Traditional Chinese medicine preparation fermentation liquor, preparation method thereof and application of traditional Chinese medicine preparation fermentation liquor in aspects of losing weight, lowering lipid and / or relieving liver injury

Abstract

The invention discloses fermentation liquor of a traditional Chinese medicine preparation, a preparation method of the fermentation liquor and application of the fermentation liquor in the aspects of losing weight, lowering lipid and / or relieving liver injury. The traditional Chinese medicine preparation is prepared from the following components in parts by weight: 3 to 6 parts of mulberries, 3 to 6 parts of raspberries, 3 to 6 parts of fructus corni, 12 to 20 parts of fructus lycii, 15 to 25 parts of rhizoma polygonati, 15 to 25 parts of fructus crataegi and 2 to 5 parts of semen cassiae. The traditional Chinese medicine preparation is prepared by extracting, sterilizing, cooling, inoculating a leavening agent and fermenting. The mulberry, the raspberry and the dogwood which are medicinal and edible materials are used for replacing fleece-flower root, the original formula of the lipid-lowering drug is combined, the fermentation liquor prepared after fermentation can effectively retain the obesity adjusting effect of the classic formula of the lipid-lowering drug, meanwhile, the biological activity of the lipid-lowering drug is remarkably improved through the optimized compatibility and the probiotic fermentation technology, and the lipid-lowering drug has a good application prospect. A safe and efficient innovative scheme is provided for functional food to intervene in obesity / overweight.

Description

Technical Field

[0001] The present invention relates to a traditional Chinese medicine preparation fermentation broth, a preparation method thereof, and applications in weight loss and lipid reduction and / or alleviating liver injury, belonging to the field of traditional Chinese medicine preparations. Background Art

[0002] Obesity is a state caused by the long-term imbalance between calorie intake and consumption, and its main characteristics include overweight, unbalanced body proportions, abnormal blood lipids, and abnormal accumulation of body fat. Long-term obesity causes excessive fat accumulation in liver cells, especially the abnormal accumulation of triglycerides, which can lead to liver cell dysfunction and trigger insulin resistance, oxidative stress, and inflammatory responses under the action of multiple molecular mechanisms.

[0003] Currently, the main intervention methods for obesity include lifestyle regulation, drug treatment, and functional food intervention, etc. Among them, the treatment of obesity with traditional Chinese medicine compounds has a long history and remarkable effects. However, due to its safety, the use of traditional Chinese medicine compounds must be taken under the guidance of traditional Chinese medicine practitioners, which greatly limits the wide application of traditional Chinese medicine compounds in the treatment of obesity. How to rationally and scientifically develop the traditional Chinese medicine compounds into food homologous products based on traditional Chinese medicine theory, combined with modern medical and nutritional research methods, and explore its regulatory mechanism on obesity is of great significance for the control and intervention of obesity / overweight and the popularization of traditional Chinese medicine compounds.

[0004] The classic traditional Chinese medicine compound Yishou Jiangzhiling is composed of Polygonum multiflorum, Lycium barbarum, Polygonatum sibiricum, Hawthorn, and Cassia obtusifolia. In the formula, Polygonum multiflorum is bitter and warm, tonifying the liver and kidney, and benefiting essence and blood, serving as the monarch drug; Polygonatum sibiricum and Lycium barbarum are sweet and flat in taste. The two nourish the liver and kidney, tonify qi and nourish yin, and assist Polygonum multiflorum in tonifying the liver and kidney together, serving as the minister drugs; Hawthorn promotes digestion and strengthens the stomach, activates qi and disperses stasis, and reduces turbidity and lipid; Cassia obtusifolia clears heat and improves eyesight, and moistens the intestines and promotes defecation, serving as the assistant drugs; all the drugs are combined to achieve the effects of tonifying the liver and kidney, nourishing blood and reducing lipid. Currently, this formula has been widely used clinically for the treatment of hyperlipidemia. However, Polygonum multiflorum has hepatotoxicity to some susceptible populations. Therefore, Jiangzhiling formula needs to be taken under the guidance of a doctor when used, which greatly limits the application of this formula. There are huge challenges in how to scientifically and effectively improve the formula.

[0005] In recent years, with the wide application of probiotics in the fields of functional foods and pharmaceuticals, their functions in regulating the intestinal flora structure, enhancing the host immunity, and regulating the disorders of glucose and lipid metabolism have received more and more attention. Probiotic fermentation of Chinese herbal medicines can not only improve the release and biotransformation of active ingredients of Chinese herbal medicines, but also improve the taste and absorbability of herbs through the fermentation process. In addition, organic acids, enzyme substances, and other bioactive substances produced by probiotic metabolism can interact with the active ingredients in Chinese herbal medicines to exert a synergistic effect and further enhance the health care effect of herbs. Summary of the Invention

[0006] Object of the Invention: The object of the present invention is to provide a traditional Chinese medicine preparation fermentation broth capable of reducing weight and lipid, and alleviating liver injury, as well as its preparation method and application.

