Dendrobium officinale polysaccharide-containing composition for reducing blood sugar as well as preparation method and application of dendrobium officinale polysaccharide-containing composition

Abstract

The invention discloses a composition containing dendrobium officinale polysaccharide for reducing blood sugar as well as a preparation method and application of the composition, and belongs to the technical field of medicines for reducing blood sugar. The composition comprises the following raw materials in parts by weight: 10-15 parts of ginsenoside, 15-20 parts of dendrobium officinale polysaccharide, 2-5 parts of inulin, 5-8 parts of pachymaran, 2-5 parts of plantain herb extract, 1-2 parts of vitamin B6, 3-5 parts of spirulina extract, 1-2 parts of itaconic acid-coenzyme A, and recombinant escherichia coli for expressing tryptophan decarboxylase gene, wherein the viable count per gram of recombinant escherichia coli is 1.1 * 10 < 5 >-1.2 * 10 < 5 > CFU / g. The viable count of tryptophan metabolic bacteria per gram is 1.2 * 10 < 2 >-1.5 * 10 < 3 > CFU / g. The hypoglycemic composition containing the dendrobium officinale polysaccharide can improve the hypoglycemic effect of the dendrobium officinale polysaccharide from different action ways, the hypoglycemic effect is more efficient, and the content of butyric acid in intestinal tracts is increased.

Description

Technical Field

[0001] The present invention relates to the technical field of anti - hyperglycemic drugs, and particularly relates to a hypoglycemic composition containing polysaccharides of Dendrobium officinale, and a preparation method and application thereof. Background Art

[0002] Diabetes is a metabolic disease characterized by hyperglycemia. With the improvement of living standards, people's lifestyle and dietary structure have also changed. In recent years, diabetes has seriously endangered human health. Diabetes has become one of the three major lethal diseases in humans with a high prevalence and low treatment rate, and its mortality rate is second only to cardiovascular and cerebrovascular diseases and cancer. Dendrobium officinale is a functional food popular among healthy people and diabetes patients. As its main component, Dendrobium officinale polysaccharide (DOP) is mainly utilized by the gut microbiota. Although the gut microbiota composition differs between healthy and diabetes individuals, no study has compared the interaction between Dendrobium officinale polysaccharide and the gut microbiota in healthy and diabetes states. This study aimed to investigate and compare the metabolic characteristics and regulatory potential of Dendrobium officinale polysaccharide on the gut microbiota in healthy and diabetes states. Our series of in vitro fermentation studies found that compared with the healthy state, the gut microbiota in the diabetes state utilized more mannose in Dendrobium officinale polysaccharide, with a higher propionate production and a lower butyrate production. In addition, metabolomics analysis showed that the effects of Dendrobium officinale polysaccharide on gut microbial metabolites were different in healthy and diabetes states, and the upregulation of taurine and the downregulation of 2-hydroxybutyric acid only occurred in the diabetes state. Biodiversity analysis indicated that the treatment with Dendrobium officinale polysaccharide could only significantly improve the gut microbiota diversity in the diabetes state, but had no significant effect on the gut microbiota diversity in the healthy state. Further gut microbiota composition analysis showed that the treatment with Dendrobium officinale polysaccharide could promote probiotics (Dubosiella, Bifidobacterium, and Akkermansia) in both healthy and diabetes states, while only inhibiting pathogens (Escherichia-Shigella) in the diabetes state. (Song Q, Zou J, Cheng S W, et al. Insights Into Metabolic Signatures and Regulatory Effect of Dendrobium officinale Polysaccharides in Gut Microbiota: A Comparative Study of Healthy and Diabetic Status[J]. Food Science&Nutrition, 2025, 13(1):e4651.). In summary, our current study on the metabolic characteristics and regulatory effects of Dendrobium officinale polysaccharide in the gut microbiota in healthy and diabetes states provides a scientific basis for its wide application as a functional food. Dendrobium officinale polysaccharide remains intact after digestion in the stomach and small intestine, thus highly suggesting that the large intestine is an important site for DOP.

[0003] Dendrobium officinale polysaccharide helps to improve hepatic glycogen metabolism in type 2 diabetic mice, relieve hyperglycemia in diabetic mice, and its hypoglycemic mechanism may be to affect the glucagon-mediated signaling pathways CAMP-PKA and Akt / FOXO1, regulate hepatic glycogen metabolism; and reverse the instability of hepatic glycogen structure and inhibit glycogen degradation (LIU Y G, YANG L L, ZHANG Y, et al. Dendrobium officinale polysaccharide ameliorates diabetic hepatic glucosemetabolism via glucagon-mediated signaling pathways and modifying liver-glycogen structure[J]. JEthnopharmacol, 2020, 248:112308.).

[0004] Short-chain fatty acids (SCFAs) are mainly produced by the gut microbiota in the colon through the fermentation of carbohydrates (including dietary and endogenous carbohydrates, mainly resistant starch and dietary fiber) and some microbial-available proteins. Short-chain fatty acids are mainly acetic acid, propionic acid and butyric acid, and the concentration ratio in the colon is approximately 60-70%:20-30%:10-20%. These metabolites can be utilized by the host, especially butyric acid, to exert a series of functions promoting intestinal health. Butyric acid can bind to G protein-coupled receptors on host cells (such as GPR41, GPR43, GPR109A) to regulate immune responses and inflammation levels. For example, butyric acid inhibits inflammatory responses and protects intestinal mucosa by activating GPR109A.

