Probiotic composition for reducing blood pressure, blood sugar and blood lipid and application thereof

Abstract

The invention belongs to the field of probiotics, and particularly relates to a probiotic composition capable of reducing blood pressure, blood sugar and blood lipid, raw materials of a probiotic fermented product comprise traditional Chinese medicinal materials and probiotics, the traditional Chinese medicinal materials comprise radix puerariae, hawthorn, rhizoma polygonati, lotus leaves and semen cassiae, and the probiotics are compound lactic acid bacteria and bifidobacteria. The preparation method of the probiotic fermented product comprises the following steps: crushing and sieving traditional Chinese medicinal materials to obtain mixed traditional Chinese medicinal material powder; adding a proper amount of water for extracting and separating to obtain an extracting solution for later use; inoculating compound lactic acid bacteria and bifidobacteria, and fermenting; and filtering, centrifuging, and removing thalli and impurities to obtain the probiotic fermented product. The composition for assisting in reducing blood pressure, blood sugar and lipid is developed, the application scenes of medicinal and edible traditional Chinese medicinal materials are enriched, a fermentation method is adopted to replace a traditional high-temperature decoction and solvent extraction method, the yield of active substances is remarkably increased, and reliable guarantee is provided for product development.

Description

Technical Field

[0001] The present invention relates to the field of probiotics, and particularly relates to a probiotic composition for reducing blood pressure, blood sugar and blood lipid. Background Art

[0002] Metabolic syndrome involves multiple metabolic disorders, and refers to a syndrome of clinical manifestations related to metabolic disorders such as obesity, hypertension, hyperglycemia, and hyperlipidemia. After suffering from the disease, the risk of death of patients will increase by two times, and at the same time, the risk of heart disease or stroke will also increase by three times. In recent years, due to factors such as population aging and bad living habits, the incidence of metabolic diseases has increased significantly. Especially, the number of patients with hypertension, diabetes and hyperlipidemia has suddenly increased, which has caused a heavy burden on the medical systems and economies around the world.

[0003] At present, although there are many drugs for treating hypertension, diabetes and hyperlipidemia, such as antihypertensive drugs, hypoglycemic drugs and statins, most of the above chemical drugs will have side effects such as gastrointestinal discomfort, liver function damage, and muscle pain, and there are still certain safety risks in their long-term use. Although exercise rehabilitation has good effects and safety, it is also limited by factors such as age and environment.

[0004] Traditional Chinese medicine has significant advantages in treating hypertension, diabetes and hyperlipidemia, such as comprehensive conditioning and small side effects. It not only targets the disease itself, but also pays more attention to the overall health conditioning of patients. By adjusting the balance of yin and yang and the operation of qi and blood in the body, it promotes the self-healing ability of the body. However, traditional Chinese medicine cannot be taken for a long time, and some products cannot be accepted by patients due to poor taste. "Medicine and food homology", as high-end cash crops, has good safety and functionality, and is a kind of food resource with more advantages than ordinary food and irritating medical drugs. However, most of these resources are currently processed by traditional processing technologies such as decoction pieces and steaming, with low yield of active ingredients and large damage, and the efficacy cannot be fully manifested, which to a certain extent limits the promotion of traditional Chinese medicine health products.

[0005] Based on the above problems, it is of great significance to develop a traditional Chinese medicine material with lipid regulation, weight loss, blood sugar reduction and blood lipid reduction and reasonably prepare a medicine and food preparation, which is suitable for long-term use by patients on the one hand, and can effectively improve metabolic syndrome and has the characteristics of safety and effectiveness on the other hand. Summary of the Invention

[0006] In order to solve the above technical problems, the inventor of the present invention prepares a functional product by fermenting raw materials of medicine and food homology with probiotics in a specific combination, which can increase the decomposition and release of active ingredients in the raw materials and promote the absorption of the body. The organic combination of the two can exert better effects.

[0007] The technical solution of the present invention is as follows:

[0008] The present invention provides a probiotic fermentation product. Through pharmacological experiments, it is confirmed that the probiotic fermentation product has good effects of reducing blood pressure, blood sugar and blood lipid in hypertensive mouse models, diabetic mouse models and hyperlipidemic mouse models.

[0009] The first object of the present invention is to provide a probiotic fermentation product. The raw materials of the probiotic fermentation product include traditional Chinese medicines and probiotics. The probiotics are compound lactic acid bacteria and bifidobacteria. The traditional Chinese medicines include the following components in parts by weight:

[0010] 20-40 parts by weight of Pueraria lobata, 20-30 parts by weight of Hawthorn fruit, 20-40 parts by weight of Polygonatum sibiricum

[0011] 8-25 parts by weight of Lotus leaf, 20-30 parts by weight of Cassia seed.

