Application of astragalus mongholicus fermentation liquor in prevention and treatment of lung cancer

Abstract

The invention provides application of astragalus mongholicus fermentation liquor in prevention and treatment of lung cancer, and belongs to the technical field of traditional Chinese medicine fermentation. The prepared astragalus membranaceus fermentation liquor can reduce tumor volume, increase body weight, regulate body immunity, regulate intestinal flora, specifically enrich Akkermansia bacteria and promote biosynthesis of short-chain fatty acid, and effectively prevent and treat lung cancer. Meanwhile, liver and kidney injury and immune organ injury in the lung cancer can also be protected.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine fermentation, and in particular relates to application of astragalus fermentation liquid in preventing and treating lung cancer. Background Art

[0002] With the vigorous development of traditional Chinese medicine, the research on traditional Chinese medicine treatment or traditional Chinese medicine-assisted treatment of lung cancer has been increasing. Compared with conventional chemotherapy drugs, traditional Chinese medicine has shown advantages in alleviating treatment-related side effects, improving patients' quality of life, and enhancing physical fitness and immunity. Astragalus has been used as a tonic in China for more than 2,000 years and has the effects of strengthening the spleen and replenishing qi and promoting the production of body fluids and nourishing blood.

[0003] In recent years, the application of probiotic deep fermentation in traditional Chinese medicine has become a hot topic to broaden the boundaries of medicine and food. There is a close interaction between microorganisms and traditional Chinese medicine. Microbial-mediated fermentation of traditional Chinese medicine can produce important microbial and traditional Chinese medicine metabolites, degrading macromolecules into small compounds to improve the efficacy of traditional Chinese medicine. On the other hand, fermented traditional Chinese medicine shows stronger potential prebiotic properties than traditional Chinese medicine, and can effectively regulate the structure and function of intestinal flora to regulate host health. Studies have shown that fermentation of astragalus with Lactobacillus plantarum can improve the growth performance of broilers, increase serum antioxidant capacity, and reduce harmful microorganisms in feces. Fermentation of astragalus with Bacillus subtilis can be used to treat hyperuricemia, reduce renal inflammation, regulate the expression of uric acid transporters, and increase the abundance of butyrate-producing bacteria. It is not clear whether fermented astragalus can prevent and treat lung cancer. Summary of the invention

[0004] In view of this, the object of the present invention is to provide an application of astragalus fermentation liquid in preventing and treating lung cancer.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides application of astragalus fermentation liquid in preparing medicine for preventing and treating lung cancer.

[0007] The present invention also provides the use of the astragalus fermentation liquid in preparing a medicine for reversing liver and kidney tissue damage.

[0008] The present invention also provides the use of the astragalus fermentation liquid in preparing a medicine for reversing immune organ damage.

[0009] The present invention also provides the use of astragalus fermented liquid in the preparation of a drug for regulating intestinal flora, wherein the astragalus fermented liquid reduces harmful bacterial genera in the intestinal flora, increases beneficial bacterial genera in the intestinal flora, and enriches specific bacterial genera in the intestinal flora.

[0010] The present invention also provides the use of astragalus fermented liquid in the preparation of functional food that helps to regulate intestinal flora. The astragalus fermented liquid reduces harmful bacterial genera in the intestinal flora, increases beneficial bacterial genera in the intestinal flora, and enriches specific bacterial genera in the intestinal flora.

[0011] Preferably, the harmful bacterial genera include Prevotella, Oscillospira, and Bacteroides, the beneficial bacterial genera include Lactobacillus, Bifidobacterium, and Turicibacter, and the specific bacterial genera include Akkermansia.

[0012] The present invention also provides the use of the astragalus fermentation liquid in the preparation of a medicine for promoting the synthesis of short-chain fatty acids, wherein the short-chain fatty acids include acetic acid, valeric acid, propionic acid, butyric acid, isobutyric acid or isovaleric acid.

[0013] Preferably, the method for preparing the astragalus fermentation broth comprises the following steps:

[0014] 1) Soaking the astragalus root in water, boiling it, filtering it, and obtaining the residue and the first medicinal liquid;

[0015] 2) adding water to the medicinal residue and boiling to obtain a second medicinal solution;

[0016] 3) combining the first medicinal solution and the second medicinal solution, concentrating them, and obtaining an astragalus water extract;

[0017] 4) Lactobacillus plantarum LZU-S-ZCJ and Lactobacillus plantarum LZU-J-TSL6 are mixed, and the mixture is inoculated into the astragalus water extract for fermentation to obtain an astragalus fermentation liquid.

[0018] Preferably, in step 1), the mass ratio of astragalus to water is 1:8-12, the soaking time is 3-5 hours, the decoction temperature is 94-97°C, the decoction time is 0.5-1.5 hours, and the filtering times are 1-3 times;

[0019] Step 2) The mass ratio of the medicinal residue to water is 1:6-10, the decoction temperature is 94-97° C., and the decoction time is 0.5-1.5 h.

