Leonurine, polygonatum polysaccharide and deoxynojirimycin composition and application thereof

Abstract

The invention discloses a leonurine, polygonatum polysaccharide and deoxynojirimycin pharmaceutical composition and application thereof. The pharmaceutical composition can prevent and treat diabetes and osteoporosis and other bone metabolism abnormalities caused by diabetes. Pharmacodynamic experiments show that the composition can effectively reduce blood sugar and has a definite curative effect and positive significance on improvement of juvenile zebra fish osteoporosis symptoms established by prednisolone, and the prescription proportion of the composition is optimized. The pharmaceutical composition disclosed by the invention has the advantages of simple formula, high active ingredient content, safety, effectiveness and the like.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology and relates to a pharmaceutical composition, in particular to a pharmaceutical composition of leonurine, polygonatum sibiricum polysaccharide and deoxynojirimycin and its application in treating diseases such as diabetes and osteoporosis caused by diabetes. Background Art

[0002] Diabetes mellitus (DM) is a metabolic disease characterized by hyperglycemia caused by insulin dysfunction. It is one of the most common chronic diseases in the world after major diseases such as tumors and cardiovascular and cerebrovascular diseases. Persistent hyperglycemia can lead to various complications.

[0003] Osteoporosis (OP) is a systemic metabolic disease characterized by bone loss, decreased bone content, decreased bone density, increased bone fragility, and susceptibility to brittle fractures. In bone tissue, the balance between osteoclasts that perform bone absorption and osteoblasts that dominate bone formation is disrupted, leading to bone metabolic diseases. There are two types of osteoporosis, primary and secondary. Primary osteoporosis is a physiological degenerative disease that occurs with age. The second type is caused by endocrine metabolic diseases or the use of drugs, which can lead to a decrease in bone mass.

[0004] Hyperglycemia is an important factor leading to abnormal bone metabolism. When blood sugar rises, urine osmotic pressure rises, and calcium ions are excreted in large quantities along with calcium in urine, causing serum Ca 2+ The expression of 2 + First, the calcium stored in the bones is released into the blood to compensate for the dysfunction caused by the decrease in blood calcium level, which leads to bone decalcification and decreased bone density. Secondly, the decrease in blood calcium caused by high blood sugar promotes the secretion of thyroid hormones, thereby changing bone metabolism and promoting the activation of osteoclasts, which ultimately leads to a decrease in bone mass. Surveys show that about 30% of people with diabetes have osteoporosis. Currently, diabetes and osteoporosis are two chronic non-infectious diseases that pose a great threat to human health.

[0005] There are many drugs for the treatment of type 2 diabetes on the market, most of which are Western medicines, such as sulfonylureas and biguanides. Although they have significant therapeutic effects, their toxic side effects are also very obvious, such as flatulence and diarrhea, etc. In addition, traditional Chinese medicine has the unique advantages of comprehensive efficacy, safety and reliability in the treatment of osteoporosis, which has attracted more and more researchers' attention. Therefore, based on the theory of traditional Chinese medicine, it is of great significance to use modern scientific and technological methods to develop a drug for high sugar combined with osteoporosis with clear effective ingredients, controllable quality, and small toxic side effects, which reflects the overall effect of traditional Chinese medicine. Summary of the invention

[0006] The object of the present invention is to provide a pharmaceutical composition of de-leonurine, polygonatum polysaccharide and deoxynojirimycin, which is obtained by using modern scientific technology on the basis of traditional Chinese medicine theory, has a simple formula, high content of active ingredients, and is safe and effective. Moreover, the pharmaceutical composition has the effects of lowering blood sugar, inhibiting bone absorption, and reducing bone loss through pharmacodynamics experiments, thereby achieving the effect of preventing and treating diseases such as high sugar and osteoporosis caused by high sugar.

[0007] Another object of the present invention is to provide the application of the above-mentioned leonurine, polygonatum polysaccharide and deoxynojirimycin pharmaceutical composition.

[0008] The purpose of the present invention can be achieved by the following technical solutions:

[0009] A pharmaceutical composition of leonurine, polygonatum polysaccharide and deoxynojirimycin, comprising leonurine, polygonatum polysaccharide and deoxynojirimycin, wherein the weight ratio of leonurine, polygonatum polysaccharide and deoxynojirimycin is 1:(1-3):(2-6).

[0010] Furthermore, the weight ratio of leonurine, polygonatum polysaccharide and deoxynojirimycin is 1:3:6.