[0012] The above-mentioned traditional Chinese medicine formula is guided by traditional Chinese medicine theory. The selected Pueraria lobata, Hawthorn fruit, Polygonatum sibiricum, Lotus leaf, Cassia seed, etc. are all resources of both medicine and food. Among them:

[0013] Pueraria lobata has the functions of relieving muscle fever, promoting eruption, promoting fluid production to quench thirst, ascending yang to stop diarrhea, dredging channels and collaterals, and relieving alcohol toxicity. The soy isoflavones it contains can dilate blood vessels, reduce vasoconstriction, promote the secretion and utilization of insulin, and have a strong antioxidant effect, which can scavenge free radicals and protect cells from oxidative damage.

[0014] Hawthorn fruit can promote digestion of food retention, disperse stasis, expel tapeworms, and treat meat retention, infantile milk food stagnation, phlegm retention, fullness and stuffiness, acid regurgitation, intestinal wind, etc. It is mainly used for meat food retention, hyperlipidemia, fullness and distension in the stomach, diarrhea and abdominal pain, stabbing pain in the heart and abdomen, flatulence pain, etc.

[0015] Polygonatum sibiricum has the effects of invigorating qi and nourishing yin, strengthening the spleen, moistening the lungs, and tonifying the kidneys. Its active ingredient polysaccharide has an inhibitory effect on α-glucosidase.

[0016] Lotus leaf has the effects of clearing away heat and relieving summer heat, ascending the clear yang, cooling blood and stopping bleeding, etc. "Zhengzhi Yaojue" states that "it can make people thin and emaciated. Taking it alone can eliminate the qi of yang edema." "Yilin Zuanyao" says that "Lotus leaf has similar effects to lotus root and lotus seed core, but it mostly enters the liver meridian, clearing heat, removing dampness, promoting the flow of qi, because green enters the liver. However, its bitter and astringent taste can actually purge the liver and gallbladder and clear the lung and consolidate water, so it can remove stasis, protect essence, eliminate excessive heat, and balance qi and blood."

[0017] Cassia seed has the effects of clearing heat and improving eyesight, moistening the intestines and relieving constipation, etc.

[0018] In order to achieve the best effect of reducing blood pressure, blood sugar and blood lipid of the composition, further, through a large number of experiments, the inventor found that in a preferred embodiment, the traditional Chinese medicines include the following components in parts by weight:

[0019] 30 parts by weight of Pueraria lobata, 30 parts by weight of Hawthorn fruit, 35 parts by weight of Polygonatum sibiricum

[0020] 20 parts by weight of lotus leaf and 25 parts by weight of cassia seed.

[0021] Meanwhile, through careful analysis and experimental verification by the inventor, when the compound lactic acid bacteria are a compound bacterium of Clostridium butyricum, Lactobacillus bulgaricus, and Lactobacillus reuteri, it is beneficial to the experimental effect. At the same time, the inventor also found that when calculated by weight ratio, the Clostridium butyricum, Lactobacillus bulgaricus, and Lactobacillus reuteri are 1:1 - 3:3 - 5, it is more beneficial to solve the technical problems of the present invention.

[0022] The second object of the present invention is to provide a preparation method of the probiotic fermentation product, which specifically includes the following steps:

[0023] (1) Crush and sieve the Chinese medicinal materials to obtain a mixed Chinese medicinal material powder for later use;

[0024] (2) Add an appropriate amount of water to the mixed Chinese medicinal material powder for extraction and separation, and keep the extract for later use;

[0025] (3) Remove impurities from the extract, add an appropriate amount of water, adjust the pH value, inoculate compound lactic acid bacteria and bifidobacteria, and ferment;

[0026] (4) After the fermentation is completed, filter and centrifuge to remove the bacteria and impurities to obtain the probiotic fermentation product.

[0027] Specifically, the operation in step (2) is: add an appropriate amount of water to the mixed Chinese medicinal material powder, adjust the pH value to 4.8 - 5.5, then add a mixed enzyme, enzymatically hydrolyze at 45 - 55 °C for 1 - 3 hours, centrifuge and separate to remove part of the impurities and insoluble substances, and keep the extract for later use.

[0028] Furthermore, the inventor found that the mixed enzyme in step (2) is a mixed enzyme of papain, chitinase, and ligninase. Preferably, calculated by weight ratio, the papain, chitinase, and ligninase in the specific enzyme are 1:6 - 15:9 - 20.

