Novel pill for treating diabetes and preparation method thereof

Abstract

The invention relates to the technical field of traditional Chinese medicine preparations, and discloses a novel pill for treating diabetes mellitus and a preparation method, and the novel pill comprises the following raw materials in parts by mass: 30-40 parts of Chinese yam, 10-15 parts of poria cocos, 20-25 parts of radix polygonati officinalis, 25-35 parts of guava, 30-35 parts of radix rehmanniae recen, 28-32 parts of radix puerariae, 30-35 parts of corn stigma, 20-30 parts of radix trichosanthis, 25-30 parts of acanthopanax and 20-30 parts of rhizoma polygonati. The preparation method comprises the following steps: S1, screening, cleaning, crushing and homogenizing; s2, performing protein denaturation treatment, performing multi-stage enzymolysis, performing enzyme inactivation and filtration, and performing ultrafiltration membrane separation; s3, carrying out gene fusion to construct a fusion gene expression vector, transferring into pichia pastoris for expression, and purifying; s4, wrapping the target peptide fragment with the human albumin fusion protein; and S5, by taking the hydroxypropyl methylcellulose aqueous solution as an adhesive, molding in a pill pan, pelleting, shaping and drying.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine preparations, in particular to a novel pill for treating diabetes and a preparation method thereof. BACKGROUND

[0002] Diabetes is a metabolic disease characterized by high blood sugar, and has become a major global public health problem. Long-term high blood sugar can cause chronic damage and dysfunction of the eyes, kidneys, heart, blood vessels, nerves, and other multiple tissues and organs, seriously affecting the quality of life of patients. The treatment of diabetes mainly relies on western medicines, including insulin, sulfonylureas, biguanides, and thiazolidinediones. These drugs have certain efficacy in controlling blood sugar, but also have many limitations. For example, insulin needs to be injected subcutaneously, which is inconvenient to use and can easily cause hypoglycemia; sulfonylureas can cause weight gain and hypoglycemia; biguanides can cause gastrointestinal discomfort and other adverse reactions. In addition, long-term use of western medicines often fails to effectively prevent the occurrence and development of diabetes complications.

[0003] Traditional Chinese medicine has a long history and rich experience in treating diabetes. Traditional Chinese medicine classifies diabetes as the category of "consumption" and believes that its onset is related to insufficient endowment, improper diet, emotional disorders, and improper work and rest, and its pathogenesis involves yin deficiency and dryness-heat, qi and yin deficiency, and yin and yang deficiency. The main principles of treatment are clearing heat and moistening dryness, nourishing yin and generating fluid, tonifying qi and invigorating the spleen, and tonifying the kidney and warming yang. Traditional Chinese medicine treatment of diabetes has the advantages of overall regulation, multi-target action, small side effects, and effective prevention and delay of complications, and is suitable for long-term use.

[0004] However, the current traditional Chinese medicine preparations for the treatment of diabetes still face many technical bottlenecks in clinical application, which seriously restrict their efficacy and popularization and application. The specific problems are as follows:

[0005] 1. Large fluctuations in the quality of raw materials

[0006] The efficacy of traditional Chinese medicine pills depends on the quality of traditional Chinese medicinal materials, but differences in the origin, harvesting time, and processing methods of traditional Chinese medicinal materials can cause fluctuations in the content of effective ingredients. For example, the polysaccharide and saponin content of commonly used diabetes-reducing medicinal materials such as Astragalus and Rehmannia can differ by more than 30% in different producing areas, directly affecting the stability of the efficacy of the pills.

[0007] 2. Delayed onset compared to the patient's needs

[0008] Diabetes patients often need to quickly control blood sugar fluctuations (such as postprandial hyperglycemia), but the existing pills have a slow dissolution rate, with an onset time of more than 2-4 hours, which is difficult to meet the immediate need for blood sugar reduction and requires the use of western medicines to meet the standard.

[0009] 3. Lack of clear quality control indicators

[0010] Most of the traditional Chinese medicine pills still rely on the traditional indicators such as appearance, weight difference to control the quality, and lack of quantitative detection of effective components (such as puerarin, ginsenosides, etc.). Some varieties even have not established the overall quality evaluation method such as fingerprint, and it is difficult to ensure the batch quality consistency.

[0011] 4. Insufficient safety risk assessment

[0012] Some pills contain heavy metals (such as cinnabar, realgar) or toxic components (such as aconite, Chuanwu), and long-term use may cause accumulation poisoning. However, the existing research lacks assessment of long-term toxicity and liver and kidney function, and the safety warning is missing.

[0013] 5. Lack of evidence-based medicine

[0014] Most of the traditional Chinese medicine pills rely on small sample, non-random controlled studies for efficacy verification, lack of multi-center, large sample, randomized double-blind clinical trial data, and it is difficult to prove its exact efficacy better than existing treatment options.

[0015] 6. Large dose and poor taste

[0016] Traditional pills can reach 6-10g (about 30-50 pills) for single use to ensure efficacy, and the sugar content of honey pills is high (the sucrose content of some honey pills is more than 20%), which not only increases the burden of patients taking medicine, but also may affect blood sugar control, especially for patients sensitive to blood sugar fluctuations. SUMMARY

[0017] (1) The technical problem solved

[0018] In view of the deficiencies in the prior art, the present application provides a new type of pill for treating diabetes and a preparation method thereof.

[0019] (2) Technical scheme

[0020] In order to achieve the above-mentioned purpose, the present application provides the following technical scheme: a new type of pill for treating diabetes, comprising the following mass parts of raw materials:

[0021] 30-40 parts of yam, 10-15 parts of poria cocos, 20-25 parts of polygonatum, 25-35 parts of psidium guajava, 30-35 parts of radix rehmanniae, 28-32 parts of pueraria, 30-35 parts of corn silk, 20-30 parts of trichosanthes, 25-30 parts of acanthopanax, and 20-30 parts of polygonatum.

[0022] Preferably, the following mass parts of raw materials are included:

[0023] 30 parts of yam, 10 parts of poria cocos, 20 parts of polygonatum, 30 parts of psidium guajava, 30 parts of radix rehmanniae, 30 parts of pueraria, 30 parts of corn silk, 20 parts of trichosanthes, 30 parts of acanthopanax, and 20 parts of polygonatum.

[0024] Preferably, the following mass parts of raw materials are included:

[0025] Shanyao 35 parts, Fuling 12 parts, Yuzhu 22 parts, Ban-shiliu 25 parts, Shengdihuang 30 parts, Gegen 28 parts, Yushu 30 parts, Tianhuafen 25 parts, Ciqi 26 parts, Huangjing 22 parts.

[0026] Preferably, the following mass parts of raw materials are included:

[0027] Shanyao 40 parts, Fuling 15 parts, Yuzhu 25 parts, Ban-shiliu 35 parts, Shengdihuang 35 parts, Gegen 32 parts, Yushu 35 parts, Tianhuafen 30 parts, Ciqi 30 parts, Huangjing 30 parts.

[0028] Preferably, a preparation method of a new type of diabetic pill comprises the following steps:

[0029] S1, raw material pretreatment: screening, cleaning, crushing, homogenization of each raw material;

[0030] S2, enzyme extraction: protein denaturation treatment, multi-stage enzymolysis, enzyme inactivation filtration, ultrafiltration membrane separation of the homogenized raw material to obtain a target peptide segment extract;

[0031] S3, human albumin fusion protein preparation: a fusion gene expression vector is constructed by gene fusion, and then expressed in Pichia pastoris and purified to obtain a human albumin fusion protein;

[0032] S4, protein coating: using the human albumin fusion protein as a coating material, coating the above-mentioned target peptide segment, cross-linking and solidifying, washing and purifying, and low-temperature pre-drying to obtain wet microspheres;

[0033] S5, pill forming: using hydroxypropyl methyl cellulose aqueous solution as a binder, the wet microspheres are molded, panned, and dried to form pills with a diameter of 3.5-4.0 mm.

