Application of Chinese yam glycoprotein in preparation of medicine for treating obesity

Abstract

The invention relates to an application of Chinese yam glycoprotein in preparation of a medicine for treating obesity, wherein the obesity is caused by high fat diet. The Chinese yam glycoprotein is derived from a natural plant Chinese yam, the biomacromolecule biological activity of the Chinese yam glycoprotein can be kept to the maximum extent through low-temperature grinding, separation and purification, and the product obtained through extraction and purification can be prepared into various dosage forms such as capsules, tablets, powder and oral liquid. Screening and verification are carried out through animal experiments, and it is proved that the Chinese yam glycoprotein has a remarkable fat reducing effect on obesity caused by high-fat diet, can improve the histological morphology of white adipose tissue, reduces the hypertrophy degree of adipose cells, meanwhile, improves abnormal glucose tolerance induced by the high-fat diet, and has an obvious fat reducing effect on obesity caused by the high-fat diet. And the expression of key factors related to adipogenesis and metabolic homeostasis in adipose tissues is regulated and controlled.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the use of a natural active biological macromolecule, yam glycoprotein, in the preparation of drugs for treating obesity. Background Technology

[0002] Adipose tissue is not only an energy storage organ but also a highly dynamic metabolic and endocrine organ, playing a crucial role in energy and metabolic homeostasis. However, abnormal fat deposition can lead to dysfunction of adipose tissue, interfering with energy homeostasis, increasing the risk of metabolic syndrome, and disrupting the endocrine function of adipose tissue. The development of mammalian adipose tissue begins in the embryonic period, involving two physiological processes: adipocyte proliferation and hypertrophy. This process involves complex network interactions among various factors, including transcription factors, signaling pathways, epigenetic modifications, the microenvironment, and nutritional conditions. Several key transcription factors are involved in adipose tissue development. For example, peroxisome proliferator-activated receptor gamma (PPARγ) acts as a core transcription factor regulating adipocyte differentiation and lipid storage; glucose transporter 4 (GLUT4), a core molecule for insulin resistance, regulates glucose uptake by adipose tissue; and adiponectin (AdipoQ), an important secretory factor of adipose tissue, participates in maintaining adipocyte metabolic homeostasis.

[0003] Yam glycoprotein (YGP) is a natural active ingredient found in the tuber of Chinese yam (Dioscorea opposita), a traditional food and medicinal plant. It is a glycosylated protein with a molecular weight of approximately 30-40 kDa, composed of a protein core and glycans linked by O- or N-glycosidic bonds. It possesses biological functions such as scavenging intracellular reactive oxygen species and anti-inflammatory responses. Previous published studies have shown that yam glycoprotein has significant effects in treating sleep apnea syndrome (ZL201010171536.1), anti-hypoxia and anti-fatigue (ZL201010171533.8), increasing the activity of mitochondrial oxidative metabolic enzymes in brain cells (ZL201210513861.0), and treating nephritis and renal hypertension (ZL201210513862.5). However, there is currently a lack of systematic research and clear applications of yam glycoproteins in improving fat deposition induced by a high-fat diet, and their effects on adipose tissue morphology and key regulatory factors remain unclear. Summary of the Invention

[0004] This invention provides the use of yam glycoprotein in the preparation of a drug for treating obesity, characterized in that...

[0005] The yam glycoprotein mentioned above is derived from Dioscorea opposita, a plant belonging to the Dioscoreaceae family and the Dioscorea genus. The preparation of the yam glycoprotein is carried out according to the following steps:

[0006] (1) Peel the fresh yam, cut it into small pieces of about 1 cubic centimeter, and freeze-dry it under vacuum at -69 ℃ for about 48 h;

[0007] (2) Place the freeze-dried sample from step (1) into a mortar, add a small amount of liquid nitrogen to the mortar, and grind it into powder;

[0008] (3) Collect the powder from step (2) into a centrifuge tube, add protein extraction solution to the centrifuge tube, the protein extraction solution contains 0.5 M Tris-HCl, 5 mM EDTA, 150 mM NaCl, 10 mM MgCl2, 1 mM PMSF and 2% β-mercaptoethanol, pH 8, use an ultrasonic homogenizer to break up the solution in the centrifuge tube, and place it on ice for 30 min.

[0009] (4) Centrifuge the sample from step (3) at 15000 ×g for 15 min and collect the supernatant.

[0010] (5) Load the supernatant from step (4) into a ConA-agarose gel 4B affinity chromatography column, and elute with deionized water and elution buffer (10 M Tris-HCl (pH = 4), 0.5 M NaCl) in sequence, and collect the eluent.

[0011] (6) Load the eluent collected in step (5) into a dextran gel G-75 affinity chromatography column, elute with deionized water, and collect the eluent.

