阅读说明：本技术 海参磷脂在制备防治糖尿病及其并发抑郁症药物中的应用 (Application of sea cucumber phospholipid in preparation of medicine for preventing and treating diabetes and depression complicated with diabetes ) 是由 张永平 王佳佳 李勇斌 宋采 张翼 聂影影 胡雪琼 于 2019-09-30 设计创作，主要内容包括：本发明提供海参磷脂在制备防治糖尿病及其并发抑郁症药物中的应用。本发明通过实验发现,海参磷脂的添加会使得动物糖尿病症状及相关的抑郁样行为得到改善。模型组与对照组相比,其组织葡萄糖、总胆固醇和低密度脂蛋白胆固醇均有显著性的增高(均为P＜0.05),组织甘油三酯和高密度脂蛋白胆固醇含量亦有明显升高,而海参磷脂处理组可显著改善模型组的上述异常指标,提示海参磷脂对糖尿病及其并发抑郁症有改善作用。(The invention provides application of sea cucumber phospholipid in preparing a medicament for preventing and treating diabetes and concurrent depression thereof. Experiments show that the animal diabetes symptoms and related depression-like behaviors can be improved by adding the sea cucumber phospholipid. Compared with a control group, the tissue glucose, the total cholesterol and the low-density lipoprotein cholesterol of the model group are obviously increased (P is less than 0.05), the tissue triglyceride and the high-density lipoprotein cholesterol are also obviously increased, and the sea cucumber phospholipid treatment group can obviously improve the abnormal indexes of the model group and prompt that the sea cucumber phospholipid has the function of improving the diabetes and the concurrent depression thereof.)

1. Application of sea cucumber phospholipids in preparation of medicines for preventing or treating diabetes and/or diabetes complicated with depression.

2. Application of sea cucumber phospholipid in preparing medicine for reducing glucose content is provided.

3. Application of sea cucumber phospholipids in preparing medicines for reducing triglyceride content is provided.

4. Application of sea cucumber phospholipids in preparing medicines for reducing total cholesterol content is provided.

5. Application of sea cucumber phospholipids in preparing medicines for reducing high-density lipoprotein cholesterol is provided.

6. Application of sea cucumber phospholipids in preparing medicines for reducing low-density lipoprotein cholesterol is provided.

7. The use according to any one of claims 1 to 6, wherein the sea cucumber phospholipid is an ethanol-soluble sea cucumber phospholipid.

8. The use according to any one of claims 1 to 6, wherein the medicament further comprises a pharmaceutically acceptable excipient.

9. The use according to any one of claims 1 to 6, wherein the pharmaceutical formulation comprises injection, tablet, powder, granule, capsule, oral liquid, ointment, cream.

Technical Field

The invention relates to the field of biological medicine, in particular to application of sea cucumber phospholipid in preparing a medicament for preventing or treating diabetes and depression complicated with the diabetes.

Background

Diabetes Mellitus (Diabetes mellitis) caused by disturbances of glucose regulation in the body is a metabolic disease characterized by persistent hyperglycemia. Diabetes is classified into gestational diabetes, type 1 diabetes, type 2 diabetes, and the like. The development of economy and the advancement of science and technology have enabled the living standard of people to be further improved, and the prevalence rate of diabetes, which is a global epidemic disease, to be increased year by year. In 2017, 4.5 million people worldwide have diabetes, and predictions made by the international diabetes union (IDF) indicate that the number of people with diabetes will reach 5.87 million in 2045 years, accounting for 8.3% of the adult population. Diabetes brings significant economic burden to patients, and the outpatient cost of people caused by diabetes reaches 3000-20000 yuan per year. A diabetic patient may incur about $ 35,900 to $ 124,600 more medical expenses in their lifetime than a non-diabetic patient. Meanwhile, the system also causes huge burden on a medical security system, in particular to an imperfect basic health service system.