[0007] Technical Solution: The present invention provides a traditional Chinese medicine preparation fermentation broth. The traditional Chinese medicine preparation comprises the following components in parts by weight: 3-6 parts of mulberry, 3-6 parts of raspberry, 3-6 parts of cornel, 12-20 parts of wolfberry, 15-25 parts of polygonatum, 15-25 parts of hawthorn, and 2-5 parts of semen cassiae; the traditional Chinese medicine preparation is prepared by extraction, sterilization, cooling, and inoculating a fermenting agent for fermentation.

[0008] Further, the fermenting agent is composed of one or more combinations of lactobacillus fermentum, lactobacillus rhamnosus, lactobacillus paracasei, lactobacillus plantarum, bifidobacterium, lactococcus lactis, and lactobacillus acidophilus.

[0009] Further, the fermenting agent is composed of one or more combinations of lactobacillus fermentum grx08, lactobacillus rhamnosus grx10, lactobacillus paracasei grx06, lactobacillus plantarum grx401, lactobacillus plantarum grx402, bifidobacterium grx05, lactococcus lactis grx602, and lactobacillus acidophilus grx95.

[0010] Specifically, lactobacillus fermentum grx08 (deposit number: CGMCC No.7965), lactobacillus rhamnosus grx10 (deposit number: CGMCC No.2526), lactobacillus paracasei grx06 (deposit number: CGMCC No.33560), lactobacillus plantarum grx401 (deposit number: CGMCC No.23544), lactobacillus plantarum grx402 (deposit number: CGMCC No.28585), bifidobacterium grx05 (deposit number: CGMCC No.21589), lactococcus lactis grx602 (deposit number: CGMCC No.22692), and lactobacillus acidophilus grx95 (deposit number: CGMCC No.8875).

[0011] The present invention also provides a preparation method of the above-mentioned traditional Chinese medicine preparation fermentation broth. The preparation method comprises the following steps: weighing each component raw material according to the weight ratio, pulverizing, sieving, fully mixing, extracting, sterilizing, cooling, inoculating the fermenting agent, and fermenting at 37°C for 24-48 h to obtain the fermentation broth.

[0012] Further, the inoculation amount of the fermentation broth is 2%-5%, and the strain number is 1×10 9 CFU / mL.

[0013] Further, the ratio of material to liquid during extraction is 1:15 - 1:30; the soaking conditions are a stirring speed of 50 - 80 rpm and a time of 10 min - 20 min; the extraction temperature is 50°C - 80°C, and the extraction time is 0.5 h - 3 h.

[0014] The present application also provides the use of the above-mentioned traditional Chinese medicine preparation fermentation broth in the preparation of products for weight loss and lipid reduction and / or alleviating liver injury.

[0015] Further, the weight loss and lipid reduction include inhibiting weight gain, alleviating liver hypertrophy and epididymal fat accumulation, inhibiting the activity of glycolipid digestive enzymes, and increasing the total sugar content in feces.

[0016] Further, the alleviation of liver injury includes reducing the content of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum, reducing oxidative stress injury of the liver, and alleviating inflammatory responses.

[0017] Further, the reduction of oxidative stress injury of the liver includes reducing the content of malondialdehyde (MDA) and increasing the activities of superoxide dismutase (SOD enzyme) and glutathione peroxidase (GSH-Px enzyme).

[0018] Further, the alleviation of inflammatory responses includes reducing the content of inflammatory factors TNF-α, IL-6, and NF-κB.

[0019] The scientific basis for the compatibility of traditional Chinese medicines in the present invention is as follows:

[0020] (1) Theoretical basis of traditional Chinese medicine:

[0021] ① Basis for substitution: Polygonum multiflorum is bitter, sweet, and astringent, warm in nature, and belongs to the liver, heart, and kidney meridians, with the effects of tonifying the liver and kidney and benefiting essence and blood. Mulberry is sweet, sour, and cold, belonging to the heart, liver, and kidney meridians, with the effects of tonifying the liver, nourishing the kidney, calming endogenous wind, and nourishing body fluids. Rubus chingii is sweet, slightly sour, warm in nature, belonging to the liver, kidney, and bladder meridians, with the effects of tonifying the kidney and consolidating essence, reducing urination, and nourishing the liver and improving eyesight. Cornus officinalis is sour, astringent, slightly warm in nature, belonging to the liver and kidney meridians, with the effects of tonifying the liver and kidney and astringing and consolidating to prevent leakage.

[0022] ② Basis for the use of herbs in the formula: In this formula, mulberry and cornus officinalis are used as the monarch herbs to tonify the liver and kidney and replenish essence and blood; rubus chingii, wolfberry fruit, and polygonatum sibiricum are used as the minister herbs to assist the monarch herbs in exerting the effect of nourishing the liver and kidney and essence and blood; hawthorn is used as the assistant herb to aid digestion, promote qi movement, and remove stasis; cassia seed is used as the guiding herb to direct the herbs into the liver and kidney meridians and harmonize all the herbs. Overall, this formula has the effects of nourishing the liver and kidney and moistening dryness with blood, and is suitable for people with abnormal blood lipids caused by liver and kidney yin deficiency and essence and blood deficiency.