[0034] Preferably, the enzyme extraction comprises the following steps:

[0035] A1, raw material crushing and homogenization: the pretreated raw materials are crushed to a particle size of 0.5-1 mm with a high-speed crusher, and then homogenized with a colloid mill at a solid-liquid ratio of 1:8-10 with deionized water to obtain raw material homogenate;

[0036] A2, protein denaturation treatment: the above-mentioned raw material homogenate is transferred into a constant-temperature reaction tank, the temperature is controlled at 60-90°C, the stirring speed is 150-200 r / min, and the heat preservation treatment is performed for 30-45 min;

[0037] A3, multi-stage enzymolysis:

[0038] First stage enzymolysis: temperature 45-55℃, enzyme dosage 0.3-0.5%, pH value 6.5-7.0, enzymolysis 2-3h;

[0039] Second stage enzymolysis: temperature 40-50℃, enzyme dosage 0.2-0.4%, pH value 5.0-5.5, enzymolysis 1.5-2h;

[0040] A4, enzyme inactivation and filtration: after enzymolysis, the reaction system is heated to 90-95℃, inactivated for 10-15min, then filtered with 200 mesh filter cloth to remove residue and collect filtrate;

[0041] A5, ultrafiltration membrane separation: the collected filtrate is separated by ultrafiltration membrane with molecular weight of 3000-5000Da, operating pressure 0.15-0.2MPa, temperature 30-35℃, and the permeate is collected, i.e. the target molecular weight peptide extract of each raw material is obtained.

[0042] Preferably, the preparation of human albumin fusion protein comprises the following steps:

[0043] B1, gene fusion construction: the human blood albumin gene sequence is extracted, recombined with the functional gene sequence of the target peptide of Puerariae radix, corn stigma and Dioscorea, and a fusion gene expression vector is constructed by restriction endonuclease digestion and DNA ligase ligation;

[0044] B2, yeast transformation and expression: the constructed fusion gene expression vector is transformed into Pichia pastoris competent cells by electroporation, then the transformed yeast is inoculated into YPD medium for shaking culture, and the fusion gene is induced to express in the yeast to produce human albumin fusion protein;

[0045] B3, fusion protein purification: the yeast fermentation broth is centrifuged to remove the bacterial precipitate, and the supernatant is collected, then the supernatant is purified by DEAE-52 ion exchange chromatography column, the target protein peak is collected by gradient elution, and the collected target protein peak solution is further purified by Sephadex G-75 gel filtration chromatography column to collect human albumin fusion protein solution.

[0046] Preferably, the protein coating comprises the following steps:

[0047] C1, the human albumin fusion protein solution after purification is prepared into protein powder by freeze-drying method: the protein solution is divided into freeze-drying bottles and pre-frozen for 2-3 hours in an ultra-low temperature refrigerator at-60 to-80℃, then transferred into a freeze-drying machine and freeze-dried for 36-48 hours under the conditions of-30 to-50℃ and vacuum degree of 0.1 to 0.2 Pa, to obtain human albumin fusion protein powder; the target molecular weight peptide segment extract of each raw material is extracted by freeze-drying, freeze-dried for 24-48 hours at-20 to-40℃ and vacuum degree of 0.5 to 0.8 Pa, mixed according to the above mass fraction to obtain mixed small molecule peptide powder, and the granular impurities are removed through an 80-mesh sieve;

[0048] C2, the mixed small molecule peptide powder is added into PBS buffer solution to prepare a peptide segment suspension with a mass concentration of 10-15%, and stirred for 5-10 minutes; the human albumin fusion protein powder is slowly added into PBS buffer solution at 30℃, stirred and dissolved to prepare a protein solution with a mass concentration of 8-12%;

[0049] C3, the peptide segment suspension is slowly dropped into the human albumin fusion protein solution at a speed of 0.5-1 mL / min by using a peristaltic pump, and the mass ratio of the peptide segment to the encapsulating protein is 1:2-3, while stirring at 30-35℃, and after the dropping is completed, the stirring is continued for 30-60 minutes to form a human albumin fusion protein-small molecule peptide microsphere suspension, and a 1% genipin solution with a dosage of 1.8% of the total mass of the system is slowly added thereto, and stirred at 30℃ for 30 minutes;

[0050] C4, the solidified microsphere suspension is centrifuged at 3000-4000 r / min at 4℃ for 10 minutes, the supernatant is discarded, the precipitate is washed with PBS buffer solution for 3 times, and finally washed with deionized water once to obtain pure microsphere precipitate;

[0051] C5, the microsphere precipitate is laid on a stainless steel tray with a thickness of ≤5 mm, placed in a vacuum drying oven, set at a temperature of 25℃ and a vacuum degree of 50 Pa, and dried for 10-12 hours to obtain wet microsphere particles, and the wet microsphere particles are collected through a standard sieve to collect particles with a mesh size of 14-20.

[0052] Preferably, the pill forming includes the following steps:

[0053] D1, 95% of the wet microsphere particles by mass fraction are taken and added into a pan coater, the rotation speed is 30 r / min, the inclination angle is 35°, after the device is started, 3% of the hydroxypropyl methyl cellulose aqueous solution by mass fraction is slowly sprayed, and the dosage is 15% of the total mass of the particles, the particles are rolled into spherical pellet cores with a diameter of 1.5-2.0 mm while stirring;

[0054] D2, keep the rotation speed of the pan 35 r / min and the temperature 28℃, evenly sprinkle the remaining 5% of the wet microspheres on the spherical core, and spray the hydroxypropyl methylcellulose aqueous solution after each sprinkling, and repeat the sprinkling-liquid spraying operation until the diameter of the pills reaches 3.5-4.0 mm;

[0055] D3, place the formed pills in a hot air circulating drying box and dry them at 30-35℃ for 8-12h.

[0056] (III) Beneficial technical effects

[0057] Hypoglycemic effect: rapid hypoglycemic effect, precise response to immediate blood glucose fluctuations. The pills can rapidly exert a hypoglycemic effect within 1 hour after administration, inhibit α-glucosidase activity and promote insulin secretion through the effective components, directly address the problem of postprandial hyperglycemia, and reduce blood glucose by 20-30% within 1 hour, which is significantly better than the 2-4 hour lag in the onset of traditional Chinese medicine pills, can timely block the damage of postprandial blood glucose surge to target organs, and meet the clinical needs of diabetic patients (especially type 2 diabetic patients) for rapid blood glucose control without relying on the use of Western medicines.

[0058] Thanks to the sustained-release micro-pellet preparation technology, the effective components of Rehmannia glutinosa in the pills can continuously improve insulin resistance within 12 hours, stabilize fasting blood glucose in the ideal range, and significantly reduce the amplitude of blood glucose fluctuations. At the same time, the pills can reduce the risk of hypoglycemia and avoid the drawbacks of traditional hypoglycemic drugs (such as insulin and sulfonylureas) that are prone to cause hypoglycemia, providing a safer long-term blood glucose management solution for patients.

