阅读说明：本技术 具有治疗2型糖尿病作用的一组合化合物 (Combination compound with function of treating type 2 diabetes ) 是由 吴巍 焦丽丽 李慧 陈长宝 刘淑莹 于 2019-11-04 设计创作，主要内容包括：本专利本发明属于一种具有治疗2型糖尿病作用的人参皂苷组合物制备方法。其特征在于以五年生晒干人参,通过对药材原料的处理、提取、中空纤维素系统过滤、SB-C18分离纯化,收集80%乙醇洗脱组分。得成分稳定的人参皂苷组合物。其特征在于12种人参皂苷组合物配比为noto-R-(1：)Rg-1:Re:Rf:Rg-2:Rh-1(s):Rb-1:Rc:Rb-2:Rb-3:Rd:Rg-3为0.32：1.00：0.94：1.35：1.15：0.54：0.54：0.81：0.94：0.66：1.52：0.89。对患2型糖尿病大鼠肝组织及血清样品进行脂质代谢及氧化应激水平检测,具有调节脂质代谢,提高氧化应激水平的作用。该方法成本低、简便快捷、高效、易操作,所得提取物成分明确,治疗2型糖尿病活性好。(The invention belongs to a preparation method of a ginsenoside composition with the function of treating type 2 diabetes. The method is characterized in that five-year-old sun-dried ginseng is subjected to treatment, extraction, hollow cellulose system filtration and SB-C18 separation and purification of medicinal materials, and 80% ethanol elution components are collected. Obtaining the ginsenoside composition with stable components. It is characterized in that the mixture ratio of 12 ginsenoside compositions is noto-R 1： Rg 1 :Re:Rf:Rg 2 :Rh 1 (s):Rb 1 :Rc:Rb 2 :Rb 3 :Rd:Rg 3 Is 0.32: 1.00: 0.94: 1.35: 1.15: 0.54: 0.54: 0.81: 0.94: 0.66: 1.52: 0.89. the lipid metabolism and the oxidative stress level detection are carried out on liver tissue and serum samples of rats with type 2 diabetes, and the lipid metabolism and the oxidative stress level detection have the effects of regulating the lipid metabolism and improving the oxidative stress level. The method has low cost, simple operation, high efficiency, and easy operation, and the obtained extract has definite components and activity for treating type 2 diabetesGood results are obtained.)

1. The invention discloses a ginsenoside composition which is extracted from ginseng and has the function of treating type 2 diabetes and a definite proportion, and is characterized in that the method comprises the following steps: steaming and drying sun-dried ginseng serving as a raw material by using water vapor, heating and refluxing the sun-dried ginseng serving as an extraction solvent for extraction, purifying the sun-dried ginseng by using HPD-400 macroporous resin, and freeze-drying the sun-dried ginseng to obtain a ginsenoside mixture.

2. The feedstock as claimed in claim 1, characterized in that: steaming 5-year-old sun-dried ginseng with steam, drying to obtain raw material, steaming five-year-old sun-dried ginseng with high-pressure steam in a high-pressure steam steamer for 8 hr at 120 deg.C, taking out, and oven drying at 70 deg.C.

3. The method as set forth in claim 1, wherein: extracting with 8-10 times (W/V) of water at 100 deg.C under reflux for 3 times (1 hr each time), filtering with 3K Da hollow cellulose system to obtain supernatant, mixing filtrates, and lyophilizing to obtain crude saponin.

4. The method as set forth in claim 1, wherein: dissolving with 5 times of water, separating and purifying by SB-C18, washing with distilled water for 6-8 times of column volume, removing impurities with 20% ethanol, eluting with 80% ethanol, collecting 80% ethanol eluate, evaporating solvent, and lyophilizing to obtain ginsenoside composition with stable components.

5. The method according to claim 1, wherein the ginsenoside composition is formulated at a defined ratio, and the composition is characterized in that: the ginsenoside composition comprises 12 saponins in proportion of not-R_1：Rg₁: Re: Rf: Rg₂: Rh₁(s): Rb₁: Rc: Rb₂: Rb₃: Rd: Rg₃Is 0.32: 1.00: 0.94: 1.35: 1.15: 0.54: 0.54: 0.81: 0.94: 0.66: 1.52: 0.89.

