A molecular ecological liquid extract of *Smilax china* obtained through specific fermentation, its preparation method and application

Abstract

This invention provides a *Tai Sui* molecular ecological fluid extract obtained through specific fermentation, its preparation method, and its applications, belonging to the field of microbial fermentation technology. The invention uses a modified fermentation medium, introduces 5-8% CO2, maintains a dissolved oxygen concentration of 20-30%, and ferments at 30-32℃ for 90-180 days. After purification, the *Tai Sui* molecular ecological fluid extract is obtained. The *Tai Sui* molecular ecological fluid prepared using the method provided by this invention significantly increases the content of active ingredients that alleviate symptoms of chronic obstructive pulmonary disease (COPD). Furthermore, this invention verifies through in vitro cell experiments that the extract of this *Tai Sui* molecular ecological fluid has an inhibitory effect on the A549 cell line. Further animal experiments demonstrate that the extract can regulate the expression level of pro-inflammatory cytokines, thereby inhibiting lung inflammation, alleviating pathological damage to lung tissue, and improving local lung abnormalities.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, and in particular to a Tai Sui molecular ecological liquid extract obtained through specific fermentation, its preparation method, and its application. Background Technology

[0002] Tai Sui is an ancient symbiotic microorganism, with core groups including Proteobacteria, Bradyrhizobium, and Sphingomonas. Studies have shown that Tai Sui contains various bioactive components such as nucleic acids, polysaccharides, and trace elements, possessing certain medicinal value. For example, the nucleic acids and their degradation products, nucleotides, contained in Tai Sui have therapeutic effects on cognitive disorders, while polysaccharides have anti-tumor and immune-enhancing effects.

[0003] Chinese patent application CN111249317A discloses a method for preparing a fermented liquid of *Tai Sui* (a type of fungus), the fermented liquid itself, and its applications. This technical solution uses kiwi, plum, strawberry, and *Ganoderma lucidum* juice, adds honey, mixes it with *Tai Sui*, ferments it, removes the *Tai Sui*, and filters it to obtain the fermented liquid. This invention uses a fermented liquid that increases the content of active ingredients in the fermented liquid, can prolong the lifespan of *C. elegans*, and has anti-wrinkle functions.

[0004] Currently, the development and application of targeted fermentation technology and active ingredients of *Tai Sui* (a type of fungus) remain unexplored, and its bioactivity in combating chronic obstructive pulmonary disease (COPD) has not yet been fully explored. COPD is a preventable and treatable chronic respiratory disease characterized by persistent airflow limitation, which is often progressive and closely related to enhanced chronic inflammatory responses of the airways and lung tissue to harmful gases or particles such as tobacco smoke. Its core pathological changes involve chronic inflammation of the airways, lung parenchyma, and pulmonary vessels, ultimately leading to airway narrowing, lung tissue destruction, and impaired lung ventilation and gas exchange. Therefore, there is an urgent need to develop a targeted fermentation process for *Tai Sui* to obtain novel, naturally derived, and highly safe drugs for improving and prognosing COPD. Summary of the Invention

[0005] This invention aims to provide a *Tai Sui* molecular ecological fluid extract obtained through specific fermentation, its preparation method, and its applications. The *Tai Sui* molecular ecological fluid of this invention is fermented using a modified fermentation medium under specific fermentation conditions, and purified to obtain the *Tai Sui* molecular ecological fluid extract. This extract is an ester-based component. When formulated with pharmaceutically acceptable excipients into tablets, capsules, injections, or oral liquids, it can be used to inhibit cellular inflammatory responses in chronic obstructive pulmonary disease and improve lung damage.

[0006] To achieve the above objectives, in a first aspect, the present invention provides a method for preparing a *Tai Sui* molecular ecological liquid extract obtained through specific fermentation, comprising:

[0007] S1. Inoculate the *Tai Sui* into the fermentation medium, introduce a mixture of CO2 and air, and ferment in a shaker in the dark to obtain *Tai Sui* molecular ecological liquid.

[0008] S2. The Tai Sui molecular ecological liquid is centrifuged, filtered, and the filtrate is adsorbed by macroporous resin, eluted with ethanol, concentrated, and dried to obtain Tai Sui molecular ecological liquid extract.

