Ganoderma lucidum molecular ecological liquid extract obtained through specific fermentation as well as preparation method and application of ganoderma lucidum molecular ecological liquid extract

Abstract

The invention provides a ganoderma lucidum molecular ecological liquid extract obtained through specific fermentation and a preparation method and application thereof, and belongs to the technical field of microbial fermentation. According to the method, an improved fermentation culture medium is adopted, 5-8% of CO2 is introduced, fermentation is performed for 90-180 days at the temperature of 30-32 DEG C under the condition that the concentration of dissolved oxygen in the environment is kept to be 20-30%, and after purification, the extract of the ganoderma lucidum molecular ecological liquid is obtained. According to the myxomycete molecular ecological liquid prepared by the preparation method provided by the invention, the content of active ingredients for relieving the symptoms of the chronic obstructive pulmonary disease can be remarkably increased; moreover, an in-vitro cell experiment verifies that the extract of the ganoderma lucidum molecular ecological liquid has an inhibition effect on an A549 cell line, and an animal experiment further proves that the extract of the ecological liquid can regulate and control the expression level of proinflammatory cytokines, so that lung inflammation is inhibited, pathological injury of lung tissues is relieved, and local abnormal conditions of the lung are improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial fermentation, in particular to a taizi molecular ecological liquid extract obtained through specific fermentation and a preparation method and application thereof. BACKGROUND

[0002] Taizi is an ancient microbial symbiotic body, and the core groups include Proteobacteria, Bradyrhizobium, Sphingomonas, etc. Studies have shown that taizi contains nucleic acids, polysaccharides, trace elements and other bioactive ingredients, and has certain medicinal value. For example, nucleic acids and their degradation products nucleotides contained in taizi have the effect of treating brain consciousness disorders, and polysaccharides have the effects of anti-tumor and improving immunity.

[0003] Chinese patent application with publication number CN111249317A discloses a preparation method of taizi fermentation liquid, taizi fermentation liquid and application. The technical solution adopts kiwi fruit, plum, strawberry and ganoderma lucidum juice, adds honey, mixes with taizi, and then ferments to remove taizi and filter to obtain taizi fermentation liquid. The taizi fermentation liquid improves the content of active ingredients in the fermentation liquid, can prolong the lifespan of Caenorhabditis elegans, and has wrinkle-removing function.

[0004] At present, the directional fermentation process of taizi and the development and application of active ingredients are still blank, and the biological activity of taizi in resisting chronic obstructive pulmonary disease has not been explored. Chronic obstructive pulmonary disease (COPD) is a preventable and treatable chronic respiratory disease characterized by persistent airflow limitation, which is closely related to the enhancement of chronic inflammatory response of airway and lung tissue to harmful gases or particles such as tobacco smoke. Its core pathological changes involve chronic inflammation of airway, lung parenchyma and pulmonary vessels, which eventually leads to airway stenosis, lung tissue destruction and affects the ventilation and gas exchange function of the lung. Therefore, it is urgent to develop a taizi directional fermentation process to obtain a new type of anti-chronic obstructive pulmonary disease improvement and prognosis drug with natural source and high safety. SUMMARY

[0005] The present application aims to provide a taizi molecular ecological liquid extract obtained through specific fermentation and a preparation method and application thereof. The taizi molecular ecological liquid in the present application is fermented in a specific fermentation condition using a modified fermentation medium, and a taizi molecular ecological liquid extract is obtained through purification. The taizi molecular ecological liquid extract is an ester component, and can be used to inhibit chronic obstructive pulmonary disease cell inflammatory response and improve lung damage when it is added into a tablet, a capsule, an injection or an oral liquid prepared by adding a pharmaceutically acceptable excipient.

[0006] To achieve the above object, in a first aspect, the present application provides a preparation method of a Taizi molecular ecological liquid extract obtained through specific fermentation, comprising:

[0007] S1, inoculating Taizi into a fermentation medium, passing in a mixed gas of CO2 and air, and fermenting in a shaker in the dark to obtain a Taizi molecular ecological liquid;

[0008] S2, centrifuging the Taizi molecular ecological liquid, filtering, adsorbing the filtrate through a macroporous resin, eluting with ethanol, concentrating, drying to obtain a Taizi molecular ecological liquid extract.