[0020] Preferably, in step 3), the concentration is performed to obtain a mass of 0.3 to 0.8 g of the original drug per milliliter of the drug solution;

[0021] Step 4) The total viable count of Lactobacillus plantarum LZU-S-ZCJ and Lactobacillus plantarum LZU-J-TSL6 is 10 6 ~10 10 CFU / mL, the ratio of the number of viable bacteria of Lactobacillus plantarum LZU-S-ZCJ to that of Lactobacillus plantarum LZU-J-TSL6 is 1:2-4;

[0022] The inoculation amount of the mixed bacteria is 2-4% of the volume of the astragalus water extract;

[0023] The fermentation temperature is 35-38° C., and the fermentation time is 35-40 hours.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The astragalus fermented liquid prepared by the present invention can reduce tumor volume, increase body weight, regulate body immunity, regulate intestinal flora, specifically enrich Akkermansia bacteria and promote the biosynthesis of short-chain fatty acids, effectively prevent and treat lung cancer, and can also protect liver and kidney damage and immune organ damage in lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The effect of Astragalus fermented liquid on tumor volume, tumor weight, tumor inhibition rate, and body weight of lung cancer mice (where A is tumor volume; B is tumor weight; C is tumor inhibition rate; D is body weight change; E is effective body weight; and F is effective body weight change);

[0027] Figure 2 The effect of Astragalus fermented liquid on the pathological morphology of tumor tissue in lung cancer mice (HE staining, ×400);

[0028] Figure 3 The effect of Astragalus fermented liquid on liver and kidney of lung cancer mice (where A is liver index; B is kidney index; C is HE staining section of liver and kidney tissue, ×400);

[0029] Figure 4 The effect of Astragalus fermented liquid on immune organs and immune cytokines in mice with lung cancer (A is spleen index; B is thymus index; C is HE staining section of spleen tissue, ×400; D is IL-1β; E is TNF-α; F is IFN-γ; G is TGF-β; H is IL-6; I is IL-12);

[0030] Figure 5The effect of fermented Astragalus on the composition of intestinal flora in mice with lung cancer (where A is the composition at the phylum level; B is the composition at the genus level; C is the analysis of differential bacterial genera of fermented Astragalus based on Lefse; D is the significance analysis of the main bacterial genera);

[0031] Figure 6 The effect of Astragalus fermentation liquid on the production of short-chain fatty acids in mice with lung cancer (where A is acetic acid, B is propionic acid, C is butyric acid, D is isobutyric acid, E is valeric acid, and F is isovaleric acid);

[0032] Figure 7 The effect of Astragalus fermented liquid on the proliferation of lung cancer cells.

[0033] Biological Deposit Description

[0034] Lactobacillus plantarum LZU-S-ZCJ, the Latin name is Lactobacillus plantarum LZU-S-ZCJ, the depository is Guangdong Microbiological Culture Collection Center, the address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, the deposit date is December 31, 2020, and the deposit number is GDMCC NO: 61402;

[0035] Lactobacillus plantarum LZU-J-TSL6, the Latin name is Lactobacillus plantarum LZU-J-TSL6, the preservation unit is Guangdong Microbiological Culture Collection Center, the address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, the preservation date is October 23, 2020, and the preservation number is GDMCC NO:61242. DETAILED DESCRIPTION

[0036] The invention provides application of astragalus fermentation liquid in preparing medicine for preventing and treating lung cancer.

[0037] The present invention also provides the use of the astragalus fermentation liquid in preparing a medicine for reversing liver and kidney tissue damage.

[0038] The present invention also provides the use of the astragalus fermentation liquid in preparing a medicine for reversing immune organ damage.

[0039] The present invention also provides the use of astragalus fermented liquid in the preparation of a drug for regulating intestinal flora, wherein the astragalus fermented liquid reduces harmful bacterial genera in the intestinal flora, increases beneficial bacterial genera in the intestinal flora, and enriches specific bacterial genera in the intestinal flora.

[0040] The present invention also provides the use of astragalus fermented liquid in the preparation of functional food that helps to regulate intestinal flora. The astragalus fermented liquid reduces harmful bacterial genera in the intestinal flora, increases beneficial bacterial genera in the intestinal flora, and enriches specific bacterial genera in the intestinal flora.

[0041] In the present invention, the harmful bacterial genera include Prevotella, Oscillospira, and Bacteroides, the beneficial bacterial genera include Lactobacillus, Bifidobacterium, and Turicibacter, and the specific bacterial genera include Akkermansia.

[0042] The present invention also provides the use of the astragalus fermentation liquid in the preparation of a medicine for promoting the synthesis of short-chain fatty acids, wherein the short-chain fatty acids include acetic acid, valeric acid, propionic acid, butyric acid, isobutyric acid or isovaleric acid.

[0043] In the present invention, the method for preparing the astragalus fermentation broth comprises the following steps:

[0044] 1) Soaking the astragalus root in water, boiling it, filtering it, and obtaining the residue and the first medicinal liquid;

[0045] 2) adding water to the medicinal residue and boiling to obtain a second medicinal solution;

[0046] 3) combining the first medicinal solution and the second medicinal solution, concentrating them, and obtaining an astragalus water extract;

[0047] 4) Lactobacillus plantarum LZU-S-ZCJ and Lactobacillus plantarum LZU-J-TSL6 are mixed, and the mixture is inoculated into the astragalus water extract for fermentation to obtain an astragalus fermentation liquid.