[0009] Preferably, in step S1, the volume concentration of CO2 in the mixed gas is 5-8%, the ventilation rate is 0.3-0.5 vvm, and the dissolved oxygen concentration in the environment is maintained at 20-30%.

[0010] Preferably, in step S1, the amount of *Tai Sui* inoculated into the fermentation medium is 5-10% (w / v), the fermentation temperature is 30-32℃, the fermentation time is 90-180 days, and the shaking speed is 50-80 rpm.

[0011] Preferably, in step S1, the fermentation medium is a modified fermentation medium comprising: maca 1g / L, ginseng peptide powder 1g / L, cordyceps militaris 1g / L, wolfberry 5g / L, jujube 10g / L, gorgon fruit 3g / L, bergamot 3g / L, honeysuckle 3g / L, sophora japonica 3g / L, and dandelion 3g / L, with sterile water added to a volume of 1L, and sterilized at 121℃ for 15min.

[0012] Preferably, in step S2, the centrifugation speed is 8000~10000 rpm, the centrifugation temperature is 20~25℃, and the centrifugation time is 10~15 min.

[0013] Preferably, in step S2, the filtration uses a filter membrane with a diameter of 0.22 μm.

[0014] Preferably, in step S2, the macroporous resin is AB-8 type macroporous resin.

[0015] Preferably, in step S2, the macroporous resin adsorption and ethanol elution steps are as follows: the filtrate is loaded onto a macroporous resin column with a diameter-to-height ratio of 1:8 and a filtrate flow rate of 1 BV / h. After adsorption saturation, impurities are eluted with 3 column volumes of 30 wt.% ethanol, and the active ingredient is eluted with 5 column volumes of 70 wt.% ethanol. The eluent is then collected.

[0016] Preferably, in step S2, the concentration temperature is 40~45℃, the vacuum degree is -0.085~0.095MPa, and the volume is concentrated to 1 / 10 of the original volume.

[0017] Preferably, in step S2, the drying temperature is -80°C and the drying time is 10~12 hours.

[0018] Secondly, the present invention provides a Tai Sui molecular ecological liquid extract obtained by the preparation method described above through specific fermentation.

[0019] Thirdly, the present invention provides the application of a Tai Sui molecular ecological liquid extract obtained through specific fermentation in the preparation of a drug for treating chronic obstructive pulmonary disease.

[0020] Compared with the prior art, the beneficial effects of the present invention are reflected in:

[0021] This invention employs a specific fermentation process in a modified fermentation medium to directionally activate the metabolic pathways of the *Tai Sui* symbiotic microbiota, particularly the secondary metabolism of core groups such as *Rhizobium* and *Sphingomonas*. This significantly increases the yield of anti-chronic obstructive pulmonary disease (COPD) active ingredients in the *Tai Sui* molecular ecological solution. Experiments have verified a large number of significantly different metabolites between the *Tai Sui* molecular ecological solution and *Tai Sui* water, fully demonstrating that fermentation is a key factor in reshaping the metabolite profile of *Tai Sui*. This reveals multi-dimensional and significant differences in metabolic pathways between the *Tai Sui* molecular ecological solution and *Tai Sui* water. These pathway alterations not only explain the differences in the post-fermentation metabolite profiles but also provide pathway-level mechanistic support for the bioactivity of the *Tai Sui* molecular ecological solution. In vitro cell experiments have demonstrated that the extract of the *Tai Sui* molecular ecological solution has a good inhibitory effect on the A549 lung cancer cell line. The 100 μg / mL extract of the *Tai Sui* molecular ecological solution showed an inhibition rate of 76.2% on A549 cells, with an IC50 of 38.5 μg / mL. Further animal experiments have demonstrated that the extract of *Smilax china* molecular ecological fluid can regulate the expression levels of pro-inflammatory cytokines in serum and lung tissue, thereby reducing inflammatory response, regulating lung tissue condition, improving local lung abnormalities, and ultimately promoting functional recovery of lung lesion areas. Attached Figure Description

[0022] Figure 1 The image shows the anion mode base peak chromatogram of the Tai Sui molecular ecological liquid prepared in Example 3.

[0023] Figure 2 The image shows the cation mode base peak chromatogram of the Tai Sui molecular ecological liquid prepared in Example 3.