[0029] In a preferred embodiment, the inventor found that the mixed enzyme in step (2) is a mixed enzyme of papain, chitinase, and ligninase. Calculated by weight ratio, the papain, chitinase, and ligninase in the specific enzyme are 1:10:15.

[0030] Specifically, the impurity removal operation in step (3) is: first add an appropriate amount of ethanol to the extract, let it stand after the ethanol concentration reaches 50% - 70% to remove the precipitate, add hot water at 80 - 90 °C to the remaining liquid, stir and extract for 1 - 2 hours, and remove the precipitate again to obtain the leaching solution for later use.

[0031] The fermentation operation in step (3) is as follows: concentrate the extract, then dissolve it in an appropriate amount of water, adjust the pH value to 5.0 - 6.0, inoculate with compound lactic acid bacteria and bifidobacteria, ferment at 38 - 45°C, filter and centrifuge to obtain a probiotic fermentation product.

[0032] Specifically, the preparation method of the probiotic fermentation product includes the following steps:

[0033] (1) Crush and sieve the Chinese medicinal materials to obtain a mixed Chinese medicinal material powder for later use;

[0034] (2) Add an appropriate amount of water to the mixed Chinese medicinal material powder, adjust the pH value to 4.8 - 5.5, then add a mixed enzyme. By weight, the mixed enzyme is papain, chitinase, and ligninase at a ratio of 1:10:15, enzymatically hydrolyze at 45 - 55°C for 1 - 3 hours, centrifuge and separate to remove some impurities and insoluble substances, and obtain an extract for later use;

[0035] (3) First add an appropriate amount of ethanol to the extract. After the ethanol concentration reaches 50% - 70%, let it stand for 2 - 4 hours to remove the precipitate. Add hot water at 80 - 90°C to the remaining liquid, stir and extract for 1 - 2 hours, centrifuge to remove the precipitate to obtain an extract; concentrate the extract, then dissolve it in an appropriate amount of water, adjust the pH value to 5.0 - 6.0, inoculate with compound lactic acid bacteria and bifidobacteria, ferment at 38 - 45°C, filter and centrifuge to obtain a probiotic fermentation product;

[0036] (4) After the fermentation is completed, filter and centrifuge to remove the bacteria and impurities to obtain a probiotic fermentation product.

[0037] The third object of the present invention is to provide the use of the probiotic fermentation product, specifically its application in the preparation of medicines and functional foods. Specifically, the present invention provides the application of the probiotic fermentation product in the preparation of medicines for treating hypertension, hypoglycemia, and hyperlipidemia, as well as the application of the probiotic fermentation product in the preparation of functional foods.

[0038] Furthermore, the present invention provides the application of the probiotic fermentation product in the preparation of medicines for treating metabolic syndrome.

[0039] Furthermore, the pharmaceutical dosage form of the probiotic fermentation product is an oral preparation, preferably one or more of granules, tablets, pills, and mixtures.

[0040] Furthermore, the dosage form of the functional food is one or more of powder, granule, dry tablet, beverage, or canned congee that is convenient for consumption.

[0041] The beneficial effects of the present invention are as follows:

[0042] (1) The composition of the present invention is a probiotic product for assisting in reducing blood pressure, blood sugar, and blood lipid, developed with traditional Chinese medicinal materials that are both edible and medicinal as the core, enriching the application scenarios of traditional Chinese medicinal materials that are both edible and medicinal and enhancing the added value of the product;

[0043] (2) The composition of the present invention is based on enzymatic hydrolysis, extraction, and probiotic fermentation technologies, replacing traditional high-temperature decoction and solvent extraction methods, which can significantly increase the yield of active substances, enhance the stability of compound nutrients, and comprehensively improve the efficacy and quality of the product;

[0044] (3) The composition of the present invention achieves relatively optimal therapeutic or improvement effects by optimizing the preparation method of the composition and through established hypertensive mouse models, diabetic mouse models, and hyperlipidemic mouse models. Description of the Drawings

[0045] Figure 1 Effect of the composition of the present invention on the SBP value of the hypertensive mouse model (compared with the blank control group, *P < 0.05, **P < 0.01; compared with the model group, # P < 0.05, ## P < 0.01);

[0046] Figure 2 Effect of the composition of the present invention on the DBP value of the hypertensive mouse model (compared with the blank control group, *P < 0.05, **P < 0.01; compared with the model group, # P < 0.05, ## P < 0.01);

