0065]

[0066] The effect of different treatments on the behavior of LPS-induced depression model mice was explored by elevated plus maze experiment, and the results are shown in Table 2. The results show that the open arm residence time of the blank group mice is 175.36±13.39s, the entering frequency is 16.67±2.16 times, the residence and entering frequency percentages are 73.06%±5.38% and 52.11%±7.29% respectively, indicating that the normal mice have strong open space exploration ability; the above indexes of the blank+antibiotic group are significantly reduced (P<0.01), suggesting that antibiotic treatment can inhibit the exploration behavior of mice. After LPS modeling, the open arm residence time of the model group is reduced to 59.03±7.72s, the entering frequency is 2.00±0.63 times, and the percentage indexes are also significantly decreased, confirming that the depression model is successfully constructed (P<0.001); the behavior of mice in the model+antibiotic group is further abnormal, and the entering frequency of some individuals in the open arm is 0 times. After intervention, the open arm residence time and entering frequency of mice in the Roy's lactobacillus and Dendrobium huoshanense alcohol extract groups are significantly increased compared with the model+antibiotic group (P<0.01), and the effect of the Dendrobium huoshanense alcohol extract+Roy's lactobacillus combined intervention group is better (P<0.001), and the indexes of the group are close to or exceed the blank group level, and are significantly better than single drug intervention, indicating that the combination of the two has a synergistic improvement effect on the depression-like behavior of LPS+antibiotic-induced mice.

[0067] Table 2 The values of open arm entering frequency and open arm residence time of mice in elevated plus maze experiment

[0068]

[0069]

[0070] The influence of different treatments on the immobility time of LPS-induced depression model mice was detected by tail suspension test, and the results are shown in Table 3. The results show that the immobility time of blank group mice is 66.17±14.36s, indicating that the normal mice have strong struggle resistance in the tail suspension test and no obvious depression-like behavior; the immobility time of blank+antibiotic group is 94.67±19.23s, which is significantly longer than that of blank group, suggesting that antibiotic treatment may induce depression-like behavior in mice by interfering with intestinal flora or neurotransmitter metabolism. After LPS modeling, the immobility time of model group increased to 121.00±26.43s, which was significantly increased compared with blank group (P<0.001), confirming the success of the depression model, and the mice showed typical passive hopeless state; the immobility time of model+antibiotic group was further prolonged to 129.00±22.05s, indicating that the combined treatment of antibiotics and LPS can exacerbate the degree of depression in mice, which may be related to the destruction of the bidirectional regulation of the gut-brain axis. After intervention, the immobility time of Lactobacillus reuteri+antibiotic group and Dendrobium huoshanense alcohol extract+antibiotic group was significantly reduced compared with model+antibiotic group (P<0.01), and the immobility time of Dendrobium huoshanense alcohol extract+Lactobacillus reuteri+antibiotic group was 82.83±14.99s, which was close to the level of blank group and significantly better than single drug intervention (P<0.01), indicating that the combination of the two can effectively improve the depression-like behavior of mice by regulating the balance of intestinal flora, inhibiting neuroinflammation and other pathways, providing a microbial-traditional Chinese medicine combined intervention scheme for the prevention and treatment of depression.

[0071] Table 3 Immobility time of mice in tail suspension test

[0072]

[0073] Table 4 Immobility time of mice in forced swimming test

[0074]

[0075] Table 4 presents the behavioral performance of mice in the forced swimming test in different experimental treatment groups, specifically recording the immobility time of each group of animals (n = 6 / group) in the last 4 minutes of the test period. The data shows that the average immobility time of the model group animals is 137.83 ± 18.57 s, showing a significant prolongation of immobility time (P < 0.001), and the blank group (66.0 ± 9.8 s) shows typical depressive behavior. It is worth noting that antibiotics treatment caused an increase in immobility time to 112.2 ± 29.3 s (P < 0.01) in the blank group, while in the model group it further exacerbated depressive behavior. Among the two interventions, the Dendrobium huoshanense ethanol extract + antibiotic group showed a more significant reduction in immobility time (mean 100.50 ± 26.22 s) (P < 0.05), and the combined intervention of Dendrobium huoshanense ethanol extract and Lactobacillus reuteri showed a more obvious modulation trend (mean 92.2 ± 12.5 s) (P < 0.001), indicating that the combination of the two can improve the depressive mood of mice by regulating the gut microbiota, providing experimental evidence for the development of "microbial-traditional Chinese medicine" composite preparations.