[0048] In the present invention, after the astragalus is soaked in water, it is decocted and filtered to obtain the residue and the first liquid medicine. The mass ratio of the astragalus to water is preferably 1:8-12, and more preferably 1:9-11; the soaking time is preferably 3-5 hours, and more preferably 3.5-4.5 hours; the decocting temperature is preferably 94-97°C, and more preferably 95-96°C; the decocting time is preferably 0.5-1.5 hours, and more preferably 0.8-1.2 hours; the number of filtrations is preferably 1-3 times, and more preferably 2 times;

[0049] In the present invention, water is added to the medicinal residues for decoction to obtain a second medicinal liquid; the mass ratio of the medicinal residues to water is preferably 1:6-10, more preferably 1:7-9; the decoction temperature is preferably 94-97°C, more preferably 95-96°C; the decoction time is preferably 0.5-1.5h, more preferably 0.8-1.2h.

[0050] In the present invention, the first medicinal solution and the second medicinal solution are combined and concentrated to obtain the Astragalus membranaceus aqueous extract, wherein the concentration is such that the original drug mass per milliliter of the medicinal solution is preferably 0.3 to 0.8 g, and more preferably 0.4 to 0.7 g.

[0051] In the present invention, Lactobacillus plantarum LZU-S-ZCJ and Lactobacillus plantarum LZU-J-TSL6 are mixed and inoculated into an astragalus water extract for fermentation to obtain an astragalus fermentation liquid. The total viable count of the Lactobacillus plantarum LZU-S-ZCJ and the Lactobacillus plantarum LZU-J-TSL6 is preferably 10 6 ~10 10 CFU / mL, more preferably 10 7 ~10 9 CFU / mL; the ratio of the number of live bacteria of Lactobacillus plantarum LZU-S-ZCJ to Lactobacillus plantarum LZU-J-TSL6 is preferably 1:2-4, and more preferably 1:3; the inoculation amount of the mixed strains is preferably 2-4% of the volume of the astragalus water extract, and more preferably 2.5-3.5% of the volume of the astragalus water extract; the fermentation temperature is preferably 35-38°C, and more preferably 36-37°C; the fermentation time is preferably 35-40h, and more preferably 36-39h.

[0052] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0053] The materials used in the present invention are as follows:

[0054] Astragalus: purchased from Lanzhou Deshengtang Co., Ltd. (batch number: 20230108), identified by Chief Chinese Medicine Practitioner Ni Lin of Gansu Provincial Institute of Drug Inspection as the dried root of Astragalus of the genus Astragalus of Leguminosae. The Astragalus was crushed and passed through a No. 3 sieve (50 mesh) for later use.

[0055] Reagents: MRS broth medium (lot number 20210605, Qingdao Haibo Biotechnology Co., Ltd.), DMEM medium (lot number 12100-046, Thermo Fisher Scientific), fetal bovine serum (lot number 900-108, Thermo Fisher Scientific), PBS buffer (lot number PM5090-500, Beijing Coolbo Technology Co., Ltd.), dimethyl sulfoxide (lot number 67-68-5, Sigma-Aldrich, USA), trypsin (lot number SH30042.01, Thermo Fisher Scientific), penicillin-streptomycin Double antibody solution (lot number SV30010, Thermo Fisher Scientific), MTT powder (lot number 298-93-1, Beijing Solebow Technology Co., Ltd.), mouse interferon-γ ELISA kit (lot number F2182-A, Shanghai Fanke Industrial Co., Ltd.), mouse interleukin-1β ELISA kit (lot number F2040-A, Shanghai Fanke Industrial Co., Ltd.), mouse interleukin-4 ELISA kit (lot number F2165-A, Shanghai Fanke Industrial Co., Ltd.), mouse interleukin-6 ELISA kit (lot number F2163-A, Shanghai Fanke Industrial Co., Ltd.), mouse interleukin 10 ELISA kit (lot number F2176-A, Shanghai Fanke Industrial Co., Ltd.), mouse interleukin 12 ELISA kit (lot number F30084-A, Shanghai Fanke Industrial Co., Ltd.), mouse tumor necrosis factor α ELISA kit (lot number F2132-A, Shanghai Fanke Industrial Co., Ltd.), mouse tumor transforming growth factor β ELISA kit (lot number F2686-A, Shanghai Fanke Industrial Co., Ltd.), acetic acid (lot number 64- 19-7, Thermo Fisher Scientific), Propionic acid (Lot No. 79-09-4, Thermo Fisher Scientific), Butyric acid (Lot No. 107-92-6, Thermo Fisher Scientific), Isobutyric acid (Lot No. 79-31-2, Thermo Fisher Scientific), Valeric acid (Lot No. 109-52-4, Thermo Fisher Scientific), Isovaleric acid (Lot No. 503-74-2, Thermo Fisher Scientific), Hexanoic acid (Lot No. 142-62-1, Thermo Fisher Scientific), Isohexanoic acid (Lot No. 646-07-1, Thermo Fisher Scientific)

[0056] Instruments and equipment: electronic balance (model JA2003, Shanghai Hengping Scientific Instrument Co., Ltd.), vertical high-pressure steam autoclave (model LDZF-50L-Ⅲ, Shanghai Shen'an Medical Equipment Factory), vortex freezer grinder (model KZ-Ⅲ-FP, Wuhan Sewell Biotechnology Co., Ltd.), microplate reader (model Epoch2, Berton Instrument Co., Ltd., USA), clean bench (model SW-CJ-2FD, Suzhou Purification Equipment Co., Ltd.), cell culture incubator (model 51026280, Thermo Fisher Scientific, USA), cell counting chamber (model C070150, Cida (Guangzhou) Biotechnology Co., Ltd.), inverted fluorescence microscope (model ECLIPSE, Nikon, Japan), high-speed centrifuge (model sigma1-14, Sigma).