[0005] Studies have shown that ginsenosides (especially PPD type and PPT type such as Rg1, Rg2, Rg3) have shown significant effects in improving insulin sensitivity, anti-inflammation and regulating glucose and lipid metabolism. Processed ginseng products or single ginsenoside preparations can directly regulate blood glucose and the expression of TNF-α and eNOS, confirming that they can regulate blood glucose and insulin resistance through anti-inflammatory pathways. This application proposes a hypoglycemic composition containing Dendrobium officinale polysaccharide and matsutake polysaccharide. Summary of the Invention

[0006] As described in the above prior art, one of the objectives of the present invention is to provide a hypoglycemic composition containing Dendrobium officinale polysaccharide and matsutake extract, which can improve the hypoglycemic effect of Dendrobium officinale polysaccharide from different action pathways, has a more efficient hypoglycemic effect, and increases the content of butyric acid in the intestine.

[0007] The second object of the present invention is to provide a method for manufacturing the hypoglycemic composition containing polysaccharides from Dendrobium officinale, and this method has simple steps and can industrially produce the hypoglycemic composition containing polysaccharides from Dendrobium officinale.

[0008] The third object of the present invention is to provide the application of the hypoglycemic composition containing polysaccharides from Dendrobium officinale in the preparation of hypoglycemic drugs.

[0009] The first object of the present invention is achieved by adopting the following technical solutions:

[0010] A hypoglycemic composition containing polysaccharides from Dendrobium officinale, and the raw materials by weight include: 10 - 15 parts of ginsenoside, 15 - 20 parts of polysaccharides from Dendrobium officinale, 2 - 5 parts of inulin, 5 - 8 parts of pachyman polysaccharide, 2 - 5 parts of plantain extract, 1 - 2 parts of vitamin B6, 3 - 5 parts of spirulina extract, 1 - 2 parts of itaconic acid - coenzyme A, recombinant Escherichia coli expressing tryptophan decarboxylase gene with viable count of 1.1×10 5 ~1.2×10 5 CFU / g, and tryptophan - metabolizing bacteria with viable count of 1.2×10 2 ~1.5×10 3 CFU / g.

[0011] Further, the raw materials by weight include: 10 parts of ginsenoside, 20 parts of polysaccharides from Dendrobium officinale, 5 parts of inulin, 5 parts of pachyman polysaccharide, 3 parts of plantain extract, 1 part of vitamin B6, 5 parts of spirulina extract, 1 part of itaconic acid - coenzyme A, recombinant Escherichia coli expressing tryptophan decarboxylase gene with viable count of 1.2×10 5 CFU / g, and tryptophan - metabolizing bacteria with viable count of 1.2×10 3 CFU / g.

[0012] Further, the tryptophan - metabolizing bacteria are one or two of Lactobacillus reuteri and Gastrococcus.

[0013] Further, the construction steps of the recombinant Escherichia coli expressing tryptophan decarboxylase gene include the following steps:

[0014] S1. Gene cloning and vector construction: Extract DNA from Saccharomyces cerevisiae, and amplify the tryptophan decarboxylase gene by PCR reaction;

[0015] S2. Expression vector construction: Insert the tryptophan decarboxylase gene into the pET series expression vector suitable for Escherichia coli to ensure the expression of the tryptophan decarboxylase gene under the control of the strong promoter T7 promoter;

[0016] S3. Host cell transformation: Select a suitable host cell (such as Escherichia coli BL21(DE3)), and introduce the constructed expression vector into the host cell by heat shock method;

[0017] S4. Induced expression and enzyme activity detection: In the host cell, activate the T7 promoter by adding an inducer (such as IPTG) to initiate the expression of the tryptophan decarboxylase gene.

[0018] S5. Lyse the host cell Escherichia coli, extract the tryptophan decarboxylase protein from the host cell lysate, use tryptophan as a substrate, and detect the production of acetyl 5-hydroxytryptamine by high performance liquid chromatography (HPLC) to evaluate the enzyme activity of tryptophan decarboxylase.

[0019] Furthermore, the preparation method of the polysaccharide of Dendrobium officinale is as follows: Cut the fresh stems of Dendrobium officinale into slices, bake them in an oven at 40 °C, boil the obtained dried stems of Dendrobium officinale in distilled water at a ratio of 0.1 kg / L for 1 h, repeat the extraction 2 times, then mix the extracts, and then centrifuge at a centrifugal speed of 4500 rpm for 15 min. After centrifugation, lyophilize the supernatant to obtain the polysaccharide of Dendrobium officinale.

