CN116327798A

CN116327798A - Application of lycium barbarum polysaccharide in preparing health care product for preventing hyperglycemia and hyperlipidemia

Info

Publication number: CN116327798A
Application number: CN202310244439.8A
Authority: CN
Inventors: 曾晓雄; 周望庭
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2023-03-14
Filing date: 2023-03-14
Publication date: 2023-06-27

Abstract

The invention discloses application of lycium barbarum polysaccharide in preparing health care products for preventing hyperglycemia and hyperlipidemia. The neutral sugar content of the wolfberry polysaccharide is 43.65%, the uronic acid content is 29.03%, and the wolfberry polysaccharide is a heteropolysaccharide with molecular weight of 4.4X104 Da, which mainly consists of rhamnose, galacturonic acid, glucose, galactose and arabinose with the molar ratio of 8.71: 50.29:3.13:17.92:19.96. Experimental results in the embodiment show that the lycium barbarum polysaccharide provided by the invention has obvious improvement effects on fasting plasma blood sugar, glucagon-like peptide (GLP-1), oral glucose tolerance, insulin resistance and abnormal hyperplasia of islet beta cells of a high-fat diet induced mouse, and can obviously reduce the weight gain of the mouse and the contents of Total Cholesterol (TC), total Triglyceride (TG), low-density lipoprotein (LDL-C) and free fatty acid (NEAA) in the plasma. In addition, the lycium barbarum polysaccharide reduces the relative abundance of Clostridium XIVb, clostridium _iv and Eisenbergiella by increasing the relative abundance of allobaculom and romidepa in intestinal flora, and promotes the production of acetic acid and n-butyric acid which are beneficial metabolites, thereby playing a role in regulating and controlling hyperglycemia and hyperlipidemia. The wolfberry polysaccharide provided by the invention can be used for preparing various preparations with hyperglycemia and hyperlipidemia preventing effects.

Description

Application of lycium barbarum polysaccharide in preparing health care product for preventing hyperglycemia and hyperlipidemia

Technical Field

The invention relates to the technical field of natural plant polysaccharide, in particular to application of lycium barbarum polysaccharide in preparing health care products for preventing hyperglycemia and hyperlipidemia.

Background

In the past few decades, the number of people suffering from global diabetes is rapidly increasing, and currently reaches 5.37 hundred million, and it is estimated that in 2030, the number of people will reach 6.43 hundred million, and in 2045, the number of people reaches 7.83 hundred million. In view of the enormous socioeconomic burden associated with diabetes, there is great concern about early prevention, which may reduce the likelihood of developing diabetes in the future. Diet, especially high fat diet, which can lead to obesity, is a major risk factor for the development of diabetes. Long-term intake of high-fat foods not only causes weight gain and blood lipid imbalance in the body, but also causes occurrence of blood glucose abnormality (blood glucose level higher than normal level but lower than diagnostic standard for diabetes), glucose intolerance, insulin resistance, and the like. Methods for treating diabetes mellitus have been widely reported, and chemical drug intervention is mainly adopted, such as insulin secretagogues, insulin sensitizers, metformin, alpha-glycosidase inhibitors, GLP-1 receptor agonists, DPP-4 inhibitors, SGLT-1/SGLT-2 inhibitors and the like. However, studies for preventing diabetes by controlling glucose metabolism and lipid metabolism disorders caused by high-fat diet are relatively slow, and more effective prevention and control agents are lacking.

The polysaccharide is a polymer carbohydrate formed by connecting 10 or more monosaccharides through glycosidic bonds, has no obvious toxic or side effect, can exert various biological activities on multiple ways and multiple layers, such as reducing blood sugar and blood fat, resisting oxidation, resisting tumor, regulating immunity, protecting nerves and the like, and has wide application prospect in the fields of health foods and biological medicines. The medlar is a common medicinal material in the Chinese traditional medicine and food homologous substances, has excellent nutritive value, and is favored and promoted by the health preserving families and medical families in the past. Modern researches have shown that the extract of Lycium barbarum can exert the effects of reducing blood sugar and blood lipid by reducing oxidative stress, maintaining the normal function of mitochondria, inhibiting the transduction of inflammatory signal channels, regulating and controlling the occurrence of abnormal autophagy, and the like. The wolfberry polysaccharide is an important component in wolfberry, can account for 5% -8% of dried wolfberry fruits, and is a material basis for the wolfberry to exert a plurality of biological effects. Therefore, the wolfberry polysaccharide is clear to be significant for the development of preparations for preventing diabetes by regulating and controlling blood sugar and blood fat induced by high-fat diet.

Disclosure of Invention

The invention applies the matrimony vine polysaccharide to the regulation and control of high-fat diet induced blood sugar and blood fat disorder, and aims to prepare the healthcare product for preventing hyperglycemia and hyperlipidemia.