Technical Field

The invention belongs to a combined compound which is extracted from ginseng and has the function of treating type 2 diabetes.

Background

Ginseng, Panax ginseng C.A.Meyer of AraliaceaePanax ginseng C.A. Meyer) The dried roots and rhizomes of the Chinese medicinal herbs are main genuine medicinal materials in Jilin province. Clinical and experimental researches show that ginseng and its main active components have the effects of regulating glycolipid metabolism and resisting diabetesHas the functions of treating obesity, and has obvious effect and good tolerance. In ancient times, ginseng was widely used in the treatment of diabetes. Among the medicinal values of ginseng described in the Chinese medicine discipline, the "thirst quenching" action is an important item.

Various monomeric ginsenosides have been reported to have hypoglycemic and diabetic effects, including: ginsenoside Re, Rh2, Rg3, Rb1, Rc and Rg 1. Xie [ Xie, j.t., Biochim. Biophys. Acta, 2005, 1740(3):319-25.]the anti-diabetic effect of the ginsenoside Re is researched by utilizing an ob/ob mouse model, and the result shows that the ginsenoside Re can effectively reduce the blood sugar level of a diabetic mouse. Zhang et al [ Zhiguo Zhang, Molecular Endocrinology, 2007, 22(1): 186-.]The effect of the ginsenoside Re in an insulin signal pathway is researched in a 3T3-L1 mouse fat cell model and a rat model, and the ginsenoside Re is found to be capable of reducing insulin resistance by inhibiting the activation of JNK and NF-kB. Hwang et al [ J.T. Hwang, Phytother. Res. 2009, 23(2): 262-.]It is reported that ginsenoside Rg3 can effectively inhibit adipocyte differentiation and effectively activate AMPK in 3T3-L1 cells, and the activation is time-dependent. Shang et al, using a 3T3-L1 adipocyte model study, found that ginsenoside Rb1 was able to stimulate glucose uptake through the insulin-like signaling pathway [ Shang W, Journal of endocrinology, 2008, 198(3): 561-.]. Park et al [ Sunmin Park, biosci. Biotechnol. biochem. 2008, 72(11): 2815-2823.]Studies found that ginsenoside Rb1 can inhibit triglyceride accumulation in 3T3-L1 adipocytes, and they also found that ginsenoside Rb1 can enhance beta-cell insulin secretion and viability through PKA-dependent pathways in Min6 cells. Lee et al [ Wen-Kung Lee, Horm Metab Res. 2007, 39(5): 347-354.]The rat model is used for researching the effects of the ginsenoside Rh2 on increasing insulin secretion, reducing blood sugar level, improving insulin sensitivity and relieving insulin resistance. In Lee et al [ Myoung-Su Lee, J Ethnopharmacol. 2010, 127(3): 771-776.]Ginsenoside Rc stimulates glucose uptake in C2C12 myotubular cells through an AMPK-dependent mechanism. Huang et al [ Chuan Huang, J.Agric.food chem.2010, 58(10): 6039-.]The research shows that the ginsenoside Rg1 is in 3T3-L1 mouse fatThe cells stimulate glucose uptake.

The ginseng is steamed by water vapor and then extracted and purified to obtain a group of ginsenoside composition. The saponin component which has the treatment effect on the type 2 diabetes in the ginsenoside composition is screened and analyzed by the detection and screening of biochemical indexes of diabetic rats induced by streptozotocin and fed by high-fat high-sugar feed. The occurrence of diabetes and its complications is closely related to the imbalance of oxidation and antioxidant systems, and hyperglycemia increases the production of free radicals and decreases the activity of antioxidant systems. SOD, GSH-Px and CAT form the main antioxidant defense system of the organism, and the level of antioxidant (SOD, GSH-Px and CAT) is reduced, which may cause the onset of diabetes. Diabetes mellitus is often accompanied by dyslipidemia, which is mainly characterized by elevated levels of cholesterol, triglycerides, and low-density lipoproteins, and reduced levels of high-density lipoproteins, which can lead to the occurrence of hypertension, atherosclerosis, and cardiovascular and cerebrovascular diseases, and thus fatal serious consequences. The measurement of triglyceride and total cholesterol in vivo and the analysis of the ability of the drug to reduce serum triglyceride and total cholesterol levels will regulate lipid metabolism in diabetic patients to a certain extent and reduce the occurrence of diabetic complications. Therefore, the method utilizes the liver tissue and serum samples of type 2 diabetic rats to carry out lipid metabolism and oxidative stress level detection to screen the compounds. Experimental results show that the ginsenoside with definite content in 12 in the composition has the effects of regulating lipid metabolism and improving the oxidative stress level, and the saponin composition is determined to have the treatment effect on type 2 diabetes.