[0059] Clinical trial data have confirmed that after 8 weeks of continuous administration of the pills, the glycosylated hemoglobin (HbA1c) of type 2 diabetic patients decreased by an average of 1.0-1.5%. As a core indicator reflecting long-term blood glucose control, the significant decrease in glycosylated hemoglobin indicates that the pills can fundamentally improve the metabolic disorder state of patients and achieve long-term stable control of blood glucose, which is better than the blood glucose control effect of some traditional Chinese medicine pills.

[0060] Effectively improve the core symptoms of diabetes: for the symptoms of polydipsia, polyphagia, polyuria, and fatigue commonly seen in diabetic patients, the pills can significantly alleviate the above discomforts by regulating the body's metabolic environment. After administration, the patients' water intake, food intake, and urination frequency are significantly reduced, the fatigue symptoms are greatly improved, and the quality of life is substantially improved, which is in line with the treatment philosophy of "differential treatment and overall conditioning" in traditional Chinese medicine.

[0061] Formulation and quality control: In view of the problem that the traditional Chinese medicine pill is affected by the difference in raw material origin and processing, the pill is optimized by adjusting the proportion of medicinal materials and combining modern extraction and purification technology, so as to accurately control the content of effective components, reduce the unstable factors of the pill caused by the difference in raw materials and the rough process, ensure the uniformity of the effective component composition and proportion of different batches of pills, realize the stable and consistent curative effect, and avoid the influence of the uneven curative effect on the clinical treatment effect.

[0062] In view of the defects of slow disintegration and incomplete absorption of the traditional pill, the pill is processed by using the advanced preparation process such as micronization and solid dispersion technology, so that the dissolution performance of the pill is significantly improved, the absorption speed of the drug in the body is accelerated, the effective time is greatly shortened, the bioavailability is improved, the hypoglycemic effect is enhanced, and the urgent needs of diabetic patients for rapid control of blood sugar are better met.

[0063] Drug experience: reduce the dosage, improve the taste and applicability. The disadvantages of the traditional honey pill, such as 6-10g (30-50 pills) for single use and more than 20% of sucrose content, are abandoned, the dosage is greatly reduced under the premise of ensuring the curative effect by optimizing the dosage form design, the influence of high sugar addition on blood sugar control is avoided, especially for diabetic patients with sensitive blood sugar fluctuation; in addition, the taste of the pill is improved, the swallowing difficulty is reduced, and the burden of the elderly and patients with weak swallowing function is solved. The pill is small and easy to carry after optimization, which is convenient for patients to take the dose when going out, meets the treatment characteristics of long-term management and regular medication of diabetes, reduces the problems of missed medication and wrong medication caused by inconvenient dosage form, and significantly improves the long-term medication compliance of patients.

[0064] Provide scientific basis and practical support for the treatment of diabetes with traditional Chinese medicine, verify the efficacy and safety of the pill through the development of multi-center and large-sample clinical controlled trials, clarify the mechanism of action of the pill in inhibiting alpha-glucosidase activity, promoting insulin secretion and improving insulin resistance, perfect the evidence-based medicine evidence, make up for the defects of weak evidence-based medicine of traditional Chinese medicine pills, lay a scientific foundation for the standardized application of Chinese medicine pills in the treatment of diabetes, and promote the modernization and standardization development of traditional Chinese medicine in the treatment of diabetes. DETAILED DESCRIPTION

[0065] The technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor belong to the scope of protection of the present application.

[0066] The novel treatment of diabetes pill formula component of the present application is commercially available without special instructions;

[0067] A new type of therapeutic diabetes pill, comprising the following mass parts of raw materials:

[0068] Shanyao 30-40 parts, Fuling 10-15 parts, Yuzhu 20-25 parts, Ban-shih-lyu 25-35 parts, Shengdihuang 30-35 parts, Gegen 28-32 parts, Corn silk 30-35 parts, Tianhuafen 20-30 parts, Ciwujia 25-30 parts, Huangjing 20-30 parts.

[0069] A preparation method of a new type of therapeutic diabetes pill, comprising the following steps:

[0070] S1, raw material pretreatment: screening, cleaning, crushing, homogenization of each raw material;

[0071] S2, enzyme extraction: protein denaturation treatment, multi-stage enzymolysis, enzyme inactivation filtration, ultrafiltration membrane separation of the homogenized raw material to obtain the target peptide segment extract;

[0072] S3, human albumin fusion protein preparation: construct a fusion gene expression vector by gene fusion, express in Pichia pastoris and purify to obtain human albumin fusion protein;

[0073] S4, protein wrapping: using human albumin fusion protein as wrapping material, wrapping the above target peptide segment, cross-linking and solidifying, washing and purifying, low temperature pre-drying to obtain wet microspheres particles;

[0074] S5, pill forming: using hydroxypropyl methyl cellulose aqueous solution as adhesive, wet microspheres particles are molded, panned, and dried to form pills with a diameter of 4.0mm.

[0075] Enzyme extraction includes the following steps:

[0076] A1, raw material crushing and homogenization: the pretreated raw materials are crushed to a particle size of 0.5mm with a high-speed crusher, then deionized water is added at a solid-liquid ratio of 1:10, and homogenized with a colloid mill to obtain raw material homogenate;

[0077] A2, protein denaturation treatment: the above raw material homogenate is transferred into a constant temperature reaction tank, the temperature is controlled at 80℃, the stirring speed is 200r / min, and the heat preservation treatment is 30min;

[0078] A3, multi-stage enzymolysis:

[0079] First stage enzymolysis: at a temperature of 45℃, 0.3% of neutral protease is added, the pH value is adjusted to 6.5, and the enzymolysis is carried out for 2h;

[0080] Second stage enzymolysis: after enzymolysis, the temperature is 50℃, 0.2% of cellulase is added, the pH value is adjusted to 5.0, and the enzymolysis is continued for 2h;

[0081] A4, enzyme inactivation and filtration: after the enzymatic hydrolysis is completed, the reaction system is warmed to 95°C, and enzyme inactivation is performed for 10 min, then 200-mesh filter cloth is used for filtration to remove residues, and the filtrate is collected;

[0082] A5, ultrafiltration membrane separation: the collected filtrate is separated by an ultrafiltration membrane with a molecular weight of 3000-5000 Da, the operating pressure is controlled at 0.15 MPa, and the temperature is 30°C, and the permeate is collected, i.e. the target molecular weight peptide segment extract of each raw material is obtained.

[0083] The preparation of the human albumin fusion protein comprises the following steps:

[0084] B1, gene fusion construction: the human blood albumin gene sequence is extracted, and the functional gene sequences of the target peptide segments of pueraria, corn silk and yam are recombined to construct a fusion gene expression vector, which is subjected to restriction endonuclease digestion and DNA ligase ligation;

[0085] B2, yeast transformation and expression: the constructed fusion gene expression vector is transformed into a Pichia pastoris competent cell by an electroporation method, then the transformed yeast is inoculated into a YPD culture medium for shaking culture, and the fusion gene is induced to express in the yeast to produce a human albumin fusion protein;

[0086] B3, fusion protein purification: the yeast fermentation broth is centrifuged to remove the bacterial precipitate, and the supernatant is collected, which is subjected to DEAE-52 ion exchange chromatography column purification, gradient elution is performed to collect the target protein peak, and the collected target protein peak solution is further subjected to Sephadex G-75 gel filtration chromatography column purification to collect the human albumin fusion protein solution.

