Hypoglycemic enzyme-bacterium symbiotic traditional Chinese medicine composition and preparation method thereof

Abstract

This invention belongs to the field of traditional Chinese medicine technology, specifically the field of probiotics technology, and specifically relates to a hypoglycemic enzyme-bacterial symbiotic traditional Chinese medicine composition and its preparation method. This invention employs an enzyme-bacterial symbiotic process. First, cellulase and pectinase disrupt plant cell walls, releasing intracellular polysaccharides, flavonoids, and other active ingredients. Subsequently, β-glucosidase and other enzymes produced during the fermentation of probiotics (especially *Lactobacillus plantarum*) can convert bound glycosides into more active aglycones (such as converting isoflavones and ginsenosides into aglycones), while simultaneously producing short-chain fatty acids. Experiments have shown that compared with conventional water extraction, the total flavonoid content in the composition of this invention is increased by more than 35%, and the molecular weight of total polysaccharides is reduced, making it more easily absorbed. Animal experiments show that, at the same dosage, the composition of this invention reduces fasting blood glucose in alloxan-induced diabetic mice by 42% more than that of the traditional mixing process.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically the field of probiotics technology, and specifically relates to a blood sugar-lowering enzyme-bacterial symbiotic traditional Chinese medicine composition and its preparation method. Background Technology

[0002] Diabetes mellitus is a metabolic disease characterized by hyperglycemia. Long-term hyperglycemia can lead to chronic damage and dysfunction of multiple organs, including the eyes, kidneys, heart, and blood vessels. Currently, commonly used hypoglycemic drugs in clinical practice include biguanides, sulfonylureas, alpha-glucosidase inhibitors, and insulin. Although these drugs can effectively control blood sugar, long-term use may be accompanied by side effects such as gastrointestinal reactions, liver and kidney damage, risk of hypoglycemia, and weight gain.

[0003] In recent years, traditional Chinese medicine and natural products have shown unique advantages in assisting in lowering blood sugar. Codonopsis pilosula, Eucommia ulmoides, Camellia chrysantha, Eucommia ulmoides male flowers, and Inonotus obliquus (chaga mushroom) have all been reported to regulate blood sugar, improve insulin resistance, or protect pancreatic β-cells. Meanwhile, regulating the gut microbiota has become a new target for intervention in type 2 diabetes, and probiotic supplementation can improve host glucose metabolism.

[0004] Traditional Chinese medicine (TCM) compound prescriptions often employ water extraction, alcohol extraction, or direct pulverization and mixing processes. However, these methods struggle to fully release the active ingredients (such as polysaccharides, flavonoids, and terpenes) in the herbs and fail to biotransform the large molecules within the herbs, thus limiting their bioavailability and efficacy. Enzyme-microbial symbiosis is a novel biotechnology combining enzymatic hydrolysis and microbial fermentation, but its application in TCM compound prescriptions for adjuvant hypoglycemia is currently limited.

[0005] Defects and shortcomings of existing technology:

[0006] (1) Insufficient release of active ingredients and slow onset of action: Traditional decoction or alcohol extraction processes are difficult to effectively destroy the cell wall structure of medicinal plants, resulting in low dissolution rates of large molecular active ingredients such as polysaccharides and flavonoids. Direct consumption of medicinal powder results in limited digestion and absorption by the human body, leading to insignificant hypoglycemic effects and slow onset of action.

[0007] (2) The bitter taste and irritating components of Chinese medicine affect patient compliance: Some medicinal materials (such as Codonopsis pilosula and Inonotus obliquus) have special odors or bitter tastes. Traditional preparations have poor taste, and patients (especially diabetic patients who need to take them for a long time) have poor compliance.

[0008] (3) Lack of synergistic effect: In the existing technology, the traditional Chinese medicine composition and probiotics are usually simply mixed. Before the probiotics colonize in the gastrointestinal tract, they may be inhibited by certain components in the traditional Chinese medicine extract; at the same time, the unfermented traditional Chinese medicine components are not pre-converted by the probiotics, and the two fail to form a real synergistic effect.

[0009] (4) Problems with strain stability and activity retention: In ordinary physical mixing processes, probiotics are mixed with traditional Chinese medicine ingredients treated at high temperatures. Due to the influence of moisture, temperature or antibacterial ingredients, the survival rate may be low, resulting in insufficient number of live bacteria in the product, making it difficult to play the role of regulating intestinal flora. Summary of the Invention

[0010] To address the problems existing in the prior art, this invention aims to achieve the following technical objective: to provide a preparation method that can fully release the active hypoglycemic components in medicinal materials such as Codonopsis pilosula and Eucommia ulmoides. This method achieves synergistic effects between the medicinal components and probiotics, generates new active metabolites through biotransformation, and improves the survival rate and colonization ability of probiotics.

