CN115806574A - A preparation method and application of snow chrysanthemum extract - Google Patents

Abstract

The invention relates to a preparation method and application of a coreopsis tinctoria extract. The method comprises the steps of extracting the coreopsis tinctoria flower heads with ethanol, and performing macroporous resin column chromatography to obtain seven main active ingredients Huang Nuoma glycoside and quercetagetin-7-O‑βGlucopyranoside, eriodictyol-7-O‑βGlucopyranoside, quercetin-7-O‑βThe content of glucopyranoside, malinoside, echinacoside and ocandin is 70 to 79 percent, and animal experiments prove that: the coreopsis tinctoria extract has the capability of improving non-alcoholic fatty liver disease, can obviously improve liver fat deposition and inflammatory reaction of high-fat-induced non-alcoholic fatty liver disease mice, reduces weight gain and hematology indexes, improves liver function and regulates lipid metabolism disorder, and shows the effect of improving non-alcoholic fatty liver disease; has effects of improving diabetic nephropathy, improving diabetic nephropathy mouse lipid metabolism and renal function, and improving diabetic nephropathy. Namely the application of the extract in the drugs for non-alcoholic fatty liver disease and diabetic nephropathy.

Description

A preparation method and application of snow chrysanthemum extract

technical field

The invention relates to the technical field of medicine, and relates to a preparation method of a snow chrysanthemum extract and its application in medicine and health products for improving alcoholic fatty liver disease and diabetic nephropathy.

Background technique

Snow Chrysanthemum, scientific name Snakeeye Chrysanthemum, is the inflorescence head of Coreopsis tinctoria in the family Asteraceae. Snow chrysanthemum originated in the Midwest of the United States, and was introduced and cultivated in my country. It is mainly distributed in high-altitude areas at the northern foot of Kunlun Mountains. It is commonly known as "Kunlun Snow Chrysanthemum". The wild snow chrysanthemum, which grows in the snow area above 3,000 meters above sea level at the northern foot of Kunlun Mountain in Hotan, Xinjiang, has unique effects of lowering blood pressure, fat and sugar due to its special alpine growth environment, and it is difficult to pick because of its low yield. Especially precious, it is a characteristic rare medicinal plant resource in Xinjiang. For a long time, snow chrysanthemum has been used as scented tea among the people. In recent years, it has been developed as a health care product to prevent and treat diseases such as hypertension, hyperlipidemia and hyperglycemia. It is also a common medicinal material in Uighur hospitals in Xinjiang. Liu Jiangyun et al. used ethanol reflux extraction, and then refined the macroporous resin to obtain the snow chrysanthemum extract, in which the total content of mariside and yellow nomaside was greater than 10%, and used the in vivo experiment to study the regulation of the snow chrysanthemum extract on hyperlipidemia The effect and antioxidative effect, the results show that the large dose group of the snow chrysanthemum extract can reduce the TG level in the serum (P<0.05), and reduce the TC in the serum but there is no statistical difference (CN 104173400 A). Up to now, there is no report about the treatment of non-alcoholic fatty liver disease by Xueju. Lu Nan et al. reported a preparation method of a snow chrysanthemum extract for improving diabetic nephropathy. It used water to extract the snow chrysanthemum powder, and then used a strong electric field to extract the snow chrysanthemum aqueous solution to obtain the snow chrysanthemum extract. The snow chrysanthemum extract contained flavonoids, green Original acid and saponin, in vivo experiments suggest that the snow chrysanthemum extract has no toxicity to liver and kidney functions, and can improve hyperlipidemia (CN 109010406 A). Mao Xinmin et al. used high glucose-induced rat glomerular mesangial cells to establish an in vitro model of diabetic nephropathy, and found that the extract of chrysanthemum ethyl acetate may play its role in inhibiting the proliferation of rat glomerular mesangial cells and the expression of fibrosis factors. Renal protection in diabetic nephropathy [Chinese Journal of Experimental Formulas]. 2017, 23(19): 134]. Maliside is the main active ingredient in snow chrysanthemum. Mao Xinmin et al. reported that it can inhibit the activity of sodium-glucose cotransporter 2 (SGLT2) in diabetic mice and high glucose-induced human renal tubular epithelial cells (HK-2). Activating the adenylate-activated protein kinase (AMPK) signaling pathway can improve diabetic nephropathy (Biomedicine & Pharmacotherapy, 2020, 131:110684).