[0087] The protein packaging comprises the following steps:

[0088] C1, the purified human albumin fusion protein solution is used to prepare a protein powder by a freeze-drying method: the protein solution is divided into freeze-drying bottles, pre-frozen in a -60°C ultra-low temperature refrigerator for 3 h, transferred into a freeze-drying machine, and freeze-dried at -40°C and a vacuum degree of 0.1 Pa for 36 h to obtain a human albumin fusion protein powder; the target molecular weight peptide segment extract of each raw material is selected, freeze-dried at -20°C and a vacuum degree of 0.5 Pa for 48 h, mixed according to the above mass ratio, and sieved through an 80-mesh sieve to remove particulate impurities;

[0089] C2, the mixed small molecular peptide powder is added into a PBS buffer to prepare a peptide segment suspension with a mass concentration of 10%, and stirred for 10 min; the human albumin fusion protein powder is slowly added into a PBS buffer at 30°C, stirred and dissolved to prepare a protein solution with a mass concentration of 12%;

[0090] C3, the peptide segment suspension is slowly dropped into the human albumin fusion protein solution at a speed of 1 mL / min by using a peristaltic pump, the mass ratio of the peptide segment to the encapsulating protein is 1:3, and stirring is performed at 30 DEG C at the same time, after the dropping is completed, the stirring is continuously performed for 60 min, a human albumin fusion protein-small molecule peptide microsphere suspension is formed, a 1% (mass fraction) genipin solution is slowly added into the suspension, the amount of the genipin solution is 1.8% of the total mass of the system, and stirring is performed at 30 DEG C for 30 min;

[0091] C4, the microsphere suspension after solidification is centrifuged at 3000 r / min for 10 min at 4 DEG C, the supernatant is discarded, the precipitate is washed with PBS buffer for 3 times, and finally the precipitate is washed with deionized water for 1 time, so that pure microsphere precipitate is obtained;

[0092] C5, the microsphere precipitate is laid on a stainless steel tray with a thickness of less than or equal to 5 mm, and is placed in a vacuum drying box, the temperature is set to 25 DEG C, the vacuum degree is set to 50 Pa, and the drying is performed for 12 h, so that wet microsphere particles are obtained, and the wet microsphere particles are passed through a standard sieve, and particles with a size of 14-20 are collected.

[0093] The pill forming includes the following steps:

[0094] D1, 95% (mass fraction) of the wet microsphere particles are taken and added into a pan-granulator, the rotation speed is 30 r / min, the inclination angle is 35 DEG, after the device is started, 3% (mass fraction) of a hydroxypropyl methyl cellulose aqueous solution is slowly sprayed, the amount of the hydroxypropyl methyl cellulose aqueous solution is 15% of the total mass of the particles, the particles are rolled to form spherical core particles with a diameter of 2.0 mm under the condition that the spraying and stirring are simultaneously performed;

[0095] D2, the rotation speed of the pan-granulator is kept at 35 r / min, and the temperature is kept at 28 DEG C, the remaining 5% of the wet microsphere particles are uniformly scattered on the spherical core particles, the hydroxypropyl methyl cellulose aqueous solution is sprayed after each scattering, and the scattering-powder spraying and liquid spraying are repeatedly performed until the diameter of the pills reaches 4.0 mm;

[0096] D3, the formed pills are placed in a hot air circulating drying box and dried at 35 DEG C for 10 h.

[0097] The preparation method of the human albumin fusion protein includes the following steps:

[0098] I. gene fusion construction

[0099] 1.1 gene sequence acquisition and amplification

[0100] 1.1.1 human blood albumin gene sequence extraction

[0101] The total RNA was extracted from the peripheral blood mononuclear cells of healthy people by using TRIzol method: 1 mL of TRIzol reagent was added to the cell precipitate, and after being fully lysed for 5 min, 0.2 mL of chloroform was added, and the mixture was vigorously shaken for 15 s, and was allowed to stand at room temperature for 3 min; centrifugation was performed at 12000 r / min for 15 min at 4℃, and the upper water phase was taken to a new centrifugal tube, and an equal volume of isopropanol was added, and the mixture was inverted and mixed, and was allowed to stand at room temperature for 10 min; centrifugation was performed at 4℃ and 12000 r / min for 10 min, and the supernatant was discarded, and the precipitate was washed with 1 mL of 75% enzyme-free ethanol for two times, each time for 5 min at 4℃ and 7500 r / min; after the ethanol was discarded, the precipitate was air-dried at room temperature, 20 μL of enzyme-free water was added for dissolving, and the total RNA was obtained.

[0102] The human blood albumin gene was amplified by RT-PCR: the total RNA was used as a template, and the first strand of cDNA was synthesized by using a reverse transcriptase (M-MLV reverse transcriptase), the reaction system was 20 μL (containing 2 μg of total RNA, 1 μL of Oligo(dT) 18 primer, 2 μL of dNTP mixture, 1 μL of reverse transcriptase, 4 μL of buffer, and enzyme-free water was added to 20 μL), and reaction was performed at 37℃ for 60 min, and inactivation was performed at 70℃ for 15 min.

[0103] The specific primer was used for PCR amplification with the cDNA as a template, the reaction system was 50 μL (containing 2 μL of cDNA, 2 μL of upper and lower primers, 4 μL of dNTP mixture, 0.5 μL of Taq DNA polymerase, 5 μL of buffer, and 34.5 μL of enzyme-free water), and the amplification conditions were as follows: pre-denaturation at 94℃ for 5 min; denaturation at 94℃ for 30 s, annealing at 58℃ for 30 s, and extension at 72℃ for 1.5 min, for a total of 35 cycles; and final extension at 72℃ for 10 min, and the human blood albumin gene fragment was obtained.

[0104] The specific primer was used for PCR amplification with the cDNA as a template, the reaction system was 50 μL (containing 2 μL of cDNA, 2 μL of upper and lower primers, 4 μL of dNTP mixture, 0.5 μL of Taq DNA polymerase, 5 μL of buffer, and 34.5 μL of enzyme-free water), and the amplification conditions were as follows: pre-denaturation at 94℃ for 5 min; denaturation at 94℃ for 30 s, annealing at 58℃ for 30 s, and extension at 72℃ for 1.5 min, for a total of 35 cycles; and final extension at 72℃ for 10 min, and the human blood albumin gene fragment was obtained.

[0105] The specific primer was used for PCR amplification with the cDNA as a template, the reaction system was 50 μL (containing 2 μL of cDNA, 2 μL of upper and lower primers, 4 μL of dNTP mixture, 0.5 μL of Taq DNA polymerase, 5 μL of buffer, and 34.5 μL of enzyme-free water), and the amplification conditions were as follows: pre-denaturation at 94℃ for 5 min; denaturation at 94℃ for 30 s, annealing at 58℃ for 30 s, and extension at 72℃ for 1.5 min, for a total of 35 cycles; and final extension at 72℃ for 10 min, and the human blood albumin gene fragment was obtained.

[0106] 1.1.2 Pueraria, corn silk, and yam target peptide segment functional gene amplification

[0107] The target molecular weight peptide segment extract of pueraria, corn silk, and yam was taken, and the total RNA of the three raw materials was extracted by using a DP441 polysaccharide and polyphenol plant RNA extraction kit of Tiangeng Biochemical, and the operation was strictly performed according to the kit instruction.

[0108] The cDNA of the three raw materials was synthesized respectively according to the RT-PCR method, and the specific primers (isoflavone synthesis related gene, corn silk polysaccharide synthesis related gene, and Dioscorea saponin synthesis related gene) were designed for the target peptide segment functional gene. The PCR amplification was performed to obtain the target peptide segment functional gene of pueraria, corn silk, and Dioscorea, and the amplification products were verified by 1% agarose gel electrophoresis and purified by a gel recovery kit.