[0011] Furthermore, in the pharmaceutical composition, the pharmaceutically acceptable carrier can take various forms. In the case of solid oral administration, suitable carriers include, but are not limited to: diluents, such as starch, corn starch, modified starch, lactose, powdered cellulose, microcrystalline cellulose, anhydrous calcium hydrogen phosphate, tricalcium phosphate, mannitol, sorbitol, sugar, maltodextrin, etc.; in addition, the carrier can also be sterile water, physiological saline or glucose aqueous solution; each carrier must be acceptable, compatible with other ingredients in the pharmaceutical composition formula and harmless to the patient. Further, the pharmaceutical composition can be prepared into capsules, tablets or granules. The preferred dosage form is tablets.

[0012] A use of a pharmaceutical composition of leonurine, polygonatum polysaccharide and deoxynojirimycin in the preparation of a drug for preventing and / or treating diabetes-related diseases.

[0013] Furthermore, the disease is diabetic osteoporosis and / or diabetic osteoporosis caused by the progression of diabetes.

[0014] Furthermore, the disease is a wound healing difficulty disease caused by diabetes.

[0015] Furthermore, the dosage of the pharmaceutical composition is 50-150 μg / day (based on a daily dosage volume of 3 ml). A pharmaceutical composition of leonurine, polygonatum sibiricum polysaccharide and deoxynojirimycin is used in the preparation of a hypoglycemic drug.

[0016] The formulation of the present invention is based on the theory of traditional Chinese medicine, combined with modern research results, to further extract the active ingredients of Leonurus japonicus, Polygonatum sibiricum and mulberry leaves, and organically combine them, so as to develop a new Chinese medicine for treating diabetes and diseases such as diabetic foot caused by diabetes, which has high content of effective ingredients, controllable quality, small toxic and side effects and can be taken for a long time and has the function of treating diabetes and wound healing difficulties caused by diabetes, such as diabetic foot.

[0017] Motherwort can lower blood pressure, promote blood circulation and regulate menstruation, clear the liver and improve eyesight, nourish blood and dispel wind, remove blood stasis and relieve pain. Leonurine is an important active ingredient in motherwort. Modern research shows that it has good hypoglycemic and anti-osteoporosis effects. Animal experiments have confirmed that motherwort can reduce fasting blood sugar in type 2 diabetic mice, improve oral glucose tolerance, and increase fasting plasma insulin. At the same time, an explanation of the regulatory mechanism is made. Motherwort regulates the expression of glucokinase, glucose-6-phosphatase and phosphoenolpyruvate carboxylase in a protein kinase B (Akt)-dependent manner, so motherwort can be used as a drug to improve the symptoms of type 2 diabetes.

[0018] Polygonatum sibiricum is a relatively precious Chinese herbal medicine. It can nourish qi and yin, strengthen the spleen, moisten the lungs, and benefit the kidneys. It can be used for symptoms such as spleen and stomach qi deficiency, fatigue, insufficient stomach yin, dry mouth and poor appetite, and lung deficiency and dry cough. After screening dozens of hypoglycemic Chinese medicines, it was found that Polygonatum sibiricum had the best hypoglycemic effect. Further using modern technology to separate the active parts of Polygonatum sibiricum, the part with hypoglycemic activity was screened out, which was Polygonatum sibiricum polysaccharide. It can strongly inhibit the activity of α-glucosidase and is used as a hypoglycemic drug. The hypoglycemic effect is also very obvious. Polygonatum sibiricum polysaccharide and the two single components PSP-1 and PSP-2 obtained by purification can effectively reduce the blood sugar of diabetic mice, regulate the insulin level in the blood, and reduce their insulin resistance, and significantly improve the oral glucose tolerance level of type II diabetic mice. Therefore, Polygonatum sibiricum polysaccharide has important practical significance for the development of hypoglycemic Chinese medicines.

[0019] Deoxynojirimycin, first extracted from the cortex of mulberry roots, is an effective inhibitor of intestinal α-glucosidase. It can be used as a hypoglycemic drug to delay the decomposition of carbohydrates into monosaccharides and slow down the absorption of glucose, thereby effectively reducing blood sugar after meals. It can also alleviate various complications of diabetes and has great therapeutic advantages. Deoxynojirimycin inhibits intestinal glucose absorption and accelerates liver glucose metabolism by directly regulating the expression of proteins involved in the glucose transport system, glycolysis and gluconeogenesis enzymes. It can transfer glucose from the blood to peripheral tissues to promote the utilization and conversion of sugar, increase blood insulin and reduce blood sugar levels.