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

[0010] Preferably, in the S1, the inoculation amount of the Taizi inoculated into the fermentation medium is 5-10% (w / v), the fermentation temperature is 30-32℃, the fermentation time is 90-180d, and the rotation speed of the shaker is 50-80rpm.

[0011] Preferably, in the S1, the fermentation medium is a modified fermentation medium, comprising: maca 1g / L, ginseng peptide powder 1g / L, cordyceps militaris 1g / L, medlar 5g / L, red dates 10g / L, gordon euryale 3g / L, bergamot 3g / L, honeysuckle 3g / L, sophora flower 3g / L, and dandelion 3g / L, supplemented with sterile water to a volume of 1L, and sterilized at 121℃ for 15min.

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

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

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

[0015] Preferably, in the S2, the steps of macroporous resin adsorption and ethanol elution are as follows: loading the filtrate onto a macroporous resin column with a diameter-height ratio of 1:8, and a flow rate of the filtrate of 1BV / h, eluting the impurities with 3 times the column volume of 30wt.% ethanol after adsorption saturation, and then eluting the active ingredients with 5 times the column volume of 70wt.% ethanol, and collecting the eluate.

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

[0017] Preferably, in S2, the drying temperature is -80 DEG C, and the drying time is 10-12h.

[0018] In a second aspect, the application provides a specific fermentation obtained Taizi molecular ecological liquid extract prepared by the preparation method.

[0019] In a third aspect, the application provides application of the specific fermentation obtained Taizi molecular ecological liquid extract in preparation of a drug for resisting chronic obstructive pulmonary disease.

[0020] Compared with the prior art, the beneficial effects of the application are embodied in that:

[0021] The application adopts a specific fermentation process, ferments in a modified fermentation medium, and directionally activates the metabolic pathways of the symbiotic flora of Taizi, especially the secondary metabolism of core groups such as Bradyrhizobium, Sphingomonas, etc., thereby significantly improving the yield of active ingredients for resisting chronic obstructive pulmonary disease in the Taizi molecular ecological liquid, and experimentally verifying that there are a large number of significantly different metabolites between the Taizi molecular ecological liquid and Taizi water, which fully proves that fermentation treatment is a key factor for remodeling the metabolite spectrum of Taizi, and reveals the multidimensional significant differences in metabolic pathways between the Taizi molecular ecological liquid and Taizi water. The change in the pathway not only explains the difference in the metabolite spectrum after fermentation, but also provides mechanism support for the biological activity of the Taizi molecular ecological liquid at the pathway level; the application proves by in vitro cell experiments that the Taizi molecular ecological liquid extract has a good inhibitory effect on the A549 lung cancer cell line, and the inhibition rate of 100 μg / mL Taizi molecular ecological liquid extract on A549 cells reaches 76.2%, and the IC50 is 38.5 μg / mL. Further animal experiments prove that the Taizi molecular ecological liquid extract can regulate the expression level of pro-inflammatory cytokines in serum and lung tissue, thereby reducing the inflammatory response, regulating the state of lung tissue, improving the local abnormal condition of the lung, and ultimately promoting the functional recovery of the lung lesion area. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Anion mode base peak chromatogram of the Taizi molecular ecological liquid prepared in Example 3.

[0023] Figure 2 Cation mode base peak chromatogram of the Taizi molecular ecological liquid prepared in Example 3.

[0024] Figure 3 Bar graph of the difference experiment of the number of metabolites of the Taizi molecular ecological liquid prepared in Example 3 and the Taizi water prepared in the comparative example.

[0025] Figure 4Differential plot of metabolite number between the Taesu molecular eco-liquid prepared in Example 3 and the Taesu water prepared in Comparative Example.

[0026] Figure 5 Data plot of multivariate differential analysis between the Taesu molecular eco-liquid prepared in Example 3 and the Taesu water prepared in Comparative Example.

[0027] Figure 6 Data plot of partial least squares discriminant analysis between the Taesu molecular eco-liquid prepared in Example 3 and the Taesu water prepared in Comparative Example.

[0028] Figure 7 Data plot of orthogonal partial least squares discriminant analysis between the Taesu molecular eco-liquid prepared in Example 3 and the Taesu water prepared in Comparative Example.