Non-alcoholic fatty liver disease (NAFLD) refers to a clinicopathological syndrome characterized by excessive fat deposition in liver cells caused by alcohol and other definite liver-damaging factors. Its occurrence and development are related to lipid metabolism disorders and increased inflammatory factors. , type II diabetes, metabolic syndrome, hypertension, atherosclerosis and other risk factors are closely related, among which insulin resistance, excessive intake of calories and fatty substances are the most important factors inducing NAFLD. NAFLD is a progressive disease that progresses from nonalcoholic fatty liver disease (NAFL) to nonalcoholic steatohepatitis (NASH) to liver fibrosis and finally to possible development of cirrhosis (NAFLD-LC) and liver cancer (HCC) , is a risk factor for increased mortality from liver-related diseases. The global prevalence of NAFLD continues to rise, and it has become the most common liver disease in my country.

Diabetic nephropathy is an important complication of diabetic patients, the most characteristic of which is diabetic glomerulosclerosis, the so-called diabetic nephropathy (diabetic nephropathy, DN). The etiology and pathogenesis of DN are not very clear at present, and it is generally believed that it may be caused by multiple factors, mainly including metabolic disorders, changes in glomerular hemodynamics, and genetic susceptibility, among which metabolic disorders may be its prerequisite. DN is the most important microvascular complication of diabetes. About one-third of diabetic patients are complicated with DN. Now it has replaced chronic glomerulonephritis as the most important cause of end-stage renal disease worldwide. There are three main methods for establishing traditional animal models of Ⅱ diabetic nephropathy, including spontaneous, induced and transgenic animal models. Clinical diabetic nephropathy is generally caused by living habits and environmental factors, only a small part of which is caused by genetic factors, so spontaneous and transgenic animal models do not completely match the pathological characteristics of clinical diabetic nephropathy. At the same time, spontaneous and transgenic animal models are relatively rare and expensive, which greatly limits the development of related research. Therefore, considering the pathogenesis and operability, among the induced animal models, the animal model induced by high-energy diet combined with streptozotocin (STZ) is currently an ideal model of type Ⅱ diabetic nephropathy.

Contents of the invention

The object of the present invention is to provide a preparation method and application of a snow chrysanthemum extract, which uses ethanol extraction and macroporous resin column chromatography to obtain seven main active components in the snow chrysanthemum extract Flavonomaside, quercetin-7-O-β-glucopyranoside, eriodictyol-7-O-β-glucopyranoside, quercetin-7-O-β-glucopyranoside The content of glucoside, maridin, seacoside and occanin is 70-79%. It has been proved by animal experiments that the snow chrysanthemum extract has the ability to improve non-alcoholic fatty liver disease, and can significantly improve the non-alcoholic liver disease induced by high fat. Hepatic fat deposition and inflammatory response in mice with fatty liver disease can reduce weight gain and hematological indicators, improve liver function and regulate lipid metabolism disorders, and show the effect of improving non-alcoholic fatty liver disease; at the same time, animal experiments show that the snow chrysanthemum extract It has the effect of improving diabetic nephropathy, can improve the lipid metabolism of sugar and kidney function in mice with diabetic nephropathy, and shows the effect of improving diabetic nephropathy. That is, the use of the extract in medicines for non-alcoholic fatty liver disease and diabetic nephropathy.

A kind of preparation method of snow chrysanthemum extract of the present invention, carry out according to the following steps:

a. Extraction: after crushing the dried snow chrysanthemum, use 50-80% ethanol to extract by weight volume ratio snow chrysanthemum: ethanol = 1:10-30 heat reflux extraction, temperature 60-90 ° C, 0.5-3 hours each time , repeat the extraction 2-3 times, and combine the extracts;

b. Concentration: The extract obtained in step a is subjected to vacuum distillation until there is no alcohol smell, and ethanol is removed to obtain a concentrated solution;

c. Macroporous resin column chromatography: Dilute the concentrated solution obtained in step b with water, load the sample to the pretreated HPD-300, HPD-100, D101 or AB-8 macroporous resin column, and then use water, 40-100 Gradient elution with % ethanol, collecting 40-80% ethanol eluted parts, concentrating, and drying to obtain the snow chrysanthemum extract, in which the seven main active ingredients in the snow chrysanthemum extract are flavonomaside and quercetin-7 -O-β-glucopyranoside, eriodictyol-7-O-β-glucopyranoside, quercetin-7-O-β-glucopyranoside, maritin, speicoside and orca The content of Ning is 70-79%.

