Composition for improving fatigue resistance of intestinal tract combined with lactobacillus and application

Abstract

The invention discloses a composition for improving fatigue resistance of intestinal combined lactobacillus and application, and belongs to the technical field of biological medicine. The traditional Chinese medicine composition comprises the following raw materials in parts by weight: 2-6 parts of Chinese yam, 0.5-3 parts of pulp of dogwood fruit, 1.5-4 parts of prepared rehmannia root, 1.5-4 parts of poria cocos, 0.5-3 parts of moutan bark and 0.5-4 parts of rhizoma alismatis. The composition can reduce the content of blood lactic acid and improve the liver glycogen reserve level, so that the effects of resisting and relieving fatigue can be achieved; the composition disclosed by the invention can be used for improving the abundance of beneficial fungi Saccharomyces in the intestinal tract and improving the abundance of combined bacillus in the intestinal tract.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine. Background Art

[0002] Physical fatigue refers to the temporary decline in body functions that occurs after continuous or high-intensity physical activities. Its occurrence is closely related to energy metabolism disorders, oxidative stress damage, and neuro-muscular regulation imbalance. In traditional Chinese medicine, it mostly belongs to the categories of "fatigue due to overwork", "consumptive fatigue", "five kinds of fatigue", etc. Its theoretical system can be traced back to "Huangdi Neijing". Traditional Chinese medicine believes that physical fatigue is a syndrome of "deficiency in origin and excess in superficiality", with the three viscera of the spleen, liver, and kidney as the core. Physical fatigue has now become a common health problem in society, especially more prominent among people with high-pressure jobs and those who sit in the office for a long time. Data shows that 75% of people globally are in a sub-healthy state, and more than 80% of Chinese working people suffer from fatigue. Currently, Western medicine is the main means to relieve physical fatigue, but adverse reactions often occur during the treatment process. As a representative of traditional medicine, traditional Chinese medicine has fewer adverse reactions compared to Western medicine, and thus has received increasing attention.

[0003] Combined Lactobacillus can exist in the human intestine and has the functions of regulating the intestinal flora, promoting digestion, protecting the gastrointestinal mucosa, and relieving constipation for the body. Lactobacillus can regulate the dysbacteriosis, promote the growth and proliferation of beneficial bacteria in the intestine, inhibit harmful bacteria in the intestine, and play a role in protecting the stability of the intestinal flora; it can also promote digestion. Combined Lactobacillus can inhibit the growth and reproduction of harmful bacteria in the digestive tract, reduce or avoid the stimulation and damage of harmful bacteria to the gastrointestinal mucosa. At the same time, combined Lactobacillus can enhance the body's immune defense ability by regulating the intestinal immune system, stimulating the activity of immune cells, and helping the body resist the invasion of pathogens.

[0004] When the number of combined intestinal Bacillus is low, the number of Lactobacillus can be increased by improving eating habits. While increasing the intake of foods rich in prebiotics, the intake of high-sugar and processed foods should be reduced, as these foods may disrupt the balance of the intestinal flora. It is also possible to consider using probiotic supplements under the guidance of a doctor to increase the number of combined Lactobacillus. Common probiotic drugs include Bifidobacterium, Lactobacillus acidophilus, and Lactobacillus rhamnosus, etc. These drugs can help restore the balance of the intestinal flora. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a composition and application for improving the anti-fatigue effect of combined intestinal Lactobacillus. The composition of the present invention can reduce the blood lactic acid content and increase the hepatic glycogen reserve level, thereby playing a role in resisting and relieving fatigue; the composition of the present invention can also increase the abundance of the beneficial intestinal fungus Saccharomyces and the abundance of combined intestinal Bacillus.

[0006] To solve the above technical problems, the technical solution adopted by the present invention is as follows: A composition for enhancing the anti-fatigue effect of Lactobacillus intestinalis consortium, the raw materials of which are composed of the following substances by weight: 2-6 parts of Chinese yam, 0.5-3 parts of dogwood fruit, 1.5-4 parts of prepared rehmannia root, 1.5-4 parts of poria cocos, 0.5-3 parts of moutan bark, 0.5-4 parts of alisma rhizome.