[0011] To achieve the above-mentioned objectives, the present invention employs the following technical solution:

[0012] The first aspect of this invention discloses a method for preparing an enzyme-bacterial symbiotic traditional Chinese medicine composition, comprising the following steps:

[0013] (1) After pulverizing the Chinese herbal raw materials, add water and adjust the pH to 5.0-6.5, then add the compound enzyme for enzymatic hydrolysis;

[0014] The Chinese medicinal materials are formulated in the following proportions by weight: Codonopsis pilosula 10-20 parts, Eucommia ulmoides 15-30 parts, Camellia chrysantha 5-15 parts, Eucommia ulmoides male flower 5-12 parts, and Inonotus obliquus 8-18 parts.

[0015] The complex enzyme includes cellulase and pectinase;

[0016] (2) After the enzymatic hydrolysis is completed, the enzyme is inactivated, and after cooling, the compound probiotic powder is added;

[0017] The compound probiotic powder contains at least two of the following: Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus rhamnosus, and Lactobacillus reuteri.

[0018] (3) Under constant temperature of 36-38℃, anaerobic fermentation is carried out for 24-72 hours to obtain the fermentation broth of the enzyme-containing bacterial symbiotic traditional Chinese medicine composition.

[0019] The amount of the compound probiotic powder added is 2-8 parts by weight.

[0020] Preferably, in step (1), the enzymatic hydrolysis temperature is 45-55℃ and the enzymatic hydrolysis time is 1-3 hours;

[0021] Preferably, in step (1), the mass ratio of cellulase to pectinase in the compound enzyme is 2:1, and the total amount of enzyme added is 0.5%-2% of the mass of the Chinese herbal raw materials;

[0022] Preferably, in step (2), the enzyme inactivation temperature is 85-95℃ and the enzyme inactivation time is 10-20 minutes;

[0023] Preferably, the fermentation broth obtained in step (3) is sterilized after separating the medicinal residue to obtain an enzyme-bacterial symbiotic traditional Chinese medicine composition.

[0024] The second aspect of this invention discloses an enzyme-bacterial symbiotic traditional Chinese medicine composition prepared by the above-mentioned preparation method.

[0025] The third aspect of this invention discloses the application of the above-mentioned enzyme-bacterial symbiotic traditional Chinese medicine composition in the preparation of drugs or functional foods that help lower blood sugar.

[0026] Compared with the prior art, the beneficial effects of this invention are as follows:

[0027] 1. Significantly Increased Active Ingredients and Enhanced Blood Glucose-Lowering Effect: This invention employs an enzyme-bacterial symbiotic process. First, cellulase and pectinase disrupt plant cell walls, releasing intracellular polysaccharides, flavonoids, and other active ingredients. Subsequently, β-glucosidase and other enzymes produced during the fermentation of probiotics (especially *Lactobacillus plantarum*) can convert bound glycosides into more active aglycones (e.g., converting isoflavones and ginsenosides into aglycones), while simultaneously producing short-chain fatty acids. Experiments have shown that compared to conventional water extraction, the total flavonoid content in the composition of this invention is increased by more than 35%, and the molecular weight of total polysaccharides is reduced, making them more easily absorbed. Animal experiments show that, at the same dosage, the composition of this invention reduces fasting blood glucose in alloxan-induced diabetic mice by 42% more than that of the traditional mixing process.

[0028] 2. Improved taste and increased compliance: The lactic acid, acetic acid and other organic acids produced by probiotic fermentation give the product a mild sour taste, while metabolizing some bitter and astringent components in traditional Chinese medicine (such as certain alkaloids and tannins), which significantly improves the taste of the product and makes it easier for patients to accept long-term use.

[0029] 3. Achieving Synergistic Effects of Traditional Chinese Medicine and Probiotics: During fermentation, the components of traditional Chinese medicine act as prebiotics, promoting the proliferation of probiotics; simultaneously, probiotics produce new active substances through biotransformation. The added probiotics ensure a high live bacteria count in the final product. The final product contains both small-molecule active substances produced during fermentation and highly active probiotics, which can synergistically lower blood sugar through two pathways: improving insulin resistance and regulating gut microbiota.