[0023] (2) Theoretical basis of modern food nutrition, functions, medicine, etc.

[0024] Anthrone compounds such as chrysophanol, emodin, and physcion in processed Polygonum multiflorum Thunb. can inhibit adipocyte differentiation and reduce the expression of fat synthase. Flavonoid compounds such as quercetin and rutin have the effects of antioxidant, anti-inflammatory and reducing obesity-related inflammatory responses. Flavonoid compounds such as quercetin, kaempferol, and isoquercitrin in Rubus idaeus L. have the effects of antioxidant, anti-inflammatory, and improving lipid metabolism. Iridoid compounds such as morroniside and loganin in Cornus officinalis Sieb. et Zucc. can reduce blood lipid, decrease fat accumulation, and regulate lipid metabolism. Flavonoid compounds and anthocyanins in Morus alba L. can regulate fatty acid synthesis and reduce fat accumulation.

[0025] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages: The present invention uses three kinds of medicine and food homology substances, namely Morus alba L., Rubus idaeus L., and Cornus officinalis Sieb. et Zucc., to replace Polygonum multiflorum Thunb., and combines with the original formula of Jiangzhiling. The fermented liquid prepared after fermentation can not only effectively retain the obesity regulation effect of the classic formula Jiangzhiling, but also significantly improve its biological activity through optimizing the compatibility and probiotic fermentation technology, providing a safe and efficient innovative solution for functional food to intervene in obesity / overweight. Brief Description of the Drawings

[0026] Figure 1 Regulatory effect of the compound on the body weight of rats (a) Dynamic change curve of rat body weight, (b) Body weight of rats in the eighth week, (c) Weight gain during the intervention period.

[0027] Figure 2 Effect of the compound on liver index and epididymal fat index (a) Liver index, (b) Epididymal fat index.

[0028] Figure 3 Effect of the compound on the digestion of energy substances (a) Amylase activity, (b) Glucosidase activity, (c) Lipase activity, (d) TC content in feces, (e) TG content in feces, (f) Total sugar content in feces.

[0029] Figure 4 Regulatory effect of the compound on liver injury (a) ALT content in blood, (b) AST content in blood.

[0030] Figure 5 Regulatory effect of the compound on liver oxidative stress injury (a) MDA content, (b) SOD enzyme activity, (c) GSH-Px activity.

[0031] Figure 6 Regulatory effect of the compound on the inflammatory response of rats (a) TNF-α content, (b) IL-6 content, (c) NF-κB content. Detailed Description of the Invention

[0032] The technical solution of the present invention will be further described below with reference to the drawings.

[0033] Example 1

[0034] 1. In this example, first, Jiangzhiling (JZL) was used as the control group, and the food-grade Jiangzhiling without Polygonum multiflorum was used as the food-grade basic formula control. The properties, flavors, meridians entered, four natures and five flavors, cold, heat, warmth, and coolness, and main therapeutic effects of 106 medicated and edible homologous substances were statistically analyzed. Finally, the medicated and edible homologous substances with the efficacy of nourishing the liver and kidney were determined to be Lycium barbarum, Morus alba fruit, Rubus idaeus, Macrocarpium officinale, and Eucommia ulmoides leaves. Considering the significant inhibition rate of the individual replacement of Morus alba fruit and Rubus idaeus, the combination replacement of the remaining components with Morus alba fruit and Rubus idaeus was considered, including the replacement of 2, 3, and 4 medicated and edible homologous substances. The specific compound raw materials and weight ratios are shown in Table 1.

[0035] Table 1 List of compound preparations

[0036]

[0037]

[0038] Preparation method: In 2000 kg of deionized water, while stirring, 100 kg of the above-mentioned 13 groups of medicated and edible homologous compound powder mixtures were added separately, mixed evenly, stirred and soaked at room temperature for 30 min, then heated to 85 °C and extracted for 1 h. Then, the extracted mixture was centrifuged (6500 rpm, 20 min) to obtain the extract, sterilized at 135 °C / 5S and 95 °C / 15 min, and cooled for standby to obtain the traditional Chinese medicine liquid preparation.

[0039] 2. Determination of in vitro digestive enzyme inhibitory ability

[0040] (1) α-Amylase inhibition rate

[0041] The above-mentioned 13 groups of traditional Chinese medicine liquid preparations were diluted 10 times respectively. 50 μL of each was mixed with 50 μL of 6 U / mL α-amylase aqueous solution, incubated at 37 °C for 10 min, and then 50 μL of 5 mg / mL soluble starch solution was added and incubated for 10 min. Then, it was placed in a 90 °C water bath to inactivate the enzyme for 1 min, 100 μL of 3,5-dinitrosalicylic acid (DNS) reagent solution was added to the above solution, and it was continued to be placed in a 90 °C water bath for 10 min. Finally, the mixture was diluted with 900 μL of distilled water. 200 μL of the diluted solution was taken to detect its absorbance at 540 nm.