[0007] Preferably, the raw materials are composed of the following substances by weight: 3-5 parts of Chinese yam, 1-2 parts of dogwood fruit, 2-3 parts of prepared rehmannia root, 2-3 parts of poria cocos, 1-2 parts of moutan bark, 1-2 parts of alisma rhizome.

[0008] More preferably, the raw materials are composed of the following substances by weight: 4.28 parts of Chinese yam, 1.71 parts of dogwood fruit, 2.14 parts of prepared rehmannia root, 2.14 parts of poria cocos, 1.71 parts of moutan bark, 1.42 parts of alisma rhizome.

[0009] The above-mentioned Lactobacillus intestinalis consortium includes Lactobacillus acidophilus ( Lactobacillus acidophilus ), Lactobacillus plantarum ( Lactobacillus plantarum ), Helicobacter pylori ( Lactobacillus helveticus ).

[0010] The present invention also provides the application of the above composition in the preparation of a preparation for increasing the abundance of Lactobacillus intestinalis consortium.

[0011] The present invention also provides the application of the above composition in the preparation of a preparation for resisting and alleviating fatigue; after administering the preparation, the blood lactic acid content decreases and the hepatic glycogen reserve level increases.

[0012] The present invention also provides the application of the above composition in the preparation of a preparation for increasing the abundance of the beneficial intestinal fungus Saccharomyces.

[0013] The present invention also provides the application of the above composition in the preparation of a preparation for enhancing the richness, comprehensive diversity of normal intestinal bacterial species and changing the intestinal microbial community.

[0014] The present invention also provides the application of the above composition in the preparation of a preparation for improving intestinal barrier function and enhancing intestinal immunity; after administering the above preparation, the number of intestinal goblet cells increases and the expression levels of Occludin and ZO-1 proteins in the colon tissue increase.

[0015] The beneficial effects produced by adopting the above technical solutions are as follows: The composition of the present invention can reduce the blood lactic acid content and increase the hepatic glycogen reserve level, thereby playing a role in resisting and alleviating fatigue; the composition of the present invention can increase the abundance of the beneficial intestinal fungus Saccharomyces and the abundance of Lactobacillus intestinalis consortium; it can also enhance the richness, comprehensive diversity of normal intestinal bacterial species and change the intestinal microbial community; improve intestinal barrier function and enhance intestinal immunity. Description of the Drawings

[0016] Figure 1-4 It is the anti - fatigue effect of the B - group formula on mice in each group; (1) Swimming time; (2) Liver glycogen; (3) Lactic acid; (4) Serum urea nitrogen (BUN). The data are shown as mean ± standard deviation. Asterisks ( ) represent p < 0.05.

[0017] Figure 5-9 It is the effect of the B - group formula on the intestinal flora; (5) pCoA plot; (6) Alpha - diversity analysis; (7) Rarefaction curve; (8) Phylum - level plot; (9) Genus - level plot; In the figure, Z represents the normal group, LI represents the B - group, and T represents the positive - control taurine group.

[0018] Figure 10-14 It is the effect of the B - group formula on intestinal fungi; (10) pCoA plot; (11) Rarefaction curve; (12) Alpha - diversity analysis; (13) Phylum - level plot; (14) Genus - level heatmap; In the figure, Z represents the normal group, LI represents the B - group, and T represents the positive - control taurine group.

[0019] Figure 15-18 It is the effect of the B - group formula on the intestinal barrier; (15) H&E staining; (16) PAS staining and the number of goblet cells; (17 - 18) Immunohistochemical detection of the expression of ZO - 1 and Occludin. Detailed Implementation Modes Example 1

[0020] The formula of this example contains 6 herbs: Chinese yam, cornel, rehmannia root, poria, moutan bark, and alisma rhizome. The raw materials are provided by Beijing Tongrentang. The adult dosage of the raw medicinal materials is converted to the mouse dosage according to the body - surface - area algorithm. The converted dosages from the adult dose are: Chinese yam 4.28 g / kg, cornel 1.71 g / kg, rehmannia root 2.14 g / kg, poria 2.14 g / kg, moutan bark 1.71 g / kg, and alisma rhizome 1.42 g / kg (the following B - group formula).