As a metabolic disorder disease, diabetes causes chronic damage and dysfunction of various tissues, organs, nerves and the like of the body, seriously harms the health of the human body, and even finally develops disability and death. Among them, type 2 diabetes accounts for about 90% of the total number of diabetic patients. Genetic factors, insulin resistance, and dysfunction of insulin secretion, among others, cause type 2 diabetes. In addition to factors such as obesity, lack of physical exercise, and age, environmental factors such as chemical toxins including polychlorinated biphenyls and organochlorine pesticides also increase their prevalence. In addition to insulin resistance, more than 90% of type 2 diabetes is caused by a defect in insulin function. Type 2 diabetes typically causes elevated blood glucose levels, increased levels of triglycerides, free fatty acids, abnormal lipid metabolism, and the like. In addition, diabetes also causes various complications, such as retinopathy, vasculopathy, diabetic foot, diabetic peripheral neuropathy, diabetic nephropathy, depression and the like, and brings great pressure to patients.

Depression refers to a syndrome of mental state in long-term depression, mental state disorder, or chest distress, difficulty in falling asleep, etc., depression is affected by various factors including gene, environment, physiology, and personal cognition, etc., and involved metabolites such as neurotransmitters including 5-hydroxytryptamine (5-HT), Dopamine (DA), Norepinephrine (NE), etc., which affect emotional response, thermoregulation, mental activity, sleep, etc., thereby comprehensively affecting the overall mental state. Because the factors related to depression are more, the hypothesis about the pathogenesis of depression is more, at present, the 5-HT hypothesis, the cytokine theory, the acetylcholine hypothesis, the norepinephrine hypothesis, the dopamine hypothesis, the comprehensive psychosocial factors and the like are mainly considered, and the research on the pathogenesis and the treatment of depression is more prone to be comprehensively considered by combining the psychological, social and other factors.

Diabetic patients have a higher probability of developing symptoms of depression than non-diabetic patients, and in a study of Mukut et al on patients with type 2 diabetes, 38.8% of patients were found to have depression, of which 45.36% showed moderate depression, 29.9% major depression and 24.74% mild depression. Also, depression increases the prevalence of diabetes. It has been considered that the association of type 2 diabetes with depressive-like behavior is associated with functional disorders of the hypothalamic-pituitary-adrenal axis (HPA axis), mainly manifested by an increase in the secretion of Cortisol (Cortisol) and by a disorder of the secretory rhythm of adrenocorticotropic hormone releasing hormone (ACTH). Therefore, in the treatment improvement of the depressive-like behavior caused by type 2 diabetes, it is necessary to comprehensively determine an appropriate treatment regimen in consideration of both the factors affecting diabetes and depression.

At present, the treatment of type 2 diabetes mainly focuses on insulin and the action thereof, and mainly comprises biguanides, sulfonylureas, insulin sensitizer, glucosidase inhibitor and the like. However, the potential risks brought by the drugs are not negligible, for example, lactic acid septicemia is easily caused by taking biguanide drugs, sulfonylureas cause certain burden on the kidney, and hypoglycemia and even death are easily caused by using insulin. Moreover, the clinical treatment medicines have almost no improvement effect on the concurrent depression symptoms of the diabetes.

Disclosure of Invention

The invention aims to solve the technical problems that the existing hypoglycemic drugs for diabetes have large side effects and poor curative effects, and cannot improve concurrent depression, and the like, and provides application of sea cucumber phospholipid in preparation of drugs for preventing or treating diabetes and relevant depression.

The above purpose of the invention is realized by the following technical scheme:

the invention provides application of sea cucumber phospholipid in preparation of a medicine for preventing or treating diabetes and/or diabetes complicated depression.

The invention also provides application of the sea cucumber phospholipid in preparing the medicament for reducing the glucose content.

The invention also provides application of the sea cucumber phospholipid in preparing the medicine for reducing the content of triglyceride.

The invention also provides application of the sea cucumber phospholipid in preparing a medicine for reducing the total cholesterol content.

The invention also provides application of the sea cucumber phospholipid in preparing the medicine for reducing the high-density lipoprotein cholesterol.

The invention also provides application of the sea cucumber phospholipid in preparing the medicine for reducing low-density lipoprotein cholesterol.

Optionally, the sea cucumber phospholipid is an ethanol-soluble sea cucumber phospholipid.