[0029] Figure 8 Data plot of metabolic pathway enrichment analysis between the Taesu molecular eco-liquid prepared in Example 3 and the Taesu water prepared in Comparative Example.

[0030] Figure 9 In vitro cell experiment result plot of the Taesu molecular eco-liquid extract prepared in Example 3.

[0031] Figure 10 Picture of H&E staining of the lung of a mouse in the CON group.

[0032] Figure 11 Picture of H&E staining of the lung of a mouse in the CON group.

[0033] Figure 12 Picture of H&E staining of the lung of a mouse in the COPD group.

[0034] Figure 13 Picture of H&E staining of the lung of a mouse in the COPD group.

[0035] Figure 14 Picture of H&E staining of the lung of a mouse in the administration group.

[0036] Figure 15 Picture of H&E staining of the lung of a mouse in the administration group.

[0037] Figure 16 Expression amount of IL-6 in the serum of a mouse in the CON group, the COPD group, and the administration group.

[0038] Figure 17 Expression amount of TNF-α in the serum of a mouse in the CON group, the COPD group, and the administration group.

[0039] Figure 18 Expression amount of IL-6 in the lung tissue of a mouse in the CON group, the COPD group, and the administration group.

[0040] Figure 19 The expression amount of TNF-α in the lung tissue of the CON group, the COPD group and the administration group mice. DETAILED DESCRIPTION

[0041] The following examples are only used to more clearly illustrate the technical solutions of the present application, and cannot be used to limit the protection scope of the present application.

[0042] The taizi sample (containing relative abundance of phylum Proteobacteria > 60%, Bradyrhizobium and Sphingomonas as dominant genera) used in the examples was collected in northeast China and stored at 4°C.

[0043] The culture medium used in the examples is as follows: maca 1 g / L, ginseng peptide powder 1 g / L, cordyceps militaris 1 g / L, medlar 5 g / L, red date 10 g / L, gordon euryale 3 g / L, bergamot 3 g / L, honeysuckle 3 g / L, sophora flower 3 g / L and dandelion 3 g / L, supplemented with sterile water to a volume of 1 L, sterilized at 121°C for 15 min.

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

[0045] Example 1

[0046] A taizi molecular ecological liquid extract obtained by specific fermentation, the preparation method comprising:

[0047] S1, 100 g of taizi was washed with sterile PBS (phosphate buffer) for 3 times. 25 g of taizi was inoculated into 500 mL of fermentation medium, the inoculation amount of taizi into the fermentation medium was 5% (w / v), the mixed gas of CO2 with a volume concentration of 5% CO2 and air was introduced, the aeration rate was 0.3 vvm, the dissolved oxygen concentration in the environment was maintained at 20%, under the condition of 30°C, the shaking speed was 50 rpm, and the fermentation was carried out in the dark for 180 d to obtain a taizi molecular ecological liquid.

[0048] S2, the taizi molecular ecological liquid was centrifuged at 8000 rpm for 15 min at 20°C, filtered with a 0.22 μm filter membrane, the filtrate was loaded onto an AB-8 type macroporous resin, the loading flow rate was maintained at 1 BV / h, after adsorption saturation, the impure proteins were eluted with 3 times the column volume of ethanol with a concentration of 30 wt.%, and then the active ingredients were eluted with 5 times the column volume of ethanol with a concentration of 70 wt.%, the active ingredient eluent was collected, vacuum concentration was performed at a vacuum degree of -0.085 MPa and a temperature of 40°C to 1 / 10 of the original volume, and then dried at -80°C for 10 h to obtain a taizi molecular ecological liquid extract.

[0049] Example 2

[0050] A taizi molecular ecological liquid extract obtained by specific fermentation, the preparation method comprising:

[0051] S1, 100g of Adesmia fruticosa was washed with sterile PBS for 3 times. 40g of Adesmia fruticosa was inoculated into 500mL of fermentation medium, the inoculation amount of Adesmia fruticosa into the fermentation medium was 8% (w / v), a mixed gas of CO2 with a CO2 volume concentration of 5% and air was introduced, the aeration rate was 0.4vvm, the dissolved oxygen concentration in the environment was maintained at 25%, the fermentation was carried out at 30°C under the condition of 60rpm of the shaking speed, and the fermentation was carried out for 120d in the dark to obtain Adesmia fruticosa molecular ecological liquid.