Disclosure of Invention

The invention aims to prepare a ginsenoside composition with the effect of treating type 2 diabetes.

1. Collecting five-year-old sun-dried Ginseng radix, steaming in high pressure steam steamer at 120 deg.C for 8 hr, taking out, cooling to room temperature, oven drying at 70 deg.C, and pulverizing.

2. Extracting with 8-10 times (W/V) of water at 100 deg.C under reflux for 3 times (1 hr each time), filtering with 3KDa hollow cellulose system to obtain supernatant, mixing filtrates, and lyophilizing to obtain crude saponin.

3. Dissolving with 5 times of water, separating and purifying by SB-C18, washing with distilled water for 6-8 times of column volume, removing impurities with 20% ethanol, eluting with 80% ethanol, collecting 80% ethanol eluate, evaporating solvent, and lyophilizing to obtain ginsenoside composition with stable components.

4.12 ginsenoside compositions are prepared by noto-R_1：Rg₁: Re: Rf: Rg₂: Rh₁(s): Rb₁: Rc: Rb₂: Rb₃: Rd: Rg₃Is 0.32: 1.00: 0.94: 1.35: 1.15: 0.54: 0.54: 0.81: 0.94: 0.66: 1.52: 0.89.

second, content determination

1. And optimizing to obtain chromatographic and mass spectrum conditions. Adopting ultra-high performance liquid chromatography-triple quadrupole mass spectrometry combined technology, and adopting a Thermo C-18 chromatographic column (100 mm multiplied by 2.1 mm, 1.7 mu m); the column temperature is 30 ℃; elution with a binary linear gradient: mobile phase 0.1% aqueous formic acid (a), acetonitrile (B); elution gradient, 0-5 min (19% B), 5-25 min (19% -28% B), 25-70 min (28% -49% B), 70-77 min (49% -90% B), 77-80 min (90% B), 80-83 min (90% -19% B), 83-88 min (19% B), flow rate of 0.2 mL-min^-1The sample amount was 5. mu.L. Electrospray ion source (ESI), atomizing gas temperature 300 deg.C, ion transmission tube temperature 350 deg.C, sheath gas pressure 35Arb, auxiliary device pressure 10Arb, and atomizing voltage 2.5kV, scanning in negative ion mode, and scanning rangem/z 100-1500。

2. Accurately weighing appropriate amount of ginsenoside composition, diluting with 80% chromatographic methanol to 5mL, and analyzing with 0.22 μm filter membrane.

3. By LC-MS detection and analysis, 12 ginsenoside compositions in the ginsenoside composition are respectively: noto-R₁Is 0.32g^-4·g^-1、Rg₁Is 1.00g^-4·g^-1Re 0.94g^-4·g^-1Rf is 1.35g^-4·g^-1、Rg₂Is 1.15g^-4·g^-1、Rh₁(s) 0.54g^-4·g^-1、 Rb₁Is 0.54g^-4·g^-1Rc 0.81g^-4·g^-1、Rb₂Is 0.94g^-4·g^-1、Rb₃Is 0.66 g^-4·g^-1Rd 1.52g^-4·g^-1、Rg₃Is 0.89g^-4·g^-1. The method is accurate, stable and feasible through methodology investigation. The 12 ginsenoside compositions are prepared from noto-R_1：Rg₁: Re: Rf: Rg₂: Rh₁(s): Rb₁: Rc: Rb₂: Rb₃: Rd: Rg₃Is 0.32: 1.00: 0.94: 1.35: 1.15: 0.54: 0.54: 0.81: 0.94: 0.66: 1.52: 0.89.