The sea cucumber phospholipid is extracted from sea cucumber, and preferably, the extraction method of the sea cucumber phospholipid comprises the following steps:

1) removing oil by acetone for the first time: adding acetone into sea cucumber powder, soaking, performing solid-liquid separation, and performing rotary evaporation on liquid obtained by the solid-liquid separation to obtain a dry product; and continuously soaking and extracting the solid obtained by solid-liquid separation with ethanol, carrying out solid-liquid separation again, carrying out rotary evaporation on the obtained liquid to obtain a phospholipid crude product, and combining the dried product and the phospholipid crude product to obtain a mixture.

2) Removing oil by secondary acetone: extracting the mixture obtained in the step 1) by using acetone to obtain a part which is insoluble in acetone as a refined phospholipid component, taking the refined phospholipid component, adding ethanol, and carrying out solid-liquid separation to obtain the ethanol-soluble sea cucumber phospholipid.

Optionally, in step 1), the volume ratio of the sea cucumber powder to the acetone is 1: 1-3.

Optionally, in the step 1), the volume ratio of the solid to the ethanol is 1: 2-4, wherein the ethanol is absolute ethanol.

Optionally, in the step 1), the ethanol soaking temperature is 35-45 ℃, and the soaking extraction is repeated for 3-5 times.

Optionally, in the step 2), the ratio of the amount of the mixture to the amount of the acetone is 1g: 1-8mL, preferably 1g: 3-5mL, more preferably 1g:4 mL.

Optionally, in the step 2), the extraction temperature is 30-70 ℃, preferably 40-60 ℃, and more preferably 50 ℃.

Optionally, in the step 2), each extraction time is 50-70min, preferably 60 min.

Optionally, in the step 2), the number of times of extraction is 2 to 4, preferably 3.

Optionally, the medicament further comprises pharmaceutically acceptable auxiliary materials, wherein the auxiliary materials comprise diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants and the like which are conventional in the pharmaceutical field.

Optionally, the dosage form of the medicament is various forms such as injection, tablets, powder, granules, capsules, oral liquid, ointment, cream and the like. The medicines in the above various dosage forms can be prepared according to the conventional method in the pharmaceutical field.

The invention has the following beneficial effects:

experiments show that the addition of the sea cucumber phospholipid can improve the depression-like behavior of diabetes model animals. Compared with a control group, the tissue glucose, the total cholesterol and the low-density lipoprotein cholesterol of the model group are obviously increased (P is less than 0.05), the tissue triglyceride and the high-density lipoprotein cholesterol are also obviously increased, the abnormal indexes of the model group can be obviously improved by the sea cucumber phospholipid treatment group, and the sea cucumber phospholipid has the effect of improving the diabetes and the concurrent depression. The invention finds that the sea cucumber phospholipid has important industrial value in the aspect of preparing related products for preventing and treating diabetes and depression.

Drawings

FIG. 1 is a flow chart of the extraction process of phospholipids from Stichopus japonicus in the example.

FIG. 2 is a graph showing the effect of acetone dosage on phospholipid yield (254 nm).

FIG. 3 is a graph showing the effect of temperature on phospholipid yield (254 nm).

FIG. 4 shows TLC images (254nm) of different components of Holothuria scabra.

FIG. 5 shows Al₂O₃TLC image (254nm) of phospholipid fraction after column separation.

FIG. 6 is a graph showing the results of measurement of body mass index according to the embodiment of the present invention.

FIG. 7 is a graph showing the results of measurement of tissue glucose levels according to the example of the present invention.

FIG. 8 is a graph showing the results of tissue triglyceride measurement in examples of the present invention.

FIG. 9 is a graph showing the results of measurement of total cholesterol in tissues according to the example of the present invention.

FIG. 10 is a graph showing the results of measurement of low density lipoprotein cholesterol in tissues according to an example of the present invention.

FIG. 11 is a graph showing the results of measurement of high-density lipoprotein cholesterol in tissues according to the example of the present invention.

FIG. 12 is a diagram illustrating the first time use from bottom to Top (Latency to Top) of an embodiment of the present invention.

FIG. 13 is a Top part number (Top Transition) chart according to an embodiment of the present invention.

FIG. 14 is a graph of the Time in Top-of-run (Time in Top) according to an embodiment of the present invention.

Fig. 15 is a graph showing the average fish school distance at time 10s according to the embodiment of the present invention.

Fig. 16 is a graph showing the average fish school distance at 20s according to the embodiment of the present invention.