[0020] The second object of the present invention is achieved by the following technical solution:

[0021] A manufacturing method of a hypoglycemic composition containing the polysaccharide of Dendrobium officinale, comprising the following steps:

[0022] S1. Raw material preparation: Weigh the raw materials of each component according to the formula amount and set aside; Dissolve the ginsenoside and the polysaccharide of Dendrobium officinale in deionized water respectively, stir evenly and set aside; Mix the inulin, the polysaccharide of Poria cocos and the extract of Plantago asiatica evenly, add deionized water and stir to dissolve; Dissolve the vitamin B6 and itaconic acid - coenzyme in deionized water and set aside; Dissolve the extract of Spirulina platensis in deionized water and stir evenly;

[0023] S2. Mixing and dissolving: Mix the dissolved ginsenoside, the polysaccharide of Dendrobium officinale, the inulin, the polysaccharide of Poria cocos, the extract of Plantago asiatica, the vitamin B6, the extract of Spirulina platensis and itaconic acid - coenzyme together, stir evenly to ensure that each component is fully dissolved and mixed to obtain a mixed solution;

[0024] S3. Adding probiotics and recombinant Escherichia coli: Add the recombinant Escherichia coli expressing the tryptophan decarboxylase gene, the probiotics and the tryptophan - metabolizing bacteria to the mixed solution respectively, stir gently to make the bacterial cells evenly distributed, and use a homogenizer to homogenize the mixed solution to ensure that each component is evenly dispersed to form a stable mixed solution;

[0025] S4. Drying treatment: The homogenized mixture is made into powder by spray drying or freeze drying to obtain the composition for reducing blood sugar containing polysaccharide of Dendrobium officinale.

[0026] The third object of the present invention is to provide an application of the composition for reducing blood sugar containing polysaccharide of Dendrobium officinale in the preparation of drugs for reducing blood sugar.

[0027] Furthermore, the composition for reducing blood sugar containing polysaccharide of Dendrobium officinale can also be used as an active ingredient to prepare the drug for reducing blood sugar together with pharmaceutically acceptable drug excipients; the drug excipients are one of excipients, disintegrants, sweeteners, binders, coating agents and swelling agents.

[0028] Furthermore, the drug for reducing blood sugar is one of solution, emulsion, suspension, injection, powder, granule, tablet and pill.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0030] (1) A hypoglycemic composition containing polysaccharides from Dendrobium officinale provided by the present invention. The polysaccharides from Dendrobium officinale can regulate the intestinal flora, increase the abundance of beneficial bacteria, and more focus on improving the intestinal microenvironment by regulating the intestinal flora; while ginsenosides can improve gastric emptying speed and reduce gastrointestinal discomfort by regulating the release of neurotransmitters and enhancing gastrointestinal motility; ginsenosides can regulate the secretion of gastric acid and mucus and enhance the protective function of the intestinal mucosa; intestinal inflammation releases pro-inflammatory cytokines such as TNF-α and IL-6, which will exacerbate insulin resistance. Ginsenosides alleviate intestinal inflammation by regulating the immune response and inflammatory signaling pathways (such as the NF-κB pathway), thereby optimizing the intestinal microenvironment, improving intestinal health, and promoting the absorption and utilization of nutrients. By inhibiting the NF-κB signaling pathway, butyric acid can reduce the production of these inflammatory factors. Poria cocos polysaccharide can reduce the abundance of Firmicutes and Bacteroidetes, and improve the microbial population in the intestines of diabetic patients. Inulin enriches the raw material source for butyric acid production. The activated form of itaconic acid, namely itaconyl-CoA, directly blocks the vitamin B12-dependent metabolic pathway, that is, prevents the succinate pathway. Microorganisms in the intestine use propionic acid metabolized by the succinate pathway to grow, increasing the content of butyric acid in the intestine; by adding Spirulina extract, tryptophan-metabolizing bacteria, and recombinant Escherichia coli expressing the tryptophan decarboxylase gene, it can effectively increase the metabolites in the intestine, promote the secretion of glucagon-like peptide-1 (GLP-1), and at the same time regulate the intestinal immune response and enhance the intestinal barrier function; Plantain extract has good water solubility and swelling properties, forms a gel-like substance in the intestine, increases the viscosity of the intestinal contents, and thus delays the absorption of glucose, helping to control the rapid rise of postprandial blood sugar; the above respective effects improve the hypoglycemic effect of polysaccharides from Dendrobium officinale from different action pathways, making blood sugar lowering more efficient. Detailed implementation mode

[0031] Next, in combination with specific embodiments, the present invention will be further described. It should be noted that on the premise of no conflict, the following described embodiments or technical features can be arbitrarily combined to form new embodiments.

[0032] Example 1

[0033] This example provides a hypoglycemic composition containing polysaccharides from Dendrobium officinale. The raw materials by weight include: 10 parts of ginsenosides, 20 parts of polysaccharides from Dendrobium officinale, 5 parts of inulin, 5 parts of Poria cocos polysaccharide, 3 parts of plantain extract, 1 part of vitamin B6, 5 parts of Spirulina extract, 1 part of itaconyl-CoA, recombinant Escherichia coli expressing the tryptophan decarboxylase gene with a viable count of 1.2×10 5 CFU / g, and Lactobacillus reuteri, a tryptophan-metabolizing bacterium, with a viable count of 1.2×10 3 CFU / g.

[0034] In this example, pachyman polysaccharide can change the microbial community structure in the intestines of ob / ob diabetic mice, increase the butyrate-producing bacteria in the Lachnospiraceae and Carboxysphaera genera, increase the butyric acid content in the intestines of ob / ob diabetic mice, improve the damage of the intestinal mucosa, protect the integrity of the intestinal mucosa, reduce the proportions of Firmicutes and Bacteroidetes, increase the abundance of polysaccharide-utilizing bacteria, and promote sugar metabolism in the body.