[0109] 1.2 Fusion gene construction

[0110] Vector enzyme digestion: The pPIC9K plasmid (Pichia pastoris expression vector) was selected as the basic vector, and the pPIC9K plasmid was double-digested with restriction endonuclease EcoR I and Xho I (10 U each). The reaction system was 50 μL (containing 10 μg of plasmid, 5 μL of enzyme digestion buffer, 1 μL of each of the two enzymes, and 33 μL of enzyme-free water), and the incubation was performed at 37°C for 4 h. The enzyme digestion products were recovered by a gel recovery kit to obtain the linearized pPIC9K vector.

[0111] Gene fragment enzyme digestion: EcoR I and Xho I were used to double-digest the human blood albumin gene fragment and the target peptide segment functional gene fragment of pueraria / corn silk / Dioscorea (the reaction system was the same as that of the vector enzyme digestion), and the incubation was performed at 37°C for 3 h. The gene fragments after enzyme digestion were recovered.

[0112] Connection reaction: The T4 DNA ligase (5 U) and the ligation buffer were added according to the molar ratio (linearized vector: human blood albumin gene fragment: pueraria target peptide segment gene: corn silk target peptide segment gene: Dioscorea target peptide segment gene = 1:3:3:3:3) to construct the total reaction system of 20 μL, and the ligation was performed at 16°C overnight to obtain the fusion gene expression vector (pPIC9K-albumin-pueraria-corn silk-Dioscorea fusion vector).

[0113] 1.3 Verification of fusion gene sequence

[0114] The ligation product was transformed into E. coli DH5α competent cells: 10 μL of the ligation product was mixed with 100 μL of the competent cells, and the mixture was subjected to ice bath for 30 min, 42°C heat shock for 90 s, and then ice bath for 2 min. Then, 800 μL of LB medium was added, and the mixture was cultured at 37°C and 200 r / min for 1 h. 200 μL of the culture was spread on LB solid medium containing ampicillin (100 μg / mL), and the mixture was cultured at 37°C for 16 h. Then, a single colony was selected.

[0115] The single colony was subjected to colony PCR identification (the primers were pPIC9K vector universal primers), and the positive colony was inoculated into LB liquid medium (containing ampicillin) and cultured at 37°C and 200 r / min for 12 h. Then, the recombinant plasmid was extracted by a plasmid extraction kit.

[0116] The sequence of the recombinant plasmid was determined by Sanger sequencing method. The sequencing results were compared with the designed fusion gene sequence to ensure that there were no base mutations, deletions or insertions. The correct fusion gene expression vector was selected and stored at -20°C for standby.

[0117] II. Yeast transformation and expression

[0118] 2.1 Preparation of Pichia pastoris competent cells

[0119] Select Pichia pastoris GS115 strain, inoculate into YPD solid medium (yeast extract 1%, peptone 2%, glucose 2%, agar 2%), 30°C inverted culture for 48h, pick single colony inoculation into 50mL YPD liquid medium, 30°C, 250r / min shaking culture for 12h, OD 600 1.0.

[0120] Take 10mL of the above bacterial solution and transfer it to 500mL YPD liquid medium. Continue to culture at 30°C, 250r / min until OD 600 2.5, 4°C, 5000r / min centrifugation for 5min, discard the supernatant, resuspend the bacterial body with 500mL ice-precooled sterile water, repeat centrifugation once; resuspend with 250mL ice-precooled sterile water, 4°C, 5000r / min centrifugation for 5min; resuspend with 20mL ice-precooled 1mol / L sorbitol solution, 4°C, 5000r / min centrifugation for 5min; finally resuspend the bacterial body with 1mL ice-precooled 1mol / L sorbitol solution, obtain Pichia pastoris GS115 competent cells, store on ice for standby.

[0121] 2.2 Electroporation operation

[0122] Linearization of fusion gene expression vector: take 10μg of correct fusion gene expression vector, use restriction endonuclease SacⅠ(10U) for single enzyme digestion, reaction system 50μL, 37°C incubation for 4h, the enzyme digestion product is purified by phenol-chloroform method, and the concentration is adjusted to 1μg / μL with ice-precooled 1mol / L sorbitol solution, and stored on ice.

[0123] Electroporation: Take 80 μL of Pichia pastoris competent cells and mix with 10 μL of linearized fusion gene expression vector, transfer to pre-cooled 0.2 cm electroporation cup, ice bath for 10 min; place the electroporation cup into the electroporation instrument (Bio-Rad Gene Pulser Xcell), set the parameters: voltage 1.5 kV, capacitance 25 μF, resistance 200 Ω, perform electric pulse treatment (pulse time 30 μs); immediately after the pulse, add 1 mL of ice-precooled 1 mol / L sorbitol solution, mix gently, transfer to a 1.5 mL centrifuge tube, and incubate at 30°C for 2 h.

[0124] 2.3 Positive strain screening and fusion protein expression

[0125] Positive strain screening: Take 200 μL of culture and spread on MD solid medium (glucose 2%, yeast nitrogen base minimal medium 1.34%, biotin 4×10 -5 2%, agar 2%) containing 100 μg / mL Geneticin (G418), invert culture at 30°C for 5 d, pick single colonies with a diameter greater than 2 mm, inoculate into MD solid medium containing 500 μg / mL G418, and culture at 30°C for 3 d to screen high-copy positive strains.

[0126] Shake flask induction expression: inoculate the high-copy positive strain into 50 mL of BMGY medium (yeast extract 1%, peptone 2%, glycerol 1%, yeast nitrogen base minimal medium 1.34%, biotin 4×10 -5 2%), culture at 30°C with 250 r / min shaking for 24 h until OD 600 is 5.0; centrifuge at 4°C and 5000 r / min for 5 min, discard the supernatant, resuspend the bacterial cells with 10 mL of BMMY medium (replace glycerol in BMGY with 0.5% methanol), culture at 30°C with 250 r / min shaking, add methanol (final concentration 0.5%) every 24 h, induce fusion gene expression for 72 h, take samples every 12 h during the period, and detect the expression amount of fusion protein.

[0127] Expression detection: SDS-PAGE electrophoresis was used to detect the expression of fusion protein: 1 mL of fermentation broth was centrifuged at 4°C and 12000 r / min for 10 min, 20 μL of supernatant was taken, 5 μL of 5×SDS loading buffer was added, and it was boiled at 100°C for 5 min, and then it was loaded into 10% separation gel and electrophoresed at 80V until bromophenol blue entered the separation gel. The voltage was adjusted to 120V, and the electrophoresis was continued until bromophenol blue reached the bottom of the gel; Coomassie brilliant blue R-250 staining for 2h, decolorizing with decolorizing solution (methyl alcohol 45%, glacial acetic acid 10%, water 45%) until the bands were clear. By comparing with standard protein Marker, the molecular weight of the fusion protein (about 75kDa) was determined, and the expression amount was preliminarily judged according to the brightness of the band; at the same time, Western Blot was used for verification: the protein after SDS-PAGE electrophoresis was transferred to PVDF membrane, blocked with 5% skim milk for 2h, and then human serum albumin specific primary antibody (1:5000 dilution) was added and incubated at 4°C overnight. The membrane was washed with TBST for 3 times (10 min each time), and then HRP-labeled secondary antibody (1:10000 dilution) was added and incubated at room temperature for 1h. The membrane was washed with TBST for 3 times, and then ECL chemiluminescence color development was performed. Exposure imaging confirmed that the fusion protein was successfully expressed.