[0076] In summary, compared with the model group, the combination of DHEE + L. reuteri significantly increased the activity time of mice in the central area of the open field, improved the exploration of mice in the open arms of the elevated plus maze, and shortened the immobility time of mice in the forced swimming and tail suspension tests, with a more obvious effect than the experimental groups using DHEE and L. reuteri alone.

[0077] 4. Microscopic sectioning and observation

[0078] After the last gavage, the mice were sacrificed, and the colon tissue was taken for HE staining and AB-APS staining, respectively, and observed under a microscope. The results are shown in Figures 4-5 .

[0079] Figure 4HE staining was performed on the colon tissues of LPS-induced mice to analyze the effects of different interventions on the structure of the colon. The results showed that the mucosal epithelium of the colon tissue of the blank group mice was complete, the glands were arranged regularly, the structure of each layer of the intestinal wall was clear, and the normal tissue architecture in the physiological state was presented. The colon of the blank + antibiotic group did not show severe damage as the model group, but compared with the blank group, the mucosal epithelium continuity had slight abnormalities, suggesting that antibiotics or the normal intestinal microecology and epithelial homeostasis were potentially interfered. The model group showed obvious pathological changes due to LPS stimulation, including mucosal epithelial damage, gland disorder and deformation, infiltration of inflammatory cells in the lamina propria, and destruction of the structural integrity of the intestinal wall, verifying the success of the modeling. The model + antibiotic group further aggravated the intestinal inflammation of the mice. Lactobacillus reuteri + antibiotic and Dendrobium huoshanense ethanol extract + antibiotic groups had certain repair function on the intestine, the mucosal epithelium integrity was improved, the gland arrangement was gradually regular, and the infiltration of inflammatory cells was reduced. The Dendrobium huoshanense ethanol extract + Lactobacillus reuteri + antibiotic group had a colon tissue morphology closer to the blank group, with continuous mucosal epithelium and clear gland structure, indicating that Lactobacillus reuteri and Dendrobium huoshanense ethanol extract synergistic treatment could improve the LPS-induced colon tissue damage by regulating mucosal epithelial repair, inhibiting excessive inflammatory response, and remodeling gland structure.

[0080] In summary, LPS successfully induced pathological damage to the colon tissue of mice, and simple probiotics or drug intervention had limitations in colon repair. The strategy of using both could play a role in protecting the morphology of the colon tissue and maintaining the structural integrity, providing morphological evidence and potential strategy direction for the intervention of intestinal inflammatory diseases.

[0081] Figure 5The experiment shown utilized AB-APS staining to observe the distribution and morphology of acidic glycoproteins such as mucin in colonic tissue, exploring the effects of different interventions (LPS, LPS combined with antibiotics, etc.) on the colonic mucosal barrier. Morphological results showed that in the control group, colonic glands were regularly arranged, acidic glycoproteins stained evenly, and the mucosal structure was normal. In the model group, due to LPS induction, glands were disordered, acidic glycoproteins were unevenly distributed, and the mucosal barrier was significantly damaged. Antibiotic intervention alone exacerbated mucosal damage in the model group, indicating that antibiotics have a negative effect on colonic mucosal repair under pathological conditions. However, each treatment group showed repair potential: the Lactobacillus reuteri + antibiotic group showed a gradual improvement in gland arrangement and a high degree of recovery in acidic glycoprotein staining. Among them, the Dendrobium officinale ethanol extract + Lactobacillus reuteri + antibiotic group had a mucosal structure closer to the control group, indicating that probiotics and traditional Chinese medicine extracts can synergistically repair LPS-induced colonic mucosal damage by regulating mucosal glycoprotein secretion and remodeling glandular structure. The above experimental results indicate that LPS modeling successfully induced abnormal colonic mucosal morphology and acidic glycoprotein distribution. Simple antibiotic intervention is not conducive to mucosal repair, while the combined administration of probiotics (such as Lactobacillus reuteri) and natural products (Dendrobium huoshanense alcohol extract) can play a role in mucosal barrier repair by protecting the structure of colonic mucosal glycoproteins and restoring glandular morphology, providing morphological basis for mucosal protection strategies under intestinal pathological conditions.