[0035] In this example, the succinate pathway mainly exists in Bacteroidetes and Firmicutes. Some bacteria in Bacteroidetes utilize dietary carbohydrates to generate propionic acid, and the relative abundance of Bacteroidetes is correlated with the relative level of propionate in human feces.

[0036] Succinic acid is a precursor of propionic acid, but under conditions such as high carbon dioxide partial pressure and high dilution rate, it can accumulate in Bacteroides cultures with inhibited phosphoenolpyruvate carboxykinase. The conversion of succinic acid to propionic acid also requires vitamin B12. If B12 is lacking, succinic acid may not be converted into propionate. In this case, intestinal microorganisms may utilize this accumulated succinic acid through other metabolic pathways (such as the conversion of succinic acid to butyric acid), thereby increasing the production of butyric acid. For example, the acetyl-CoA pathway: This is one of the main pathways for butyric acid production. Two molecules of acetyl-CoA condense to form butyryl-CoA, which is then converted to butyric acid through phosphotransbutyrylase and butyrate kinase.

[0037] Some Firmicutes bacteria (such as Phascolarctobacterium succinatutens) in the human colon can convert succinic acid to propionic acid; other Gram-negative bacteria convert lactic acid to propionate through the succinate pathway (such as Veillonella spp.) or the acrylate pathway (Megasphaera elsdenii).

[0038] Bacteroidetes: Bacteroides thetaiotaomicron is one of the main propionic acid-producing bacteria in the human intestine and produces propionic acid by fermenting dietary fiber.

[0039] Firmicutes: Prevotellaceae: Some strains are capable of producing propionic acid.

[0040] Clostridium propionicum: Converts lactic acid to propionic acid through the acrylate pathway, but lactic acid is not metabolized under normal human conditions.

[0041] Veillonella spp.: Produces propionic acid through the succinate pathway.

[0042] Propionibacterium freudenreichii subsp. freudenreichii: It can produce propionic acid and acetic acid, and its main pathway is the succinic acid pathway.

[0043] In this example, the activated form of itaconic acid, that is, itaconyl-CoA, directly blocks the vitamin B12-dependent metabolic pathway and prevents them from growing using propionic acid.

[0044] In this example, inulin is a type of fructan fiber and belongs to one of the dietary fibers. It can be fermented by microorganisms in the intestine to produce short-chain fatty acids, including butyric acid, thereby improving the intestinal barrier function. Therefore, inulin can indirectly increase the production of butyric acid by regulating the metabolic activities of the intestinal flora, but inulin itself is not a direct chemical precursor of sodium butyrate.

[0045] In this example, the plantain extract has good water solubility and swelling properties and is commonly used in dietary fiber supplements. The plantain extract has good water absorption and swelling properties, can increase the volume of intestinal contents, promote intestinal peristalsis, relieve constipation, form a gel-like substance in the intestine, increase the viscosity of intestinal contents, and thus delay the absorption of glucose, helping to control the rapid rise of postprandial blood glucose.

[0046] In this example, ginsenosides can enhance gastrointestinal peristalsis and improve gastric emptying speed by regulating the release of neurotransmitters, reducing gastrointestinal discomfort; ginsenosides can regulate the secretion of gastric acid and mucus and enhance the protective function of the intestinal mucosa; intestinal inflammation releases pro-inflammatory cytokines such as TNF-α and IL-6, which will exacerbate insulin resistance. Ginsenosides alleviate intestinal inflammation by regulating the immune response and inflammatory signaling pathways (such as the NF-κB pathway), thereby optimizing the intestinal microenvironment, improving intestinal health, and promoting the absorption and utilization of nutrients. These effects are not limited to blood sugar lowering but also involve preventing and alleviating various complications caused by diabetes, such as diabetic nephropathy, diabetic cognitive dysfunction, fatty liver, cerebral infarction, and myocardial lesions. The comprehensive therapeutic effect of ginsenosides makes it have potential clinical value in diabetes management.

[0047] Tryptophan is mainly metabolized by microorganisms in the intestine, producing a variety of derivatives (such as indole, indole-3-acetic acid, indole-3-propionic acid, etc.), and these metabolites play important roles in intestinal barrier function and immune response. N-acetyl-5-hydroxytryptamine (NAS), as a tryptophan metabolite, can regulate intestinal immune response and enhance intestinal barrier function by activating the aryl hydrocarbon receptor (AhR). N-acetyl-5-hydroxytryptamine (NAS) can improve glucose metabolism disorders by promoting the secretion of GLP-1.

[0048] In this example, Spirulina is a type of cyanobacteria microalgae rich in various nutrients, and the content of tryptophan in it is relatively abundant. According to relevant research, the content of tryptophan in Spirulina can reach 0.97 grams per 100 grams, and this content is close to the 1.0-gram standard recommended by the Food and Agriculture Organization of the United Nations (FAO), which makes Spirulina one of the good sources of tryptophan.