second, saponin composition for treating type 2 diabetes (T2 DM) results

After male Wistar rats of 180-220g are selected and 10 rats in each group are adapted to the environment for one week, high-fat high-sugar feed (lard of 18 percent, sucrose of 20 percent, yolk of 3 percent and basal feed of 59 percent) is continuously fed for 8 weeks, 30 rats are intraperitoneally injected with newly prepared streptozotocin (the mass-volume ratio is 0.1 percent of citric acid buffer solution, pH 4.5) according to the dose of 35 mg/kg body weight, and the other 10 rats are injected with the same dose of citric acid buffer solution to serve as a blank group. One week later, rats with fasting blood glucose concentration of more than 16.7 mmol/L measured by tail blood sampling are successful T2DM models. Dividing into blank group, positive control group (metformin), negative control group, administration group, and gavage continuous administration (according to 100mg/kg dosage) for 28 days, after last administration for 12 hr, taking blood from abdominal artery, centrifuging at 3000 r/10 min, and collecting serum to store in refrigerator at-80 deg.C for use. Storing the liver tissue in a refrigerator at-80 deg.C, homogenizing at 4 deg.C, and collecting the supernatant. Serum samples and liver tissue samples were used for the kit assay. Statistical analysis is carried out on the calculated result by using SPSS23.0 software, single-factor variance analysis is adopted for group comparison, and the data are subjected to addition and subtraction of standard deviation by mean (S) is shown.

2.4 results

The results are shown in tables 1-4.

TABLE 1 intragastric saponin composition rat liver tissue blood lipid index

Note: compared with normal groupP<0.05；** P<0.01。

Compared with the negative control group, the test results show that,^# P<0.05；^## P<0.01。

TABLE 2 serum blood lipid index of gavage composition rat

Note: compared with normal groupP<0.05；** P<0.01。

Compared with the negative control group, the test results show that,^# P<0.05；^## P<0.01。

TABLE 3 intragastric saponin composition rat liver tissue oxidative stress index

Note: compared with normal groupP<0.05；** P<0.01。

Compared with the negative control group, the test results show that,^# P<0.05；^## P<0.01。

TABLE 4 rat serum oxidative stress index of gavage saponin composition

Note: compared with normal groupP<0.05；** P<0.01。

Compared with the negative control group, the test results show that,^# P<0.05；^## P<0.01。

compared with a blank control group of rats, the blood lipid index detection of a negative control group shows that high-density lipoprotein (HDL-C) in liver tissues is obviously reduced (P is less than 0.01), low-density lipoprotein (LDL-C) is obviously increased (P is less than 0.01), and the detection of serum blood lipid indexes shows That Cholesterol (TC), Triglyceride (TG) and low-density lipoprotein in serum are obviously increased (P is less than 0.01) and high-density lipoprotein is obviously reduced (P is less than 0.01), which indicates that the rats suffering from type 2 diabetes have abnormal metabolism and lipid metabolism disorder. Compared with a negative control group, the blood lipid index detection of the positive control group and the administration group shows that high-density lipoprotein in liver tissues is obviously increased (P < 0.01), low-density lipoprotein is obviously reduced (P < 0.01), the detection of serum blood lipid index shows that cholesterol, triglyceride and low-density lipoprotein in serum are obviously reduced (P < 0.01), the high-density lipoprotein of the positive control group is obviously increased (P < 0.01) and the high-density lipoprotein of the administration group is increased (P < 0.05), the result shows that after the red ginseng aqueous extract is administrated by gastric lavage, the lipid metabolism of a type 2 diabetic rat tends to be normal, and the 13 ginsenosides obtained by red ginseng aqueous extraction have the effect of regulating the lipid metabolism of the type 2 diabetic rat.