[0042] α-Amylase inhibition rate % = 1 - (A sample - A blank ) / (A test - A control )

[0043] A sample represents the absorbance of the sample (traditional Chinese medicine liquid preparation + enzyme + soluble starch); A blankis the blank absorbance (traditional Chinese medicine liquid + enzyme + distilled water); A test is the test absorbance (enzyme + soluble starch + distilled water); A control is the model absorbance (enzyme + 100 μL distilled water).

[0044] (2) Inhibitory ability of α-glucosidase

[0045] Dilute the above 13 groups of traditional Chinese medicine liquids 10-fold respectively. Take 50 μL each and 50 μL of α-glucosidase with a concentration of 4 U / mL and incubate at 37 °C for 10 min. Then add 50 μL of 2.5 mmol / L 4-nitrophenyl-α-D-glucopyranoside (pNPG) and incubate at 37 °C for 20 min. Add 80 μL of 0.2 mol / L Na2CO3 to the mixture to terminate the reaction. Measure the absorbance of the mixture at 405 nm.

[0046] α-glucosidase inhibition rate % = 1 - (A sample - A blank ) / (A test - A control )

[0047] A sample represents the absorbance of the sample (traditional Chinese medicine liquid + enzyme + pNPG); A blank is the blank absorbance (traditional Chinese medicine liquid + enzyme + PBS); A test is the test absorbance (enzyme + pNPG + distilled water); A control is the model absorbance (enzyme + PBS + distilled water).

[0048] (3) Inhibitory ability of pancreatic lipase

[0049] Dilute the above 13 groups of traditional Chinese medicine liquids 10-fold respectively. Take 50 μL each and add 50 μL of pancreatic lipase solution to a 96-well plate. Incubate at 37 °C for 10 min, then add 50 μL of 4-nitrophenyl butyrate (pNPB) with a concentration of 2 mg / mL and continue to incubate for 20 min. Detect the absorbance value at 405 nm.

[0050] Inhibition rate (%) = 1 - (A sample - A blank ) / (A test - A control )

[0051] A sample is the absorbance after the sample reaction (traditional Chinese medicine liquid + enzyme + pNPB); A blank is the blank absorbance (traditional Chinese medicine liquid + buffer + pNPB); A test is the test absorbance (distilled water + enzyme + pNPB); A controlTo control the absorbance (distilled water + buffer + pNPB).

[0052] (4) Inhibitory ability of cholesterol esterase

[0053] Dilute the above 13 groups of traditional Chinese medicine liquid preparations by 10 times respectively. Take 50 μL and 50 μL of pNPB solution and mix them, then incubate at 37 °C for 10 min. Then add 50 μL of cholesterol esterase with a concentration of 1.63 U / mL and continue to incubate at 37 °C for 20 min. Detect the absorbance of the above mixture at 405 nm.

[0054] Inhibition rate (%) = 1 - (A sample - A blank ) / (A test - A control )

[0055] A sample is the absorbance after the sample reaction (traditional Chinese medicine liquid preparation + enzyme + pNPB); A blank is the blank absorbance (traditional Chinese medicine liquid preparation + buffer + pNPB); Atest is the test absorbance (fermentation broth + enzyme + pNPB); Acontrol is the control absorbance (distilled water + buffer + pNPB).

[0056] The specific results are shown in Table 2.

[0057] Table 2 List of inhibitory abilities of different formula compounds on glycolipid digestive enzymes

[0058]

[0059]

[0060] Different letters represent significant differences (p < 0.05), the same below.

[0061] As can be seen from Table 2, for amylase, compared with the original Jiangzhiling formula (40.34%) and the food-grade basic formula (32.48%), Experimental Group 1 (48.70%), Experimental Group 2 (51.85%), Experimental Group 3 (49.24%) and Experimental Group 4 (46.85%) all showed significant improvements (p < 0.05). Among them, Experimental Group 2 was the highest (51.85%), with an increase of 28.42%. For α-glucosidase, Experimental Group 2 (93.47%) had the highest inhibition rate, which was significantly higher than that of the original Jiangzhiling formula (88.74%) and the food-grade basic formula (72.46%) (p < 0.05). In addition, for Experimental Group 1 (86.14%), Experimental Group 3 (88.45%) and Experimental Group 4 (89.98%), although there was no significant difference compared with the original formula, they were significantly higher than the basic formula group (p < 0.05). For the inhibition rate of cholesteryl esterase, the inhibition rates of Experimental Group 1 (83.22%), Experimental Group 3 (79.45%) and Experimental Group 4 (81.21%) were much higher than those of the original Jiangzhiling formula (67.85%) and the food-grade basic formula (64.36%). Among them, Experimental Group 1 had the largest increase range (p < 0.05). For the inhibition rate of pancreatic lipase, the inhibition rates of Experimental Group 1 (70.21%), Experimental Group 3 (65.48%) and Experimental Group 4 (66.85%) were much higher than those of the original Jiangzhiling formula (34.75%) and the food-grade basic formula (40.35%). Among them, Experimental Group 1 increased by 102% and showed the best performance. Combining the above results, Experimental Groups 1, 2, 3 and 4 could significantly improve their digestive enzyme inhibitory activities (p < 0.05). Generally speaking, Experimental Group 4 had the best effect. Therefore, Experimental Group 4 was used for the next fermentation research in the follow-up.