[0020] In summary, the combination of leonurine, polygonatum polysaccharide and deoxynojirimycin is safe and effective, with complementary advantages. It can not only achieve a significant blood sugar lowering effect, but also promote the prevention and treatment of osteoporosis, and has little toxicity and side effects. It is expected to become a drug required for the long-term treatment of bone metabolic disorders such as high sugar and osteoporosis caused by high sugar. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The effects of the drug combination of different concentrations and the same ratio on the fasting blood glucose (FBG) of diabetic zebrafish;

[0022] Figure 2 The effects of the drug composition of different concentrations and the same ratio on the bone mineralization of diabetic zebrafish;

[0023] Figure 3 The effects of the drug composition of different concentrations and the same ratio on the bone density of diabetic zebrafish;

[0024] Figure 4 The effects of drug compositions of different proportions and the same concentration on fasting blood glucose (FBG) in diabetic zebrafish;

[0025] Figure 5 The effects of different proportions and the same concentration of drug compositions on bone mineralization in diabetic zebrafish;

[0026] Figure 6 The figure shows the effect of drug compositions of different concentrations and the same ratio on the bone density of diabetic zebrafish.

[0027] Among them, **P<0.001, ***P<0.0001, compared with the negative control group. DETAILED DESCRIPTION

[0028] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art.

[0029] The present invention is further described in detail below in conjunction with specific preparation examples and with reference to data. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0030] In the following examples, various processes and methods not described in detail are conventional methods known in the art. The sources of the reagents used, their trade names and, if necessary, their components are listed, are indicated when they first appear. The same reagents used thereafter are the same as those first indicated unless otherwise specified.

[0031] The leonurine, polygonatum sibiricum polysaccharide and deoxynojirimycin in the present invention are all plant-extracted or semi-synthetic products.

[0032] Among them, the extraction method of leonurine includes but is not limited to solvent reflux ultrasonic and microwave extraction methods; the synthesis of leonurine includes but is not limited to: using syringic acid as a raw material, using ethyl chloroformate to protect and acylate its active carbon group, tetrahydrofuran ring opening under Lewis acid catalysis, alcoholysis of acyl chloride and other reactions to prepare carbonyl products (4-ethoxycarbonyl-oxy-syringic acid-butyl ester), using the Gabriel amination method to prepare leonuramine, and finally reacting with methylisothiourea to prepare leonurine. The obtained leonurine includes but is not limited to leonurine or leonurine hydrochloride or leonurine sulfate, etc.

[0033] Common extraction methods of Polygonatum sibiricum polysaccharide include but are not limited to solvent extraction, flash extraction, microwave / ultrasonic assisted extraction, enzyme extraction, etc.

[0034] The extraction methods of deoxynojirimycin include but are not limited to decoction, maceration, percolation, reflux, microwave, ultrasound, flash, enzyme extraction, etc., and the solvents used are water, ethanol and methanol of different concentrations.

[0035] The specific preparation method of leonurine, polygonatum polysaccharide and deoxynojirimycin in this embodiment is:

[0036] Leonurine: extracted from Leonurine herbs. Brief description of the extraction method: Dry and crush the Leonurine herbs, add an aqueous solution containing 1% hydrochloric acid, with a solid-liquid ratio of 1:8-12, reflux extraction 3 times, combine the filtrates, and adjust the pH of the filtrate to an appropriate level after concentration, alcohol precipitation, and impurity removal, and pass it through a 732 cationic resin column for treatment to produce Leonurine.

[0037] Polygonatum sibiricum polysaccharide: extracted from Polygonatum sibiricum medicinal materials. The extraction method is briefly described as follows: Take Polygonatum sibiricum medicinal materials, dry and crush them, add water, the solid-liquid ratio is 1:10-15, reflux extraction three times, combine the filtrate, concentrate, extract crude protein, repeatedly precipitate with alcohol, remove impurities, and obtain the total Polygonatum sibiricum polysaccharide extract.

[0038] Deoxynojirimycin: It is extracted from mulberry leaves and mulberry branches. The extraction method is briefly described as follows: Crush the dried mulberry leaves or mulberry branches, add 65% ethanol solution, the solid-liquid ratio is 1:10-20, and extract by ultrasonic method at 60-70℃. Combine the extracts, remove impurities, treat with 101 macroporous resin column, collect the eluate, and dry it.

[0039] Embodiment 1:

[0040] The preparation method of the pharmaceutical composition of leonurine, polygonatum polysaccharide and deoxynojirimycin of the present invention is as follows: weigh 10 mg of leonurine, 10 mg of polygonatum polysaccharide, 20 mg of deoxynojirimycin, add appropriate amount of auxiliary materials, granulate to prepare a preparation, and the preparation is in the form of capsules.