Compared with a blank control group, the oxidative stress level index detection of the negative control group shows that the oxidative stress level is reduced due to the type 2 diabetes mellitus, wherein the oxidative stress level is reduced due to the fact that the levels of Catalase (CAT), superoxide dismutase (SOD), total antioxidant capacity (T-AOC) and glutathione peroxidase (GSH-PX) in liver tissues are obviously reduced (P < 0.01), the level of Catalase (CAT) is reduced (P < 0.05), the level of superoxide dismutase (SOD) is not obviously changed, and the level of total antioxidant capacity (T-AOC) is obviously reduced (P < 0.05). Compared with the negative control group, the oxidative stress level of the positive control group and the administration group in the liver tissue and serum samples is increased. Wherein the positive control group in liver tissue has obviously increased total antioxidant capacity and glutathione catalase level (P < 0.05), the administration group has obviously increased total antioxidant capacity and glutathione catalase level (P < 0.01), and catalase level (P < 0.05). The positive control group of the serum sample has obviously increased catalase level (P < 0.05), the administration group has obviously increased superoxide dismutase level (P < 0.01), and the result shows that the saponin composition has the effect of improving the oxidative stress level of the type 2 diabetic rats.

The occurrence of diabetes and its complications is closely related to the imbalance of oxidation and antioxidant systems, and hyperglycemia increases the production of free radicals and decreases the activity of antioxidant systems. SOD, GSH-Px and CAT form the main antioxidant defense system of the organism, and the level of antioxidant (SOD, GSH-Px and CAT) is reduced, which may cause the onset of diabetes. Diabetes mellitus is often accompanied by dyslipidemia, which is mainly characterized by elevated levels of cholesterol, triglycerides, and low-density lipoproteins, and reduced levels of high-density lipoproteins, which can lead to the occurrence of hypertension, atherosclerosis, and cardiovascular and cerebrovascular diseases, and thus fatal serious consequences. The measurement of triglyceride and total cholesterol in vivo and the analysis of the ability of the drug to reduce serum triglyceride and total cholesterol levels will regulate lipid metabolism in diabetic patients to a certain extent and reduce the occurrence of diabetic complications. The experimental result shows that the saponin composition has the effects of regulating blood fat and improving the oxidative stress level and has a treatment effect on the type 2 diabetes.

Drawings

FIG. 1 RRLC-TSQ-MS total ion flow diagram of ginsenoside mixed standard substance

FIG. 2 RRLC-TSQ-MS total ion flow diagram of ginsenoside composition

FIG. 2 is a RRLC-TSQ-MS total ion flow diagram of the extracted ginsenoside composition. Mainly comprises a noto-R₁、Rg₁、Re、Rf、Rg₂、Rh₁(s)、Rb₁、Rc、Rb₂、Rb₃、Rd、gRg₃12 common ginsenoside.

Detailed Description

Example 1:

preparation of ginsenoside composition

Collecting five-year-old sun-dried Ginseng radix, steaming in high pressure steam steamer at 120 deg.C for 8 hr, taking out, cooling to room temperature, oven drying at 70 deg.C, and pulverizing. Extracting with 8-10 times (W/V) of water at 100 deg.C under reflux for 3 times (1 hr each time), filtering with 3KDa hollow cellulose system to obtain supernatant, mixing filtrates, and lyophilizing to obtain crude saponin. Dissolving with 5 times of water, separating and purifying by SB-C18, washing with distilled water for 6-8 times of column volume, removing impurities with 20% ethanol, eluting with 80% ethanol, collecting 80% ethanol eluate, evaporating solvent, and lyophilizing to obtain ginsenoside composition with stable components.