[0062] Determination of the Ability of the Fermented Compound Prescription in Example 2 to Inhibit Digestive Enzymes

[0063] Using Experimental Group 4 as the raw material of the fermented compound prescription, prepare the traditional Chinese medicine liquid preparation according to the method in Example 1, and then prepare the fermented compound prescription according to Table 3. The inoculation amount of the strain is 3% (v / v), and the number of strains is 1×10 9 CFU / mL. When performing mixed bacteria fermentation, first obtain the bacterial sludge by centrifugal washing, and adjust the number of strains to 1×10 9 CFU / mL. After mixing equal volumes of multiple strains, inoculate them into the compound at a ratio of 3% and then carry out fermentation.

[0064] Table 3 List of the Preparation of the Fermented Compound Prescription

[0065]

[0066] Among them, the 8-strain consortium consists of Lactobacillus fermentum grx08, Lactobacillus rhamnosus grx10, Lactobacillus paracasei grx06, Lactobacillus plantarum grx401, Lactobacillus plantarum grx402, Bifidobacterium grx05, Lactococcus lactis grx602, and Lactobacillus acidophilus grx95. After centrifugation, washing, and resuspension, the viable cell count of each strain reached 1×10 9 CFU / mL. After mixing equal volumes, it was inoculated into the compound extract at 3% (v / v) for fermentation.

[0067] The fermented compound was diluted 40-fold and tested for its ability to inhibit digestive enzyme activity in vitro. The results are shown in Table 4.

[0068] Table 4 List of the inhibitory ability of the fermented compound on digestive enzyme activity

[0069]

[0070] As can be seen from Table 4, experimental groups 5-8 showed significant synergistic effects compared with the original JZL formula and experimental group 4 (p<0.05). Specifically, compared with the original JZL formula, experimental groups 5-8 had significantly higher inhibition rates on α-amylase, α-glucosidase, cholesterol esterase, and pancreatic lipase. For example, the α-amylase inhibition rate of experimental group 8 reached 57.66%, which was 217.9% higher than that of the original JZL formula, and the α-glucosidase inhibition rate increased to 84.55%, far higher than 48.43% of the original JZL formula. Similarly, the cholesterol esterase and pancreatic lipase inhibition rates of experimental group 8 reached 70.48% and 69.87% respectively, which were 80.98% and 216.1% higher than those of the original JZL formula, showing extremely significant functional improvement (p<0.05). In addition, compared with experimental group 4, experimental groups 5-8 showed better inhibitory effects in terms of all enzyme inhibition rates. For example, the α-amylase, α-glucosidase, cholesterol esterase, and pancreatic lipase inhibition rates of experimental group 7 were increased by 190.5%, 76.0%, 71.0%, and 164.4% respectively compared with experimental group 4, indicating significantly enhanced biological activity after optimizing the fermentation process.

[0071] In summary, experimental groups 5-8 not only showed significant synergistic effects compared with the original JZL formula and experimental group 4, but also had better enzyme inhibitory activity compared with the control fermented compound, further highlighting the innovation and application value of this study.

[0072] Example 3 Regulatory effect of the compound fermented liquid on obese rats

[0073] In this example, a simple obesity rat model induced by high-fat diet was used. Orlistat (H20143119) was used as positive control 1, the original formula of Jiangzhiling (JZL) and the substitute formula of Jiangzhiling (experimental group 4, TJZL) were used as control groups, and the fermented compound in the present invention was used for intervention research. The experiment was conducted by intragastric administration to systematically evaluate its anti-obesity and metabolic regulation effects. Specifically, the activities of intestinal digestive enzymes in rats were measured, including α-amylase (Solarbio, BC0615), α-glucosidase (Solarbio, BC0615) and lipase (Solarbio, BC2340), to evaluate the regulatory effects on glucose and lipid metabolism. At the same time, the content of energy substances in feces was analyzed, and the body weight and food intake of rats were recorded. The organ index was calculated by combining the liver and epididymal fat weights to evaluate the weight loss effect at the overall level. Further, the lipid content in blood and liver was detected, and the serum ALT and AST levels (provided by Meikang Biotech Co., Ltd.) were measured to evaluate liver function. In addition, the content of MDA (malondialdehyde, Solarbio, BC0025) in liver and the activities of SOD (Solarbio, BC5165) and GSH-Px (Solarbio, BC1195) were measured to evaluate the level of oxidative stress damage in liver. Finally, the contents of inflammatory factors TNF-α, IL-6 and NF-κB (provided by Shanghai Hualan Chemical Technology Co., Ltd.) were measured to reflect the inflammatory level and further explore the anti-inflammatory effect of the fermented compound. The details are as follows:

[0074] (1) Preparation of the gastric infusion solution for positive control group 1: Take 1 capsule (0.12 g) of Shuerjia Orlistat Capsule, and mix it thoroughly in 72 mL of sterile physiological saline to prepare a 1.65 mg / mL Orlistat solution for standby.