[0047] Figure 3 Effect of the composition of the present invention on the MBP value of the hypertensive mouse model (compared with the blank control group, *P < 0.05, **P < 0.01; compared with the model group, # P < 0.05, ## P < 0.01);

[0048] Figure 4 Effect of the composition of the present invention on the heart rate of the hypertensive mouse model (compared with the blank control group, *P < 0.05, **P < 0.01; compared with the model group, # P < 0.05, ## P < 0.01);

[0049] Figure 5 Effect of the composition of the present invention on the glucose consumption level;

[0050] Figure 6 Effect of the composition of the present invention on the glycogen accumulation level;

[0051] Figure 7 Effect of the composition of the present invention on the detection of G6P activity level;

[0052] Figure 8 Effect of the composition of the present invention on the activity level of PEPCK enzyme;

[0053] Figure 9 Effect of the composition of the present invention on the activity level of GYS2 enzyme;

[0054] Figure 10 Observation of the organs of hyperlipidemic mice by the composition of the present invention (from top to bottom: spleen, stomach, kidney, heart; from left to right: normal control group, model group, positive control drug group, Example 3 group, Comparative Example 2 group);

[0055] Figure 11 Effect of the composition of the present invention on the organ weights of hyperlipidemic mice. Detailed implementation mode

[0056] The technical solutions of the present application will be further elaborated below in the form of examples. It should be noted that the following examples are only used to illustrate the technical solutions of the present invention and do not limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered by the protection scope of the present invention.

[0057] Preparation of granule in Example 1

[0058] 40 g of Pueraria lobata, 40 g of Hawthorn fruit, 40 g of Polygonatum sibiricum

[0059] 50 g of Lotus leaf, 60 g of Cassia seed

[0060] (1) The Chinese medicinal materials are pulverized and sieved to obtain a mixed Chinese medicinal material powder for standby;

[0061] (2) An appropriate amount of water is added to the mixed Chinese medicinal material powder, the pH value is adjusted to 5.0, and then a mixed enzyme is added. By weight, the mixed enzyme is papain, chitinase, and ligninase at a ratio of 1:6:20. Enzymolysis is carried out at 55 °C for 3 hours, followed by centrifugation and separation to remove part of the impurities and insoluble substances, and the obtained extract is reserved for standby;

[0062] (3) First, an appropriate amount of ethanol is added to the extract. After the ethanol concentration reaches 70%, it is left standing for 4 hours to remove the precipitate. The remaining liquid is added with hot water at 90 °C and stirred for extraction for 2 hours, and the precipitate is removed by centrifugation to obtain an extraction solution; the extraction solution is concentrated, then dissolved in an appropriate amount of water, the pH value is adjusted to 5.0, and composite lactic acid bacteria and bifidobacteria are inoculated, and fermentation is carried out at 40 - 45 °C, followed by filtration and centrifugation to obtain a probiotic fermentation product;

[0063] (4) After the fermentation is completed, filtration and centrifugation are carried out to remove the bacteria and impurities, and a solid granule is prepared.

[0064] Preparation of Granules in Example 2

[0065] 60 g of Pueraria lobata, 50 g of Crataegus pinnatifida, 50 g of Polygonatum sibiricum

[0066] 16 g of Nelumbo nucifera leaf, 40 g of Cassia obtusifolia

[0067] (1) The Chinese medicinal materials are pulverized and sieved to obtain a mixed powder of Chinese medicinal materials for standby;

[0068] (2) Appropriate amount of water is added to the mixed powder of Chinese medicinal materials, the pH value is adjusted to 4.8 - 5.5, and then a mixed enzyme is added. By weight, the mixed enzyme is papain, chitinase, and ligninase in a ratio of 1:10:15. Enzymolysis is carried out at 45 °C for 1 hour, followed by centrifugation and separation to remove part of the impurities and insoluble substances, and the extract is obtained for standby;

[0069] (3) First, appropriate amount of ethanol is added to the extract. After the ethanol concentration reaches 65%, it is left standing for 2 hours to remove the precipitate. The remaining liquid is added with hot water at 80 °C, stirred and extracted for 2 hours, and the precipitate is removed by centrifugation to obtain an extract. The extract is concentrated, then dissolved in an appropriate amount of water, the pH value is adjusted to 6.0, and compound lactic acid bacteria and bifidobacteria are inoculated, and fermentation is carried out at 38 - 45 °C, followed by filtration and centrifugation to obtain a probiotic fermentation product;

[0070] (4) After fermentation, filtration and centrifugation are carried out to remove the bacterial cells and impurities, and solid granules are prepared.