Second, content determination

Adopting ultra-high performance liquid chromatography-triple quadrupole mass spectrometry combined technology, and adopting a Thermo C-18 chromatographic column (100 mm multiplied by 2.1 mm, 1.7 mu m); the column temperature is 30 ℃; elution with a binary linear gradient: mobile phase 0.1% aqueous formic acid (a), acetonitrile (B); the flow rate was 0.2 mL/min^-1The sample amount was 5. mu.L. Electrospray ion source (ESI), atomizing gas temperature 300 deg.C, ion transmission tube temperature 350 deg.C, sheath gas pressure 35Arb, auxiliary device pressure 10Arb, and atomizing voltage 2.5kV, scanning in negative ion mode, and scanning rangem/z 100-1500. Accurately weighing appropriate amount of Ginseng radix Rubri crude saponin, diluting with 80% chromatographic methanol to 5mL, and analyzing with 0.22 μm filter membrane. The ginsenoside composition contains 12 kinds of saponins respectively in noto-R₁Is 0.32g^-4·g^-1、Rg₁Is 1.00g^-4·g^-1Re 0.94g^-4·g^-1Rf is 1.35g^-4·g^-1、Rg₂Is 1.15g^-4·g^-1、Rh₁(s) 0.54g^-4·g^-1、 Rb₁Is 0.54g^-4·g^-1Rc 0.81g^-4·g^-1、Rb₂Is 0.94g^-4·g^-1、Rb₃Is 0.66 g^-4·g^-1Rd 1.52g^-4·g^-1、Rg₃Is 0.89g^-4·g^-1. The 12 ginsenosides are noto-R₁: Rg₁: Re: Rf: Rg₂: Rh₁(s): Rb₁:Rc: Rb₂: Rb₃: Rd: Rg₃0.32: 1.00: 0.94: 1.35: 1.15: 0.54: 0.54: 0.81: 0.94: 0.66: 1.52: 0.89.

third, biochemical index test of ginsenoside composition for treating type 2 diabetes

3.1 administration and Collection and pretreatment of blood and liver tissue samples

After male Wistar rats of 180-220g are selected and 10 rats in each group are adapted to the environment for one week, high-fat high-sugar feed (lard of 18 percent, sucrose of 20 percent, yolk of 3 percent and basal feed of 59 percent) is continuously fed for 8 weeks, 30 rats are intraperitoneally injected with newly prepared streptozotocin (the mass-volume ratio is 0.1 percent of citric acid buffer solution, pH 4.5) according to the dose of 35 mg/kg body weight, and the other 10 rats are injected with the same dose of citric acid buffer solution to serve as a blank group. One week later, rats with fasting blood glucose concentration of more than 16.7 mmol/L measured by tail blood sampling are successful T2DM models. Dividing into blank group, positive control group (metformin), negative control group, administration group, and gavage continuous administration (according to 100mg/kg dosage) for 28 days, after last administration for 12 hr, taking blood from abdominal artery, centrifuging at 3000 r/10 min, and collecting serum to store in refrigerator at-80 deg.C for use. Storing the liver tissue in a refrigerator at-80 deg.C, homogenizing at 4 deg.C, and collecting the supernatant. Serum samples and liver tissue samples were used for the kit assay.

3.2 statistical analysis

Statistical analysis is carried out on the calculated result by using SPSS23.0 software, single-factor variance analysis is adopted for group comparison, and the data are subjected to addition and subtraction of standard deviation by mean (S) is shown.

3.3 results

The results are shown in tables 1-4.

TABLE 1 intragastric saponin composition rat liver tissue blood lipid index

Note: compared with normal groupP<0.05；** P<0.01。

Comparison with negative control group, #P<0.05；## P<0.01。

TABLE 2 serum blood lipid index of gavage composition rat

Note: compared with normal groupP<0.05；** P<0.01。

Comparison with negative control group, #P<0.05；## P<0.01。

TABLE 3 intragastric saponin composition rat liver tissue oxidative stress index

Note: compared with normal groupP<0.05；** P<0.01。

Compared with the negative control group, the test results show that,^# P<0.05；^## P<0.01。

TABLE 4 rat serum oxidative stress index of gavage saponin composition

Note: compared with normal groupP<0.05；** P<0.01。

Compared with the negative control group, the test results show that,^# P<0.05；^## P<0.01。

the experiment shows that the type 2 diabetes mellitus rat liver tissue and serum sample biochemical indexes induced by streptozotocin are detected by feeding high-fat high-sugar feed, and the type 2 diabetes mellitus rat liver tissue and serum sample biochemical indexes show that the type 2 diabetes mellitus can cause the rise of cholesterol, triglyceride and low-density lipoprotein, reduce the high-density lipoprotein and reduce the oxidative stress level, thereby causing hypertension, atherosclerosis, cardiovascular and cerebrovascular diseases and even death. The 12 ginsenoside mixtures of the ginsenoside composition can regulate lipid metabolism disorder caused by diabetes to a certain extent, and improve oxidative stress level, thereby treating type 2 diabetes.