Use of the snow chrysanthemum extract obtained by the method in preparing a medicine for improving non-alcoholic fatty liver disease.

Use of the snow chrysanthemum extract obtained by the method in the preparation of medicines for improving diabetic nephropathy.

The advantage of the invention is that: the snow chrysanthemum extract obtained by the invention can be applied to the preparation of medicines for non-alcoholic fatty liver disease and diabetic nephropathy. Compared with the prior art, the snow chrysanthemum extract has simpler preparation process, stable chemical composition, high purity of active ingredients and stable efficacy.

Description of drawings

Fig. 1 is the high performance liquid chromatogram of the snow chrysanthemum extract CE that the present invention obtains;

Fig. 2 is the effect of the snow chrysanthemum extract CE obtained by the present invention on the body weight change of NAFLD mice;

Fig. 3 is the effect of the snow chrysanthemum extract CE that the present invention obtains on NAFLD mouse liver index, epididymis fat and subcutaneous fat index change, wherein a, b, c, d, e are used to show the ANOVA statistics by Tukey's between groups Differences, P<0.05; the same letter between each group means that there is no significant difference between the two groups, and the absence of the same letter between each group means that there is a significant difference between the two groups.

Fig. 4 is the effect of the snow chrysanthemum extract CE obtained by the present invention on ALT and AST in the serum of NAFLD mice, wherein A is the ALT content, B is the AST content, ND is the normal control group, HFD is the high-fat model group, and SI100 is In the silibinin group, CE150 is the low-dose group of chrysanthemum extract, CE300 is the middle-dose group of chrysanthemum extract, CE600 is the high-dose group of chrysanthemum extract, a, b, c, d, e are used to indicate the difference between groups Statistical difference by Tukey's ANOVA, P<0.05; the same letter in each group means no significant difference between the two groups, and the absence of the same letter in each group means significant difference between the two groups.

Fig. 5 is the effect of the snow chrysanthemum extract CE obtained by the present invention on TC and TG in the serum of NAFLD mice, wherein A is the TC content, B is the TG content, ND is the normal control group, HFD is the high-fat model group, and SI100 is In the silibinin group, CE150 is the low-dose group of chrysanthemum extract, CE300 is the middle-dose group of chrysanthemum extract, and CE600 is the high-dose group of chrysanthemum extract; The ANOVA statistical difference, P<0.05; the same letter in each group means no significant difference between the two groups, and the absence of the same letter in each group means significant difference between the two groups.

Figure 6 shows the histopathological changes of the liver of mice in the normal control group, high fat model group, silibinin group, and various dosage groups of snow chrysanthemum extract CE (HE staining, 200 times light microscope).

Fig. 7 is the effect of CE of the extract of Snow Chrysanthemum obtained in the present invention on the kidney index of rats with diabetic nephropathy ((x)-±s, n=10). , where a,b,c,d are used to indicate the statistical difference between the groups through Tukey's ANOVA, P<0.05; the same letter between the groups means that there is no significant difference between the two groups, and there is no significant difference between the groups Indicates a significant difference between the two groups.

n＝10)。Fig. 8 is the effect of the snow chrysanthemum extract CE obtained in the present invention on the blood sugar level of rats with diabetic nephropathy (

n=10).

n＝10)，其中Control为正常对照组，DN为模型组，ME200为二甲双胍组，CE150为雪菊提取物低剂量组，CE300为雪菊提取物中剂量组，CE600为雪菊提取物高剂量组；a,b,c,d用于表明组间通过Tukey's的ANOVA统计学差异，P<0.05；各组间有相同字母即代表两组之间无显著性差异，各组间没有相同字母则代表两组之间差异显著。Fig. 9 is the effect of the snow chrysanthemum extract CE obtained in the present invention on blood creatinine SCr and blood urea nitrogen BUN in the serum of diabetic nephropathy rats (

n=10), wherein Control is the normal control group, DN is the model group, ME200 is the metformin group, CE150 is the low-dose group of C. chrysanthemum extract, CE300 is the middle-dose group of C. chrysanthemum extract, and CE600 is the high-dose group of C. chrysanthemum extract. Groups; a, b, c, d are used to indicate the statistical difference between the groups through Tukey's ANOVA, P<0.05; the same letter between the groups means that there is no significant difference between the two groups, and there is no significant difference between the groups Indicates a significant difference between the two groups.