The invention is realized by the following technical scheme:

the preparation of the wolfberry polysaccharide comprises the following steps:

(1) Pulverizing dried fructus Lycii, adding into 80% ethanol (v/v) and water bath at 90deg.C for 2 hr, filtering with gauze to remove supernatant, collecting fructus Lycii residue, and treating for 2 times

(2) Drying the medlar residue in the step (1) to constant weight, extracting for 3 times according to the feed-liquid ratio of 1:30 (w/v) at the extraction temperature of 90 ℃ for 2 hours, mixing the extracting solutions, and concentrating to a proper volume;

(3) Mixing the concentrated solution in the step (2) with 4 times of absolute ethyl alcohol, and standing at 4 ℃ overnight. Centrifuging (4000 rpm,15 min) to obtain precipitate, redissolving in pure water, repeatedly extracting for 5 times by using Sevage method to remove protein, and retaining water phase extract.

(4) Concentrating the solution obtained in the step (3) to a proper volume under reduced pressure at 50 ℃, dialyzing for 2 days by using a 8000-14000Da dialysis bag, concentrating the solution in the dialysis bag to a proper volume, and freeze-drying for 2 days in vacuum to obtain the wolfberry crude polysaccharide LBPs.

(5) And (3) subjecting the wolfberry crude polysaccharide obtained in the step (4) to DEAE-Sepharose Fast Flow column chromatography, eluting with deionized water, 0.05M NaCl solution, 0.1M NaCl solution and 0.3M NaCl solution in sequence, and eluting 3 column volumes for each elution grade.

(6) And (3) collecting and combining the components eluted by the 0.3M NaCl solution in the step (5), concentrating to a proper volume, carrying out deionized dialysis for 2 days, and carrying out vacuum freeze drying for 2 days to finally obtain the lycium barbarum polysaccharide acidic sugar component LBPs-4.

(7) Detecting the purity of the LBPs-4 obtained in the step (6) by using a molecular exclusion chromatography to obtain a single symmetrical peak, which shows that the obtained Lycium barbarum polysaccharide is a pure product.

The LBPs-4 prepared according to the technical proposal has the neutral sugar content of 43.65 percent and the uronic acid content of 29.03 percent, and mainly comprises rhamnose, galacturonic acid, glucose, galactose and arabinose with the molar ratio of 8.71: 50.29:3.13:17.92:19.96 and the molecular weight of 4.4X10 ⁴ Heteropolysaccharide of Da.

The invention adopts a high-fat diet induced mouse model, and the results of the intragastric administration of the lycium barbarum polysaccharide LBPs-4 prepared by the invention show that the LBPs-4 obviously reduces fasting plasma blood sugar and insulin resistance index HOMA-IR of the mouse, reverses abnormal hyperplasia of islet beta cells, improves oral glucose tolerance and plasma GLP-1 level, obviously inhibits weight increase of the mouse, reduces the content of TC, TG, LDL-C and NEAA in the plasma, prompts that the LBPs-4 has the functions of reducing blood sugar and blood fat, and can be applied to preparing health care products for preventing hyperglycemia and hyperlipidemia.

Specifically, the invention also shows that the lycium barbarum polysaccharide LBPs-4 has a regulating effect on the intestinal microorganisms of the high-fat diet induced mice, particularly, the relative abundance of the allobaculom and Romboutsia in the intestinal flora is obviously increased, the relative abundance of Clostridium XIVb, clostridium IV and Eisenbergiella is reduced, and the content of beneficial metabolites acetic acid and n-butyric acid in the intestinal tract is increased.

Drawings

FIG. 1 is a gradient elution profile of a DEAE-Sepharose Fast Flow gel column in accordance with the present invention for preparing LBPs-4

FIG. 2 is a HPGPC chart of LBPs-4 prepared in the present invention.

FIG. 3 shows the effect of LBPs-4 prepared in accordance with the present invention on blood glucose-related parameters of high fat diet-induced mice.

FIG. 4 shows the effect of LBPs-4 prepared according to the present invention on islet beta cell function.

FIG. 5 shows the effect of LBPs-4 prepared in accordance with the present invention on the body weight and dietary intake of high fat diet-induced mice.

FIG. 6 shows the effect of LBPs-4 prepared according to the present invention on lipid profile of high fat diet-induced mice

FIG. 7 shows the results of the analysis of the level control of intestinal flora in high-fat diet-induced mice by LBPs-4 prepared in the present invention.

Detailed Description

In order to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. The described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1: the preparation method of the wolfberry polysaccharide comprises the following technical steps:

(5) And (3) subjecting the wolfberry crude polysaccharide obtained in the step (4) to DEAE-Sepharose Fast Flow column chromatography, eluting with deionized water, 0.05M NaCl solution, 0.1M NaCl solution and 0.3M NaCl solution in sequence, and eluting 3 column volumes for each elution grade. The elution profile is shown in figure 1.