[0052] S2, the Adesmia fruticosa molecular ecological liquid was centrifuged at 9000rpm for 15min at 25°C, filtered with a 0.22μm filter membrane, and then the filtrate was loaded onto AB-8 type macroporous resin, the loading flow rate was maintained at 1BV / h, after adsorption saturation, impurity proteins were eluted with 3 times the column volume of ethanol with a concentration of 30wt.%, then active ingredients were eluted with 5 times the column volume of ethanol with a concentration of 70wt.%, the active ingredient eluate was collected, concentrated to 1 / 10 of the original volume under the condition of a vacuum degree of -0.09MPa and 45°C, and dried at -80°C for 12h to obtain Adesmia fruticosa molecular ecological liquid extract.

[0053] Example 3

[0054] An Adesmia fruticosa molecular ecological liquid extract obtained through specific fermentation, a preparation method thereof comprising:

[0055] S1, 100g of Adesmia fruticosa was washed with sterile PBS for 3 times. 40g of Adesmia fruticosa was inoculated into 500mL of fermentation medium, the inoculation amount of Adesmia fruticosa into the fermentation medium was 8% (w / v), a mixed gas of CO2 with a CO2 volume concentration of 5% and air was introduced, the aeration rate was 0.4vvm, the dissolved oxygen concentration in the environment was maintained at 25%, the fermentation was carried out at 30°C under the condition of 60rpm of the shaking speed, and the fermentation was carried out for 120d in the dark to obtain Adesmia fruticosa molecular ecological liquid.

[0056] S2, the Adesmia fruticosa molecular ecological liquid was centrifuged at 9000rpm for 15min at 25°C, filtered with a 0.22μm filter membrane, and then the filtrate was loaded onto AB-8 type macroporous resin, the loading flow rate was maintained at 1BV / h, after adsorption saturation, impurity proteins were eluted with 3 times the column volume of ethanol with a concentration of 30wt.%, then active ingredients were eluted with 5 times the column volume of ethanol with a concentration of 70wt.%, the active ingredient eluate was collected, concentrated to 1 / 10 of the original volume under the condition of a vacuum degree of -0.09MPa and 45°C, and dried at -80°C for 12h to obtain Adesmia fruticosa molecular ecological liquid extract.

[0057] Comparative Example 1

[0058] An Adesmia fruticosa water, a preparation method thereof comprising: 100g of Adesmia fruticosa was washed with sterile PBS for 3 times, 100g of Adesmia fruticosa was inoculated into 1L of ultrapure water, and the culture was carried out at 25°C for 180d under the condition of being sealed and stationary.

[0059] Differential analysis of the components of the Tai Sui molecular ecological liquid obtained in Example 3 and the Tai Sui water prepared in Comparative Example 1:

[0060] (1) Base peak chromatogram: the response value of the ion with the strongest signal intensity in the LC-MS mass spectrum at each time point was taken as the vertical coordinate, and the retention time was taken as the horizontal coordinate. The base peak chromatogram (BPC) in the negative ion mode (NEG) and the positive ion mode (POS) was drawn, respectively, and the results are shown in Figure 1 and Figure 2 .

[0061] (2) Differential analysis of the Tai Sui molecular ecological liquid and the Tai Sui water was performed. According to the three indicators of fold change, multiple test significance level, and VIP value of multivariate analysis, the results are shown in Figure 3 and Figure 4 .

[0062] Figure 3 The bar graph of the difference in the number of metabolites between the Tai Sui molecular ecological liquid and the Tai Sui water is shown, in which there are 520 up-regulated metabolites and 378 down-regulated metabolites. The metabolites of the Tai Sui molecular ecological liquid and the Tai Sui water are significantly different, which proves that the fermentation process has greatly changed the composition of metabolites, and the fold change is >4, the multiple test significance level is <0.05, and the VIP value of multivariate analysis is >1, which has clear statistical significance and biological significance, and excludes false positive differences caused by random fluctuations.