[0030] 4. Simple operation and easy to industrialize: The process of this invention is relatively simple, the equipment requirements are not high, the fermentation cycle is short (24-72 hours), which is suitable for large-scale industrial production and the cost is controllable. Detailed Implementation

[0031] The technical solution of the present invention will be described in detail below with reference to embodiments, but this does not limit the present invention to the scope of the embodiments described. Process parameters not specified in the embodiments of this application can be performed according to conventional methods, and all raw materials used can be obtained through commercial channels.

[0032] Example 1: Preparation of an enzyme-bacterial symbiotic traditional Chinese medicine composition (Tangrenyin)

[0033] (1) Raw material ratio (parts by weight): 15 parts of Codonopsis pilosula, 20 parts of Eucommia ulmoides, 10 parts of Camellia chrysantha, 8 parts of Eucommia ulmoides male flowers, 12 parts of Inonotus obliquus. 5 parts of compound probiotic powder (of which the ratio of Lactobacillus plantarum to Lactobacillus acidophilus is 1:1, and the total number of live bacteria is 2×10^11 CFU / g).

[0034] (2) Pretreatment: The above-mentioned Chinese herbal raw materials are dried, pulverized and passed through a 60-mesh sieve to obtain mixed powder.

[0035] (3) Enzymatic hydrolysis: Add sterile water at 5 times the total mass of the mixed powder, adjust the pH to 5.8, add compound enzyme (cellulase: pectinase = 2:1, total enzyme amount 1%), enzymatic hydrolysis at 50℃ for 2 hours, after enzymatic hydrolysis, inactivate the enzyme at 90℃ for 15 minutes.

[0036] (4) Sterilization and cooling: After enzyme inactivation treatment, the enzyme hydrolysate is cooled to 37°C, compound probiotic powder is added, and the powder is stirred to mix it thoroughly. The mixture is then anaerobic fermented at 37°C for 48 hours.

[0037] (6) Post-processing: After fermentation, the residue and fermentation juice are filtered and separated. The fermentation juice is heated to 95℃ and kept for 30 minutes for sterilization. It is then filled into 30mL glass oral liquid bottles in a sterile room and sterilized again. After cooling, it is labeled, boxed, and packed into cartons to make the finished product.

[0038] Comparative Example 1: Traditional Water Extraction Method

[0039] Take the same proportion of Chinese herbal raw materials as in Example 1, add 10 times the amount of sterile water, reflux extract at 100°C for 2 hours, filter, and concentrate to the same volume as the fermented juice of the separated medicinal residue in Example 1 of this invention.

[0040] Comparative Example 2: Enzymatic hydrolysis

[0041] Using only steps (1)-(3) of Example 1, the enzymatic hydrolysis product was obtained by direct filtration after enzyme inactivation.

[0042] Example 2: Detection of active ingredients in the composition

[0043] 1. Determination of total flavonoid content

[0044] The total flavonoid content in the sample was determined by the sodium nitrite-aluminum nitrate colorimetric method.

[0045] 2. Determination of total polysaccharide content

[0046] The total polysaccharide content in the samples was determined using the phenol-sulfuric acid method.

[0047] 3. Determination of polysaccharide molecular weight distribution

[0048] The molecular weight distribution of polysaccharides in the samples was determined by high performance liquid chromatography-size exclusion chromatography (HPLC-SEC).

[0049] 4. The results of the content of active ingredients by different preparation methods are shown in Table 1;

[0050] Group Total flavonoids (mg / g) Total polysaccharides (mg / g) Percentage of low molecular weight polysaccharides (<10 kDa) (%) Example 1 16.78 58.67 52 Comparative Example 1 12.35 45.13 15 Comparative Example 2 13.32 54.25 28

[0051] Compared with the control group, the total flavonoid content in the group of this invention was increased by more than 30%, and the proportion of low molecular weight polysaccharides increased from 15% to 52%. This may be because the fermentation process designed in this invention consumes monosaccharides, disaccharides, etc., and generates extracellular polysaccharides. The results show that the enzyme-bacterial symbiosis process significantly promotes the production and transformation of polysaccharides, as well as the increase in the total flavonoid content of biological components.

[0052] Example 2: Efficacy Verification Experiment

[0053] Experimental subjects: SPF-grade Kunming mice, weighing 20±2g, male, 50 mice in total.

[0054] Model establishment: A diabetes model was established by intraperitoneal injection of alloxan (200 mg / kg). A fasting blood glucose level ≥11.1 mmol / L after 7 days was considered a successful model.