[0057] Cells and animals: (1) Cells: Lewis lung cancer cell line (LCC), purchased from Prosai Life Science Technology Co., Ltd.

[0058] (2) Animals: 40 SPF female C57BL / 6 mice, 4-5 weeks old, weighing 20±2 g, were purchased from Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. All mice were housed in an SPF clean area laboratory with free access to normal feed and sterile distilled water. The laboratory temperature was 22℃±2℃, the humidity was 35%-55%, and the circadian rhythm was 12h light / dark alternation. All animal experiments were conducted in accordance with the Regulations on Laboratory Animal Management and approved by the Ethics Committee of Lanzhou University (approval number: EAF2022035).

[0059] Example 1

[0060] Place the astragalus powder in a round-bottom flask, add 10 times the amount of sterile water and soak for 4 hours, then boil at 96°C for 1 hour, filter the liquid into a beaker with gauze, filter twice to obtain the residue and the first liquid;

[0061] Add 8 times the volume of sterile water to the residue and continue to boil at 96°C for 1 hour to obtain a second medicinal solution; combine the first medicinal solution and the second medicinal solution, concentrate to 0.5 g / mL, collect the supernatant by centrifugation and sterilize to obtain an astragalus water extract;

[0062] Lactobacillus plantarum LZU-S-ZCJ and Lactobacillus plantarum LZU-J-TSL6 were mixed at a ratio of 1:3 in terms of the number of live bacteria. The total number of live bacteria of Lactobacillus plantarum LZU-S-ZCJ and Lactobacillus plantarum LZU-J-TSL6 was 10 8CFU / mL, inoculated into the Astragalus water extract at a rate of 3%, fermented at 36°C for 36 h, and after centrifugation, the supernatant was sterilized to obtain the Astragalus fermentation liquid.

[0063] Example 2

[0064] Place the astragalus powder in a round-bottom flask, add 8 times the amount of sterile water and soak for 3 hours, then boil at 94°C for 0.5 hours, filter the liquid into a beaker with gauze, filter twice to obtain the residue and the first liquid;

[0065] Add 6 times the volume of sterile water to the residue and continue to boil at 94°C for 0.5h to obtain a second medicinal solution; combine the first medicinal solution and the second medicinal solution, concentrate to 0.3g / mL, collect the supernatant by centrifugation and sterilize to obtain an astragalus water extract;

[0066] Lactobacillus plantarum LZU-S-ZCJ and Lactobacillus plantarum LZU-J-TSL6 were mixed at a ratio of 1:2 in terms of the number of live bacteria. The total number of live bacteria of Lactobacillus plantarum LZU-S-ZCJ and Lactobacillus plantarum LZU-J-TSL6 was 10 6 CFU / mL, inoculated into the Astragalus water extract at a 2% inoculation rate, fermented at 35°C for 35h, and after centrifugation, the supernatant was sterilized to obtain the Astragalus fermentation liquid.

[0067] Example 3

[0068] Place the astragalus powder in a round-bottom flask, add 12 times the amount of sterile water and soak for 5 hours, then boil at 97°C for 1.5 hours, filter the liquid into a beaker with gauze, filter twice to obtain the residue and the first liquid;

[0069] Add 10 times the volume of sterile water to the residue and continue to boil at 97°C for 1.5 hours to obtain a second medicinal solution; combine the first medicinal solution and the second medicinal solution, concentrate to 0.8 g / mL, collect the supernatant by centrifugation and sterilize to obtain an astragalus water extract;

[0070] Lactobacillus plantarum LZU-S-ZCJ and Lactobacillus plantarum LZU-J-TSL6 were mixed at a ratio of 1:4 in terms of the number of live bacteria. The total number of live bacteria of Lactobacillus plantarum LZU-S-ZCJ and Lactobacillus plantarum LZU-J-TSL6 was 10 10CFU / mL, inoculated into the Astragalus water extract at a 4% inoculation rate, fermented at 38°C for 40h, and sterilized the supernatant after centrifugation to obtain the Astragalus fermentation liquid.

[0071] Experimental Example 1

[0072] Effects of Astragalus Fermentation Broth on Body Weight, Tumor Volume and Tumor Inhibition Rate in Mice

[0073] 1. Culture of lung cancer cells

[0074] Lewis lung carcinoma, LLC cell line was revived and cultured in complete DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO. 2 , and cultured continuously in an incubator with saturated humidity.

[0075] 2. Animal Experiment Grouping and Intervention

[0076] Lewis lung cancer cells in the logarithmic growth phase were digested and centrifuged, resuspended in sterile PBS, counted, and the cell concentration was adjusted to 2×10 6 40 female C57BL / 6 mice aged 4-5 weeks and weighing 18g-22g were raised in the SPF animal room of Lanzhou University. Modeling was performed after one week of adaptation, and 200μL of evenly dispersed Lewis lung cancer cell suspension was inoculated subcutaneously into the right armpit of the mice. Within 24h after the modeling, each group of mice was given the corresponding drug.