[0063] Figure 4 The volcano plot of the difference in the number of metabolites between the Tai Sui molecular ecological liquid and the Tai Sui water reveals that there are a large number of significantly different metabolites between the Tai Sui molecular ecological liquid and the Tai Sui water, and most of the differential metabolites have important contributions to the separation between groups (VIP≥1). The core metabolites that are significantly up-regulated are the material basis of fermentation function, which preliminarily verifies that the fermentation treatment has produced essential changes in the composition of metabolites of Tai Sui.

[0064] (3) Differential analysis of the components of the Tai Sui molecular ecological liquid and the Tai Sui water by multivariate analysis. The PCA model between the Tai Sui molecular ecological liquid prepared in Example 3 and the Tai Sui water obtained in Comparative Example 1 was established, and the distribution and separation trend of the two groups of samples were observed, and the results are shown in Figure 5 .

[0065] Figure 5The middle ellipse is the 95% confidence interval of the samples of the Taiyin molecular ecological liquid and the Taiyin water, all the points of the two groups of samples fall within the respective 95% confidence ellipse, there is no obvious abnormal point deviating from other samples in the group, and the sample points of the Taiyin molecular ecological liquid are highly aggregated in the negative interval of PCA1, with a small range, indicating that the metabolite profiles of the samples in the group are very consistent, the experimental repeatability is good, and the sample points of the Taiyin water are relatively dispersed in the positive interval of PCA1, but are still contained in the 95% confidence ellipse, indicating that there is a certain difference in the group, but the whole still has distinguishable group characteristics, indicating that the detection process of the experimental samples is stable, and the data is reliable.

[0066] According to Figure 5 As shown by the data, the 95% confidence ellipses of the Taiyin molecular ecological liquid group and the Taiyin water group are completely separated without overlapping areas, proving that the fermentation treatment has caused essential changes in the metabolite composition of the Taiyin, so there is a highly significant overall difference in the metabolite composition between the Taiyin molecular ecological liquid and the Taiyin 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 Taiyin molecular ecological liquid and the Taiyin water to establish a relationship model between the metabolite expression and the sample category, which can realize the modeling and prediction of the sample category, and the results are shown in Figure 6 .

[0068] Figure 6 The middle ellipse is the 95% confidence interval of the samples of the Taiyin molecular ecological liquid and the Taiyin water, the 95% confidence ellipses of the Taiyin molecular ecological liquid and the Taiyin water are completely separated without overlapping areas, indicating that the PLS-DA model can significantly distinguish the Taiyin molecular ecological liquid and the Taiyin water, indicating that the difference in the metabolite composition of the two groups of samples has a clear category distinguishability, the change in the metabolite profile caused by the fermentation treatment has a predictable regularity, the sample points of the Taiyin molecular ecological liquid are highly aggregated in the negative interval of T score, with a very small ellipse range, indicating that the metabolite expression patterns of the samples in the group are very consistent, the experimental repeatability is good, and the sample points of the Taiyin water are relatively dispersed in the positive interval of T score, but are still contained in the 95% confidence ellipse, indicating that there is a certain difference in the group, but the whole still has distinguishable group characteristics.

[0069] The completely separated results of PLS-DA prove that there is a strong correlation between the metabolite expression and the category attribute of whether it has been fermented, and the model can accurately predict whether the sample belongs to the Taiyin molecular ecological liquid or the Taiyin water through the metabolite profile, further verifying the substantial change in the metabolite composition of the Taiyin caused by the fermentation treatment.

[0070] (5) The two groups of biological samples were subjected to orthogonal partial least squares discriminant analysis, and a relationship model between the expression of metabolites and the sample categories was established to realize the modeling prediction of the sample categories. Meanwhile, the variable projection importance (VIP) was calculated to measure the ability of each metabolite to classify and discriminate the samples of each group, thereby assisting the screening of metabolic markers. Generally, when VIP>1, it indicates that the variable has a significant effect on the classification of the sample categories. The results are shown in Figure 7 .