[0128] III. Purification of fusion protein

[0129] 3.1 Centrifugal treatment of yeast fermentation broth

[0130] Low-temperature centrifugal separation: the Pichia pastoris fermentation broth induced for 48h was transferred to a centrifugal bottle, and the centrifugal bottle was placed in a high-speed refrigerated centrifuge (Beckman Avanti J-E). The temperature was set to 4°C, the speed was set to 10000 r / min, and the centrifugal time was set to 15 min. The temperature and speed were monitored in real time during centrifugation to ensure stable parameters.

[0131] Collection and pretreatment of supernatant: after centrifugation, the centrifugal bottle was slowly taken out, the state of the cell precipitate was observed, the upper supernatant was carefully taken out with a sterile pipette and transferred to a sterile storage tank, and the volume of the supernatant was recorded. 1 mL of supernatant was filtered with a 0.22 μm sterile filter membrane.

[0132] 3.2 Buffer preparation before purification and equipment preparation

[0133] Buffer preparation:

[0134] Equilibrium liquid: 0.02 mol / L Tris-HCl buffer (pH 8.0): weigh Tris 2.42 g, add 800 mL of deionized water, adjust the pH to 8.0 with concentrated hydrochloric acid, make up to 1000 mL, filter sterilize with a 0.22 μm filter membrane, and store at 4°C.

[0135] Elution buffer: 0.02 mol / L Tris-HCl buffer (pH 8.0) containing 0.1–0.3 mol / L NaCl: Weigh 5.84 g (0.1 mol / L), 11.69 g (0.2 mol / L), and 17.53 g (0.3 mol / L) of NaCl respectively, add 1000 mL of the above equilibration buffer, stir to dissolve, filter through a 0.22 μm filter membrane for sterilization, and store at 4 °C.

[0136] Preparation of eluent gradient: Mix 0.1 mol / L, 0.2 mol / L and 0.3 mol / L NaCl eluents at a volume ratio of 1:1:1.

[0137] Equipment preparation:

[0138] Chromatography column: Select DEAE-52 ion exchange chromatography column (size 2.6×50cm). Before use, rinse the column bed with deionized water to remove air bubbles, and then equilibrate the chromatography column with equilibration buffer at a flow rate of 1mL / min until the column efficiency is stable.

[0139] Detection system: Connect the ultraviolet detector (detection wavelength 280nm), conductivity meter and recorder. Calibrate the ultraviolet detector (zero with deionized water, verify the response value with 1mg / mL bovine serum albumin solution) to ensure accurate detection data.

[0140] Aseptic operation: The entire purification process is carried out in a sterile clean room. The chromatography column, storage tank, pipeline, etc. are all sterilized by high-pressure steam at 121℃ for 30 minutes and rinsed 3 times with sterile equilibration solution to avoid contamination by other microorganisms.

[0141] Example 1

[0142] A novel pill for treating diabetes comprises the following ingredients in parts by weight:

[0143] 30 parts yam, 10 parts poria cocos, 20 parts polygonatum odoratum, 30 parts guava, 30 parts rehmannia glutinosa, 30 parts kudzu root, 30 parts corn silk, 20 parts trichosanthes kirilowii, 30 parts eleutherococcus senticosus, and 20 parts polygonatum sibiricum.

[0144] Example 2

[0145] A novel pill for treating diabetes comprises the following ingredients in parts by weight:

[0146] 35 parts of Chinese yam, 12 parts of Poria cocos, 22 parts of Polygonatum odoratum, 25 parts of guava, 30 parts of Rehmannia glutinosa, 28 parts of Pueraria lobata, 30 parts of corn silk, 25 parts of Trichosanthes kirilowii, 26 parts of Acanthopanax senticosus, and 22 parts of Polygonatum sibiricum.

[0147] Example 3

[0148] A novel pill for treating diabetes comprises the following ingredients in parts by weight:

[0149] Shanyao 40 parts, Fuling 15 parts, Yuzhu 25 parts, Ban-shiliu 35 parts, Shengdihuang 35 parts, Gegen 32 parts, Corn silk 35 parts, Tianhuafen 30 parts, Ciwujia 30 parts, Huangjing 30 parts.

[0150] Therapeutic effect test:

[0151] Reversing type 2 hyperglycemia experience program:

[0152] 1. Applicable population: type 2 hyperglycemia patients who have not yet developed complications.

[0153] 2. Experience method:

[0154] (1) Blood sugar exceeds normal indicators and has not yet taken medicine, insulin patients:

[0155] a. The highest value of fasting blood sugar is more than 10 mmol / L, take 1 pill 5-10 minutes before each meal, 3 times a day.

[0156] b. The highest value of fasting blood sugar is not more than 10 mmol / L, take 1 pill 5-10 minutes before each meal, 2 times a day.

[0157] (2) Patients whose fasting blood sugar has not been controlled within the normal range after taking medicine and insulin:

[0158] a. The highest value of fasting blood sugar is more than 10 mmol / L, take 1 pill 5-10 minutes before each meal, 3 times a day.

[0159] b. The highest value of fasting blood sugar is not more than 10 mmol / L, take 1 pill 5-10 minutes before each meal, 2 times a day.

[0160] (3) Patients whose fasting blood sugar has been controlled within the normal range after taking medicine and insulin. Stop taking medicine and insulin for 1 day before experience, then measure fasting blood sugar and take pills according to the following requirements:

[0161] a. The highest value of fasting blood sugar is more than 10 mmol / L, take 1 pill 5-10 minutes before each meal, 3 times a day.

[0162] b. The highest value of fasting blood sugar is not more than 10 mmol / L, take 1 pill 5-10 minutes before each meal, 2 times a day.

[0163] Test results:

[0164] (1) Group: blood sugar exceeds normal indicators and has not yet taken medicine, insulin patients

[0165] a: fasting blood glucose history highest value more than 10 mmol / L (30 cases)

[0166] 1. Fast onset and long-term glycemic control effect:

[0167] Short-term rapid reduction of blood sugar: 1 h after taking the medicine, the average postprandial blood glucose decreased from baseline (15.6±2.1 mmol / L) to 11.7±1.5 mmol / L, with a decrease of 25.0%;

[0168] Long-term stable glycemic control: within 12 h after taking the medicine, the fasting blood glucose fluctuation range was only 0.8±0.2 mmol / L (maintained at 7.0-7.8 mmol / L), with no obvious rapid rise and fall of blood glucose;

[0169] Long-term improvement of glycosylation: after 8 weeks, the glycosylated hemoglobin (HbA1c) decreased from baseline (8.9±0.6%) to 7.4±0.4%, with an average decrease of 1.5%.

[0170] 2. Improvement of symptoms of diabetes:

[0171] After 8 weeks, 27 patients had significant improvement in symptoms of polydipsia, polyphagia and polyuria (daily water intake decreased from 2500±300 mL to 1500±200 mL, food intake decreased by 15-20%, and urination frequency decreased from 8-10 times / day to 4-5 times / day); 29 cases had significant improvement in symptoms of fatigue; 1 case had no significant improvement.

[0172] 3. Safety:

[0173] Liver and kidney function: ALT and Scr were detected regularly within 8 weeks, and all maintained within the normal range (ALT <40 U / L, Scr <110 μmol / L), with no liver and kidney function impairment;

[0174] Adverse reactions: 2 cases had mild abdominal distension (within 1-2 weeks of taking the medicine), which was relieved without stopping the medicine; no hypoglycemia (blood glucose <3.9 mmol / L) occurred.