[0077] The groups were as follows: ① normal group (group C), subcutaneous injection of sterile saline and continuous gavage of 200 μL sterile saline for 21 days;

[0078] ② Model group (M group), subcutaneous injection of lung cancer cells, and continuous gavage of 200 μL sterile saline for 21 days;

[0079] ③ The group of Astragalus membranaceus aqueous extract prepared in Example 1 (Group A) was subcutaneously injected with lung cancer cells and continuously gavaged with 200 μL of sterile Astragalus membranaceus aqueous extract for 21 days, which is equivalent to 5 g / kg / day of Astragalus membranaceus raw drug according to the recommendation of Chinese Pharmacopoeia;

[0080] ④ The Astragalus fermented liquid group (FA group) prepared in Example 1 was injected subcutaneously with lung cancer cells and continuously gavaged with 200 μL of sterile Astragalus fermented liquid for 21 days;

[0081] ⑤ Positive drug group (P group): lung cancer cells were injected subcutaneously, and the dosage was calculated according to the body surface area of ​​humans and animals. Cisplatin 5 mg / kg was injected intraperitoneally once every 7 days.

[0082] (1) Observe the condition of the mice 24 hours after the last administration.

[0083] (2) 24 hours after the last administration, mice were killed by spinal dislocation, tumor tissues were aseptically removed, and tumor volume and tumor inhibition rate were measured. Animal experiments passed the experimental animal ethics review of Lanzhou University (review number: EAF2023057).

[0084] Tumor volume = (length × width 2 ) / 2;

[0085] Tumor inhibition rate = (tumor weight of model group - tumor weight of drug-treated group) / average tumor weight of model group × 100%;

[0086] (3) Measure the weight changes of mice 24 h after the last administration;

[0087] Body weight change = body weight at sacrifice - body weight at first administration;

[0088] Effective body weight = body weight at sacrifice - tumor weight;

[0089] Effective body weight change = body weight at sacrifice - tumor weight - body weight at first administration;

[0090] (4) Statistical analysis

[0091] Data are expressed as mean ± standard deviation. All experiments were independent and repeated at least 3 times. One-way ANOVA was used to compare the statistical significance of data, followed by Dunnett-t test or t test using GraphPad Prism 8.0. P < 0.05 was considered statistically significant.

[0092] Experimental results: After modeling, the status of mice in each group was different. The size of the axillary tumors in the model group mice gradually increased, and some mice had ulcerated tumors. The increase in tumor size in the model group mice caused difficulty in movement, accompanied by decreased body temperature, drowsiness, malaise, hair loss, etc. After the intervention of fermented astragalus, the tumor burden was significantly smaller than that in the model group, and the mice were able to move freely, with normal body temperature and shiny fur.

[0093] like Figure 1 As shown in A, the tumor volume of mice in the model group was larger, with an average tumor weight of 5.39±0.42g. The tumor volume of mice in the fermented astragalus group and the positive drug group was similar, with tumor weights of 2.85±0.50g and 2.94±0.41g, respectively, which were significantly smaller than those in the model group (P<0.0001). The tumor weight of mice in the astragalus water extract group was 4.78±0.50g, which was smaller than that in the model group, but there was no statistical difference ( Figure 1 B).

[0094] like Figure 1As shown in Figure C, the inhibition rate of Astragalus water extract on mouse tumors is 11.4%, the inhibition rate of Astragalus fermentation liquid on mouse tumors is 47.2%, and the inhibition rate of cisplatin on mouse tumors is 45.6%. Astragalus fermentation liquid has a significant anti-tumor effect compared with Astragalus water extract.

[0095] like Figure 1 As shown in D to F in the figure, the weight change, effective weight and effective weight change were calculated according to the initial weight, weight at sacrifice and tumor weight of each group of mice. The weight of mice in each group increased compared with the initial weight, especially the weight of mice in the model group was affected by tumor, and the weight increase was extremely significant (P<0.0001). After deducting the tumor weight, the effective weight of mice in the Astragalus fermented liquid group was higher than that of other drug-treated groups and close to that of the normal group. The results of effective weight change showed that the actual weight of mice in the model group and the Astragalus water extract group decreased, while the Astragalus fermented liquid and cisplatin could increase the weight of mice with lung cancer.

[0096] Experimental Example 2

[0097] Effect of Astragalus Fermentation Liquid on Pathological Morphology of Tumors in Mice with Lung Cancer

[0098] The tumor tissues of mice were fixed with 4% paraformaldehyde, embedded in paraffin, sliced, dewaxed, stained with hematoxylin and eosin, dehydrated and sealed, and the morphological changes of the tumor tissues were observed under a light microscope.

[0099] Experimental results: Figure 2 As shown in the figure. In the model group, tumor cells were densely arranged with normal morphology, the nuclei were deeply stained and unevenly distributed, nuclear division was common, and no obvious necrosis was observed in the cells. Compared with the model group, the tumor cells in the Astragalus water extract group were relatively loosely arranged, with different morphologies, reduced density, and more obvious immune cell infiltration. The tumor cells in the Astragalus fermentation liquid group and the positive drug group were further loosely arranged, with a large number of tumor cells necrotizing, further increased cavitation, and nuclear fragmentation.