[0024] Figure 3 The bar graph shows the difference in the amount of metabolites between the Tai Sui molecular ecological liquid prepared in Example 3 and the Tai Sui water prepared in the comparative example.

[0025] Figure 4The experimental volcano diagram shows the difference in the amount of metabolites between the Tai Sui molecular ecological liquid prepared in Example 3 and the Tai Sui water prepared in the comparative example.

[0026] Figure 5 This is a graph showing the multivariate difference analysis data of the Tai Sui molecular ecological liquid prepared in Example 3 and the Tai Sui water prepared in the comparative example.

[0027] Figure 6 The partial least squares discriminant analysis data are shown for the Tai Sui molecular ecological liquid prepared in Example 3 and the Tai Sui water prepared in the comparative example.

[0028] Figure 7 The graph shows the orthogonal partial least squares discriminant analysis data of the Tai Sui molecular ecological liquid prepared in Example 3 and the Tai Sui water prepared in the comparative example.

[0029] Figure 8 The graph shows the metabolic pathway enrichment analysis data of the Tai Sui molecular ecological liquid prepared in Example 3 and the Tai Sui water prepared in the comparative example.

[0030] Figure 9 The image shows the in vitro cell experiment results of the Tai Sui molecular ecological liquid extract prepared in Example 3.

[0031] Figure 10 Images of H&E staining in the lungs of CON group mice.

[0032] Figure 11 This is a magnified view of a portion of the lungs of CON group mice stained with H&E.

[0033] Figure 12 Images of H&E staining in the lungs of COPD group mice.

[0034] Figure 13 This is a magnified view of a portion of the H&E staining image of the lungs of COPD group mice.

[0035] Figure 14 Images of H&E staining in the lungs of mice in the drug-treated group.

[0036] Figure 15 This is a magnified view of a portion of the H&E staining image of the lungs of mice in the drug-treated group.

[0037] Figure 16 The expression level of IL-6 in the serum of mice in the CON group, COPD group and drug treatment group is shown.

[0038] Figure 17 The expression levels of TNF-α in the serum of mice in the CON group, COPD group, and drug-treated group are shown.

[0039] Figure 18 The expression level of IL-6 in the lung tissue of mice in the CON group, COPD group and drug treatment group.

[0040] Figure 19 The expression level of TNF-α in the lung tissue of mice in the CON group, COPD group and drug-treated group is shown. Detailed Implementation

[0041] The following embodiments are only used to illustrate the technical solutions of the present invention more clearly, and should not be used to limit the scope of protection of the present invention.

[0042] The *Tai Sui* samples used in the examples (containing a relative abundance of Proteobacteria >60%, with *Rhizobium* and *Sphingomonas* as the dominant genera) were collected from Northeast China and stored at 4°C.

[0043] The culture medium used in the example was: maca 1g / L, ginseng peptide powder 1g / L, cordyceps militaris 1g / L, wolfberry 5g / L, red dates 10g / L, gorgon fruit 3g / L, citron 3g / L, honeysuckle 3g / L, sophora japonica flower 3g / L, and dandelion 3g / L, with sterile water added to a volume of 1L, and sterilized at 121℃ for 15min.

[0044] The main compounds used in the examples and comparative examples were all commercially available products and were not subjected to any further purification treatment.

[0045] Example 1

[0046] A molecular ecological liquid extract of *Ulva prolifera* obtained through specific fermentation, the preparation method of which includes:

[0047] S1. Wash 100g of *Smilax china* three times with sterile PBS (phosphate buffer). Inoculate 25g of *Smilax china* into 500mL of fermentation medium, with an inoculum of 5% (w / v). Purge with a mixture of CO2 and air at a volume concentration of 5% at a purge rate of 0.3 vvm, maintaining a dissolved oxygen concentration of 20%. Ferment at 30℃ on a shaker at 50 rpm in the dark for 180 days to obtain *Smilax china* molecular ecological liquid.

[0048] S2. The *Saccharum sibiricum* molecular ecological solution was centrifuged at 8000 rpm for 15 min at 20℃, filtered through a 0.22 μm filter membrane, and the filtrate was loaded onto AB-8 macroporous resin at a flow rate of 1 BV / h. After adsorption saturation, impurities were eluted with 3 column volumes of 30 wt.% ethanol, followed by 5 column volumes of 70 wt.% ethanol to elute the active ingredient. The eluent containing the active ingredient was collected, concentrated to 1 / 10 of its original volume under vacuum of -0.085 MPa at 40℃, and dried at -80℃ for 10 h to obtain the *Saccharum sibiricum* molecular ecological solution extract.