n＝10)，其中Control为正常对照组，DN为模型组，ME200为二甲双胍组，CE150为雪菊提取物低剂量组，CE300为雪菊提取物中剂量组，CE600为雪菊提取物高剂量组；a,b,c,d,e用于表明组间通过Tukey's的ANOVA统计学差异，P<0.05；各组间有相同字母即代表两组之间无显著性差异，各组间没有相同字母则代表两组之间差异显著。Figure 10 is the effect of the snow chrysanthemum extract CE obtained in the present invention on total cholesterol TC and triglyceride TG in the serum of diabetic nephropathy rats (

n=10), wherein Control is the normal control group, DN is the model group, ME200 is the metformin group, CE150 is the low-dose group of C. chrysanthemum extract, CE300 is the middle-dose group of C. chrysanthemum extract, and CE600 is the high-dose group of C. chrysanthemum extract. Group; a, b, c, d, e are used to indicate the statistical difference between the groups through Tukey's ANOVA, P<0.05; the same letter between the groups means that there is no significant difference between the two groups, and there is no difference between the groups Letters represent significant differences between the two groups.

Detailed ways

Example 1

a. Extraction: after crushing the dried capitulum of Chrysanthemum chrysanthemum, use 50% ethanol to extract 3 times under reflux at a weight-to-volume ratio of Chrysanthemum chrysanthemum: ethanol = 1:10. liquid;

b. Concentration: The extract obtained in step a is subjected to vacuum distillation until there is no alcohol smell, and ethanol is removed to obtain a concentrated solution;

c. Macroporous resin column chromatography: the concentrated solution obtained in step b is diluted with water, loaded onto a pretreated HPD-300 macroporous resin column (column volume 1.0L/BV), followed by 4BV of water, 3BV of 50% Ethanol, 2BV 70% ethanol and 3BV 95% ethanol gradient elution, flow rate 1.5-2.5BV/h, collecting 50% and 70% ethanol elution parts, concentrating, drying, to obtain snow chrysanthemum extract, wherein the snow chrysanthemum extract The seven main active ingredients are flavonomaside, quercetin-7-O-β-glucopyranoside, eriodictyol-7-O-β-glucopyranoside, quercetin-7-O- The content of β-glucopyranoside, mariside, seacoside and occanin is 70%.

Example 2

a. Extraction: After crushing the dried capitulum, use 75% ethanol to extract 2 times under reflux at a weight-to-volume ratio of chrysanthemum: ethanol = 1:20, at a temperature of 65° C., for 1 hour each time, and combine the extractions liquid;

b. Concentration: The extract obtained in step a is subjected to vacuum distillation until there is no alcohol smell, and ethanol is removed to obtain a concentrated solution;

c. Macroporous resin column chromatography: the concentrated solution obtained in step b is diluted with water, loaded onto a pretreated HPD-300 macroporous resin column (column volume 1.0L/BV), followed by 4BV of water, 3BV of 50% Ethanol, 2BV 70% ethanol and 3BV 95% ethanol gradient elution, flow rate 1.5-2.5BV/h, collecting 50% and 70% ethanol elution parts, concentrating, drying, to obtain snow chrysanthemum extract, wherein the snow chrysanthemum extract The seven main active ingredients are flavonomaside, quercetin-7-O-β-glucopyranoside, eriodictyol-7-O-β-glucopyranoside, quercetin-7-O- The content of β-glucopyranoside, mariside, seacoside and occanin is 72%.