Fig. 17 is a graph showing the average fish school distance at 30s according to the embodiment of the present invention.

Fig. 18 is a graph showing the average fish school distance at 40 th time in the example of the present invention.

Fig. 19 is a graph showing the average fish school distance at 50s according to the embodiment of the present invention.

Fig. 20 is a graph showing the average fish school distance at 60s according to the embodiment of the present invention.

FIG. 21 is a graph showing the average fish school distance in 5min according to the embodiment of the present invention.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Diabetes is a disease that endangers human health at present, often accompanied by depressive symptoms. At present, no ideal therapeutic medicine is available for diabetes and depression. The existing diabetes drugs only pay attention to the influence on the blood sugar and related blood indexes of a diabetic patient, but do not relate to the prevention and treatment of depression. The present invention focuses on the amelioration of diabetes and associated depressive symptoms.

On the screening of novel hypoglycemic drugs, the hypoglycemic natural substance has low side effect, has obvious improvement effect on diabetes, and has good development prospect.

The sea cucumber is a traditional medicinal and edible marine resource and has multiple effects of regulating immunity and the like. The invention explores the influence of the sea cucumber phospholipid extract on diabetes and relevant symptoms of depression. And (3) establishing a type II diabetes model by using zebra fish as a model animal. The zebra fish diabetes model is established by a model group and an experimental group through 2 percent of glucose water soaking and 20 percent of cholesterol feed feeding. The experimental group is added with low, medium and high concentration sea cucumber phospholipids of 10 mug/kg, 100 mug/kg and 1000 mug/kg respectively, the sea cucumber phospholipids are added into the feed of the experimental group, a behavior experiment is carried out after 14 days, and then the Body Mass Index (BMI) and the blood glucose and blood lipid indexes of the zebra fish of each group are measured.

The experimental result shows that in the behavioral experiment Novel Tank Test, the model group shows obvious tension (P < 0.05) relative to the control group. The Shoaling Test also shows more tension. The addition of sea cucumber phospholipids improves the depressive-like behavior of each group, wherein the concentration with the best improvement effect is 100 mug/kg. Although experiments show that BMI indexes of all groups have no obvious difference, compared with a control group, tissue glucose, total cholesterol and low-density lipoprotein cholesterol of a model group are obviously increased (P is less than 0.05), the contents of tissue triglyceride and high-density lipoprotein cholesterol are also obviously increased, and a sea cucumber phospholipid treatment group can obviously improve the abnormity of the indexes, wherein the improvement effect of the sea cucumber phospholipid in a 10 mu g/kg dosage group on the blood glucose and blood lipid indexes of diabetes is better than that of the other concentrations. The sea cucumber phospholipid has effect of improving diabetes or diabetes complicated with depression. The invention discloses that the sea cucumber phospholipid has wide market application value in the aspect of preparing related products for preventing and treating diabetes or diabetes complicated depression.

In the following examples, the sea cucumber phospholipid used is an ethanol-soluble sea cucumber phospholipid extract, and the extraction method is described in chinese patent application No. 2019107756578, application of sea cucumber phospholipid in preparing medicines for inhibiting neuroinflammation, specifically, the ethanol-soluble fraction obtained in step 1.2.4 in the patent, i.e., material fr.2-2 in fig. 1 of the patent.

The extraction method of the sea cucumber phospholipid comprises the following steps:

1. material

The holothuria scabra is provided by Shenzhen Taifeng Oriental ocean Biotech Limited, the dosage of which is 13kg, and the holothuria scabra is crushed into powder; other reagents are all domestic analytical purifiers.

2. Instrument for measuring the position of a moving object

WFH-201B dark box type ultraviolet transmission reflection analyzer, Shanghai precision instruments and meters Co., Ltd; KH-300ZDE ultrasonic cleaner, Kunshan Seama ultrasonic Instrument Co., Ltd; RE-6000 rotary evaporator, Shanghai Yanglong Biochemical apparatus factory; CA-1116A cooling water circulation device, EYELA; MZ2CNT chemical diaphragm vacuum pump, Vacuubrand; ME204E METTLER balance, METTLER TOLEDO; GZX-9070MBE electric heating air blast drying oven, Shanghai Boxun industries, Inc. medical equipment factory; agilent 7890A gas chromatograph: CTC headspace sampler, FID detector and Chemstation workstation.