(6) And (3) collecting and combining the components eluted by the 0.3M NaCl solution in the step (5), concentrating to a proper volume, carrying out deionized dialysis for 2 days, and carrying out vacuum freeze drying for 2 days to finally obtain the lycium barbarum polysaccharide acidic sugar component LBPs-4. Wherein the total sugar content was 72.68%.

Example 2: determination of polysaccharide purity

The purity of the polysaccharide was determined by size exclusion high performance liquid chromatography (HPGPC). A certain amount of polysaccharide sample is weighed to prepare 5mg/mL polysaccharide aqueous solution, and the polysaccharide aqueous solution is filtered by a 0.22 mu m filter membrane for standby. The pullulan polysaccharide with different molecular weights is weighed to be prepared into 5mg/mL aqueous solution, and the aqueous solution is used as standard solution for standby. HPGPC detection conditions: agilent technologies 1200 series high performance liquid chromatograph; a differential detector (RID); TSK G4000PW XL (7.8 mm. Times.300 mm, tosoh Crop., tokyo, japan) column; mobile phase: 0.1M Na ₂ SO ₄ And 0.01M PBS; flow rate: 0.5mL/min; column temperature is 35 ℃; sample injection amount: 20. Mu.L. The results are shown in FIG. 2.

Example 3: experimental study of Lycium barbarum polysaccharide for preventing hyperglycemia and hyperglycemia

Experiment 1: 24 Specific Pathogen Free (SPF) C57BL/6 male mice (5 weeks old) were placed in an experimental environment for one week, the ambient temperature was kept at 20-25 ℃, the humidity was 55-65%, and 12h of light and dark were alternately cycled. After one week of acclimation, all mice were randomly divided into 3 groups: normal Control (NC), high Fat Diet (HFD) and Lycium barbarum polysaccharide LBPs-4 in the intervention group, 8 mice each. Wherein NC groups were fed normal feed (10% of energy from fat), free diet drinking water, 9 a day in the morning: 00 lavage of 0.2mL physiological saline per mouse; HFD group was fed high fat diet (10% of energy from fat), and each mouse was perfused with 0.2mL of saline every morning; the LBPs-4 group was fed with high fat diet (10% of energy was derived from fat) and each mouse was perfused with 0.2mL of LBPs-4 solution (dose 200 mg/kg/day) every morning every day. During the experiment, the body weight and food intake of the mice were weighed and recorded weekly. All mice were sacrificed after 14 weeks of continuous treatment, fasting overnight before sacrifice, and fasting blood glucose was measured by an Accu-Chek Performance glucometer after tail vein blood collection. Fresh blood was collected and centrifuged (3000 rpm,15 min) to obtain plasma, and the blood glucose, fasting insulin, GLP-1, TC, TG, HDL-C and NEAA content in the plasma were measured. Insulin resistance is expressed by using an insulin resistance steady state index (HOMA-IR), and the calculation formula is as follows: HOMA-IR index = fasting glycemia (mmol/L) x fasting insulin (mIU/L)/22.5.

Experiment 2: an Oral Glucose Tolerance Test (OGTT) was performed three days before week 14 in experiment 1. All mice were fasted for 6 hours from 9 am to 3 pm, however, were perfused with glucose (1.5 g/kg), collected at the tail vein of the mice at 0, 15, 30, 60, 90, 120min, and blood glucose levels were measured using an Accu-Chek Performance blood glucose meter. Area under OGTT curve (AUC) was calculated using the trapezoidal rule.

Experiment 3: islet beta cell function changes were assessed by immunofluorescent staining. Fresh pancreatic tissue is fixed in 4% paraformaldehyde, cut into slices (4 μm) after operations such as dehydration and embedding, then wax cut into slices are dewaxed to water, antigen retrieval, primary antibody incubation, secondary antibody incubation, DAPI counterstaining of cell nuclei and sealing, and the images are imaged, observed and analyzed by an inverted fluorescence microscope.

Experiment 4: the 16S rRNA high throughput sequencing analysis was performed on mouse faeces. Total DNA was extracted from mouse faeces using a rapid DNA extraction kit. The extracted DNA was subjected to 1% agarose gel electrophoresis and Nanodrop 2000 for quality inspection, and then the target region (V3-V4 region of 16S rRNA gene) was amplified. And adding a specific tag sequence into the gene amplification product to obtain an original library, quantifying and mixing the library, and performing 16S rRNA gene amplicon sequencing on an Illumina Hiseq platform after the quality detection of the library is qualified. Raw data after off-press were mass filtered and pooled and all obtained sequencing Reads were clustered into Amplified Sequence Variants (ASVs) with 97% sequence similarity using UPARSE, and each AS representative sequence was aligned to the SILVA database to complete the taxonomic annotation of ASVs. The differential taxonomic differences between sample groups were analyzed using Tukey's HSD detection and LEfSe.