[0049] In this example, tryptophan is the precursor of N-acetyl-5-hydroxytryptamine (NAS), and it can be converted into N-acetyl-5-hydroxytryptamine (NAS) through the metabolism of the intestinal microbiota. Therefore, supplementing tryptophan can increase the production of N-acetyl-5-hydroxytryptamine (NAS). N-acetyl-5-hydroxytryptamine (NAS) relieves glucose metabolism disorders in HFD mice by promoting the secretion of glucagon-like peptide-1 (GLP-1).

[0050] 5-Hydroxytryptamine (5-HT): 5-Hydroxytryptamine is an intermediate product of tryptophan metabolism and can be further converted into N-acetyl-5-hydroxytryptamine (NAS). Therefore, supplementing 5-hydroxytryptamine or its precursor substances (such as L-tryptophan) can promote the production of N-acetyl-5-hydroxytryptamine (NAS).

[0051] In this example, tryptophan-metabolizing bacteria (such as Lactobacillus reuteri) can metabolize tryptophan into indole derivatives, and these derivatives can be further converted into N-acetyl-5-hydroxytryptamine (NAS).

[0052] In this example, Streptococcus contains tryptophan decarboxylase, which can convert tryptophan into N-acetyl-5-hydroxytryptamine (NAS). Tryptophan decarboxylase is an enzyme that depends on vitamin B6 (in the form of pyridoxal-5'-phosphate). Therefore, vitamin B6 can act as a coenzyme, and supplementing vitamin B6 can promote the activity of tryptophan decarboxylase.

[0053] In this example, the tryptophan decarboxylase gene (TDC) is introduced into Escherichia coli to achieve the efficient production of N-acetyltryptophan (NAS), and its application in the hypoglycemic composition containing Dendrobium officinale polysaccharide can indeed play a role in the intestine.

[0054] The construction steps of recombinant Escherichia coli expressing tryptophan decarboxylase gene include the following steps:

[0055] S1. Gene cloning and vector construction: Extract DNA from Saccharomyces cerevisiae and amplify the tryptophan decarboxylase gene by PCR reaction;

[0056] S2. Expression vector construction: Insert the tryptophan decarboxylase gene into the pET series expression vector suitable for Escherichia coli to ensure the expression of the tryptophan decarboxylase gene under the control of the strong promoter T7 promoter;

[0057] S3. Host cell transformation: Select a suitable host cell and introduce the constructed expression vector into the host cell by heat shock method;

[0058] S4. Induced expression and enzyme activity detection: In the host cell Escherichia coli BL21, activate the T7 promoter by adding the inducer IPTG to initiate the expression of the tryptophan decarboxylase gene.

[0059] S5. Lyse Escherichia coli cells, extract the tryptophan decarboxylase protein from the Escherichia coli cell lysate, use tryptophan as a substrate, and detect the production of acetyl-5-hydroxytryptamine by high performance liquid chromatography to evaluate the enzyme activity of the tryptophan decarboxylase protein.

[0060] In this example, the preparation method of Dendrobium officinale polysaccharide is as follows: Cut the fresh Dendrobium officinale stems into slices, bake them in an oven at 40 °C, boil the obtained Dendrobium officinale stem dry in distilled water at a ratio of 0.1 kg / L for 1 h, repeat the extraction 2 times, then mix the extracts, and then centrifuge at a centrifugal speed of 4500 rpm for 15 min. After centrifugation, freeze-dry the supernatant to obtain Dendrobium officinale polysaccharide.

[0061] This example also provides a preparation method of a hypoglycemic composition containing Dendrobium officinale polysaccharide, including the following steps:

[0062] S1. Raw material preparation: Weigh the raw materials of each component according to the formula amount and set aside; Dissolve ginsenoside and Dendrobium officinale polysaccharide in deionized water respectively, stir evenly and set aside; Mix inulin, pachyman polysaccharide and plantain extract evenly, add deionized water and stir to dissolve; Dissolve vitamin B6 and itaconic acid-coenzyme in deionized water and set aside; Dissolve spirulina extract in deionized water and stir evenly;

[0063] S2. Mixing and dissolving: Mix the above dissolved ginsenoside, Dendrobium officinale polysaccharide, inulin, pachyman polysaccharide, plantain extract, vitamin B6, spirulina extract and itaconic acid-coenzyme together, stir evenly to ensure that each component is fully dissolved and mixed to obtain a mixed solution;

[0064] S3. Addition of probiotics and recombinant Escherichia coli: Add recombinant Escherichia coli expressing the tryptophan decarboxylase gene, probiotics, and tryptophan-metabolizing bacteria into the mixed solution respectively, stir gently to evenly distribute the bacteria, and use a homogenizer to homogenize the mixed solution to ensure that each component is evenly dispersed, forming a stable mixed solution;

[0065] S4. Drying treatment: Make the homogenized mixed solution into powder by spray drying or freeze drying to obtain a hypoglycemic composition containing polysaccharides from Dendrobium officinale.

[0066] Example 2

[0067] This example provides a hypoglycemic composition containing polysaccharides from Dendrobium officinale. The raw materials by weight include: 15 parts of ginsenoside, 18 parts of polysaccharides from Dendrobium officinale, 2 parts of inulin, 6 parts of pachyman, 5 parts of plantain extract, 1.5 parts of vitamin B6, 3 parts of spirulina extract, 1.5 parts of itaconic acid-CoA, recombinant Escherichia coli expressing the tryptophan decarboxylase gene with 1.1×10 5 CFU / g viable bacteria count, and Lactobacillus reuteri, a tryptophan-metabolizing bacterium, with 1.2×10 2 CFU / g viable bacteria count.