[0041] Embodiment 2:

[0042] The preparation method of the pharmaceutical composition of leonurine, polygonatum polysaccharide and deoxynojirimycin of the present invention is as follows: weigh 10 mg of leonurine, 20 mg of polygonatum polysaccharide, and 40 mg of deoxynojirimycin, add appropriate amount of auxiliary materials, granulate to prepare a preparation, and the preparation is in the form of granules.

[0043] Embodiment 3:

[0044] The preparation method of the pharmaceutical composition of leonurine, polygonatum polysaccharide and deoxynojirimycin of the present invention is as follows: weigh 10 mg of leonurine, 30 mg of polygonatum polysaccharide, and 60 mg of deoxynojirimycin, add appropriate amount of auxiliary materials, granulate to form a preparation, and the preparation is in the form of tablets.

[0045] Embodiment 4:

[0046] The preparation method of the pharmaceutical composition of leonurine, polygonatum polysaccharide and deoxynojirimycin of the present invention is as follows: weigh 10 mg of leonurine, 25 mg of polygonatum polysaccharide, and 55 mg of deoxynojirimycin, add appropriate amount of auxiliary materials, granulate to form a preparation, and the preparation is in the form of tablets.

[0047] The pharmaceutical composition of leonurine, polygonatum polysaccharide and deoxynojirimycin has the effects of treating diabetes, lowering blood sugar and treating osteoporosis in diabetic patients. These pharmacological effects are confirmed by the following pharmacodynamic test examples.

[0048] 1. Therapeutic effects of drug compositions of different concentrations and the same ratio on osteoporosis in diabetic zebrafish larvae

[0049] 360 zebrafish fry 2 days after fertilization were randomly divided into a blank control group, a model group, a positive drug control group (metformin + etidronate disodium), a 50μg / mL drug composition administration group, a 100μg / mL drug composition administration group and a 150μg / mL drug composition administration group, with 60 fish in each group. In this experiment, the weight ratio of deoxynojirimycin, leonurine and polygonatum polysaccharide was 1:3:6 (i.e., the proportion of each component in Example 3 was selected). Each administration group was administered by immersion. Except for the blank group, the remaining groups of fry were injected with 0.02% streptozotocin intraperitoneally to construct a diabetic model, and the blank group was injected with the same volume of citrate buffer. After culturing to the 5th day, the blood sugar value was detected. After judging that the model was successful, all the administration groups were treated with prednisolone immersion to construct an osteoporosis model of diabetic fry.

[0050] The juveniles in the blank group and the model group were placed in water, and the other groups were placed in the corresponding drug-containing culture medium. Four days after administration, the zebrafish juveniles in all groups were stained with alizarin red, and the head bones of the zebrafish juveniles were observed using an EVOS micro-imager.

[0051] The blood glucose levels of the drug-treated group and the blank control group were statistically compared using an inter-group t-test. The results are shown in Tables 1 and Figure 1 .

[0052] Table 1 Effect of the drug combination on fasting blood glucose (FBG) in diabetic zebrafish

[0053]

[0054]

[0055] **P<0.001, ***P<0.0001, compared with the negative control group.

[0056] Leonurine, Polygonatum sibiricum polysaccharide and deoxynojirimycin all have good hypoglycemic effects, and blood sugar level is an important indicator for measuring blood sugar levels. As shown in Table 1, the combination of the three drugs has a significant hypoglycemic effect, and the higher the concentration, the better the effect. The effect of the high-concentration drug combination even exceeds that of the positive drug group.

[0057] The bone mineralization area and cumulative optical density of the drug-treated group and the blank control group were statistically compared using the inter-group t test. The results are shown in Table 2 and Figure 2 , Figure 3 .

[0058] Table 2 Effects of the drug combination on bone mineralization area and cumulative optical density in diabetic zebrafish

[0059]

[0060]

[0061] **P<0.001, ***P<0.0001, compared with the negative control group.

[0062] As can be seen from Table 2, the skull staining area of ​​diabetic zebrafish larvae in the model group was significantly reduced due to the disease, while after the intervention of the combination of the three drugs and the positive drug, the skull staining area of ​​the zebrafish larvae increased, the staining intensity increased, the staining area of ​​the high-concentration group exceeded that of the positive drug group, and the staining area of ​​the high-concentration drug combination preventive treatment group for 5 days was the largest.

[0063] In summary, the decrease in blood glucose levels and the drug composition are dose-dependent, while the skull mineralization area increases as the concentration becomes higher, and high concentrations are more conducive to reversing prednisolone-induced osteoporosis in diabetic zebrafish larvae.