As can be seen from fig. 3A, the fasting plasma blood glucose level of HFD mice was significantly higher than that of NC mice, whereas the fasting plasma blood glucose of HFD fed mice was significantly reduced after LBPs-4 intervention, indicating that LBPs-4 had a relief effect on HFD-induced hyperglycemia. Also, as shown in fig. 3B, HFD group HOMA-IR index increased significantly compared to NC group; however, the HOMA-IR index of the LBPs-4 group was significantly reduced compared to the HFD group, indicating that LBPs-4 can alleviate HFD-induced insulin resistance. GLP-1 is an intestinal hormone and controls blood glucose by enhancing various mechanisms such as insulin secretion and insulin sensitivity. As shown in FIG. 3C, the intervention with LBPs-4 can significantly increase GLP-1 content in HFD fed mice plasma, further demonstrating the regulatory capacity of LBPs-4 on blood glucose. In addition, the AUC of the LBPs-4 mice was significantly reduced compared to the AUC of the HFD mice in the OGTT test (FIG. 3D), indicating that LBPs-4 had a restorative effect on HFD-induced impaired glucose tolerance, as compared to the NC mice. Immunofluorescence analysis showed that the insulin positive region of HFD mice was 1.3 times higher than that of NC mice (FIG. 4), while after stem prognosis with LBPs-4, the insulin positive region of HFD fed mice was restored to normal levels, indicating that LBPs-4 could prevent HFD-induced proliferation of beta cells. The results show that LBPs-4 has good effect in preventing HFD-induced blood glucose disorder.

The effect of LBPs-4 on body weight and blood lipid in HFD fed mice is demonstrated from FIGS. 5 and 6. As shown in fig. 5A, the body weight gain of HFD mice increased significantly after 5 weeks compared to NC group. However, intervention with LBPs-4 significantly inhibited HFD-induced weight gain in mice from week 6 through week 14. In addition, there was no significant difference in food intake between the LBPs-4 and HFD groups of mice (FIG. 5B), indicating that the regulation of body weight by LBPs-4 was independent of food intake. As shown in fig. 6, the levels of TC, TG, LDL-C and NEAA were significantly increased in the plasma of the mice in the HFD group compared to the NC group. However, the levels of TC, TG, LDL-C and NEAA in plasma were significantly reduced in the LBPs-4 group compared to the HFD group. The results show that LBPs-4 also have good effect in preventing HFD-induced dyslipidemia.

As shown in FIG. 7, the difference in composition at the genus level of intestinal microorganisms in HFD group and LBPs-4 group was analyzed using LEfSe. As a result, LBPs-4 were found to be significantly enriched in Allobaculum and Romboutsia, which were 3.6-fold and 4.3-fold relative abundances, respectively, of HFD group. In addition, the relative abundance of Clostridium XIVb, clostridium _ IV, eisenbergiella was significantly reduced to 39.3%, 62.4% and 55.9% in the LBPs-4 group, respectively. Allobaculom and romiboutsia have been reported to produce butyric acid and acetic acid, respectively, and both have been reported to regulate blood glucose homeostasis by promoting GLP-1 release from intestinal secretory cells. Some studies have shown that Clostridium XIVb, clostridium IV and Eisenbergiella are closely related to HFD-induced metabolic syndrome. Thus, LBPs-4 may promote the growth of beneficial bacteria and inhibit the proliferation of harmful bacteria, thereby improving HFD-induced disorders of intestinal flora.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of Lycium barbarum polysaccharide as the only active component in preparing health products for preventing hyperglycemia and hyperlipidemia is provided.

2. The application of the lycium barbarum polysaccharide in preparing the health-care product for preventing hyperglycemia and hyperlipidemia as claimed in claim 1, wherein the lycium barbarum polysaccharide is prepared by the following method:

3. Use of a polysaccharide according to claim 1 or 2, wherein the polysaccharide has a neutral sugar content of 43.65% and an uronic acid content of 29.03%, and a molecular weight of 4.4x10 consisting essentially of rhamnose, galacturonic acid, glucose, galactose and arabinose in a molar ratio of 8.71: 50.29:3.13:17.92:19.96 ⁴ Heteropolysaccharide of Da.

4. Use of a matrimony vine polysaccharide according to claim 1 or 2, wherein the matrimony vine polysaccharide increases the relative abundance of allobaculom and romioutsia in the intestinal flora, decreases the relative abundance of Clostridium XIVb, clostridium IV and Eisenbergiella, and increases the content of acetic acid and n-butyric acid in the intestinal tract.

5. Use of a matrimony vine polysaccharide according to claim 1 or 2, characterized in that: the wolfberry polysaccharide is prepared into health product preparation by conventional method with or without other adjuvants.