[0055] Grouping: Forty model mice were randomly divided into four groups of 10 mice each: model control group (administered saline), positive control group (metformin, 100 mg / kg / d), traditional mixed group (equal amounts of traditional Chinese medicine powder and probiotic powder (same as in Example 1), 500 mg / kg / d), and the present invention group (the composition prepared in Example 1, 500 mg / kg / d). Ten normal mice were also included as a blank control group.

[0056] Administration method: Administered by gavage for 28 consecutive days. The model group and the blank group were given the same volume of physiological saline.

[0057] Detection indicators: Fasting blood glucose (FBG) was measured before administration and on days 7, 14, 21, and 28 after administration. Results are shown in the table below:

[0058] Group Dosage (mg / kg) Pre-drug blood glucose (mmol / L) Blood glucose (mmol / L) after 28 days of drug administration Blood glucose reduction rate (%) Blank control group - 5.62±0.34 5.48±0.41 - Model control group - 22.35±2.15 24.18±2.86 -8.2 (increase) Positive control group 100 22.01±1.98 12.54±1.76** 43.0 Traditional Mixed Group 500 21.85±2.21 17.32±2.05* 20.7 This invention group 500 22.14±2.08 10.86±1.52** ## 50.9

[0059] Note: Compared with the model control group, *P<0.05, **P<0.01; compared with the traditional mixed group, #P<0.05, ##P<0.01.

[0060] Experimental conclusion: The enzyme-bacterial symbiotic traditional Chinese medicine composition provided by this invention can significantly reduce fasting blood glucose in diabetic model mice. Its hypoglycemic effect is superior to that of traditional physical mixing processes and is similar to that of the positive control drug metformin. The results indicate that the enzyme-bacterial symbiotic process of this invention effectively enhances the auxiliary hypoglycemic effect of the traditional Chinese medicine composition.

[0061] This invention is not limited to the above-described embodiments. Any changes in shape or structure are within the scope of protection of this invention. The scope of protection of this invention is defined by the appended claims. Those skilled in the art can make various changes, modifications, substitutions, combinations, and simplifications to these embodiments without departing from the principles and essence of this invention. All such changes and simplifications should be considered equivalent substitutions and fall within the scope of protection of this invention.

Claims

1. A preparation method of an enzyme bacteria symbiotic traditional Chinese medicine composition, characterized in that, Includes the following steps: (1) After pulverizing the Chinese herbal raw materials, add water and adjust the pH to 5.0-6.5, then add the compound enzyme for enzymatic hydrolysis; The Chinese medicinal materials are formulated in the following proportions by weight: Codonopsis pilosula 10-20 parts, Eucommia ulmoides 15-30 parts, Camellia chrysantha 5-15 parts, Eucommia ulmoides male flower 5-12 parts, and Inonotus obliquus 8-18 parts. The complex enzyme includes cellulase and pectinase; (2) After the enzymatic hydrolysis is completed, the enzyme is inactivated, and after cooling, the compound probiotic powder is added; The compound probiotic powder contains at least two of the following: Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus rhamnosus, and Lactobacillus reuteri. (3) Under constant temperature of 36-38℃, anaerobic fermentation is carried out for 24-72 hours to obtain the fermentation broth of the enzyme-containing bacterial symbiotic traditional Chinese medicine composition.

2. The production method according to claim 1, characterized by, The amount of the compound probiotic powder added is 2-8 parts by weight.

3. The preparation method according to claim 1, characterized in that, In step (1), the enzymatic hydrolysis temperature is 45-55℃ and the enzymatic hydrolysis time is 1-3 hours.

4. The production method according to claim 1, characterized by, In step (1), the mass ratio of cellulase to pectinase in the compound enzyme is 2:1, and the total amount of enzyme added is 0.5%-2% of the mass of the Chinese herbal raw materials.

5. The preparation method according to claim 1, characterized in that, In step (2), the enzyme inactivation temperature is 85-95℃ and the enzyme inactivation time is 10-20 minutes.

6. The preparation method according to claim 1, characterized in that, After separating the medicinal residue from the fermentation broth obtained in step (3), the mixture is sterilized to obtain an enzyme-bacterial symbiotic traditional Chinese medicine composition.

7. The enzyme-bacterial symbiotic traditional Chinese medicine composition prepared by any one of claims 1-6.

8. The application of the enzyme-bacterial symbiotic traditional Chinese medicine composition according to claim 7 in the preparation of drugs or functional foods that assist in lowering blood sugar.