[0049] Example 2

[0050] A molecular ecological liquid extract of *Ulva prolifera* obtained through specific fermentation, the preparation method of which includes:

[0051] S1. Rinse 100g of *Tai Sui* three times with sterile PBS. Inoculate 40g of *Tai Sui* into 500mL of fermentation medium, with an inoculum size of 8% (w / v). Purge with a mixture of CO2 and air at a volume concentration of 5% at a purge rate of 0.4 vvm, maintaining a dissolved oxygen concentration of 25%. Ferment at 30℃ on a shaker at 60 rpm in the dark for 120 days to obtain *Tai Sui* molecular ecological liquid.

[0052] S2. The *Saccharum sibiricum* molecular ecological solution was centrifuged at 9000 rpm for 15 min at 25℃, filtered through a 0.22 μm filter membrane, and the filtrate was loaded onto AB-8 macroporous resin at a flow rate of 1 BV / h. After adsorption saturation, impurities were eluted with 3 column volumes of 30 wt.% ethanol, followed by 5 column volumes of 70 wt.% ethanol to elute the active ingredient. The eluent of the active ingredient was collected, concentrated to 1 / 10 of its original volume under vacuum of -0.09 MPa at 45℃, and dried at -80℃ for 12 h to obtain the *Saccharum sibiricum* molecular ecological solution extract.

[0053] Example 3

[0054] A molecular ecological liquid extract of *Ulva prolifera* obtained through specific fermentation, the preparation method of which includes:

[0055] S1. Rinse 100g of *Smilax china* three times with sterile PBS. Inoculate 50g of *Smilax china* into 500mL of fermentation medium, with an inoculum of 10% (w / v). Introduce a mixture of CO2 and air with a CO2 volume concentration of 8% at a ventilation rate of 0.5 vvm, maintaining a dissolved oxygen concentration of 30%. Ferment at 30℃ on a shaker at 80 rpm in the dark for 90 days to obtain *Smilax china* molecular ecological liquid.

[0056] S2. The *Saccharum sibiricum* molecular ecological solution was centrifuged at 10,000 rpm for 10 min at 20℃, filtered through a 0.22 μm filter membrane, and the filtrate was loaded onto AB-8 macroporous resin at a flow rate of 1 BV / h. After adsorption saturation, impurities were eluted with 3 column volumes of 30 wt.% ethanol, followed by 5 column volumes of 70 wt.% ethanol to elute the active ingredient. The eluent containing the active ingredient was collected, concentrated to 1 / 10 of its original volume under vacuum of -0.095 MPa at 45℃, and dried at -80℃ for 10 h to obtain the *Saccharum sibiricum* molecular ecological solution extract.

[0057] Comparative Example 1

[0058] A method for preparing a type of Tai Sui water includes: rinsing 100g of Tai Sui three times with sterile PBS, inoculating 100g of Tai Sui into 1L of ultrapure water, and then sealing and culturing at 25°C for 180 days.

[0059] Analysis of the differences in the components of the *Tai Sui* molecular ecological liquid obtained in S1 in Example 3 and the *Tai Sui* water prepared in Comparative Example 1:

[0060] (1) Base Peak Chromatogram: The base peak chromatogram (BPC) in anion mode (NEG) and cation mode (POS) was plotted with the response value of the ion with the strongest signal intensity in the liquid chromatography-mass spectrometry (LC-MS) at each time point as the ordinate and the retention time as the abscissa. The results are as follows: Figure 1 and Figure 2 As shown.

[0061] (2) A difference analysis was conducted on Tai Sui molecular ecological liquid and Tai Sui water. Screening was performed based on three indicators: fold change, multiple test significance level, and VIP value of multivariate analysis. The results are as follows: Figure 3 and Figure 4 As shown.