[0068] In this example, the construction steps of recombinant Escherichia coli expressing the tryptophan decarboxylase gene include the following steps:

[0069] S1. Gene cloning and vector construction: Extract DNA from Saccharomyces cerevisiae and amplify the tryptophan decarboxylase gene by PCR reaction;

[0070] S2. Expression vector construction: Insert the tryptophan decarboxylase gene into the pET series expression vector suitable for Escherichia coli to ensure that the tryptophan decarboxylase gene is expressed under the control of the strong promoter T7 promoter;

[0071] S3. Host cell transformation: Select a suitable host cell and introduce the constructed expression vector into the host cell by heat shock method;

[0072] S4. Induced expression and enzyme activity detection: In the host cell Escherichia coli BL21, activate the T7 promoter by adding the inducer IPTG to initiate the expression of the tryptophan decarboxylase gene.

[0073] S5. Lyse Escherichia coli cells, extract the tryptophan decarboxylase protein from the Escherichia coli cell lysate, use tryptophan as a substrate, and detect the production of acetyl-5-hydroxytryptamine by high performance liquid chromatography to evaluate the enzyme activity of the tryptophan decarboxylase protein.

[0074] In this embodiment, the preparation method of Dendrobium officinale polysaccharide is as follows: Cut the fresh stems of Dendrobium officinale into slices, bake them in an oven at 40 °C, boil the obtained dried Dendrobium officinale stems in distilled water at a ratio of 0.1 kg / L for 1 h, repeat the extraction 2 times, then mix the extracts, and then centrifuge at a centrifugal speed of 4500 rpm for 15 min. After centrifugation, freeze-dry the supernatant to obtain Dendrobium officinale polysaccharide.

[0075] This embodiment also provides a preparation method of a hypoglycemic composition containing Dendrobium officinale polysaccharide, including the following steps:

[0076] S1. Raw material preparation: Weigh the raw materials of each component according to the formula amount and set aside; Dissolve ginsenoside and Dendrobium officinale polysaccharide in deionized water respectively, stir evenly and set aside; Mix inulin, pachyman polysaccharide and plantain extract evenly, add to deionized water and stir to dissolve; Dissolve vitamin B6 and itaconic acid - coenzyme in deionized water and set aside; Dissolve spirulina extract in deionized water and stir evenly;

[0077] S2. Mixing and dissolving: Mix the above - dissolved ginsenoside, Dendrobium officinale polysaccharide, inulin, pachyman polysaccharide, plantain extract, vitamin B6, spirulina extract and itaconic acid - coenzyme together, stir evenly to ensure that each component is fully dissolved and mixed to obtain a mixed solution;

[0078] S3. Adding probiotics and recombinant Escherichia coli: Add recombinant Escherichia coli expressing the tryptophan decarboxylase gene, probiotics and tryptophan - metabolizing bacteria to the mixed solution respectively, stir gently to make the bacterial cells evenly distributed, and use a homogenizer to homogenize the mixed solution to ensure that each component is evenly dispersed to form a stable mixed solution;

[0079] S4. Drying treatment: Make the homogenized mixed solution into powder by spray drying or freeze drying to obtain a hypoglycemic composition containing Dendrobium officinale polysaccharide.

[0080] Example 3

[0081] The hypoglycemic composition containing Dendrobium officinale polysaccharide provided in this embodiment includes the following raw materials by weight: 12 parts of ginsenoside, 15 parts of Dendrobium officinale polysaccharide, 3 parts of inulin, 8 parts of pachyman polysaccharide, 2 parts of plantain extract, 2 parts of vitamin B6, 4 parts of spirulina extract, 2 parts of itaconic acid - coenzyme A, recombinant Escherichia coli expressing the tryptophan decarboxylase gene with 1.18×10 5 CFU / g of viable bacteria, and Lactobacillus reuteri, a tryptophan - metabolizing bacterium, with 1.5×10 3 CFU / g of viable bacteria.

[0082] In this embodiment, the construction steps of the recombinant Escherichia coli expressing the tryptophan decarboxylase gene include the following steps:

[0083] S1. Gene cloning and vector construction: Extract DNA from Saccharomyces cerevisiae, and amplify the tryptophan decarboxylase gene by PCR reaction;

[0084] S2. Expression vector construction: Insert the tryptophan decarboxylase gene into the pET series expression vector suitable for Escherichia coli to ensure the expression of the tryptophan decarboxylase gene under the control of the strong promoter T7 promoter;

[0085] S3. Host cell transformation: Select a suitable host cell, and introduce the constructed expression vector into the host cell by heat shock method;

[0086] S4. Induced expression and enzyme activity detection: In the host cell Escherichia coli BL21, activate the T7 promoter by adding the inducer IPTG to initiate the expression of the tryptophan decarboxylase gene.

[0087] S5. Lyse Escherichia coli cells, extract the tryptophan decarboxylase protein from the Escherichia coli cell lysate, use tryptophan as a substrate, and detect the production of acetyl 5-hydroxytryptamine by high performance liquid chromatography to evaluate the enzyme activity of the tryptophan decarboxylase protein.