[0075] (2) Animal grouping and establishment of the simple obesity model rats

[0076] The breeding environment was at a temperature of 25 ± 1°C and a humidity of 55 ± 5%, with a 12-hour light-dark cycle. Rats were allowed to drink water and eat normal feed freely for 1 week. The normal feed (product number: XT93M) and high-fat feed (product number: XTHF45) were provided by Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd., and the feed consisted of 10% lard, 10% egg yolk powder, 1% cholesterol, 0.2% bile salt and 78.8% basal feed.

[0077] After adaptation, male rats (4 weeks old, 140 g - 160 g, Wistar, Jinan Pengyue Laboratory Animal Breeding Co., Ltd., SCXK (Lu) 20220006) were randomly divided into 8 groups (n = 8), as shown in Table 5. Except for the blank group, the other groups were fed a high-fat diet for a 4-week modeling period. The body weight of the rats was measured and recorded weekly. When the body weight was more than 10% heavier than that of the blank group, it was confirmed that the model was successfully established. Then, a 4-week intervention was carried out. The concentration of traditional Chinese medicine in the gavage samples used for the intervention was configured according to the ratio of feed to water of 1:15 (w / v). The specific groups are shown in Table 5. The gavage dose was calculated as animal dose (mg / kg) = human dose (mg / d) × conversion factor = 150 mL / d / 60 kg * 6.17 = 1.5 mL / 100 g / d. Therefore, the gavage doses of placebo, traditional Chinese medicine, and western medicine in all groups were 1.5 mL / 100 g / d. Referring to the recommended dosage of orlistat of 0.24 g / d (taken at lunch and dinner), the dose for rats was converted as animal dose (mg / kg) = human dose (mg / kg) × 6.17. Calculated based on 60 kg, the dose was 2.468 mg / 100 g / d. Therefore, the concentration of orlistat was configured to be 1.65 mg / mL.

[0078] Table 5 Animal grouping and gavage treatment

[0079]

[0080] (3) Regulation effect on body weight

[0081] The body weight of the rats was measured at a fixed time on the last day of each week, and the body weight change curve of the rats was plotted ( Figure 1 a). As can be seen from the figure, each intervention group could effectively inhibit the rapid weight gain trend induced by high-fat diet. By the eighth week, the body weight of the model group was 511.38 g, significantly higher than that of the blank group (425.10 g, p < 0.05). After the intervention, the body weight decreased, especially for the fermented compound prescriptions, including experimental groups 5, 7, and 8. Compared with the model group, the body weight in the eighth week was significantly reduced (p < 0.05). By calculating and comparing the weight increments during the 4-week intervention period, it was found that the intervention effect of experimental group 8 was the best, with a weight increment of 77.07 g, significantly lower than that of the model group (126.38 g). In addition, the 3 fermented prescriptions (experimental groups 5, 7, and 8) had a significant improvement compared with the original Jiangzhiling prescription and the substituted Jiangzhiling prescription (experimental group 4) (p < 0.05). The above results indicate that the compound prescription and the fermented compound prescription have a significant effect on inhibiting weight gain, and the effect of the fermented prescription is more significant.

[0082] (4) Regulation effect on liver index and epididymal fat index

[0083] After the animals were sacrificed, the liver and epididymal fat were removed and accurately weighed. The organ index was calculated according to the formula organ index = organ weight (g) / body weight (100 g). The results are asFigure 2 As shown in the figure. It can be seen from the figure that feeding with high-fat diet led to a significant increase in liver index (p<0.05), increasing from 3.03 g / 100 g (blank) to 3.79 g / 100 g (model). After compound intervention, all experimental groups showed a significant decrease (p<0.05), and there was no significant difference between the fermented formula and the non-fermented formula (p>0.05). For the epididymal fat index, the model group (2.93 g / 100 g) was significantly higher than the blank group (1.33 g / 100 g), and all experimental groups could significantly reduce their epididymal fat index. The regulatory effects of experimental groups 7 and 8 were the best, reaching 1.88 g / 100 g and 1.78 g / 100 g respectively. The above results indicate that compound intervention can significantly reduce liver hypertrophy and epididymal fat accumulation caused by obesity, and mixed bacteria fermentation has better regulatory ability.