[0175] b: fasting blood glucose history highest value not more than 10 mmol / L (30 cases)

[0176] 1. Fast onset and long-term glycemic control effect:

[0177] Short-term rapid reduction of blood sugar: 1 h after taking the medicine, the average postprandial blood glucose decreased from baseline (12.3±1.3 mmol / L) to 9.2±0.9 mmol / L, with a decrease of 25.2%;

[0178] Long-term stable glycemic control: within 12 h after taking the medicine, the fasting blood glucose fluctuation range was 0.6±0.1 mmol / L (maintained at 6.5-7.1 mmol / L);

[0179] Long-term glycation improvement: After 8 weeks, HbA1c decreased from baseline (7.8±0.5%) to 6.5±0.3%, with an average decrease of 1.3%.

[0180] 2. Improvement of symptoms of diabetes:

[0181] After 8 weeks, 28 cases of polydipsia, polyphagia, and polyuria were significantly improved, and 30 cases of fatigue were significantly improved.

[0182] 3. Safety: No adverse reactions occurred, liver and kidney function indicators were normal, and blood glucose fluctuation was stable.

[0183] (2) Group: Patients whose fasting blood glucose was not controlled in the normal range after taking medicine and insulin

[0184] a: The highest value of fasting blood glucose history reached more than 10 mmol / L (30 cases)

[0185] 1. Rapid onset and long-term glycemic control effect:

[0186] Short-term rapid blood glucose reduction: 1 hour after taking medicine, the average postprandial blood glucose decreased from baseline (14.9±1.8 mmol / L) to 11.2±1.1 mmol / L, with a decrease of 24.8%;

[0187] Long-term stable glycemic control: Within 12 hours after taking medicine, the fasting blood glucose fluctuation range was 0.9±0.2 mmol / L (maintained at 7.5-8.4 mmol / L);

[0188] Long-term glycation improvement: After 8 weeks, HbA1c decreased from baseline (8.7±0.7%) to 7.2±0.5%, with an average decrease of 1.5%.

[0189] 2. Improvement of symptoms of diabetes:

[0190] After 8 weeks, 24 cases were significantly improved, 5 cases were partially improved, and 1 case was not improved, with a total improvement rate of 96.7%.

[0191] 3. Safety:

[0192] 1 case of fasting blood glucose 3.8 mmol / L (mild hypoglycemia, no dizziness, palpitations) occurred on the 3rd day, after adjusting the dose to 1.5 pills before dinner, the blood glucose was maintained at 4.2-6.0 mmol / L, and no hypoglycemia occurred subsequently;

[0193] Liver and kidney function were normal, 1 case had mild nausea, which was relieved on the 5th day, and there were no other adverse reactions.

[0194] b: The highest value of fasting blood glucose history did not exceed 10 mmol / L (30 cases)

[0195] 1. Rapid onset and long-term glycemic control effect:

[0196] Short-term rapid hypoglycemic effect: 1 hour after taking the medicine, the average postprandial blood glucose decreased from baseline (11.8 ± 1.1 mmol / L) to 8.9 ± 0.8 mmol / L, a decrease of 24.6%;

[0197] Long-term stable glycemic control: within 12 hours after taking the medicine, the fasting blood glucose fluctuation was 0.7 ± 0.1 mmol / L (maintained at 6.7-7.4 mmol / L);

[0198] Long-term glycated hemoglobin improvement: after 8 weeks, HbA1c decreased from baseline (7.5 ± 0.4%) to 6.3 ± 0.2%, an average decrease of 1.2%.

[0199] 2. Improvement of polydipsia symptoms:

[0200] After 8 weeks, 26 cases were significantly improved, 4 cases were partially improved, and the total improvement rate was 100%.

[0201] 3. Safety: no hypoglycemia, normal liver and kidney function, no adverse reactions.

[0202] (3) Group: patients whose fasting blood glucose was controlled within normal values after taking medicine and insulin

[0203] a: fasting blood glucose history maximum value above 10 mmol / L (30 cases)

[0204] 1. Rapid onset and long-term glycemic control effect:

[0205] Short-term rapid hypoglycemic effect: 1 hour after taking the medicine, the postprandial blood glucose was maintained at 6.8 ± 0.5 mmol / L, with no significant decrease;

[0206] Long-term stable glycemic control: within 12 hours after taking the medicine, the fasting blood glucose fluctuation was 0.5 ± 0.1 mmol / L (maintained at 5.5-6.0 mmol / L);

[0207] Long-term glycated hemoglobin improvement: after 8 weeks, HbA1c decreased from baseline (6.5 ± 0.3%) to 5.8 ± 0.2%, an average decrease of 0.7%.

[0208] 2. Improvement of polydipsia symptoms:

[0209] After 8 weeks, 29 cases were significantly improved, and 1 case was partially improved.

[0210] 3. Safety: no adverse reactions, liver and kidney function indicators remained normal, and there was no risk of blood glucose fluctuation.

[0211] b: fasting blood glucose history maximum value not exceeding 10 mmol / L (30 cases)

[0212] 1. Rapid onset and long-term glycemic control effect:

[0213] Short-term rapid hypoglycemic effect: 1 hour after taking the medicine, the postprandial blood glucose was maintained at 6.5±0.4 mmol / L, and the blood glucose was stable;

[0214] Long-term stable blood glucose control: within 12 hours, the fasting blood glucose fluctuation range was 0.4±0.1 mmol / L (maintained at 5.3-5.7 mmol / L), and the fluctuation was minimal;

[0215] Long-term glycosylation improvement: after 8 weeks, HbA1c decreased from baseline (6.2±0.2%) to 5.6±0.1%, with an average decrease of 0.6%.

[0216] 2. Improvement of symptoms of diabetes mellitus:

[0217] After 8 weeks, 30 cases showed significant improvement in symptoms, with no residual discomfort.

[0218] 3. Safety: no adverse reactions, normal liver and kidney function.

[0219] Although embodiments of the present application have been shown and described, it will be understood by those having ordinary skill in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the application, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A novel pill for treating diabetes, characterized in that, Includes the following quantities of raw materials: 30-40 parts of yam, 10-15 parts of poria cocos, 20-25 parts of polygonatum odoratum, 25-35 parts of guava, 30-35 parts of rehmannia glutinosa, 28-32 parts of kudzu root, 30-35 parts of corn silk, 20-30 parts of trichosanthes kirilowii, 25-30 parts of eleutherococcus senticosus, and 20-30 parts of polygonatum sibiricum.

2. The novel diabetes treatment pill according to claim 1, characterized in that, Includes the following preferred mass fractions of raw materials: 30 parts yam, 10 parts poria cocos, 20 parts polygonatum odoratum, 30 parts guava, 30 parts rehmannia glutinosa, 30 parts kudzu root, 30 parts corn silk, 20 parts trichosanthes kirilowii, 30 parts eleutherococcus senticosus, and 20 parts polygonatum sibiricum.

3. The novel diabetes treatment pill according to claim 1, characterized in that, Includes the following preferred mass fractions of raw materials: 35 parts of Chinese yam, 12 parts of Poria cocos, 22 parts of Polygonatum odoratum, 25 parts of guava, 30 parts of Rehmannia glutinosa, 28 parts of Pueraria lobata, 30 parts of corn silk, 25 parts of Trichosanthes kirilowii, 26 parts of Acanthopanax senticosus, and 22 parts of Polygonatum sibiricum.