[0071] Preparation of Granules in Example 3

[0072] 80 g of Pueraria lobata, 60 g of Crataegus pinnatifida, 80 g of Polygonatum sibiricum

[0073] 16 g of Nelumbo nucifera leaf, 40 g of Cassia obtusifolia

[0074] (1) The Chinese medicinal materials are pulverized and sieved to obtain a mixed powder of Chinese medicinal materials for standby;

[0075] (2) Appropriate amount of water is added to the mixed powder of Chinese medicinal materials, the pH value is adjusted to 4.8, and then a mixed enzyme is added. By weight, the mixed enzyme is papain, chitinase, and ligninase in a ratio of 1:15:9. Enzymolysis is carried out at 55 °C for 3 hours, followed by centrifugation and separation to remove part of the impurities and insoluble substances, and the extract is obtained for standby;

[0076] (3) First, appropriate amount of ethanol is added to the extract. After the ethanol concentration reaches 50%, it is left standing for 2 hours to remove the precipitate. The remaining liquid is added with hot water at 85 °C, stirred and extracted for 2 hours, and the precipitate is removed by centrifugation to obtain an extract. The extract is concentrated, then dissolved in an appropriate amount of water, the pH value is adjusted to 5.5, and compound lactic acid bacteria and bifidobacteria are inoculated, and fermentation is carried out at 38 - 45 °C, followed by filtration and centrifugation to obtain a probiotic fermentation product;

[0077] (4) After fermentation, filter and centrifuge to remove the thalli and impurities, and make solid granules.

[0078] Preparation of the Granules in Example 4

[0079] 60 g of Pueraria lobata, 60 g of Crataegus pinnatifida, 70 g of Polygonatum sibiricum

[0080] 40 g of Folium Nelumbinis, 50 g of Cassia obtusifolia

[0081] The preparation method is the same as that in Example 1.

[0082] Preparation of the Granules in Comparative Example 1

[0083] 65 g of Astragalus membranaceus, 80 g of Crataegus pinnatifida, 25 g of Lycium barbarum

[0084] 16 g of Folium Nelumbinis, 30 g of Plantago asiatica

[0085] The preparation method is the same as that in Example 1.

[0086] Preparation of the Granules in Comparative Example 2

[0087] 80 g of Pueraria lobata, 60 g of Crataegus pinnatifida, 80 g of Polygonatum sibiricum

[0088] 16 g of Folium Nelumbinis, 40 g of Cassia obtusifolia

[0089] For the above 12 raw medicinal materials, select and remove impurities, conduct pretreatment according to the pharmacopoeia method, decoct with water twice. For the first time, add 8 times the amount of water and decoct for 2 h, and for the second time, add 6 times the amount of water and decoct for 1 h. Combine the decoction liquids and concentrate under reduced pressure to 500 ml of concentrated decoction.

[0090] Verification Example 1 Influence of the Composition of the Invention on Blood Pressure and Heart Rate of a Hypertension Mouse Model

[0091] 1 Experimental Materials

[0092] 1.1 Experimental Animals

[0093] Healthy female BALB / c mice (provided by the Pharmacology Center of Lunan Pharmaceutical Group), with a body weight of 20 ± 2 g, and adaptively fed for 7 days before the start of the experiment.

[0094] 1.2 Animal Grouping, Modeling and Administration

[0095] Prepare a hypertensive mouse model according to the conventional modeling method. Randomly divide the successfully modeled mice into groups of 10 each, namely the model group, the perindopril (0.607 mg / kg / d) group, the low (7.5 g / kg / d), medium (15 g / kg / d), and high-dose groups (30 g / kg / d) of Example 4 (calculated based on the original crude drug amount), and the low (7.5 g / kg / d), medium (15 g / kg / d), and high-dose groups (30 g / kg / d) of Comparative Example 2 (calculated based on the original crude drug amount). Another 10 normal mice were taken as the normal control group. After the start of the experiment for each group of mice, intragastric administration was carried out. The normal control and the model control group were administered an equal volume of distilled water once a day for 4 consecutive weeks.

[0096] 2.2 Detection indicators

[0097] Record once a week and observe the changes in blood pressure and heart rate of each group of mice.

[0098] 2.3 Data processing

[0099] Use SPSS 22.0 software for statistical analysis. The data is expressed as "mean ± standard deviation". For multi-group analysis, one-way ANOVA is used, and P < 0.05 is considered statistically significant.