Example 3

a. Extraction: After crushing the dried capitulum of Chrysanthemum chrysanthemum, extract 3 times under reflux with concentration of 80% ethanol according to the ratio of weight to volume of Chrysanthemum chrysanthemum: ethanol = 1:30, temperature 80°C, 2 hours each time, combined extraction liquid;

b. Concentration: The extract obtained in step a is subjected to vacuum distillation until there is no alcohol smell, and ethanol is removed to obtain a concentrated solution;

c. Macroporous resin column chromatography: the concentrated solution obtained in step b is diluted with water, and loaded onto a pretreated HPD-100 macroporous resin column (column volume 1.0L/BV), followed by 4BV of water, 3BV of 40% Ethanol, 2BV of 60% ethanol and 3BV of 100% ethanol were eluted at a flow rate of 1.5-2.5BV/h. The eluted parts of 40% and 60% ethanol were collected, concentrated, and dried to obtain the chrysanthemum extract. The seven main active ingredients in the snow chrysanthemum extract are flavonomaside, quercetin-7-O-β-glucopyranoside, eriodictyol-7-O-β-glucopyranoside, quercetin The content of prime-7-O-β-glucopyranoside, mariside, seacoside and okanin was 74%.

Example 4

a. Extraction: After crushing the dried capitulum of Chrysanthemum chrysanthemum, use 50% ethanol to extract 3 times under reflux at a weight-to-volume ratio of chrysanthemum chrysanthemum: ethanol = 1:20, at a temperature of 90° C., for 3 hours each time, and combine the extractions liquid;

b. Concentration: The extract obtained in step a is subjected to vacuum distillation until there is no alcohol smell, and ethanol is removed to obtain a concentrated solution;

c, macroporous resin column chromatography: the concentrated solution obtained in step b is diluted with water, and loaded onto a pretreated D101 macroporous resin column (column volume 1.0L/BV), followed by 3BV of water, 3BV of 50% ethanol, 2BV 75% ethanol and 2BV 100% ethanol were eluted at a flow rate of 1.5-2.5BV/h, and the eluted parts of 50% and 75% ethanol were collected, concentrated, and dried to obtain the extract of Snow Chrysanthemum. The seven main active ingredients in the snow chrysanthemum extract are flavonomaside, quercetin-7-O-β-glucopyranoside, eriodictyol-7-O-β-glucopyranoside, quercetin The content of prime-7-O-β-glucopyranoside, mariside, seacoside and okanin is 76%.

Example 5

Take the dried one, grind it, and extract it with 50% ethanol according to the weight-volume ratio of snow chrysanthemum: ethanol = 1:20 at 90°C under reflux for 0.5 hours each time, and repeat the extraction 3 times. The remaining extraction methods are the same as in Example 1. The extracts were combined, and the ethanol in the extracts was distilled off under reduced pressure to obtain a concentrated solution. Dilute the concentrated solution with water, load the sample on AB-8 macroporous resin column (column volume 1.0L/BV), and use 3BV of water, 3BV of 50% ethanol, 2BV of 80% ethanol and 2BV of 100% ethanol to elute in sequence, with a flow rate of 1.5-2.5 BV/h, collect 50% and 80% ethanol eluted parts, concentrate, dry, and obtain the snow chrysanthemum extract, the seven main active ingredients in the snow chrysanthemum extract are flavonomaside, quercetin-7-O -β-glucopyranoside, eriodictyol-7-O-β-glucopyranoside, quercetin-7-O-β-glucopyranoside, maritin, echinoside and occanin The content is 79%.

a. Extraction: after crushing the dried capitulum, use concentration of 60% ethanol to extract 2 times under reflux according to weight volume ratio of chrysanthemum: ethanol = 1:10, temperature 90°C, 0.5 hour each time, combined extraction liquid;

b. Concentration: The extract obtained in step a is subjected to vacuum distillation until there is no alcohol smell, and ethanol is removed to obtain a concentrated solution;

c. Macroporous resin column chromatography: Dilute the concentrated solution obtained in step b with water, load the sample to the pretreated AB-8 macroporous resin column (column volume 1.0L/BV), and use 3BV water and 3BV 50% successively Ethanol, 2BV 80% ethanol and 2BV 100% ethanol elution, flow rate 1.5-2.5BV/h, collecting 50% and 80% ethanol elution parts, concentrating, drying, to obtain snow chrysanthemum extract, seven The main active ingredients are flavonomaside, quercetin-7-O-β-glucopyranoside, eriodictyol-7-O-β-glucopyranoside, quercetin-7-O-β - 79% content of glucopyranoside, mariside, trenitin and occanin.