[0071] Figure 7 The 95% confidence ellipses of the molecular ecological liquid of Zhuyesi and the water of Zhuyesi were completely separated without any overlap, indicating that the OPLS-DA model could significantly distinguish the molecular ecological liquid of Zhuyesi and the water of Zhuyesi, and that there were essential differences in the metabolite compositions of the two groups of samples. The change in the metabolite profile caused by fermentation processing had strong specificity. The sample points of the molecular ecological liquid of Zhuyesi were highly clustered in the positive interval of PC1, and the ellipse range was very small, indicating that the metabolite expression patterns of the samples within the group were highly consistent, and the experimental repeatability was excellent. The sample points of the water of Zhuyesi were relatively dispersed in the negative interval of PC1, but were still contained in the 95% confidence ellipse, indicating that there were certain differences within the group, but the overall group characteristics were still distinguishable.

[0072] Figure 7 The data further verified that there were significant differences in the metabolite compositions between the molecular ecological liquid of Zhuyesi and the water of Zhuyesi, and such differences could be accurately distinguished by the supervised model. Combined with the analysis results of PCA and PLS-DA, it was fully demonstrated that fermentation processing was the key factor for remodeling the metabolite profile of Zhuyesi, which provided strong model support for the subsequent screening of differential metabolic markers and the analysis of the biological activity mechanism of fermentation.

[0073] (6) Based on the KEGG database, the differential metabolites of the molecular ecological liquid of Zhuyesi and the water of Zhuyesi were subjected to metabolic pathway enrichment analysis, which could reveal the metabolic pathways that had undergone significant changes, thereby contributing to the interpretation of biological phenotypes. The metabolic pathways with p value (P value) <0.05 were defined as the metabolic pathways in which the differential metabolites were significantly enriched, and the results are shown in Figure 8 .

[0074] According to Figure 8 , the color of p value represents P value from extremely small (4e-04) to relatively large (0e+00, i.e., not significant) from blue to red. Except for carbon metabolism and biosynthesis of secondary metabolites, the P values of the remaining pathways were <0.05, indicating that there were statistically significant differences between the molecular ecological liquid of Zhuyesi and the water of Zhuyesi in these pathways;

[0075] The enrichment factor represents the number of differential metabolites annotated to the pathway, the larger the value, the higher the proportion of differential metabolites in the pathway, and 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, indicating that fermentation has a significant impact on phenylalanine metabolism and pentose phosphate pathway metabolism, proving that the metabolic activity of these pathways has changed substantially after fermentation;

[0076] The circle size represents the number of differential metabolites annotated to the pathway, the larger the circle size, the more metabolites involved in differential regulation in the pathway, and the biosynthesis of secondary metabolites has the largest circle (containing 60 differential metabolites), indicating that the remodeling of the secondary metabolic network by fermentation is global, and the biosynthesis of cofactors and 2-oxo carboxylic acid metabolism also have relatively large circles, thus reflecting the rich changes in metabolites of these pathways.

[0077] Figure 8 The data reveals the multidimensional significant differences between Taizi molecular ecological liquid and Taizi water in metabolic pathways, which are mainly concentrated in secondary metabolism, cofactor synthesis, sugar metabolism (pentose phosphate pathway), and amino acid metabolism (phenylalanine). The changes in these pathways not only explain the differences in the metabolite profile after fermentation, but also provide pathway-level mechanism support for the biological activity of Taizi molecular ecological liquid, and also confirm the key role of cofactors in maintaining the growth and function of Taizi.

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

[0079] In vitro cell experiment: CCK-8 method was used to detect the inhibition effect of Taizi molecular ecological liquid extract prepared in Example 3 on A549 lung cancer cells. The concentration gradient of Taizi molecular ecological liquid extract was set as 10 μg / mL, 20 μg / mL, 50 μg / mL, and 100 μg / mL. After incubating with A549 lung cancer cells for 48 h, the inhibition rate of cancer cells was calculated.

[0080] Animal experiment: 18 mice were randomly divided into 3 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 daily treatment with Taizi molecular ecological liquid extract prepared in Example 3 (dosing group, 5 mL / kg / day).

[0081] The COPD group mice were intranasally instilled with lipopolysaccharide (75 μg / mouse) on the 1st and 14th days of the experiment after the mice were anesthetized with tribromoethanol. The CON group mice were exposed to indoor air, while the other groups of mice were exposed to cigarette smoke environment twice a day, each time for 2 h; the dosing group mice were given Taizi molecular ecological liquid extract (5 mL / kg / day) intragastrically 1 hour before exposure to cigarette smoke, while the CON group and COPD group were given the same volume of normal saline.