[0084] (5) Regulatory effect on the digestion of energy substances

[0085] After slaughter, the colon of rats was taken, and the chyme was extruded with a sterile pipette tip and diluted to the same concentration with sterile saline. The activities of pancreatic lipase, amylase and glucosidase in the intestinal contents were measured. The results are as Figure 3 shown. It can be seen from the figure that experimental groups 4, 5, 7 and 8 could all significantly reduce the activity of intestinal amylase (p<0.05), and after fermentation, the inhibitory abilities of experimental groups 5, 7 and 8 were significantly higher than that of experimental group 4 (p<0.05). The same trend was observed in glucosidase. The inhibitory abilities of experimental groups 5, 7 and 8 were the strongest, and the enzyme activities were significantly lower than those of experimental group 4 and the original JZL formula (p<0.05). For the activity of pancreatic lipase, the activity of the model group was as high as 728.55 U / mg prot. The best effect was in the positive control group 1, but after compound intervention, the lipase activities all showed a significant decrease (p<0.05). The best effect was in experimental group 8, and the enzyme activity decreased to 337.28 U / mg prot, with a decrease rate of 53%. There was no significant difference among the fermented experimental groups 5, 7 and 8 (p>0.05), but the enzyme activities were all significantly lower than those of experimental group 4 and the original JZL formula (p<0.05). The above results indicate that both the compound and its fermented formula can significantly inhibit the activities of glycolipid digestive enzymes, and the inhibitory ability can be improved after fermentation.

[0086] Fresh feces of rats were collected, and the contents of total sugar, TC and TG in the dry matter were measured after drying. The results are as Figure 3As shown in the figure. It can be seen from the figure that after the compound intervention, the contents of TC and TG in feces both increased significantly (p<0.05). For the TC content, the regulatory effects of experimental groups 7 and 8 of the fermentation formula were the most significant, reaching 11.99 mmol / mL and 12.59 mmol / mL respectively, which were significantly higher than 7.11 mmol / mL in the model group (p<0.05). For the TG content, the content in the model group was 4.34 mmol / mL. All compound experimental groups could significantly increase its content. Among them, the best effect was in experimental group 7, with its TG content reaching 7.90 mmol / mL, and there was no significant difference among the experimental groups. For the total sugar content, all experimental groups and the original JZL formula could significantly increase the total sugar content in feces. Among them, the regulatory effects of experimental groups 5, 7 and 8 were the best, and the effect of the mixed bacteria fermentation group was significantly higher than that of experimental group 4 (p<0.05).

[0087] The above results show that each compound formula in this patent has excellent ability to inhibit the activities of glycolipid digestive enzymes, can effectively block the digestion and absorption of energy substances, increase the content of energy substances in feces, and the inhibitory ability of the compound can be significantly improved after fermentation.

[0088] (6) Regulatory effect on liver injury

[0089] Blood was taken from the eyeballs, and serum was obtained by centrifugation (4000 rpm, 10 min). The contents of ALT and AST in the serum were measured by an automatic biochemical analyzer to evaluate the liver injury situation. The results are as Figure 4 shown. It can be seen from the figure that the high-fat diet led to a significant increase in the contents of ALT and AST in the blood of rats (p<0.05), reaching 66.97 U / L and 196.33 U / L in the model group respectively. After the compound intervention, the contents of ALT and AST both decreased significantly (p<0.05). Among them, the regulatory effects of experimental groups 7 and 8 of the mixed bacteria fermentation were the best, reaching 36.53 U / L and 34.93 U / L respectively, with no significant difference from the blank group (p>0.05). For the AST content, the content in the model group was 196.33 U / L, which was significantly higher than the other groups (p<0.05). After the intervention of the compound examples, its content decreased significantly (p<0.05), and there was no significant difference among the groups (p>0.05). The above results show that the compound experimental groups can effectively reduce the increase in the contents of ALT and AST caused by obesity, and the regulatory effect is better after fermentation.

[0090] Accurately weigh the fresh liver, grind it at low temperature on ice, and take the supernatant for testing after centrifugation (8500 rpm, 5 min). Measure the oxidative stress product malondialdehyde (MDA) and the activities of oxidative stress enzymes including SOD enzyme and GSH-Px enzyme in the liver. The results are as Figure 5As shown. It can be seen from the figure that the MDA content in the model group was 2.41 mmol / mg prot, which was significantly higher than that in the blank group (0.44 mmol / mg prot, p < 0.05). After intervention, the MDA content in each experimental group decreased significantly compared with the model group (p < 0.05). Among them, the best regulatory effect was in experimental group 8, which was adjusted to 0.58 mmol / mg prot, and there was no significant difference among the experimental groups (p > 0.05). Regarding the SOD enzyme activity, the SOD enzyme activity in the model group was significantly lower than that in the blank group (p < 0.05). After intervention with each example, the SOD enzyme activity increased significantly (p < 0.05). Among them, the enzyme activities in experimental groups 5, 7, and 8 of the fermentation formula were higher than those in experimental group 4, but there was no significant difference (p > 0.05). Regarding the GSH-Px enzyme activity, each experimental group could significantly increase the GSH-Px enzyme activity (p < 0.05). Among them, the regulatory effect of the original JZL formula was the best. The effects of grx08 fermentation (FTJZL 08) and grx08 + 67 fermentation (FTJZL 08 + 67) in the fermented compound formula were better, but there was no significant difference from experimental group 4 (p > 0.05). Combining the above results, it shows that obesity can cause oxidative stress damage, manifested as an increase in MDA content and a decrease in the activities of SOD enzyme and GSH-Px enzyme. All experimental groups can significantly regulate the oxidative stress damage state caused by obesity, and fermentation can improve the regulatory effect of experimental group 4.