[0062] Figure 3 The bar chart shows the differences in the number of metabolites between the Tai Sui molecular ecological liquid and Tai Sui water. 520 metabolites were upregulated, and 378 were downregulated. The significant difference in metabolites between the Tai Sui molecular ecological liquid and Tai Sui water demonstrates that the fermentation process drastically altered the metabolite composition. Furthermore, the fold change was >4, the multiple test significance level was <0.05, and the VIP value of multivariate analysis was >1, indicating clear statistical significance and biological significance, thus ruling out false positives caused by random fluctuations.

[0063] Figure 4 Volcano plots showing the differences in the number of metabolites between Tai Sui molecular ecological liquid and Tai Sui water reveal a large number of significantly different metabolites between the two, and most of the differential metabolites make important contributions to the intergroup differentiation (VIP≥1). The significantly upregulated core metabolites are the material basis for fermentation function, which preliminarily verifies that fermentation treatment has a fundamental change in the metabolite composition of Tai Sui.

[0064] (3) The differences between the components of the *Tai Sui* molecular ecological solution and the *Tai Sui* water were analyzed by multivariate differential analysis. A PCA model was established between the *Tai Sui* molecular ecological solution prepared in Example 3 and the *Tai Sui* water obtained in Comparative Example 1. The distribution and separation trends of the two groups of samples were observed, and the results are as follows: Figure 5 As shown.

[0065] Figure 5The medium ellipse represents the 95% confidence interval for the Tai Sui molecular ecological liquid and Tai Sui water samples. All points from both groups of samples fall within their respective 95% confidence ellipses, with no significant outliers deviating from other samples within the group. Furthermore, the Tai Sui molecular ecological liquid sample points are highly clustered in the negative PCA1 interval, with a relatively small range, indicating that the metabolite profiles of the samples within the group are very consistent and the experiment has good repeatability. The Tai Sui water sample points are relatively dispersed in the positive PCA1 interval, but are still included in the 95% confidence ellipse, indicating that there are certain differences within the group, but the overall group still has distinguishable population characteristics, indicating that the detection process of the experimental samples is stable and the data is reliable.

[0066] according to Figure 5 The data show that the 95% confidence ellipses of the Tai Sui molecular ecological liquid group and the Tai Sui water group are completely separated with no overlapping regions, proving that the fermentation treatment has a fundamental change in the metabolite composition of Tai Sui. Therefore, there is a very significant overall difference in the metabolite composition between Tai Sui molecular ecological liquid and Tai Sui water, and the difference is mainly driven by the first principal component PCA1.

[0067] (4) Partial least squares discriminant analysis (PLS-DA) was performed on the Tai Sui molecular ecological liquid and Tai Sui water to establish a relationship model between metabolite expression levels and sample categories. This model can be used to predict sample categories. The results are as follows: Figure 6 As shown.

[0068] Figure 6 The ellipse represents the 95% confidence interval for the Tai Sui molecular ecological liquid and Tai Sui water samples. The 95% confidence ellipses for Tai Sui molecular ecological liquid and Tai Sui water are completely separated with no overlapping areas, indicating that the PLS-DA model can significantly distinguish between Tai Sui molecular ecological liquid and Tai Sui water. This suggests that the differences in metabolite composition between the two groups of samples have a clear class distinction, and the changes in metabolite profiles caused by fermentation treatment have a predictable regularity. The sample points of Tai Sui molecular ecological liquid are highly clustered in the negative T score interval, and the ellipse range is very small, indicating that the metabolite expression patterns of the samples within the group are very consistent and the experimental repeatability is good. The sample points of Tai Sui water are relatively dispersed in the positive T score interval, but are still included in the 95% confidence ellipse, indicating that there are certain differences within the group, but the overall population still has distinguishable characteristics.

[0069] The complete separation results of PLS-DA demonstrate a strong correlation between metabolite expression levels and whether fermentation has been performed. The model can accurately predict whether a sample belongs to Tai Sui molecular ecological liquid or Tai Sui water through metabolite profiles, further verifying the substantial changes in the composition of Tai Sui metabolites caused by fermentation treatment.

[0070] (5) Orthogonal partial least squares discriminant analysis was performed on the two groups of biological samples to establish a relationship model between metabolite expression levels and sample categories, thereby enabling modeling and prediction of sample categories. Simultaneously, the projected importance (VIP) of variables was calculated to measure the ability of each metabolite to classify each group of samples, thus assisting in the screening of metabolic biomarkers. It is generally considered that a VIP > 1 indicates that the variable has a significant effect on distinguishing sample categories. The results are as follows: Figure 7 As shown.