[0082] On the last day of the experiment, the serum of the experimental mice was obtained, and the expression of pro-inflammatory cytokines IL-6 and TNF-α in the serum of the mice was detected by Elisa; the mice were sacrificed, and the lung tissue was obtained, and 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] The results of in vitro cell experiments are shown in Figure 9 As shown in the figure, with the increase of the concentration of Taizi molecular ecological liquid extract from 0 to 100 μg / mL, the inhibitory effect of Taizi molecular ecological liquid extract on A549 cells showed an obvious concentration-dependent enhancement trend, and there was still no inhibition rate plateau at a higher concentration; the half inhibitory concentration (IC50) of Taizi molecular ecological liquid extract on A549 cells was 38.5 μg / mL, and only a low concentration was needed to achieve 50% growth inhibition of A549 cells; the inhibition rate of 100 μg / mL Taizi molecular ecological liquid extract on A549 cells was 76.2%, and at this concentration, Taizi molecular ecological liquid extract significantly blocked the proliferation process of A549 cells, only a small amount of cells remained active, which showed strong in vitro anti-A549 cell proliferation activity.

[0084] In the animal experiment, the H&E staining results of the lung tissue of the experimental mice are shown in Figure 10~Figure 15 . Figure 10 The lung tissue of the CON group mice is shown in Figure 11 . Figure 10 The local enlarged view of the black frame area shows that the surface covering of the lung tissue is a layer of smooth serosa, and there is no obvious abnormality. Figure 12 The lung tissue of the COPD group mice is shown in Figure 13 . Figure 12 The local enlarged view of the black frame area shows that the lung alveolar wall in the area indicated by the yellow arrow can be seen to be infiltrated by inflammatory cells mainly composed of granulocytes, the lung alveolar wall is slightly thickened, and the alveolar is narrow; the area indicated by the brown arrow has local lymphocyte and granulocyte focal infiltration; the area indicated by the green arrow has a large range of fine bronchial epithelial cell degeneration and cytoplasmic vacuolization, with few necrosis, the area indicated by the blue arrow has nuclear pyknosis and deep staining, and the areas indicated by the purple arrow and the orange arrow have multiple perivascular edema, loose arrangement of connective tissue, and point-like granulocyte and lymphocyte infiltration. Figure 14 The lung tissue of the drug administration group mice is shown in Figure 15 . Figure 14 The local enlarged view of the black frame shows that the lung tissue is covered with a layer of smooth serosa, and there is no obvious abnormality; the lung alveolar wall in the area indicated by the yellow arrow can be seen to be infiltrated by inflammatory cells mainly composed of granulocytes, and there is slight thickening of the lung alveolar wall and narrowing of the alveolar; the area indicated by the green arrow shows fine bronchial epithelial cell degeneration and cytoplasmic vacuolization, with few necrosis, and the area indicated by the blue arrow shows nuclear pyknosis and dissolution.

[0085] In the animal experiment, the expression of pro-inflammatory cytokines IL-6 and TNF-α in the serum of the experimental mice was detected by Elisa, and the results are shown in Figure 16 and Figure 17 , wherein Figure 16 is the expression amount of pro-inflammatory cytokine IL-6 in the serum of the experimental mice, Figure 17 is the expression amount of pro-inflammatory cytokine TNF-α in the serum of the experimental mice. The concentrations of IL-6 and TNF-α in the serum of the mice in the COPD group were significantly higher than those in the CON group and the drug administration group. After the intervention of the extract of the molecular ecological liquid of Cyclocelatum moluccanum, the concentrations of IL-6 and TNF-α in the serum of the mice in the drug administration group decreased and were significantly lower than those in the COPD group.

[0086] The expression of pro-inflammatory cytokines IL-6 and TNF-α in the lung tissue of the experimental mice was detected by q-PCR, and the results are shown in Figure 18 and Figure 19 , wherein Figure 18 is the expression amount of pro-inflammatory cytokine IL-6 in the lung tissue of the experimental mice, Figure 19 is the expression amount of pro-inflammatory cytokine TNF-α in the lung tissue of the experimental mice. The concentrations of IL-6 and TNF-α in the lung tissue of the mice in the COPD group were significantly higher than those in the CON group and the drug administration group. After the intervention of the extract of the molecular ecological liquid of Cyclocelatum moluccanum, the concentrations of IL-6 and TNF-α in the lung tissue of the mice in the drug administration group decreased and were significantly lower than those in the COPD group.