[0088] In this embodiment, the preparation method of Dendrobium officinale polysaccharide is as follows: Cut the fresh stems of Dendrobium officinale into slices, bake them in an oven at 40 °C, boil the obtained dried Dendrobium officinale stems in distilled water at a ratio of 0.1 kg / L for 1 h, repeat the extraction 2 times, then mix the extracts, and then centrifuge at a centrifugal speed of 4500 rpm for 15 min. After centrifugation, lyophilize the supernatant to obtain Dendrobium officinale polysaccharide.

[0089] This embodiment also provides a preparation method of a hypoglycemic composition containing Dendrobium officinale polysaccharide, including the following steps:

[0090] S1. Raw material preparation: Weigh the raw materials of each component according to the formula amount for standby; Dissolve ginsenoside and Dendrobium officinale polysaccharide in deionized water respectively, stir evenly for standby; Mix inulin, pachyman polysaccharide and plantain extract evenly, add deionized water, and stir to dissolve; Dissolve vitamin B6 and itaconic acid-coenzyme in deionized water for standby; Dissolve spirulina extract in deionized water and stir evenly;

[0091] S2. Mixing and dissolving: Mix the above-dissolved ginsenoside, Dendrobium officinale polysaccharide, inulin, pachyman polysaccharide, plantain extract, vitamin B6, spirulina extract and itaconic acid-coenzyme together, stir evenly to ensure that each component is fully dissolved and mixed to obtain a mixed solution;

[0092] S3. Adding probiotics and recombinant Escherichia coli: Add recombinant Escherichia coli expressing the tryptophan decarboxylase gene, probiotics, and tryptophan-metabolizing bacteria into the mixed solution respectively, stir gently to make the bacterial cells evenly distributed, and use a homogenizer to homogenize the mixed solution to ensure that all components are evenly dispersed, forming a stable mixed solution;

[0093] S4. Drying treatment: Make the homogenized mixed solution into powder by spray drying or freeze drying, and obtain a hypoglycemic composition containing polysaccharides from Dendrobium officinale.

[0094] Example 4

[0095] This example provides a hypoglycemic drug. According to different pharmaceutical excipients, a hypoglycemic composition containing polysaccharides from Dendrobium officinale can also be used as an active ingredient to prepare a hypoglycemic drug together with pharmaceutically acceptable pharmaceutical excipients;

[0096] The pharmaceutical excipient can be one of excipients, disintegrants, sweeteners, binders, coating agents, and swelling agents.

[0097] According to different pharmaceutical excipients, the hypoglycemic drug can be one of solution, emulsion, suspension, injection, powder, granule, tablet, and pill.

[0098] Comparative Example 1

[0099] The difference from Example 1 is that this example provided by Comparative Example 1 provides a hypoglycemic composition containing polysaccharides from Dendrobium officinale. The raw materials by weight include: 10 parts of ginsenoside and 20 parts of polysaccharides from Dendrobium officinale.

[0100] Experimental Example 1

[0101] 110 healthy mice, weighing 20 ± 5 g, were purchased from the Experimental Animal Center of Guangdong Medical University. 10 normal control mice were used as the normal control group, and the other 100 mice were used as the model group, randomly divided by gender. Dissolve 55 mg of streptozotocin in 0.05 M citric acid buffer solution with a pH of 4.5, and induce diabetes by gavage. 72 hours after gavage, take the mice in the model control group that have fasted for 12 hours to measure the fasting blood glucose. Those with a blood glucose value greater than 10 mmol / L indicate that the diabetic model mice are successfully constructed. During the model construction process, the mice in the normal control group were injected with sterilized normal saline. The model group was further divided into 5 groups, with 20 mice in each group, namely the model control group, Example 1 - 3 groups, and Comparative Example 1 group. Measure the blood glucose value of each group and take the average. The results are shown in Table 1.

[0102] Table 1 Blood Glucose Measurement Results

[0103] Group Number of mice (pcs) Blood glucose concentration (mmol / L) Normal control group 10 3.58±0.21 Model control group 20 20.35±1.56 Example 1 20 2.36±0.12 Example 2 20 2.56±0.59 Example 3 20 2.98±0.24 Comparative example 1 20 8.69±1.32

[0104] As can be seen from Table 1, the blood glucose value of the model control group was greater than 10 mmol / L, indicating that the model was successfully constructed. Compared with the model control group, the groups of Examples 1 to 3 all showed a significant blood glucose-lowering effect, and the blood glucose-lowering effect of the group of Comparative Example 1 was lower than that of the groups of Examples 1 to 3, indicating that the blood glucose-lowering drug provided by the present invention improves the blood glucose-lowering effect of polysaccharides from Dendrobium officinale from different action pathways and is more efficient in lowering blood glucose.

[0105] The above embodiments are only the preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantive changes and substitutions made by those skilled in the art based on the present invention fall within the scope of protection required by the present invention.