Example 6

Protective effect of snow chrysanthemum extract CE on liver injury in NAFLD mice induced by high fat

4-5 weeks old C57BL6 male mice were randomly divided into blank control group and model group, 12 mice in the blank control group were fed with common feed; 60 mice in the model group were fed with 60% high-fat feed, and after 16 weeks of high-fat feeding The mice in the model group were randomly divided into the model group, the positive drug group, and the CE low, medium and high dose groups of snow chrysanthemum extract, with 12 mice in each group. From the 17th week, the normal diet group and the model group were given drug blank solution by intragastric administration, the positive drug group group was given silibinin 100 mg/kg by intragastric administration, and the low, middle and high dose groups of snow chrysanthemum extract CE were given 150 mg/kg respectively. , 300mg/kg and 600mg/kg dose gavage administration of the snow chrysanthemum extract that embodiment 1-5 obtains, gavage once a day, continuous administration for 8 weeks, measure food intake every day, measure body weight every week, last gavage After administration, they were fasted without food and water for 12 hours, their body weight was weighed on an empty stomach, and their eyeballs were enucleated to take blood, and then they were killed immediately. Detect total cholesterol TC, triglyceride TG, alanine aminotransferase ALT and aspartate aminotransferase AST.

The results showed that, compared with the model group, the body weight curves of mice in each dose group of Snow Chrysanthemum extract increased slowly, and the CE high-dose 600mg/kg group had the slowest body weight increase. The body weight of the mice increased, and there was a dose-effect relationship. After 8 weeks of administration, compared with the model group, the body weight of the high-dose group decreased significantly, and the difference was statistically significant (P<0.05);

Table 1. The effect of snow chrysanthemum extract CE on the body weight change of NAFLD mice

The statistical results of the daily food intake of the mice showed that compared with the normal diet group, the daily food intake of the high-fat diet group was significantly reduced, and there was no significant difference in the food intake among the high-fat diet groups;

The organ index results showed that compared with the normal diet group, the indexes of liver, epididymis fat and subcutaneous fat of all animals induced by high-fat diet were significantly increased (P<0.05). After 8 weeks of intervention in each dose group of chrysanthemum extract CE, the liver index of each group decreased significantly (P<0.05), and after the intervention of each dose group of snow chrysanthemum extract CE, the index of epididymis fat and subcutaneous fat decreased significantly (P<0.05) , the results are shown in Figure 3 and Table 2. At the same time, compared with the high-fat diet model group, after CE administration intervention, the weight and volume of the mouse liver, epididymal fat and subcutaneous fat were also significantly reduced in varying degrees;

Table 2. Effects of Snow Chrysanthemum Extract CE on the Changes of Organ Index in NAFLD Mice

Note: The letters a, b, c, d, e are used to indicate the statistical difference between the groups through Tukey's ANOVA, P<0.05; the same letter between the groups means that there is no significant difference between the two groups, and there is no significant difference between the groups The same letters represent significant differences between the two groups.

Compared with the normal diet group, the levels of ALT and AST in the serum of mice in the high-fat diet model group were significantly increased (P<0.01); compared with the high-fat diet model group, the AST level of the positive drug group silibinin group was significantly decreased ( P<0.01), the ALT level of the high-dose group of Snow Chrysanthemum Extract significantly decreased (P<0.01); each dose group of Snow Chrysanthemum Extract CE could significantly reduce the level of AST in serum (P<0.01), the results are shown in Figure 4 and Table 3. It shows that the extract CE of snow chrysanthemum can reduce the serum ALT and AST levels of high-fat diet mice, and has a direct protective effect on the liver;

Table 3. The effect of snow chrysanthemum extract CE on ALT and AST in the serum of NAFLD mice

Note: The letters a, b, c, d, e are used to indicate the statistical difference between the groups through Tukey's ANOVA, P<0.05; the same letter between the groups means that there is no significant difference between the two groups, and there is no significant difference between the groups The same letters represent significant differences between the two groups.

All dosage groups of Snow Chrysanthemum extract CE can significantly reduce serum TC and TG levels (P<0.01), the results are shown in Figure 5 and Table 4, indicating that Snow Chrysanthemum Extract CE can reduce serum blood lipids in mice fed a high-fat diet and regulate lipid levels. Mass metabolism disorder, and was dose-related.