[0087] In summary, the extract of the molecular ecological liquid of Cyclocelatum moluccanum can effectively regulate the expression level of pro-inflammatory cytokines, inhibit lung inflammation, and thus relieve the pathological damage of lung tissue, regulate the state of lung tissue, and improve the local abnormality of the lung.

[0088] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the technical principles of the present application, a number of improvements and modifications can be made, and these improvements and modifications should also be considered as the protection scope of the present application.

Claims

1. A preparation method of a molecular ecological extract of a Taizi obtained by specific fermentation, characterized in that, The preparation method comprises the following steps: S1, inoculating Taizi into a fermentation medium, and fermenting in a dark place in a shaker by passing mixed gas of CO2 and air to obtain a Taizi molecular ecological liquid; S2, centrifuging the Taizi molecular ecological liquid, filtering, adsorbing the filtrate by a macroporous resin, eluting the macroporous resin by ethanol, concentrating, and drying to obtain a Taizi molecular ecological liquid extract.

2. The preparation method of the extract of the Taizi molecular ecological liquid obtained by specific fermentation according to claim 1, characterized in that, In the S1, the volume concentration of CO2 in the mixed gas is 5-8%, the aeration rate is 0.3-0.5vvm, and the dissolved oxygen concentration in the environment is maintained at 20-30%.

3. The preparation method of the extract of the Taizi molecular ecological liquid obtained by specific fermentation according to claim 1, characterized in that, In the S1, the inoculation amount of the Taizi inoculated into the fermentation medium is 5-10% (w / v), the fermentation temperature is 30-32℃, the fermentation time is 90-180d, and the rotation speed of the shaker is 50-80rpm.

4. The preparation method of the extract of the Taizi molecular ecological liquid obtained by specific fermentation according to claim 1, characterized in that, In the S1, the fermentation medium is a modified fermentation medium, which comprises maca 1g / L, ginseng peptide powder 1g / L, cordyceps militaris 1g / L, medlar 5g / L, red dates 10g / L, gordon euryale 3g / L, bergamot 3g / L, honeysuckle 3g / L, sophora flower 3g / L and dandelion 3g / L, and the volume is supplemented to 1L with sterile water, and sterilized at 121℃ for 15min.

5. The preparation method of the extract of the Taizi molecular ecological liquid obtained by specific fermentation according to claim 1, characterized in that, In the S2, the rotation speed of the centrifugation is 8000-10000rpm, the centrifugation temperature is 20-25℃, and the centrifugation time is 10-15min; and the filter membrane with a diameter of 0.22μm is used for the filtration.

6. The preparation method of the extract of the Taizi molecular ecological liquid obtained by specific fermentation according to claim 1, characterized in that, In the S2, the macroporous resin is AB-8 type macroporous resin; and the steps of adsorbing the macroporous resin and eluting by ethanol are as follows: loading the filtrate into a macroporous resin column with a diameter-height ratio of 1:8, and a flow rate of the filtrate of 1BV / h, eluting the impurities by 3 times the column volume of 30wt.% ethanol after adsorption saturation, and eluting the active ingredients by 5 times the column volume of 70wt.% ethanol, and collecting the eluate.

7. The method of claim 1, wherein the preparation of the extract of the Taee-sage molecular ecological liquid obtained by specific fermentation is characterized by, In the S2, the concentration temperature is 40-45℃, the vacuum degree is -0.085-0.095MPa, and the concentration is performed to 1 / 10 of the original volume.

8. The preparation method of the extract of the Taizi molecular ecological liquid obtained by specific fermentation according to claim 1, characterized in that, In the S2, the drying temperature is -80℃, and the drying time is 10-12h.

9. A Taizi molecular ecological liquid extract obtained by specific fermentation, which is prepared by the preparation method according to any one of claims 1-8.

10. Application of the Taizi molecular ecological liquid extract obtained by specific fermentation according to claim 9 in the preparation of a drug for resisting chronic obstructive pulmonary disease.

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