Claims

1. A blood sugar lowering composition containing Dendrobium officinale polysaccharide, characterized in that: The raw materials include, by weight: 10-15 parts of ginsenoside, 15-20 parts of dendrobium officinale polysaccharide, 2-5 parts of inulin, 5-8 parts of tuckahoe polysaccharide, 2-5 parts of plantain extract, 1-2 parts of vitamin B6, 3-5 parts of spirulina extract, 1-2 parts of itaconic acid-coenzyme A, and 1.1×10 viable bacteria per gram. 5 ~1.2×10 5 CFU / g of recombinant Escherichia coli expressing the tryptophan decarboxylase gene, with a viable count of 1.2×10 2 ~1.5×10 3 CFU / g of tryptophan metabolizing bacteria.

2. The blood sugar lowering composition containing Dendrobium officinale polysaccharide according to claim 1, characterized in that: The raw materials in parts by weight include: 10 parts of ginsenoside, 20 parts of dendrobium officinale polysaccharide, 5 parts of inulin, 5 parts of tuckahoe polysaccharide, 3 parts of plantain extract, 1 part of vitamin B6, 5 parts of spirulina extract, 1 part of itaconic acid-coenzyme A, and 1.2×10 viable bacteria per gram. 5 CFU / g of recombinant Escherichia coli expressing the tryptophan decarboxylase gene, with a viable count of 1.2×10 3 CFU / g of tryptophan metabolizing bacteria.

3. The blood sugar lowering composition containing Dendrobium officinale polysaccharide according to claim 1, characterized in that: The tryptophan metabolizing bacteria are one or both of Lactobacillus reuteri and Gastrococcus.

4. The blood sugar lowering composition containing Dendrobium officinale polysaccharide according to claim 1, characterized in that: The construction step of the recombinant Escherichia coli expressing the tryptophan decarboxylase gene comprises the following steps: S1. Gene cloning and vector construction: DNA was extracted from Saccharomyces cerevisiae and the tryptophan decarboxylase gene was amplified by PCR reaction; S2. Construction of expression vector: inserting the tryptophan decarboxylase gene into a pET series expression vector suitable for Escherichia coli, ensuring that the tryptophan decarboxylase gene is expressed under the control of a strong promoter, T7 promoter; S3. Host cell transformation: Select appropriate host cells and introduce the constructed expression vector into the host cells by heat shock method; S4. Induced expression and enzyme activity detection: In the host cell, the T7 promoter is activated by adding an inducer to start the expression of the tryptophan decarboxylase gene. S5. Lysing the host cells, extracting the tryptophan decarboxylase protein in the host cell lysate, using tryptophan as a substrate, detecting the generation of acetyl 5-hydroxytryptamine by high performance liquid chromatography, and evaluating the enzyme activity of the tryptophan decarboxylase protein.

5. The blood sugar lowering composition containing Dendrobium officinale polysaccharide according to claim 1, characterized in that: The preparation method of the Dendrobium officinale polysaccharide is as follows: fresh Dendrobium officinale stems are cut into slices, baked in an oven at 40°C, the obtained Dendrobium officinale stems are boiled with distilled water at a ratio of 0.1kg / L for 1h, the extraction is repeated twice, and then the extracts are mixed, followed by centrifugation at a centrifugal speed of 4500rpm for 15min. After centrifugation, the supernatant is freeze-dried to obtain the Dendrobium officinale polysaccharide.

6. A method for preparing a hypoglycemic composition containing Dendrobium officinale polysaccharide according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1. Raw material preparation: weigh the raw materials of each component according to the formula amount and set aside; dissolve the ginsenoside and the Dendrobium officinale polysaccharide in deionized water respectively, stir evenly and set aside; mix the inulin, the Poria cocos polysaccharide and the Plantain extract evenly, add them into deionized water, stir and dissolve; dissolve the vitamin B6 and itaconic acid-coenzyme in deionized water and set aside; dissolve the Spirulina extract in deionized water and stir evenly; S2, mixed dissolution: the dissolved ginsenosides, the dendrobium officinale polysaccharides, the inulin, the tuckahoe polysaccharides, the plantain extract, the vitamin B6, the spirulina extract, and itaconic acid-coenzyme are mixed together, and stirred evenly to ensure that each component is fully dissolved and mixed to obtain a mixed solution; S3, adding probiotics and recombinant Escherichia coli: adding the recombinant Escherichia coli expressing the tryptophan decarboxylase gene, the probiotics, and the tryptophan metabolizing bacteria to the mixed solution respectively, stirring gently to make the bacteria evenly distributed, and homogenizing the mixed solution using a homogenizer to ensure that the components are evenly dispersed to form a stable mixed solution; S4, drying treatment: preparing the homogenized mixed solution into powder by spray drying or freeze drying to obtain the blood sugar lowering composition containing Dendrobium officinale polysaccharide.

7. Use of a hypoglycemic composition containing Dendrobium officinale polysaccharide according to any one of claims 1 to 5 in the preparation of a hypoglycemic drug, characterized in that: The hypoglycemic composition containing Dendrobium officinale polysaccharide as an active ingredient is prepared into the hypoglycemic drug with pharmaceutically acceptable drug excipients; the drug excipient is one of an excipient, a disintegrant, a sweetener, a binder, a coating agent and a swelling agent.

8. Use of a hypoglycemic composition containing Dendrobium officinale polysaccharide according to any one of claim 7 in the preparation of a hypoglycemic drug, characterized in that: The blood sugar lowering drug is one of a solution, an emulsion, a suspension, an injection, a powder, a granule, a tablet, and a pill.