[0100] 3 Effects of the composition of the present invention on the changes in blood pressure and heart rate of each group of mice

[0101] The results are as Figures 1 - 3 shown. It can be seen from the figure that at the start of the experiment, compared with the normal control group, the blood pressure of the mice in the model group increased significantly, with a significant difference (**P < 0.01), and within the six weeks of the experiment, the blood pressure of the mice in the model group continued to increase. After intervention with the positive control drug perindopril and the administration of each group, the upward trend of blood pressure in each group of mice was alleviated to varying degrees ( # P < 0.05, ## P < 0.01). Among them, although the blood pressure of the mice in the high-dose group of the probiotic composition was higher than that of the control group, there was no significant difference (P > 0.05).

[0102] From Figure 4 it can be seen that as the blood pressure increased, the heart rate of the mice in the model group gradually accelerated. After intervention with the administration of each drug group, the heart rate of each group of mice decreased ( # P < 0.05, ## P < 0.01). Since the 4th week, the heart rate of the positive drug group and the probiotic composition group of mice has basically remained stable.

[0103] Verification Example 2 Effects of the composition of the present invention on high glucose-induced HepG2 cells

[0104] 1 Experimental method

[0105] Cell culture: HepG2 cells were cultured in DMEM medium containing 10% FBS and 1% double antibody in an incubator at 37°C and 5% CO2. When the cell confluence reached 80%-90%, they were digested with trypsin and passaged at a ratio of 1:3.

[0106] High-glucose induction: The normal control group was cultured in a medium containing 5.5 mmol / L glucose, and the model group was cultured in a medium containing 50 mmol / L glucose.

[0107] Grouping and treatment: The medium induced by high glucose was randomly divided into 8 groups, namely the metformin group (concentration 2 mM), the model group, the low (0.3 mg / ml), medium (0.6 mg / ml), and high-dose groups (1.0 mg / ml) of Example 4, the low (0.3 mg / ml), medium (0.6 mg / ml), and high-dose groups (1.0 mg / ml) of Comparative Example 2. 100 μL of cell suspension was added to each well of each group. Another 6 wells were only added with cell culture medium as the blank group, and the remaining wells were added with phosphate buffer (PBS). After culturing for 24 hours, the cells were collected for subsequent experiments.

[0108] 2.2 Detection indexes

[0109] 2.2.1 Detection of glucose consumption level

[0110] The glucose content in the cell culture medium was detected by the glucose oxidase method, and the glucose consumption within 24 hours was calculated.

[0111] 2.2.2 Detection of glycogen accumulation level, glucose-6-phosphatase (G6P), phosphoenolpyruvate carboxykinase (PEPCK), and GYS2 activity levels

[0112] Detection was carried out according to the kit method.

[0113] 3 Experimental results

[0114] 3.1 Effect of the composition of the present invention on glucose consumption level

[0115] From Figure 5 the results, it can be seen that compared with the normal control group, the glucose consumption level in high-glucose-induced HepG2 cells was significantly reduced. By administering different compositions, the glucose consumption level could be increased to a certain extent in each group, promoting glycogen synthesis, improving insulin resistance and hyperglycemic state. Among them, the glucose consumption level in the positive control drug metformin group was the highest, the high-dose group of the present invention was close to it, while the increase in glucose consumption level in the control group was poor.

[0116] 3.2 Effect of the composition of the present invention on glycogen accumulation level

[0117] The results are shown in Figure 6 , and it can be seen that after high-glucose induction, the glycogen accumulation level in HepG2 cells decreased significantly, which was consistent with the trend in the model group in the experiment. Administering different compositions could significantly increase the glycogen accumulation level, promote glycogen synthesis, thereby improving insulin resistance and hyperglycemia. Among them, the medium-dose group and high-dose group of the present invention were higher than the positive control drug group, while the comparative example groups were lower than the low-dose group of the present invention.

[0118] 3.3 Effect of the composition of the present invention on the detection of G6P activity level

[0119] From Figure 7 it can be seen that after high-glucose induction, the G6P enzyme activity in the model group increased significantly, indicating that the gluconeogenesis pathway was active. After adding the traditional Chinese medicine composition of the present invention, the G6P enzyme activity decreased. Among them, the high-dose group of the composition of the present invention was lower than the positive control drug metformin group, while the comparative example groups were higher than the positive control drug group and each group of the present invention.