Table 4. The effect of CE of snow chrysanthemum extract on TC and TG in the serum of NAFLD mice

Note: The letters a, b, c, and d are used to indicate the statistical difference between the groups through Tukey's ANOVA, P<0.05; the same letter between the groups means that there is no significant difference between the two groups, and there is no same letter between the groups It means there is a significant difference between the two groups.

The observation results of liver tissue morphology showed that the structure of liver lobules and liver cells in the normal diet group was normal, and the hepatic cords were arranged neatly; Mixed vacuoles and balloon-like changes, and neutrophil infiltration were obvious; compared with the model group, each drug intervention group had different degrees of relief, the damage area was significantly reduced, the infiltration of intracellular inflammatory cells was reduced, and the vacuoles and balloons were reduced. The abnormalities were reduced, and the structure of cells and lobules recovered to varying degrees.

The consensus on the diagnosis and treatment of non-alcoholic fatty liver disease in the Asia-Pacific region recommends that the pathological diagnostic criteria of NASH (Non-alcoholic steatohepatitis, NASH) adopt the guidelines set by the Pathology Committee of the NASH Clinical Research Network of the National Institutes of Health in 2005. The NAFLD activity score (NAFLD activity score, NAS) was evaluated. The results showed that each dose group of Xueju extract CE could inhibit hepatic steatosis, ballooning change and relieve inflammation symptoms to varying degrees. The results are shown in Figure 6 and Table 5;

n＝12)Table 5. Scores of hepatic fatty degeneration, hepatic lobular inflammation and hepatic ballooning of mice in each group (

n=12)

Note: The letters a, b, c, and d are used to indicate the statistical difference between the groups through Tukey's ANOVA, P<0.05; the same letter between the groups means that there is no significant difference between the two groups, and there is no same letter between the groups It means there is a significant difference between the two groups.

Example 7

Protective effect of snow chrysanthemum extract CE on rats with diabetic nephropathy induced by high-energy diet combined with streptozotocin (STZ)

Select 100 male SD rats of Specific Pathogen Free (SPF) grade, 6-8 weeks old, and weigh 180-220g, and randomly divide them into blank control group and model group. The rest of the animals were in the high-sugar and high-fat diet group, fed with high-sugar and high-fat feed. After feeding all the animals in the model group for 4 weeks, the body weight and random blood sugar of the rats were measured, and the animals in the high-fat and high-sugar diet group were intraperitoneally injected with 1% chain Uzotocin STZ, the injection dose is 35mg/kg, after 7 days, the tail vein is injected and blood is taken to detect the random blood sugar. If the blood sugar value is greater than 16.7mmol/L, the modeling is considered successful. Rats were divided into 5 groups by random number table method, respectively model group, metformin group (200mg/kg), snow chrysanthemum extract CE low (150mg/kg), medium (300mg/kg), high (600mg/kg) ) three dose groups; during grouping, the animals with the largest or smallest weight deviation from the mean value of fasting blood glucose value, renal function index (serum creatinine Scr and blood urea nitrogen BUN) were removed, 10 in each group. After the grouping, the rats in each group were intragastrically administered once a day at the same time according to the set dose from the ninth week, and the blank control group and the model group were intragastrically administered with the drug blank solvent, and the administration was continued for 4 weeks. After the last gavage administration, fasting without food and water for 12 hours, weighing the fasting body weight, giving 2.5% pentobarbital sodium intraperitoneal injection to anesthetize the rat, fixing the rat, dissecting, collecting blood from the abdominal aorta and testing the specimen. Serum blood glucose (FBG), triglycerides (TG), total cholesterol (TC), serum creatinine (SCr), blood urea nitrogen (BUN);

The results showed that: the kidney index of the model group increased, and there was a very significant difference compared with other groups (P<0.05); there was no significant difference between the CE high-dose group and the normal group (P>0.05); the change of the kidney index It shows that the rats in the model group have hypertrophy in the kidneys, and the early pathological changes of compound DN; the renal index can be significantly reduced after the intervention of each group, and each dose group of CE has a dose-effect relationship. (CE150) had the same effect, and the CE high-dose group had the best effect. The results are shown in Figure 7 and Table 6:

n＝10)Table 6. Effect of Snow Chrysanthemum Extract CE on Kidney Index of Rats with Diabetic Nephropathy (

n=10)

Note: The letters a, b, c, and d are used to indicate the statistical difference between the groups through Tukey's ANOVA, P<0.05; the same letter between the groups means that there is no significant difference between the two groups, and there is no same letter between the groups It means there is a significant difference between the two groups.