4. A novel pill for treating diabetes according to claim 1, characterized in that, Includes the following preferred mass fractions of raw materials: 40 parts of yam, 15 parts of poria cocos, 25 parts of polygonatum odoratum, 35 parts of guava, 35 parts of rehmannia glutinosa, 32 parts of kudzu root, 35 parts of corn silk, 30 parts of trichosanthes kirilowii, 30 parts of eleutherococcus senticosus, and 30 parts of polygonatum sibiricum.

5. A method for preparing a novel pill for treating diabetes according to any one of claims 1 to 4, characterized in that, Includes the following steps: S1. Raw material pretreatment: Screening, washing, crushing and homogenizing each raw material; S2. Enzymatic extraction: The homogenized raw material is subjected to protein denaturation treatment, multi-stage enzymatic hydrolysis, enzyme inactivation filtration, and ultrafiltration membrane separation to obtain the target peptide extract. S3. Preparation of human albumin fusion protein: A fusion gene expression vector was constructed by gene fusion, which was then transformed into Pichia pastoris for expression and purification to obtain human albumin fusion protein; S4. Protein encapsulation: Using human albumin fusion protein as the encapsulation material, the above target peptides are encapsulated, and moist microspheres are obtained by cross-linking and solidification, washing and purification, and low-temperature pre-drying. S5. Pill Forming: Using hydroxypropyl methylcellulose aqueous solution as a binder, moistened microspheres are molded, formed, sizing, and dried in a pill pan to produce pills with a diameter of 3.5–4.0 mm.

6. The method for preparing a novel pill for treating diabetes according to claim 5, characterized in that, Enzymatic extraction includes the following steps: A1. Raw material crushing and homogenization: The pretreated raw materials are crushed to a particle size of 0.5-1 mm using a high-speed crusher. Then, deionized water is added at a solid-liquid ratio of 1:8-10, and the mixture is homogenized using a colloid mill to obtain a raw material homogenate. A2. Protein denaturation treatment: Transfer the above raw material homogenate into a constant temperature reaction vessel, control the temperature at 60-90℃, the stirring speed at 150-200r / min, and keep it at the temperature for 30-45min. A3. Multi-stage enzymatic hydrolysis: First stage of enzymatic hydrolysis: At a temperature of 45-55℃, add neutral protease at a dosage of 0.3-0.5%, adjust the pH value to 6.5-7.0, and incubate for 2-3 hours for enzymatic hydrolysis; Second stage of enzymatic hydrolysis: After the enzymatic hydrolysis is completed, at a temperature of 40-50℃, add cellulase at a dosage of 0.2-0.4%, adjust the pH value to 5.0-5.5, and continue to incubate for 1.5-2 hours of enzymatic hydrolysis. A4. Enzyme inactivation and filtration: After the enzymatic hydrolysis is completed, heat the reaction system to 90-95℃ and keep it at that temperature for 10-15 minutes to inactivate the enzyme. Then filter it with a 200-mesh filter cloth to remove the residue and collect the filtrate. A5. Ultrafiltration membrane separation: The collected filtrate is separated by passing it through an ultrafiltration membrane with a molecular weight of 3000-5000 Da. The operating pressure is controlled at 0.15-0.2 MPa and the temperature is 30-35℃. The permeate is collected to obtain the target molecular weight peptide extract of each raw material.

7. The method for preparing a novel pill for treating diabetes according to claim 5, characterized in that, The preparation of human albumin fusion protein includes the following steps: B1. Gene fusion construction: The human serum albumin gene sequence was extracted and recombined with the functional gene sequences of target peptides from kudzu root, corn silk, and yam to construct a fusion gene expression vector, which was then ligated by restriction endonuclease digestion and DNA ligase. B2. Yeast Transformation and Expression: The constructed fusion gene expression vector was transformed into competent cells of Pichia pastoris using electroporation. The transformed yeast cells were then inoculated into YPD medium and cultured with shaking to induce the expression of the fusion gene in the yeast cells, producing human albumin fusion protein. B3. Fusion protein purification: The yeast fermentation broth was collected by centrifugation, the cell precipitate was removed, and the supernatant was collected. The supernatant was purified by DEAE-52 ion exchange chromatography column, and the target protein peak was collected by gradient elution. The collected target protein peak solution was further purified by Sephadex G-75 gel filtration chromatography column to collect the human albumin fusion protein solution.

8. The method for preparing a novel pill for treating diabetes according to claim 5, characterized in that, Protein encapsulation includes the following steps: C1. Take the purified human albumin fusion protein solution and prepare protein powder by freeze-drying: dispense the protein solution into freeze-drying bottles, pre-freeze in an ultra-low temperature freezer at -60 to -80℃ for 2 to 3 hours, transfer to a freeze dryer, and freeze-dry at -30 to -50℃ and a vacuum of 0.1 to 0.2 Pa for 36 to 48 hours to obtain human albumin fusion protein powder; select the target molecular weight peptide extracts of each raw material, freeze-dry at -20 to -40℃ and 0.5 to 0.8 Pa for 24 to 48 hours, mix according to the above mass parts to obtain mixed small molecule peptide powder, and remove particulate impurities by passing through an 80-mesh sieve; C2. Add the mixed small molecule peptide powder to PBS buffer to prepare a peptide suspension with a mass concentration of 10-15%, and stir for 5-10 minutes; slowly add the human albumin fusion protein powder to PBS buffer at 30°C, stir to dissolve, and prepare a protein solution with a mass concentration of 8-12%. C3. Using a peristaltic pump, slowly add the peptide suspension to the human albumin fusion protein solution at a rate of 0.5-1 mL / min. The mass ratio of peptide to encapsulated protein is 1:2-3. Stir at 30-35℃. After the addition is complete, continue stirring for 30-60 min to form a human albumin fusion protein-small molecule peptide microsphere suspension. Slowly add 1% (w / w) genipin solution to the suspension, which is 1.8% of the total mass of the system. Stir at 30℃ for 30 min. C4. Centrifuge the solidified microsphere suspension at 3000-4000 r / min for 10 min at 4℃, discard the supernatant, wash the precipitate 3 times with PBS buffer, and finally wash it once with deionized water to obtain pure microsphere precipitate. C5. Spread the microsphere precipitate evenly on a stainless steel tray with a thickness of ≤5mm, place it in a vacuum drying oven, set the temperature to 25℃ and the vacuum degree to 50Pa, and dry for 10-12h to obtain moist microsphere particles. Pass the moist microsphere particles through a standard sieve to collect particles of 14-20 mesh.

9. The method for preparing a novel pill for treating diabetes according to claim 5, characterized in that, Pill forming includes the following steps: D1. Take 95% wet microspheres by mass and add them to the pelletizing pan. Rotate at 30 r / min and tilt at 35°. After turning on the equipment, slowly spray 3% hydroxypropyl methylcellulose aqueous solution by mass, which is 15% of the total mass of the particles. Stir while spraying to roll the particles into spherical pellets with a diameter of 1.5 to 2.0 mm. D2. Keep the pan-frying pan rotating at 35 r / min and the temperature at 28℃. Sprinkle the remaining 5% of moist microspheres evenly on the spherical pellet core. Spray hydroxypropyl methylcellulose aqueous solution after each sprinkling. Repeat the powder sprinkling-liquid spraying operation until the pellet diameter reaches 3.5-4.0 mm. D3. Place the formed pills in a hot air circulating drying oven and dry at 30-35℃ for 8-12 hours.