[0071] Figure 7 The 95% confidence ellipses for the *Tai Sui* molecular ecological liquid and *Tai Sui* water were completely separated without any overlap, indicating that the OPLS-DA model can significantly distinguish between the two samples. This suggests a fundamental difference in the metabolite composition of the two groups, and that fermentation treatment has a strong specificity in altering the metabolite profile. The sample points for the *Tai Sui* molecular ecological liquid were highly clustered in the positive PC1 region, with a very small ellipse range, indicating a high degree of consistency in metabolite expression patterns within the group and excellent experimental repeatability. The sample points for the *Tai Sui* water were relatively dispersed in the negative PC1 region, but were still included in the 95% confidence ellipse, indicating some differences within the group, but the overall population still maintained distinguishable characteristics.

[0072] Figure 7 The data further validated the highly significant class differences in metabolite composition between the *Tai Sui* molecular ecological liquid and *Tai Sui* water, and these differences could be accurately distinguished using a supervised model. Combined with the results of PCA and PLS-DA analyses, this fully demonstrates that fermentation treatment is a key factor in reshaping the metabolite profile of *Tai Sui*, providing strong model support for subsequent screening of differential metabolic biomarkers and elucidation of the bioactive mechanisms of fermentation.

[0073] (6) Metabolic pathway enrichment analysis of differential metabolites in *Tai Sui* molecular ecological fluid and *Tai Sui* water based on the KEGG database can reveal metabolic pathways with significant changes, thus contributing to the interpretation of biological phenotypes. Metabolic pathways with a p-value < 0.05 were defined as those with significantly enriched differential metabolites. The results are as follows: Figure 8 As shown.

[0074] according to Figure 8 As shown, the color of the p value from blue to red represents the p value from very small (4e-04) to relatively large (0e+00, i.e. not significant). Except for carbon metabolism and biosynthesis of secondary metabolites, the p values ​​of the other pathways are all <0.05, indicating that there are statistically significant differences between these pathways in Tai Sui molecular ecological liquid and Tai Sui water.

[0075] The enrichment factor represents the number of differential metabolites annotated to the pathway. The higher the value, the higher the enrichment ratio of differential metabolites in the pathway. The enrichment factors of phenylalanine metabolism and pentose phosphate pathway are also relatively high, indicating that the proportion of differential metabolites in the pathway is large. This shows that fermentation has a significant impact on phenylalanine metabolism and pentose phosphate pathway metabolism, proving that the metabolic activities of these pathways have undergone substantial changes after fermentation.

[0076] The size of the dot represents the number of differential metabolites annotated in that pathway. The larger the dot, the more metabolites are involved in differential regulation in that pathway. The largest dot is for the biosynthesis of secondary metabolites (containing 60 differential metabolites), indicating that fermentation remodels the secondary metabolic network globally. The dots for cofactor biosynthesis and 2-oxocarboxylic acid metabolism are also relatively large, thus reflecting the rich metabolite changes in these pathways.

[0077] Figure 8 The data revealed significant multidimensional differences in metabolic pathways between Tai Sui molecular ecological liquid and Tai Sui water, with the core differences concentrated in secondary metabolism, cofactor synthesis, sugar metabolism (pentose phosphate pathway), and amino acid metabolism (phenylalanine). These changes not only explain the differences in metabolite profiles after fermentation, but also provide pathway-level mechanistic support for the bioactivity of Tai Sui molecular ecological liquid, and also confirm the key role of cofactors in maintaining the growth and function of Tai Sui.

[0078] (7) Anti-chronic obstructive pulmonary disease activity test:

[0079] In vitro cell experiments: The inhibitory effect of the *Tai Sui* molecular ecological fluid extract prepared in Example 3 on A549 lung cancer cells was detected using the CCK-8 assay. Concentration gradients of the *Tai Sui* molecular ecological fluid extract were set at 10 μg / mL, 20 μg / mL, 50 μg / mL, and 100 μg / mL. After co-incubation with A549 lung cancer cells for 48 h, the inhibition rate of cancer cells was calculated.