[0120] 3.4 Effect of the composition of the present invention on the PEPCK enzyme activity level

[0121] From Figure 8 it can be seen that in HepG2 cells induced by high glucose, the PEPCK enzyme activity increased significantly. Among them, the PEPCK enzyme activity in the model group increased significantly. After adding the traditional Chinese medicine composition of the present invention, the PEPCK enzyme activity decreased. Among them, the high-dose group of the composition of the present invention was lower than the positive control drug metformin group, while the comparative example groups were higher than the positive control drug group and each group of the present invention.

[0122] 3.5 Effect of the composition of the present invention on the GYS2 enzyme activity level

[0123] After high-glucose induction, the GYS2 enzyme activity in HepG2 cells decreased significantly, indicating the obstruction of the glycogen synthesis pathway. From Figure 9 it can be seen that the GYS2 enzyme activity in the model group decreased significantly, and the results were consistent. After adding the traditional Chinese medicine composition of the present invention, the PEPCK enzyme activity increased. Among them, the high-dose group and medium-dose group of the composition of the present invention were higher than the positive control drug metformin group, while the comparative example groups were lower than the positive control drug group and the low-dose group of the present invention.

[0124] Verification Example 3 Effect of the composition of the present invention on hyperlipidemic mice

[0125] 1 Experimental animals

[0126] Healthy female BALB / c mice (provided by the Pharmacology Center of Lunan Pharmaceutical Group), with a body weight of 20 ± 2 g. Before the experiment, they were conventionally raised at a temperature of 25 ± 1 °C and a humidity of 60 ± 2%, with free diet and housed separately in cages.

[0127] 2 Experimental Methods and Drug Administration

[0128] They were randomly divided into 5 groups with 12 mice in each group. The normal control group was fed with normal diet, and the rest were fed with high-fat diet. After 7 weeks, when the obesity model was successfully established, intragastric administration of drugs was started. The positive drug control group, Example 3 group, and Comparative Example 2 group were intragastrically administered with atorvastatin calcium tablets at 0.013 mg / mouse / d, 15 g / kg / d (calculated based on the original crude drug amount), and 15 g / kg / d (calculated based on the original crude drug amount) respectively for 8 weeks of drug intervention.

[0129] 3 Detection Indexes

[0130] 3.1 Effects on the Organ Weights of Mice

[0131] At the end of the experiment, the mice were sacrificed, and the gastrointestinal tract, heart, spleen, and kidneys of the mice in each group were removed and weighed for corresponding analysis.

[0132] 3.2 Effects on the Four Blood Lipid Levels of Mice

[0133] The serum of the mice was collected to analyze the serum lipid profile, including total triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C).

[0134] 4 Data Processing

[0135] SPSS 22.0 software was used for statistical analysis, and the data were expressed as "mean ± standard deviation". For the analysis among multiple groups, one-way analysis of variance was used, and P < 0.05 was considered to indicate a statistically significant difference.

[0136] 5 Analysis of Experimental Results

[0137] 5.1 Effects on the Organ Weights of Mice

[0138] At the end of the experiment, the chest and abdomen were carefully incised, and the organs were gently removed with forceps. The observation results were as follows:

[0139] (1) Gastric observation: The color of the stomach of the mice in the normal control group was slightly pink, and the shape was normal; the color of the stomach of the mice in the model group was dull, and swelling occurred; the stomachs of the mice in the positive control group and Example 3 group were similar, and the shape was slightly smaller. The color of the stomach observed in the Comparative Example 2 group was darker, and the shape was similar to that of the model group.

[0140] (2) Heart observation: The color of the heart of the mice in the normal control group was bright red, and the shape was normal; the color of the heart of the mice in the model group was black, slightly larger, and hypertrophic; the color of the hearts of the mice in the positive control group and Example 3 group was bright, and the color of the heart in the Comparative Example 2 group was darker.

[0141] (3) Spleen color: The spleens of the mice in the normal control group were dark red in color and normal in shape; the spleens of the mice in the model group were blackened, slightly enlarged, and had nodules in some cases; the spleens of the mice in the positive control group and Example 3 group were close to those of the normal control group, and the spleen color of the Comparative Example 2 group was darker.

[0142] (4) Kidney color: The kidneys of the mice in the normal control group were reddish-brown in color; the kidneys of the mice in the model group were blackened and slightly enlarged; the kidneys of the mice in the positive control group and Example 3 group were close to those of the normal control group, and the color of the Comparative Example 2 group was darker.

[0143] The results of visceral weighing are as Figure 11 shown. As can be seen from the figure, compared with the model group, the positive control drug group and Example 3 group of the present invention can effectively improve hyperlipidemia symptoms, and the effects are similar; the improvement effect of the Comparative Example 2 group on hyperlipidemia is not obvious.