[0100] Experimental Example 3

[0101] Effects of Astragalus Fermentation Liquid on Liver and Kidney of Lung Cancer Mice

[0102] (1) Organ index

[0103] The liver and kidney of the mouse were completely removed, and the organ index was calculated, organ index = organ index (mg) / mouse body weight (g) × 100%.

[0104] (2) Observation of pathological morphology of liver and kidney of lung cancer mice. For specific methods, see Experimental Example 2.

[0105] (3) Statistical analysis See Experimental Example 2.

[0106] Experimental results: Figure 3As shown in A and B in Figure 2, compared with the normal group, the liver and kidney indexes of the model group increased significantly, reflecting that the liver and kidney tissues of Lewis lung cancer mice were damaged to a certain extent. Compared with the model group, administration of Astragalus fermented liquid and Astragalus water extract had a certain reversal effect on the damage to liver and kidney tissues, while the positive drug cisplatin had no such effect.

[0107] From the results of liver tissue morphology ( Figure 3 C), the liver tissue structure of mice in the normal group was normal, the liver cells were arranged tightly and orderly, small in size, and no cell swelling occurred. The liver cells of mice in the model group were severely swollen and arranged loosely and disorderly. The liver cells of mice in the Astragalus fermented liquid group were the most normal compared to those in other drug-treated groups, arranged neatly and uniformly, and no swollen cells were found. From the results of renal tissue morphology, the renal tissue structure of mice in the normal group was complete and arranged regularly. Pathological changes occurred in the renal tissue of mice in the model group, with epithelial cells shedding, obvious widening of the intercellular gap, and disorderly arrangement of cells. Compared with the model group, the pathological changes in the renal tissue of mice in the Astragalus fermented liquid group were alleviated, the epithelial cells were arranged more regularly and tightly, the intercellular gaps were reduced, and the inflammatory infiltration was improved.

[0108] Experimental Example 4

[0109] Effects of Astragalus Fermentation Broth on Immune Organs and Immune Cytokines in Mice with Lung Cancer

[0110] (1) Organ index

[0111] The thymus and spleen of the mouse were completely removed, and the organ index was calculated, organ index = organ index (mg) / mouse body weight (g) × 100%.

[0112] (2) Observation of pathological morphology of spleen in mice with lung cancer. For specific methods, see Experimental Example 2.

[0113] (3) Cytokine assay

[0114] The ELISA method was used to detect the levels of serum cytokines IL-6, IL-12, IL-1β, TNF-α, TGF-β and IFN-γ in each group of mice. The specific operation was subject to the instructions of the kit.

[0115] (4) Statistical analysis See Experimental Example 2.

[0116] Experimental results: The spleen index and thymus index were used to evaluate the overall effect of fermented astragalus on the immune system. Figure 4As shown in A and B in Figure 2, compared with the normal group, the spleen index of the model group was significantly higher than that of the normal group, and the thymus index was significantly lower than that of the control group (P < 0.0001), indicating that the immune system of lung cancer mice was impaired. Compared with the model group, the spleen index of the fermented astragalus liquid group was significantly reduced, and the thymus index was significantly increased (P < 0.0001), indicating that fermented astragalus may reverse the damage to immune organs caused by modeling.

[0117] Spleen tissue sections of mice in each group were Figure 4 As shown in C. The spleen tissue structure of mice in the normal group was intact, the boundary between the red pulp and the white pulp was clear, and no pathological structure was found. Compared with the normal group, the number of lymphocytes in the model group was significantly decreased, lysed and necrotic cells appeared, and the boundary between the red pulp and the white pulp was unclear. The spleen tissue structure integrity of mice in the Astragalus fermented liquid group was relatively good, the boundary between the red and white pulp was clear, and there were fewer lysed and necrotic cells, which was similar to the positive drug group and better than the Astragalus water extract group.

[0118] The immune function of lung cancer mice was further evaluated using immune cytokines. Figure 4 As shown in D to I in Figure 2, compared with the normal group, the levels of IL-1β, TNF-α, IFN-γ, and TGF-β in the model group increased, and the level of IL-12 decreased (P < 0.05). Compared with the model group, the levels of IL-1β, TNF-α, IFN-γ, TGF-β, and IL-6 in the Astragalus fermentation liquid group showed a downward trend, and the level of IL-12 showed an increasing trend. In particular, compared with the Astragalus water extract group, the secretion level of TGF-β in the Astragalus fermentation liquid group was extremely significantly reduced (P < 0.01), and the secretion level of IL-12 was significantly increased (P < 0.05). The immune cytokine regulation of mice in the Astragalus fermentation liquid group, the Astragalus water extract group, and the positive drug group was inconsistent, which may be due to the fact that the immune regulation of cisplatin on lung cancer mice is different from that of Astragalus fermentation liquid and Astragalus water extract.