The blood sugar in the diabetic nephropathy rat model group rose to about 25mmol/L. After 4 weeks of administration in the snow chrysanthemum extract CE and metformin groups, compared with the model group, the fasting blood sugar in each administration group decreased significantly, indicating that the doses of CE and metformin groups were significantly reduced. Both the metformin group had the effect of lowering blood sugar, the results are shown in Figure 8 and Table 7.

n＝10)Table 7. The effect of snow chrysanthemum extract CE on the change of blood sugar in rats with diabetic nephropathy (

n=10)

After 4 weeks of drug intervention, the levels of serum creatinine SCr and blood urea nitrogen BUN were significantly reversed, indicating that the glomerular filtration function of rats was improved. The above results show that the snow chrysanthemum extract CE can improve the renal function damage of DN rats, the results are shown in Figure 9 and Table 8;

n＝10)Table 8. The effect of snow chrysanthemum extract CE on SCr and BUN in the serum of diabetic nephropathy rats (

n=10)

Note: The letters a, b, c, and d are used to indicate the statistical difference between the groups through Tukey's ANOVA, P<0.05; the same letter between the groups means that there is no significant difference between the two groups, and there is no same letter between the groups It means there is a significant difference between the two groups.

Compared with the normal group, the levels of TC and TG in the DN group were significantly increased (P<0.05); compared with the DN group, the levels of TC and TG in the metformin group and the different dosage groups of the snow chrysanthemum extract CE were significantly reduced (P<0.05) , and each dose group of CE has a significant dose-effect relationship on the reduction level of TC and TG, as shown in Figure 10 and Table 9. It is suggested that the extract CE of snow chrysanthemum can reduce the level of TC and TG in DN rats, and has the effect of improving blood lipid.

n＝10)Table 9. The effect of snow chrysanthemum extract CE on TC and TG in the serum of diabetic nephropathy rats (

n=10)

Note: The letters a, b, c, d, e are used to indicate the statistical difference between the groups through Tukey's ANOVA, P<0.05; the same letter between the groups means that there is no significant difference between the two groups, and there is no significant difference between the groups The same letters represent significant differences between the two groups.

The results show that the active ingredient content of the snow chrysanthemum extract obtained by the method of the invention is high, has the effect of improving non-alcoholic fatty liver disease and diabetic nephropathy, and can be applied to the pharmaceutical use of non-alcoholic fatty liver disease and diabetic nephropathy.

Claims (3)

1. A preparation method of a coreopsis tinctoria extract is characterized by comprising the following steps: the method comprises the following steps:

a. extraction: pulverizing dried coreopsis tinctoria, extracting with 50-80% ethanol under reflux at 60-90 deg.C for 0.5-3 hr for 2-3 times, and mixing extractive solutions;

b. concentration: c, distilling the extracting solution obtained in the step a under reduced pressure until no alcohol smell exists, and removing the ethanol to obtain a concentrated solution;

c. macroporous resin column chromatography: diluting the concentrated solution obtained in the step b by adding water, loading the concentrated solution to a pretreated HPD-300, HPD-100, D101 or AB-8 macroporous resin column, sequentially carrying out gradient elution by using water and 40-100% ethanol, collecting the elution part of 40-80% ethanol, concentrating and drying to obtain the coreopsis tinctoria extract, wherein seven main active ingredients Huang Nuoma glucoside and quercetagetin-7-O-βGlucopyranoside, eriodictyol-7-O-βGlucopyranoside, quercetin-7-O-β-glucopyranoside, maliside, echinacoside and ocandin are present in an amount of 70-79%.

2. Use of a coreopsis tinctoria extract obtained by the method of claim 1 for the preparation of a medicament for ameliorating non-alcoholic fatty liver disease.

3. Use of a coreopsis tinctoria extract obtained by the process of claim 1 in the manufacture of a medicament for the amelioration of diabetic nephropathy.

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