[0021] Materials and Methods 1 Materials 1.1 Experimental Animals Male C57BL / 6 mice, 6 weeks old, weighing (18±2) g, were purchased from Beijing Speywood Biotechnology Co., Ltd., with the license number: SCXK(Beijing)2024-0001. After purchase, they were adaptively raised in the Experimental Animal Center of Shanxi University of Traditional Chinese Medicine. All animals were placed in an SPF-level constant temperature (22-24°C) environment with a 12 / 12 h light-dark cycle, and had free access to food and water. This study was approved by the Animal Experiment Ethics Committee of Shanxi University of Traditional Chinese Medicine.

[0022] 1.2 Experimental drugs Taurine (batch number: C15550690); Chinese yam, fructus corni, rehmannia glutinosa, poria cocos, cortex moutan, and alisma orientale (all purchased from Beijing Tongrentang).

[0023] 1.3 Main instruments and equipment High-speed refrigerated centrifuge, lactate analyzer, fluorescence quantitative PCR instrument, multi-functional microplate reader, swimming tank, electronic balance, automatic biochemical analyzer, water bath.

[0024] 1.4 Experimental reagents Blood urea nitrogen (BUN) assay kit, lactic acid (LA) assay kit, hepatic glycogen assay kit.

[0025] 2. Methods 2.1 Animal grouping Male C57BL / 6 mice were randomly divided into 3 groups: blank control group (CON group, sterile water), positive control group (Tau group, taurine 0.426 g / kg), and B formula group (B group), with 20 mice in each group. The mice were adaptively fed for 1 week before the experiment. The CON group was intragastrically administered 0.1 mL of water every day, the B formula group was intragastrically administered the B formula, and the Tau group was intragastrically administered 0.426 g / kg of taurine every day. The intragastric administration was continued for 30 days. The body weight was measured every 3 days, and the intragastric administration volume was adjusted according to the change in body weight.

[0026] 2.2 Preparation of B formula The formula contains 6 herbs: Chinese yam, fructus corni, rehmannia glutinosa, poria cocos, cortex moutan, and alisma orientale. The raw materials were provided by Beijing Tongrentang. The adult dosage of the raw medicinal materials was converted to the mouse dosage according to the body surface area algorithm. The converted dosage for the B formula group was: Chinese yam 4.28 g / kg, fructus corni 1.71 g / kg, rehmannia glutinosa 2.14 g / kg, poria cocos 2.14 g / kg, cortex moutan 1.71 g / kg, and alisma orientale 1.42 g / kg. The above dosages were the daily dosages per mouse.

[0027] 2.3.1 Mouse load-bearing swimming experiment The load-bearing swimming experiment is an animal experiment model commonly used to evaluate anti-fatigue ability. The length of the swimming time directly reflects the endurance of the animal. The longer the swimming time, the stronger the anti-fatigue ability. 30 minutes after the last administration of the test sample, mice with a 5% body weight lead sheet attached to the root of the tail were placed in a swimming tank to swim. The water depth was not less than 30 cm, and the water temperature was 25°C ± 1.0°C. Record the time from the start of swimming to the death of the mouse, that is, the load-bearing swimming time of the mouse.

[0028] 2.3.2 Determination of serum urea nitrogen 30 minutes after the last administration of the test sample, swim in water at 30°C without load for 90 minutes, and collect blood after resting for 60 minutes. For rats, collect tail blood, and for mice, collect about 0.5 mL of whole blood by removing the eyeball (without adding anticoagulant). Place it in a 4°C refrigerator for about 3 hours. After the blood coagulates, centrifuge at 2000 r / min for 15 minutes, and take the serum for standby. Determine it by the diacetyl-oxime method.

[0029] 2.3.3 Determination of liver glycogen Euthanize the animals 30 minutes after the last dosing. Take the liver, rinse it with physiological saline, dry it with filter paper, weigh the liver, and detect the liver glycogen content according to the instructions of the glycogen content detection kit (BC0345).