[0080] Animal experiments: Eighteen mice were randomly divided into three groups (n=6): normal indoor air exposure (CON group), lipopolysaccharide combined with cigarette smoke treatment (COPD group), and lipopolysaccharide combined with cigarette smoke treatment, and were treated daily with the Tai Sui molecular ecological liquid extract prepared in Example 3 (treatment group, 5 mL / kg / day).

[0081] On days 1 and 14 of the experiment, COPD mice were anesthetized with tribromoethanol and then injected with lipopolysaccharide (75 μg / mouse) via intranasal drip. CON group mice were exposed to indoor air, while other groups were exposed to cigarette smoke twice a day for 2 hours each time. Mice in the drug treatment group were given Tai Sui molecular ecological fluid extract (5 mL / kg / day) in the stomach one hour before exposure to cigarette smoke, while CON and COPD groups were given the same volume of physiological saline.

[0082] On the last day of the experiment, serum was collected from the experimental mice, and the expression of pro-inflammatory cytokines IL-6 and TNF-α in the mouse serum was detected by ELISA. The mice were sacrificed, lung tissue was collected, H&E staining was performed, and the expression of pro-inflammatory cytokines IL-6 and TNF-α in the lung tissue was detected by q-PCR.

[0083] In vitro cell experiment results as follows Figure 9 As shown, as the concentration of the *Smilax china* molecular ecological fluid extract increased from 0 to 100 μg / mL, its inhibitory effect on A549 cells showed a significant concentration-dependent increasing trend, and no plateau phase of inhibition rate was observed at higher concentrations. The half-maximal inhibitory concentration (IC50) of the *Smilax china* molecular ecological fluid extract on A549 cells was 38.5 μg / mL, indicating that only a low concentration was needed to achieve 50% inhibition of A549 cell growth. The inhibition rate of 100 μg / mL *Smilax china* molecular ecological fluid extract on A549 cells was 76.2%. At this concentration, the *Smilax china* molecular ecological fluid extract significantly blocked the proliferation process of A549 cells, leaving only a small number of cells to maintain activity, demonstrating strong in vitro anti-A549 cell proliferation activity.

[0084] In animal experiments, the H&E staining results of lung tissue from experimental mice were as follows: Figures 10-15 As shown. Figure 10 Lung tissue from CON group mice, Figure 11 for Figure 10 The magnified view of the central area shows that the membrane structure on the surface of the lung tissue is clear, covered by a smooth serous membrane, with no obvious abnormalities. Figure 12 Lung tissue from COPD group mice. Figure 13 for Figure 12 The enlarged view of the area within the black box (No. 2) shows inflammatory cell infiltration, mainly granulocytes, on the alveolar walls in the area indicated by the yellow arrow, with mild thickening of the alveolar walls and alveolar narrowing; focal infiltration of lymphocytes and granulocytes is visible in the area indicated by the brown arrow; extensive degeneration and cytoplasmic vacuolation of bronchiolar epithelial cells are observed in the area indicated by the green arrow, with rare necrosis; deep nuclear pyknosis and staining are observed in the area indicated by the blue arrow; and perivascular edema is visible in multiple areas indicated by the purple and orange arrows, with loose connective tissue arrangement and punctate infiltration of granulocytes and lymphocytes. Figure 14 Lung tissue from mice in the drug-treated group. Figure 15 for Figure 14 The enlarged view within the black box shows that the lung tissue surface is covered with a smooth serous membrane without obvious abnormalities; the alveolar walls in the area indicated by the yellow arrow show inflammatory cell infiltration, mainly granulocytes, with mild thickening of the alveolar walls and alveolar stenosis; the area indicated by the green arrow shows degeneration of bronchiolar epithelial cells, cytoplasmic vacuolation, and rare necrosis; the area indicated by the blue arrow shows nuclear pyknosis and deep staining or dissolution.

[0085] In animal experiments, the expression results of pro-inflammatory cytokines IL-6 and TNF-α in the serum of experimental mice detected by ELISA are as follows: Figure 16 and Figure 17 As shown, Figure 16 The expression level of the pro-inflammatory cytokine IL-6 in the serum of experimental mice. Figure 17 The expression level of pro-inflammatory cytokine TNF-α in the serum of experimental mice was measured. The concentrations of IL-6 and TNF-α in the serum of COPD group mice were significantly higher than those in CON group and drug-treated group. After intervention with Tai Sui molecular ecological liquid extract, the concentrations of IL-6 and TNF-α in the serum of drug-treated group mice decreased and were significantly lower than those in COPD group.