[0144] 5.2 Effects on the four lipid levels in mice

[0145] At the end of the experiment, the mouse serum was collected, and the conditions of the mice were observed and recorded. The results are shown in Table 1:

[0146] Table 1 Four lipid levels in hyperlipidemic mice

[0147]

[0148] (Compared with the blank control group, *P < 0.05, **P < 0.01; compared with the model group, # P < 0.05, ## P < 0.01)

[0149] As shown in the above table, compared with the blank control group, the TC, TG, and LDL-C in the model group increased significantly; the HDL-C decreased somewhat. Compared with the model group, each administration group down-regulated the levels of TC, TG, and LDL-C in the mouse serum to varying degrees, and each administration group up-regulated the level of HDL-C in the mouse serum to varying degrees, and the results of the positive control drug group and Example 3 group were similar. The above results indicate that the probiotic composition of the present invention can effectively regulate the lipid levels in mice.

Claims

1. A probiotic fermented product, characterized in that, The raw materials of the probiotic fermentation product include traditional Chinese medicines and probiotics. The traditional Chinese medicines include Pueraria lobata, Hawthorn Fruit, Polygonatum sibiricum, Lotus Leaf, and Cassia Seed. The probiotics are compound lactic acid bacteria and bifidobacteria.

2. The probiotic fermentation product according to claim 1, wherein The traditional Chinese medicines include the following components in parts by weight: Pueraria lobata 20 - 40 parts by weight, Hawthorn Fruit 20 - 30 parts by weight, Polygonatum sibiricum 20 - 40 parts by weight Lotus Leaf 8 - 25 parts by weight, Cassia Seed 20 - 30 parts by weight.

3. The probiotic fermentation product according to claim 1, wherein The traditional Chinese medicines include the following components in parts by weight: Pueraria lobata 30 parts by weight, Hawthorn Fruit 30 parts by weight, Polygonatum sibiricum 35 parts by weight Lotus Leaf 20 parts by weight, Cassia Seed 25 parts by weight.

4. The probiotic fermentation product according to claim 1, wherein, The compound lactic acid bacteria are a compound bacterium of Clostridium butyricum, Lactobacillus bulgaricus, and Lactobacillus reuteri. Further, by weight ratio, Clostridium butyricum, Lactobacillus bulgaricus, and Lactobacillus reuteri are 1:1 - 3:3 - 5.

5. The probiotic fermentation product according to claim 1, wherein The preparation method of the probiotic fermentation product includes the following steps: (1) Crush the traditional Chinese medicines and sieve them to obtain a mixed powder of traditional Chinese medicines for standby; (2) Add an appropriate amount of water to the mixed powder of traditional Chinese medicines for extraction and separation, and reserve the extract; (3) Remove impurities from the extract, add an appropriate amount of water, adjust the pH value, inoculate compound lactic acid bacteria and bifidobacteria, and ferment; (4) After fermentation, filter and centrifuge to remove bacteria and impurities to obtain the probiotic fermentation product.

6. The probiotic fermentation product according to claim 5, wherein The specific operation of step (2) is as follows: Add an appropriate amount of water to the mixed powder of traditional Chinese medicines, adjust the pH value to 4.8 - 5.5, then add a mixed enzyme, enzymolyze at 45 - 55 °C for 1 - 3 hours, centrifuge and separate to remove some impurities and insoluble substances, and reserve the extract.

7. The probiotic fermentation product according to claim 6, wherein The mixed enzyme in step (2) is a mixed enzyme of papain, chitinase, and ligninase. Preferably, by weight ratio, papain, chitinase, and ligninase in the specific enzyme are 1:6 - 15:9 - 20.

8. The probiotic fermentation product according to claim 5, wherein The impurity removal operation in step (3) is as follows: First, add an appropriate amount of ethanol to the extract. After the ethanol concentration reaches 50% - 70%, let it stand to remove the precipitate. Add hot water at 80 - 90 °C to the remaining liquid, stir and extract for 1 - 2 hours, and remove the precipitate again to obtain the extracted liquid for standby.

9. The probiotic fermentation product according to claim 5, wherein The fermentation operation in step (3) is as follows: Concentrate the extracted liquid, then dissolve it in an appropriate amount of water, adjust the pH value to 5.0 - 6.0, inoculate compound lactic acid bacteria and bifidobacteria, ferment at 38 - 45 °C, filter and centrifuge to obtain the probiotic fermentation product.

10. Use of the probiotic fermentation product according to claim 1 in the preparation of medicines and functional foods.

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