[0030] 2.3.4 Determination of blood lactic acid Collect blood 30 minutes after the last dosing, then swim in water at 30°C without load for 10 minutes and stop. Before swimming, collect 20 μL of blood and add it to 40 μL of cell lysis solution, and immediately shake it vigorously to break the cells; immediately collect 20 μL of blood after swimming and add it to 40 μL of cell lysis solution and shake it; collect 20 μL of blood again after resting for 20 minutes and add it to 40 μL of cell lysis solution and shake it. Use a lactate analyzer to measure. For the self-prepared reagent measurement method, also collect 20 μL of blood at the above three time points and measure it with a lactate analyzer.

[0031] 2.3.6 Determination of intestinal flora After mice were raised for 4 weeks, collect feces and send them to Shanghai Personal Biotechnology Co., Ltd. for determination on dry ice. The rest of the data processing was carried out in the Bioinformatics Cloud of Shanghai Personal Biotechnology Co., Ltd. (https: / / www.genescloud.cn / ).

[0032] 2.3.7 HE staining to observe pathological morphological changes of the colon For the mice sacrificed in 2.3.3, quickly take part of the colon tissue and fix it in 4% paraformaldehyde. After dehydration, clearing, wax infiltration, and embedding to make wax blocks, use a microtome to cut 3 μm thin sections, perform hematoxylin-eosin (H&E) staining, and observe the pathological changes of the colon tissue.

[0033] 2.3.8 Periodic Acid-Schiff (PAS) Staining Perform PAS staining on goblet cells in the colon of mice. Observe and take images using a microscope.

[0034] 2.3.9 Immunohistochemistry (IHC) Stained Sections After dewaxing, hydration, antigen retrieval, primary antibody incubation, secondary antibody incubation, DAB color development, hematoxylin staining, and mounting, collect relevant parts of the samples and calculate the proportion of positive area.

[0035] 2.4 Data Processing The experimental data results were analyzed using IBM SPSS Statistics 27 statistical software, and graphs were made using GraphPad Prism 9 software. P<0.05 indicates significant differences in the data.

[0036] Experimental Results 1. Formula B Improves Physical Fatigue in Mice To evaluate the anti-fatigue effect of Formula B, we conducted a loaded swimming experiment. Compared with the CON group, the positive control Tau group taking taurine could significantly prolong the forced swimming time of mice. Meanwhile, Formula B increased the forced swimming time ( Figure 1 ). The average loaded swimming times of Formula B and Tau groups were 377 s and 444 s respectively, which were increased by 29.5% and 52.6% compared with the CON group ( Figure 1 ). These data indicate that Formula B has an anti-fatigue effect in the loaded swimming experiment of mice.

[0037] Compared with the CON group, the hepatic glycogen contents of the Tau group and Formula B group were significantly increased, and Formula B and Tau groups were increased by 39.5% and 40.9% respectively compared with the CON group ( Figure 2 ). There was no significant difference in BUN content between Formula B and CON groups (P>0.05), but it still showed a decrease ( Figure 3 ). Meanwhile, the blood LA content of Formula B group was significantly lower than that of the control group, and it was decreased by 28.03% compared with the CON group ( Figure 4 ).

[0038] 2. Formula B Improves the Structure of Intestinal Flora and Increases the Abundance of Lactobacillus conjugatus To further study the effect of Formula B on intestinal flora, we collected mouse fecal samples, extracted DNA and performed microbial diversity sequencing analysis on the 16S rRNA gene fragment. Among the Observed Species index and Chao1 index, Formula B group was higher than the CON group, indicating that the richness of normal intestinal bacterial species in the low-dose group of Formula B was increased ( Figure 5)。Beta diversity calculates the distance between samples using the evolutionary relationships and abundance information of the sequences of each sample to reflect whether there are significant differences in the microbial communities between samples. Principal Coordinates Analysis (PCoA) is a commonly used method for Beta diversity analysis. The results of Beta diversity analysis indicate that Group B changed the composition of the microbial community in the mouse intestine( Figure 6 );The flatness of the rarefaction curve reflects the influence of sequencing depth on the diversity of the observed samples. The flatter the curve, the more sufficient the sequencing results are to reflect the diversity contained in the current samples( Figure 7 )。The above results show that the formula of Group B can increase the richness and comprehensive diversity of normal intestinal bacterial species and change the intestinal microbial community.