[0119] Experimental Example 5

[0120] Effects of Astragalus Fermentation Broth on Intestinal Microflora in Mice with Lung Cancer

[0121] Intestinal flora analysis: Mouse fecal samples were sent to Suzhou Panomik Biomedical Technology Co., Ltd. for 16SrRNA gene sequencing. Total genomic DNA was extracted from the samples, and the DNA concentration and integrity were evaluated using Nanodrop and 1.2% agarose gel electrophoresis, respectively. According to the concentration, the DNA was diluted to 1 μg / mL with sterile water. The bacterial 16SrRNA V4 variable region was amplified (PCR primers: 515F 5′-ACTCCTACGGGAGGCAGCA-3′ (SEQ ID NO: 1), 806R 5′-GGACTACHVGGGTWTCTAAT-3′ (SEQ ID NO: 2). After PCR amplification of the target fragment, the PCR amplification recovery product was subjected to fluorescence quantification. The sequencing library was prepared using the Ion Plus Fragment Library Kit. The library was quantified using the Quant-iT PicoGreendsDNA Assay Kit on the Promega QuantiFluor fluorescence quantification system.

[0122] Experimental results: Figure 5 Compared with normal mice, it was found that the number of Bacteroidetes and Actinobacteria in the intestines of lung cancer mice increased, and the number of Firmicutes and the ratio of Firmicutes to Bacteroidetes F / B decreased ( Figure 5 A and D in the figure). Correspondingly, at the genus level, it was found that the Allobaculum genus under the phylum Firmicutes in the intestines of mice with lung cancer decreased, and the Bacteroides genus under the phylum Bacteroidetes and the Prevotella genus under the phylum Actinobacteria increased. After supplementing with Astragalus water extract and Astragalus fermentation liquid, it was found that the two had similar effects on the intestinal flora. Both Astragalus water extract and Astragalus fermentation liquid can increase F / B and Turicibacter genus in the intestines of mice with lung cancer, and reduce Prevotella genus. However, compared with the Astragalus water extract group, the effect of Astragalus fermentation liquid on increasing F / B and reducing Prevotella genus was more obvious, and there was a significant difference (P < 0.05, Figure 5 In addition, there were differences in the regulation of intestinal flora between the two groups. Compared with the model group, the intestinal actinomycetes of mice in the astragalus water extract group decreased, the Oscillospira genus increased, and the Lactobacillus, Bifidobacterium, Bacteroides and Akkermansia genera remained unchanged, while the actinomycetes, Lactobacillus, Bifidobacterium, and Akkermansia genera increased in the astragalus fermentation liquid group, and the Oscillospira and Bacteroides genera decreased ( Figure 5D in the figure). Based on the analysis of the specific intestinal bacteria of mice in the Astragalus fermentation liquid group and mice with lung cancer by Lefse, it was found that Akkermansia was a specific genus enriched in the intestines of mice supplemented with Astragalus fermentation liquid. In summary, Astragalus fermentation liquid regulates the intestinal flora of mice with lung cancer in a way different from Astragalus water extract, reduces harmful bacteria or potential pathogens such as Prevotella, Oscillospira, and Bacteroides, increases beneficial bacteria such as Lactobacillus, Bifidobacterium, and Turicibacter, and specifically enriches Akkermansia bacteria to improve mouse lung cancer.

[0123] Experimental Example 6

[0124] Effect of Astragalus Fermentation Broth on the Production of Short-Chain Fatty Acids in Lung Cancer Mice

[0125] Analysis of short-chain fatty acids: The fecal sample was transferred into a 2 mL centrifuge tube and 50 μL of 15% phosphoric acid and 100 μL of 125 μg / mL isohexanoic acid solution were added. The mixture was homogenized with 400 μL of ether for 1 min and centrifuged at 12,000 rpm for 10 min at 4°C. The supernatant was used for determination. Short-chain fatty acids in feces were analyzed using TRACE 1310 gas chromatography (HP Innowax column: 30 cm × 0.25 mm × 0.25 μm) combined with ISQ LT mass spectrometry.

[0126] Experimental results: Short-chain fatty acids (SCFAs) are important metabolites produced by intestinal bacterial fermentation of dietary fiber, mainly including acetic acid, propionic acid, butyric acid, etc. Many intestinal microorganisms are related to the production of SCFAs, which are of great significance to the prevention and treatment of lung cancer and human health. Therefore, the study detected the content of 6 SCFAs in the feces of each group of mice. Normally, acetic acid, propionic acid, and butyric acid are the three main SCFAs that can be detected in the intestines and feces.

[0127] Depend on Figure 6 It can be seen that compared with normal mice, the contents of acetic acid, propionic acid, butyric acid, isobutyric acid, and valeric acid in the fecal metabolites of lung cancer model mice were significantly decreased (P≤0.05), and the content of isovaleric acid showed a downward trend. Compared with the model group, supplementation of Astragalus fermentation broth can significantly increase the contents of acetic acid and valeric acid in the fecal metabolites of mice (P≤0.05), suggesting that there may be a potential connection between the effect of Astragalus fermentation broth and these two SCFAs. In addition, the contents of propionic acid, butyric acid, isobutyric acid, and isovaleric acid in the Astragalus fermentation broth group also showed an upward trend. In general, the ability of Astragalus fermentation broth to regulate the biosynthesis of SCFAs is ideal, and is superior to Astragalus water extract and cisplatin.