[0064] 2. Therapeutic effects of drug combinations of different proportions and the same concentration on osteoporosis in diabetic zebrafish larvae

[0065] 360 zebrafish larvae 2 days after fertilization were randomly divided into blank control group, model group, positive drug control group (etidronate disodium + metformin), 1:1:2 drug combination group, 1:2:4 drug combination group and 1:3:6 drug combination group, 60 in each group. All drugs were administered by immersion. Except for the blank group, the remaining groups of larvae were injected with 0.02% streptozotocin intraperitoneally to establish a diabetic model, and the blank group was injected with the same volume of citrate buffer. After culturing to the 5th day, the blood glucose level was tested. After judging that the model was successful, all drug groups were immersed in prednisolone to establish an osteoporosis model of diabetic larvae.

[0066] The juveniles in the blank group and the model group were placed in water, and the other groups were placed in the corresponding drug-containing culture medium. Four days after administration, the zebrafish juveniles in all groups were stained with alizarin red, and the head bones of the zebrafish juveniles were observed using an EVOS micro-imager.

[0067] The blood glucose levels of the drug-treated group and the blank control group were statistically compared using an inter-group t-test. The results are shown in Tables 3 and Figure 4 .

[0068] Table 3 Effect of the drug composition on fasting blood glucose (FBG) in diabetic zebrafish

[0069]

[0070]

[0071] **P<0.001, ***P<0.0001, compared with the negative control group.

[0072] As shown in Table 3, the drug compositions of the three ratios all have obvious hypoglycemic effects, and the hypoglycemic effects are similar at the same concentration.

[0073] The bone mineralization area and cumulative optical density of the drug-treated group and the blank control group were statistically compared using the inter-group t test. The results are shown in Table 4 and Figure 5 , Figure 6 .

[0074] Table 4 Effects of the drug combination on bone mineralization area and cumulative optical density in diabetic zebrafish

[0075]

[0076]

[0077] **P<0.001, ***P<0.0001, compared with the negative control group.

[0078] It can be seen from Table 4 that at the same concentration, the three different ratios of the drug compositions all have a certain degree of reversal effect on osteoporosis in diabetic zebrafish fry, and as the ratio of Polygonatum sibiricum polysaccharide to deoxynojirimycin increases, the reversal effect becomes stronger.

[0079] From the above pharmacodynamic tests, it can be seen that the combination of leonurine, polygonatum polysaccharide and deoxynojirimycin can effectively lower blood sugar levels and can significantly promote the prevention and treatment of osteoporosis within a controllable concentration range, indicating that this product has a significant effect in preventing and treating abnormal bone metabolism such as osteoporosis caused by diabetes and its complications, and can be used to prepare drugs for preventing and treating abnormal bone metabolism such as osteoporosis caused by diabetes and its complications (more specifically, the pharmaceutical composition of the present invention is suitable for lowering blood sugar in type 1 / type 2 diabetes and symptoms such as type 1 / type 2 diabetes combined with osteoporosis).

[0080] The above embodiments are only for illustrating the technical idea of ​​the present invention, and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the present invention.

Claims

1. A pharmaceutical composition of leonurine, polygonatum polysaccharide and deoxynojirimycin, characterized in that: The pharmaceutical composition comprises leonurine, polygonatum polysaccharide and deoxynojirimycin, and the weight ratio of leonurine, polygonatum polysaccharide and deoxynojirimycin is 1:(1-3):(2-6).

2. The pharmaceutical composition of leonurine, polygonatum sibiricum polysaccharide and deoxynojirimycin according to claim 1, characterized in that: The weight ratio of leonurine, polygonatum sibiricum polysaccharide and deoxynojirimycin is 1:3:

6.

3. A pharmaceutical composition of leonurine, polygonatum polysaccharide and deoxynojirimycin as claimed in claim 1 or 2, characterized in that: The pharmaceutical composition can be prepared into capsules, tablets or granules, and the preferred dosage form is tablets.

4. Use of a pharmaceutical composition of leonurine, polygonatum polysaccharide and deoxynojirimycin in the preparation of a drug for preventing and / or treating diabetes-related diseases.

5. The use according to claim 4, characterized in that The disease is diabetes mellitus complicated with osteoporosis and / or diabetes mellitus combined with osteoporosis.

6. The use according to claim 4, characterized in that The disease is a wound healing difficulty disease caused by diabetes.

7. Use of a pharmaceutical composition of leonurine, polygonatum polysaccharide and deoxynojirimycin in the preparation of hypoglycemic drugs.

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