[0039] After intervention with the formula of Group B, changes occurred in the intestinal flora of mice. At the phylum level, the F / B ratio (Firmicutes / Bacteroidota ratio) in Group B was up-regulated compared with the CON group( Figure 8 )。At the genus level, the relative abundance of Ligilactobacillus in Group B was up-regulated( Fig. 9 )。Some studies have shown that increasing the abundance of the intestinal microbiota Ligilactobacillus can improve energy metabolism, reduce oxidative stress and inflammation, regulate the balance of the intestinal flora, and inhibit the growth of pathogenic bacteria (such as Escherichia coli and Salmonella). After intervention with the formula of Group B, the relative abundance of Ligilactobacillus in the mouse intestine was up-regulated, which has a positive effect on intestinal health. It is speculated that the increase in its relative abundance may be related to the anti-fatigue effect of the formula of Group B.

[0040] 3. Effects of the formula of Group B on intestinal fungi The Chao1 index, Shannon index, Observed Species index, and Simpson index in Group B were higher than those in the CON group, indicating that the formula of Group B improved the richness and diversity of the intestinal fungal community in mice ( Fig.10 )。The results of Beta diversity analysis indicate that there was no obvious difference in the community composition of the intestinal fungal community between Group B and the CON group in mice( Fig.11 )。The rarefaction curve shows that the sequencing results are sufficient to reflect the diversity contained in the current samples( Fig.12 )。Changes occurred in the intestinal fungal community in Group B compared with the CON group. At the phylum level, the proportion of Mucoromycota in Group B was increased( Fig.13 );At the genus level, Group B increased the abundance of the beneficial fungus Saccharomyces( Fig.14 )。Saccharomyces is a commonly used probiotic in clinical practice and has the functions of anti-infection and enhancing the intestinal barrier.

[0041] 4. The formula of Group B improves the intestinal barrier After clarifying the changes in the intestinal flora composition of different groups of mice, in order to further explore the effects of Formula B on the intestinal tissue morphology and related functions of mice, this study performed hematoxylin-eosin (H&E) staining and periodic acid-Schiff (PAS) staining analysis on the intestines of mice. The intestinal tissue structure of each group of mice was intact, the boundaries between the tissues of each layer were clear, and the mucosa layer had abundant glands, and there were no pathological changes (Appendix Fig.15 ). Compared with the CON group, the number of goblet cells in the intestines of mice in the B group increased (Appendix Fig.16 ). The increase in the number of goblet cells is usually regarded as an important indicator of the improvement of intestinal barrier function, suggesting that Formula B may have a repair or enhancement effect on intestinal barrier function. To further explore the protective effect of Formula B on the intestinal mucosal barrier of mice, this study used immunohistochemistry to detect the expression of ZO-1 and Occludin in the colon tissue. Compared with the CON group, the protein expression of Occludin and ZO-1 in the colon tissue of the B group was significantly increased, suggesting that Formula B can enhance the intestinal barrier function of mice and improve intestinal immunity. (Appendix Figure 17-18 ).

[0042] Conclusion The experimental results of this study found that Formula B can extend the load-bearing swimming time of mice, reduce the blood lactic acid content, increase the liver glycogen reserve level of mice, and enhance the fatigue tolerance of the body.

[0043] Formula B regulates the intestinal flora and increases the abundance of the beneficial bacterium Lactobacillus. Formula B can regulate the intestinal fungal community and increase the abundance of the beneficial fungus Saccharomyces.

[0044] The main mechanisms of body fatigue generation mainly include the metabolite accumulation theory, the energy consumption theory, and the free radical theory. During anaerobic respiration of cells, lactic acid is produced during sugar metabolism, and ammonia is produced when protein metabolism hydrolysis is strengthened. It forms urea through the hepatic circulation and finally forms BUN. The more these two products, blood lactic acid and BUN, accumulate in the body, the easier it is to produce fatigue. The experimental results of this invention found that Formula B can reduce the blood lactic acid content and improve the fatigue tolerance of the body. According to the "energy consumption theory", at the beginning of exercise, glucose in cells is consumed, blood sugar decreases, and liver glycogen is quickly broken down into glucose and released into the blood to maintain blood sugar stability. When liver glycogen is insufficient, it will cause hypoglycemia and energy supply interruption, which will lead to body fatigue. The results of this study found that Formula B can increase the liver glycogen reserve level of mice and enhance the fatigue tolerance of the body.