FIG. 1 shows a process flow chart of phospholipid extraction from sea cucumber.

3. Primary oil removal by cold acetone and preparation of phospholipid crude product

Soaking the sea cucumber powder in pre-cooled acetone at 4 ℃ overnight, wherein the volume of the acetone is 2 times of that of the sea cucumber powder, performing suction filtration, performing water pump decompression and spin-drying on filtrate at 50 ℃ to obtain a material with primarily removed fat, and weighing; soaking the filter residue in 3 times volume of anhydrous ethanol overnight, performing ultrasonic treatment at 40 deg.C for 30min, continuously extracting for 4 times, mixing the filtrates obtained by 4 times of extraction, drying, and weighing to obtain phospholipid crude product.

4. Acetone secondary oil removing and refined phosphatide preparing method

The most important factors influencing the removal of the fat component of the crude phospholipid by acetone comprise temperature and feed-liquid ratio. Therefore, the extraction time is 60min, the extraction is carried out for 3 times, the influence of the acetone dosage on the phospholipid yield is researched under the condition of 30 ℃, and the material-liquid ratio is respectively set as 1g: 1mL, 1g: 2mL, 1g:4mL, 1g: 6mL, 1g: 8mL, obtaining the material-liquid ratio of 1g:4mL, and the highest oil removal rate by soaking acetone for 30min by ultrasound; the material-liquid ratio is 1g: under the condition of 4mL, the influence of temperature on the phospholipid yield is researched, the temperature is respectively set to be 30 ℃, 40 ℃, 50 ℃,60 ℃ and 70 ℃, and the highest oil removal rate of acetone at 50 ℃ is obtained. Finally, the optimal conditions for the secondary acetone deoiling are determined as follows: the material-liquid ratio is 1g: extracting with 4mL of water at 50 deg.C for 60min for 3 times. Analyzing the oil component dissolved in acetone twice by TLC spot plate to obtain the same oil component dissolved in acetone for the first time, and mixing the two to obtain acetone extract (Fr.1); the acetone-insoluble fraction was a purified phospholipid fraction (Fr.2). The refined phospholipid fraction is dissolved in 95% ethanol, filtered, and rotary-dried (applied to chromatographic column) at 4 deg.C (Fr.2-2), and the insoluble fraction (Fr.2-1) is rotary-dried at 4 deg.C.

5. Separation of ethanol soluble fraction phospholipids

Activating 1000g of alumina at 120 deg.C for about 3 hr, taking out, cooling to room temperature, adding eluting solution (90% ethanol solution), soaking, and stirring to swell completely. The column is packed by a wet method, the chromatographic column is vertically arranged, and an automatic partial collector is used as a receiver of eluent. About 20g of Fr.2-2 was dissolved in 90% ethanol solution and filtered through a 0.22 μm filter. When the solution flowed to near the upper surface of the alumina, it was immediately eluted continuously with the formulated elution solvent. The flow rate of the eluate was adjusted (0.3mL/min) and the tube was replaced every 10mL using an automatic fraction collector. Detecting the collected liquid under 254nm of ultraviolet spectrophotometer, collecting the liquids with similar absorption spectra under 254nm according to the ultraviolet detection result, concentrating the finally obtained 3 components Fr.2-2-1, Fr.2-2-2 and Fr.2-2-3 into paste with rotary evaporator, and vacuum drying.

6. GC determination of acetone solvent residue in phospholipid extracts

The material Fr.2 in figure 1, namely refined phospholipid, is weighed according to the dosage of 1.0g, precisely weighed, placed in a 25mL measuring flask, dissolved by purified water to be diluted to a scale, shaken evenly, precisely weighed to be 5mL, placed in a 20mL headspace flask, capped, sealed and used for gas phase determination. Precisely weighing appropriate amount of acetone, adding water to obtain a control stock solution containing 2.5 μ g of acetone per 1mL, precisely weighing 20mL of the control stock solution, placing in a 50mL measuring flask, adding water to dilute to scale, shaking, precisely weighing 5mL, and placing in a measuring flask with a volume of 50mL20mL headspace bottles, capped, sealed, as control solutions. A chromatographic column: HP-INNOWAX (30 m.times.0.320 mm, 0.50 μm); column temperature: the temperature is increased by program, the initial temperature is 40 ℃, the temperature is kept for 6min, and the temperature is 25 ℃ per min^-1Heating to 220 deg.C at a certain speed, and maintaining for 5 min; the temperature of a sample inlet is 220 ℃; the temperature of the detector is 250 ℃; nitrogen is taken as carrier gas, and the flow rate is 2.0 mL/min; the balance temperature of the headspace sample injection bottle is 90 ℃, the balance time is 30min, the sample injection volume is 1.5mL, and the split ratio is 5: 1.