[0128] Experimental Example 7

[0129] Cell inhibition rate determination

[0130] LLC cells in the logarithmic growth phase were selected and 100 μL of single cell suspension (5×10 4 / mL) were inoculated in a 96-well cell culture plate and incubated overnight until the cell monolayer adhered to the bottom of the well. The culture medium was removed, and the Astragalus aqueous solution (Group A) prepared in Example 1 and the Astragalus fermentation broth (Group FA) prepared in Example 1 (0, 20, 40, 80 μg / mL) were added in gradient concentrations, respectively, and 200 μL of complete culture medium was added to each well. After incubation in an incubator for 48 hours, 20 μL of freshly prepared MTT solution (5 mg / mL) was added to each well, and the culture was terminated after incubation for 4 hours. The supernatant was discarded, 150 μL DMSO was added to each well, oscillated at a low speed for 10 minutes, and allowed to stand for 5 minutes to completely dissolve the blue-purple formazan crystals. The OD of each well was measured. 490 , and calculated the cell inhibition rate.

[0131] Experimental results: Figure 7 As shown. After 48 hours of co-culture with LLC cells, different concentrations of Astragalus fermented broth had a certain direct inhibitory effect on LLC cells, and the cell proliferation rates were 80.58%, 72.05%, and 69.02% (P≤0.05), respectively. Compared with Astragalus water extract, Astragalus fermented broth had a stronger inhibitory effect on LLC cell proliferation, especially at a high concentration of 80μg / mL, there was a significant difference between the two (P≤0.05).

[0132] As can be seen from the above embodiments and experimental examples, the Astragalus fermented liquid prepared by the present invention can be applied to the prevention and treatment of lung cancer, can reduce tumors, increase body weight, and regulate immunity, and its effect is significantly better than that of the Astragalus water extract, and is equivalent to the positive drug cisplatin. Astragalus fermented liquid can also protect liver and kidney damage in lung cancer, while cisplatin has no such effect. In addition, Astragalus fermented liquid has the effect of regulating intestinal flora and promoting the production of short-chain fatty acids.

[0133] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of Astragalus fermentation broth in the preparation of drugs for the prevention and treatment of lung cancer.

2. Application of Astragalus fermented liquid in the preparation of drugs for reversing liver and kidney tissue damage.

3. Application of Astragalus fermented liquid in the preparation of drugs for reversing immune organ damage.

4. The use of Astragalus fermented liquid in the preparation of a drug for regulating intestinal flora, characterized in that: The astragalus fermented liquid reduces harmful bacteria in the intestinal flora, increases beneficial bacteria in the intestinal flora, and enriches specific bacteria in the intestinal flora.

5. The use of astragalus fermented liquid in the preparation of functional food that helps regulate intestinal flora, characterized in that: The astragalus fermented liquid reduces harmful bacteria in the intestinal flora, increases beneficial bacteria in the intestinal flora, and enriches specific bacteria in the intestinal flora.

6. The use according to claim 4 or 5, characterized in that: The harmful bacterial genera include Prevotella, Oscillospira, and Bacteroides, the beneficial bacterial genera include Lactobacillus, Bifidobacterium, and Turicibacter, and the specific bacterial genera include Akkermansia.

7. Use of Astragalus fermentation broth in the preparation of a drug for promoting the synthesis of short-chain fatty acids, characterized in that: The short-chain fatty acids include acetic acid, valeric acid, propionic acid, butyric acid, isobutyric acid or isovaleric acid.

8. The use according to any one of claims 1 to 7, characterized in that: The preparation method of the astragalus fermentation liquid comprises the following steps: 1) Soaking the astragalus root in water, boiling it, filtering it, and obtaining the residue and the first medicinal liquid; 2) adding water to the medicinal residue and boiling to obtain a second medicinal solution; 3) combining the first medicinal solution and the second medicinal solution, concentrating them, and obtaining an astragalus water extract; 4) Lactobacillus plantarum LZU-S-ZCJ and Lactobacillus plantarum LZU-J-TSL6 are mixed, and the mixture is inoculated into the astragalus water extract for fermentation to obtain an astragalus fermentation liquid.

9. The use according to claim 8, characterized in that: Step 1) The mass ratio of the astragalus to water is 1:8-12, the soaking time is 3-5 hours, the decoction temperature is 94-97° C., the decoction time is 0.5-1.5 hours, and the filtering times are 1-3 times; Step 2) The mass ratio of the medicinal residue to water is 1:6-10, the decoction temperature is 94-97° C., and the decoction time is 0.5-1.5 h.

10. The use according to claim 8, characterized in that: Step 3) Concentrating the drug to a mass of 0.3 to 0.8 g of the original drug per milliliter of the drug solution; Step 4) The total viable count of Lactobacillus plantarum LZU-S-ZCJ and Lactobacillus plantarum LZU-J-TSL6 is 10 6 ~10 10 CFU / mL, the ratio of the number of viable bacteria of Lactobacillus plantarum LZU-S-ZCJ to that of Lactobacillus plantarum LZU-J-TSL6 is 1:2-4; The inoculation amount of the mixed bacteria is 2-4% of the volume of the astragalus water extract; The fermentation temperature is 35-38° C., and the fermentation time is 35-40 hours.

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