[0091] (7) Regulatory effect on inflammatory response

[0092] To explore the regulatory effect of the compound formula on the low-inflammatory response caused by obesity, the contents of TNF-α, IL-6, and NF-κB in the serum were measured, and the results are as Figure 6As shown. It can be seen from the figure that the TNF-α content in the model group was 387.40 pg / mL, which was significantly higher than that in the blank group (166.32 pg / mL) (p < 0.05). After the compound intervention, its content decreased significantly (p < 0.05), and there was no significant difference among the experimental groups (p > 0.05). Regarding the IL-6 content, the content in the model group was 52.51 pg / mL, which was significantly different from that in the blank group (28.42 pg / mL) (p < 0.05). After the intervention of each experimental group, its content decreased significantly. Among them, the best regulatory effects were achieved in experimental groups 5 and 7 of grx08 fermentation (FTJZL 08) and grx08 + 67 mixed bacteria fermentation (FTJZL 08 + 67), reaching 34.21 pg / mL and 35.56 pg / mL respectively, but there was no significant difference among the experimental groups (p > 0.05). Regarding the NF-κB content, the content in the model group was 1248.31 pg / mL, which was significantly higher than that in the blank group. Each experimental group could significantly reduce its content. Among them, the regulatory effect of the compound after mixed bacteria fermentation (experimental group 8) was the best, which was significantly lower than that in experimental group 4 (p < 0.05), indicating that mixed bacteria fermentation significantly improved the inflammatory regulatory ability of the compound. The above results show that each experimental group has a significant alleviation of the low inflammatory response caused by obesity, specifically manifested as a decrease in the levels of inflammatory factors TNF-α, IL-6, and NF-κB.

Claims

1. A traditional Chinese medicine preparation fermentation broth, characterized in that, The traditional Chinese medicine preparation comprises the following components in parts by weight: 3 - 6 parts of mulberry, 3 - 6 parts of raspberry, 3 - 6 parts of cornel, 12 - 20 parts of wolfberry, 15 - 25 parts of polygonatum, 15 - 25 parts of hawthorn, and 2 - 5 parts of semen cassiae; the traditional Chinese medicine preparation is prepared by extraction, sterilization, cooling, and inoculating a fermentation agent for fermentation.

2. The fermented liquid of the traditional Chinese medicine preparation according to claim 1, wherein, The fermentation agent is composed of one or a combination of more than one of lactobacillus fermentum, lactobacillus rhamnosus, lactobacillus paracasei, lactobacillus plantarum, bifidobacterium, lactococcus lactis, and lactobacillus acidophilus.

3. The fermented liquid of the traditional Chinese medicine preparation according to claim 2, wherein, The fermentation agent is composed of one or a combination of more than one of lactobacillus fermentum grx08, lactobacillus rhamnosus grx10, lactobacillus paracasei grx06, lactobacillus plantarum grx401, lactobacillus plantarum grx402, bifidobacterium grx05, lactococcus lactis grx602, and lactobacillus acidophilus grx95.

4. The preparation method of the fermentation broth of the traditional Chinese medicine preparation according to any one of claims 1 to 3, characterized in that, The preparation method comprises the following steps: weighing, crushing, and sieving each component raw material according to the weight ratio, fully mixing, extracting, sterilizing, cooling, inoculating the fermentation agent, and fermenting at 37°C for 24 - 48 h to obtain the fermentation broth.

5. The preparation method according to claim 4, characterized in that, The inoculum size of the fermentation broth is 2%-5%, and the number of strains is 1×10 9 CFU / mL.

6. Use of the fermentation broth of the traditional Chinese medicine preparation according to any one of claims 1 to 3 in the preparation of a product for losing weight and reducing lipid and / or relieving liver injury.

7. The application according to claim 6, wherein The weight loss and lipid reduction include inhibiting weight gain, relieving liver hypertrophy and epididymal fat accumulation, inhibiting the activity of glycolipid digestive enzymes, and increasing the total sugar content in feces.

8. The application according to claim 6, characterized in that The relieving of liver injury includes reducing the contents of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum, reducing oxidative stress injury of the liver, and relieving inflammatory reactions.

9. The application according to claim 8, wherein The reduction of oxidative stress injury of the liver includes reducing the content of malondialdehyde and increasing the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px).

10. The application according to claim 8, characterized in that, The relieving of inflammatory reactions includes reducing the contents of inflammatory factors TNF-α, IL-6, and NF-κB.

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