[0045] The gut microbiota is known as the "second genome" of the body, and the analysis of gut microbiota diversity has become a new perspective for studying health and diseases in recent years. Intense exercise generates excessive free radicals, causing exercise-induced oxidative stress injury, increasing the deposition of harmful metabolites, inducing fatigue aggravation, and may also lead to a decrease in gut microbiota diversity and an increase in the relative abundance of pathogenic bacteria. This study found that Formula B can improve gut microbiota diversity, promote the growth of the beneficial bacterium Lactobacillus and increase the abundance of the beneficial fungus Saccharomyces, which helps to improve the body's immunity.

[0046] The intestinal barrier is mainly composed of a mechanical barrier, a chemical barrier, an immune barrier, and a biological barrier, and plays an important role in maintaining intestinal function and preventing bacteria and endotoxins from transferring from the intestinal lumen to the blood circulation. Among them, the intestinal mechanical barrier is mainly composed of intestinal epithelial cells and tight junctions between cells, and is the most important "first line of defense" of the intestinal barrier. This study found that the number of goblet cells in the intestines of mice in Group B increased and the structure was intact. This indicates that Formula B has a reparative or enhancing effect on intestinal barrier function. Occludin is a key component of tight junctions, and ZO-1 is a scaffold protein of tight junctions. The two together are responsible for forming and maintaining the integrity and stability of intestinal tight junctions. Intestinal barrier injury makes it easier for harmful substances such as bacteria, their metabolites, and endotoxins to enter the blood circulation, triggering a systemic inflammatory response, which in turn affects brain function. The results of this experiment showed that the protein expressions of Occludin and ZO-1 in Group B increased significantly, indicating that Formula B can enhance the intestinal barrier function of mice and improve intestinal immunity. In summary, Formula B is expected to become a new raw material for health care functional products with anti-fatigue, gut microbiota regulation, and intestinal barrier enhancement effects.

Claims

1. A composition for enhancing the anti-fatigue effect of Lactobacillus conjugatus in the intestine, characterized in that: The raw materials are composed of the following materials by weight: 2-6 parts of yam, 0.5-3 parts of cornus fruit, 1.5-4 parts of rehmannia root, 1.5-4 parts of tuckahoe, 0.5-3 parts of peony bark and 0.5-4 parts of oriental rhizome.

2. The composition according to claim 1, wherein: The raw materials are composed of the following materials by weight: 3-5 parts of yam, 1-2 parts of cornus fruit, 2-3 parts of rehmannia root, 2-3 parts of tuckahoe, 1-2 parts of peony bark and 1-2 parts of oriental rhizome.

3. The composition according to claim 2, characterized in that: The raw materials are composed of the following substances by weight: 4.28 parts of yam, 1.71 parts of cornus fruit, 2.14 parts of rehmannia root, 2.14 parts of tuckahoe, 1.71 parts of peony bark, and 1.42 parts of oriental rhizome.

4. The composition according to any one of claims 1-3, characterized in that: The intestinal combined lactobacilli include Lactobacillus acidophilus ( Lactobacillus acidophilus ), Lactobacillus plantarum ( Lactobacillus plantarum ), and Helicobacter pylori ( Lactobacillus helveticus ).

5. Use of the composition according to claim 4 in the preparation of a preparation for increasing the abundance of intestinal associated lactobacilli.

6. Use of the composition according to claim 4 in preparing a preparation for resisting or relieving fatigue.

7. The application according to claim 6, characterized in that: After administration of the preparation, blood lactate content decreased and liver glycogen reserve level increased.

8. Use of the composition according to claim 4 in the preparation of a preparation for increasing the abundance of Saccharomyces, a beneficial intestinal fungus.

9. Use of the composition of claim 4 in preparing a preparation for improving the species richness and comprehensive diversity of normal intestinal bacteria and changing the intestinal microbial community.

10. Use of the composition according to claim 4 in preparing a preparation for improving intestinal barrier function and enhancing intestinal immunity.

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