[0082] 5. Detection of GABA levels in mice

[0083] Serum and colon tissue were collected, and GABA levels were measured according to the GABA kit instructions.

[0084] The results are as follows Figure 6 As shown, by Figure 6 The serum GABA levels in the Model+Abx group were significantly lower than those in the Control+Abx group (P<0.05). However, after administration of L. reuteri and DHEE alone, the serum GABA levels in mice significantly increased (P<0.01), and the combined administration of L. reuteri and DHEE showed the best effect. Figure 6 The results of fecal GABA level analysis showed that the GABA level in the Model+Abx group was significantly lower than that in the Control+Abx group (P<0.01). Among the intervention groups, treatments such as L. reuteri, DHEE, and DHEE+L. reuteri could increase fecal GABA levels to varying degrees, with the combined treatment group showing the most significant difference (P<0.05).

[0085] In summary, the expression of GABA in the serum and feces of the depression model mice was significantly down-regulated, while the intervention methods such as L. reuteri and DHEE could affect the expression of GABA, restore the level of GABA to a certain extent, and play a better therapeutic effect after the combined use of D. formosanum and L. reuteri, providing a basis for explaining the occurrence and intervention mechanism of depression.

[0086] The above-described embodiments are merely preferred modes of the present application and are not intended to limit the scope of the present application. Various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art without departing from the design spirit of the present application shall fall within the scope of protection of the present application as defined by the claims.

Claims

1. A pharmaceutical composition, characterized by, The Dendrobium huoshanense extract and the Lactobacillus reuteri bacterial solution.

2. The composition of claim 1, wherein The preparation method of the Dendrobium huoshanense extract comprises the following steps: The Dendrobium huoshanense is crushed, mixed with a 70% ethanol solution, extracted by reflux for 2 hours, and filtered to collect the filtrate and the residue; The residue is extracted repeatedly for 3 times, the filtrate is combined, and concentrated under reduced pressure to a paste, so as to obtain the Dendrobium huoshanense extract.

3. The use of the pharmaceutical composition of claim 1 or 2 in the preparation of a drug for treating intestinal inflammatory diseases.

4. Use according to claim 3, wherein the compound is ###0002### The intestinal inflammatory diseases are irritable bowel syndrome, Crohn's disease or inflammatory bowel disease.

5. A medicament for the treatment of an inflammatory disease of the intestinal tract, characterized in that, The pharmaceutical composition of claim 1 or 2 is used as the main effective component.

6. The medicament according to claim 5, wherein The pharmaceutical composition further comprises pharmaceutically acceptable adjuvants.

7. The use of the pharmaceutical composition of claim 1 or 2 in the preparation of a drug for treating diseases related to the intestinal-brain axis.

8. Use according to claim 7, wherein the compound is ###0002### The diseases related to the intestinal-brain axis are Alzheimer's disease or depression.

9. A medicament for the preparation of a medicament for the treatment of a gut-brain axis related disease, characterized in that, The pharmaceutical composition of claim 1 or 2 is used as the main effective component.

10. The medicament according to claim 9, wherein The pharmaceutical composition further comprises pharmaceutically acceptable adjuvants.

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