[0086] The results of q-PCR detection of the expression of pro-inflammatory cytokines IL-6 and TNF-α in lung tissue are as follows: Figure 18 and Figure 19 As shown, Figure 18 The expression level of the pro-inflammatory cytokine IL-6 in the lung tissue of experimental mice. Figure 19 The expression level of the pro-inflammatory cytokine TNF-α in the lung tissue of experimental mice was measured. The concentrations of IL-6 and TNF-α in the lung tissue of COPD group mice were significantly higher than those in CON group and drug-treated group. After intervention with Tai Sui molecular ecological fluid extract, the concentrations of IL-6 and TNF-α in the lung tissue of drug-treated group mice decreased and were significantly lower than those in COPD group.

[0087] In summary, the extract of Tai Sui molecular ecological liquid can effectively regulate the expression level of pro-inflammatory cytokines, inhibit lung inflammation, thereby alleviating pathological damage to lung tissue, regulating lung tissue condition, and improving local abnormalities in the lungs.

[0088] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of a Taizisheng molecular ecological liquid extract obtained through specific fermentation in the preparation of a drug for resisting chronic obstructive pulmonary disease, characterized in that, The preparation method of the Tai Sui molecular ecological liquid extract includes: S1. Inoculate *Tai Sui* into a fermentation medium, introduce a mixture of CO2 and air, wherein the volume concentration of CO2 in the mixture is 5-8%, the aeration rate is 0.3-0.5 vvm, the dissolved oxygen concentration in the environment is maintained at 20-30%, and fermentation is carried out in a shaker in the dark, wherein the fermentation temperature is 30-32℃, the fermentation time is 90-180 days, and *Tai Sui* molecular ecological liquid is obtained. S2. The Tai Sui molecular ecological liquid was centrifuged, filtered, and the filtrate was adsorbed by macroporous resin, eluted with ethanol, concentrated, and dried to obtain Tai Sui molecular ecological liquid extract. In step S1, the fermentation medium is a modified fermentation medium, comprising: maca 1g / L, ginseng peptide powder 1g / L, cordyceps militaris 1g / L, wolfberry 5g / L, jujube 10g / L, gorgon fruit 3g / L, bergamot 3g / L, honeysuckle 3g / L, sophora japonica 3g / L, and dandelion 3g / L, with sterile water added to a volume of 1L, and sterilized at 121℃ for 15min.

2. The application of the *Tai Sui* molecular ecological liquid extract obtained through specific fermentation according to claim 1 in the preparation of a drug for treating chronic obstructive pulmonary disease, characterized in that... In step S1, the amount of *Tai Sui* inoculated into the fermentation medium is 5-10% w / v, and the shaking speed is 50-80 rpm.

3. The application of the *Tai Sui* molecular ecological liquid extract obtained through specific fermentation according to claim 1 in the preparation of a drug for treating chronic obstructive pulmonary disease, characterized in that... In step S2, the centrifugation speed is 8000~10000 rpm, the centrifugation temperature is 20~25℃, and the centrifugation time is 10~15 min; the filtration uses a filter membrane with a diameter of 0.22 μm.

4. The application of the *Tai Sui* molecular ecological liquid extract obtained through specific fermentation according to claim 1 in the preparation of a drug for treating chronic obstructive pulmonary disease, characterized in that... In step S2, the macroporous resin is AB-8 type macroporous resin; the macroporous resin adsorption and ethanol elution steps are as follows: the filtrate is loaded onto the macroporous resin column with a diameter-to-height ratio of 1:8 and a filtrate flow rate of 1 BV / h. After adsorption saturation, impurities are eluted with 3 column volumes of 30 wt.% ethanol, and the active ingredient is eluted with 5 column volumes of 70 wt.% ethanol. The eluent is then collected.

5. The application of the *Tai Sui* molecular ecological liquid extract obtained through specific fermentation according to claim 1 in the preparation of a drug for treating chronic obstructive pulmonary disease, characterized in that... In step S2, the concentration temperature is 40~45℃, the vacuum degree is -0.085~0.095MPa, and the volume is concentrated to 1 / 10 of the original volume.

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