The results show that the acetone residue in 3 samples is 0.23 +/-0.01%, which is less than the residue limit specified for the solvent acetone in the 2005 edition of Chinese pharmacopoeia: not more than 0.5%.

FIG. 2 shows a graph of the effect of acetone usage on phospholipid yield (254 nm): under the condition of 30 ℃, the material-liquid ratios are 1:1, 1:2, 1:4, 1:6 and 1:8 respectively, the yields of the crude phospholipid are 68.2%, 68.5%, 68.7%, 68.7% and 68.7% respectively, and the acetone component TLC results are shown in figure 6, and the acetone oil removal rate is highest under the condition of the material-liquid ratio of 1:4(m/v) (30 ℃) in view of solvent saving.

FIG. 3 is a graph showing the effect of temperature on phospholipid yield (254 nm). Under the condition of the feed-liquid ratio of 1:4(m/v), the extraction temperature is 30 ℃, 40 ℃, 50 ℃,60 ℃ and 70 ℃, the yield of the phospholipid crude product is 68.2%, 69.3%, 70.2%, 70.3% and 70.1% respectively, the yield of the phospholipid crude product is the highest at 50 ℃, and the acetone component TLC result is shown in figure 5. Therefore, the optimal conditions for the final acetone secondary de-oiling are: extracting at 50 deg.C for 60min for 3 times at a ratio of materials to liquids of 1/4.

FIG. 4 shows TLC images (254nm) of different components of Holothuria scabra. Analyzing the oil component dissolved in acetone twice by TLC spot plate to obtain the same oil component dissolved in acetone for the first time, and mixing the two to obtain acetone extract (Fr.1); the acetone-insoluble fraction was a purified phospholipid fraction (Fr.2). The purified phospholipid fraction was dissolved in 95% ethanol, wherein the insoluble fraction was presumed to be mainly lecithin (Fr.2-1) according to the literature, and the dissolved fraction was filtered (Fr.2-2) to be purified by further separation on the column, and its TLC spectrum is shown in FIG. 8.

FIG. 5 shows Al2O₃TLC image (254nm) of phospholipid fraction after column separation.

In fig. 4 and 5, chloroform: the methanol ratio is 5:1, the chloroform-methanol mixture is used as a chromatographic separation liquid, and the sample for chromatography is the material Fr.2-2 in FIG. 1.

As shown in the TLC spectrum of FIG. 5, the fraction Fr.2-2 was separated by alumina column chromatography to obtain 3 fractions Fr.2-2-1, Fr.2-2-2 and Fr.2-2-3, the fraction Fr.2-2-1 being the main phospholipid fine fraction, presumably cephalin.

7. Grouping

In step 4, the refined phospholipid component is dissolved in 95% ethanol, filtered, dried (put on a chromatographic column for later use), and stored at 4 ℃ to be an ethanol-soluble part (SCE), namely a material Fr.2-2 in figure 1; the insoluble fraction was spin-dried and stored at 4 ℃ as an ethanol insoluble fraction (SCW), i.e., material Fr.2-1 in FIG. 1.

Material Fr.2-2 in FIG. 1, namely SCE, is holothurian phospholipid soluble in ethanol, and SCE is dissolved in ethanol, sterilized and filtered to obtain ethanol solution containing holothurian phospholipid with required concentration; SCW is water soluble but ethanol insoluble sea cucumber phospholipid, and when cell experiments are carried out, SCW is dissolved in a small amount of water, and after sterilization and filtration, the SCW is added into cell suspension to prepare the cell suspension containing the sea cucumber phospholipid with the required concentration.