[0012] (7) Load the eluent collected in step (6) into a dextran gel G-50 affinity chromatography column, elute with deionized water, and collect the eluent.

[0013] (8) Place the eluent collected in step (7) at -20 °C overnight, freeze-dry at -69 °C for 72 h using a vacuum freeze dryer, collect the freeze-dried sample, and obtain yam glycoprotein.

[0014] In some embodiments, the yam glycoprotein is derived from Dioscorea opposita, a species of yam belonging to the Dioscoreaceae family and the Dioscorea genus.

[0015] In some implementations, the obesity described in the intended use is caused by a high-fat diet.

[0016] In some implementations, the yam glycoprotein can reduce the area of ​​fat cells and the weight of adipose tissue.

[0017] In some embodiments, the yam glycoprotein described in the intended use can regulate adipose tissue gene expression in subjects in need: it can reduce the expression of PPARγ in subjects in need, increase the expression of GLUT4 in subjects in need, and / or increase the expression of AdipoQ in subjects in need.

[0018] In some embodiments, the drug is a pharmaceutical preparation made by using yam glycoprotein as the active ingredient, plus pharmaceutically acceptable excipients or auxiliary ingredients.

[0019] In some implementations, the pharmaceutical preparation is an oral liquid, suspension, granules, tablets, or capsules.

[0020] The beneficial effects that this invention can achieve are:

[0021] 1) The preparation method of the yam glycoprotein described in this invention is simple and does not involve denaturation by reagents such as alcohol, thus preserving its biological activity to the greatest extent;

[0022] 2) The use of the yam glycoprotein described in this invention in the preparation of drugs for treating obesity: It can significantly reduce weight gain induced by a high-fat diet in mice, significantly improve the histological morphology of inguinal white adipose tissue, reduce adipocyte area, improve glucose tolerance abnormalities in high-fat diet mice, and regulate the expression levels of PPARγ, GLUT4, and AdipoQ in adipose tissue. This invention provides a new application for yam glycoprotein in the field of regulating fat deposition and has good application prospects. Attached Figure Description

[0023] Note: In the figure, *P<0.05, **P<0.01, ***P<0.001 vs. control group.

[0024] Figure 1 The effect of yam glycoprotein on mouse body weight is shown: Figure A represents different groups, and Figure B represents the changes in body weight of mice in different groups;

[0025] Figure 2 The effects of yam glycoprotein on the weight and morphology of white adipose tissue in mice were shown: Figure A shows the comparison of adipose tissue morphology in different groups of mice, Figure B shows the comparison of fat weight in different groups of mice, and the comparison of fat cell area in different groups of mice.

[0026] Figure 3The effects of yam glycoprotein on the expression levels of key adipose tissue development factors AdipoQ, GLUT4, and PPARγ were shown: Figure A shows the comparison of the relative mRNA expression levels of AdipoQ, GLUT4, and PPARγ in mice from different groups; Figure B shows the comparison of Western blot results of AdipoQ, GLUT4, and PPARγ in different groups; Figure C shows the comparison of the expression levels of AdipoQ, GLUT4, and PPARγ in mice from different groups.

[0027] Figure 4 The effect of yam glycoprotein on glucose tolerance in mice is shown: Figure A shows the change of blood glucose levels in different groups of mice over time, and Figure B shows the area under the glucose tolerance curve in different groups of mice.

[0028] definition

[0029] The term "treatment" includes therapeutic treatment, preventative treatment, and applications that reduce the risk of developing disease or other risk factors in a treated individual. Treatment does not require a complete cure for the disease, but rather includes implementation plans that alleviate symptoms or reduce underlying risk factors.

[0030] The term "subject" refers to a mammal, preferably a mouse or a human.

[0031] The term “about” usually means within 20%, preferably within 10%, and more preferably within 5% of a given value or range.

[0032] The term "pharmaceutical-grade excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active agent, as is well known in the art. This includes, but is not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers.

[0033] Abbreviations

[0034] YGP stands for yam glycoprotein.

[0035] PPARγ is the peroxisome proliferator-activated receptor gamma (PPARγ).

[0036] GLUT4 is glucose transporter 4.

[0037] AdipoQ is adiponectin. Detailed Implementation

[0038] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Many variations and modifications can be made based on the teachings of the present invention, and all such variations and modifications fall within the scope of protection claimed by the present invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0039] Example 1: Preparation of yam glycoprotein

[0040] 1.1 Wash and peel the fresh Chinese yam tubers, then cut them into 1 cm pieces. 3 Small pieces of yam were freeze-dried at -69℃ for 48 h using a vacuum freeze dryer. 0.1 g of the freeze-dried yam powder was weighed and placed in a 1.5 mL centrifuge tube. 200 μL of protein extraction buffer (0.5 M Tris-HCl (pH = 8), 5 mM EDTA (pH = 8), 150 mM NaCl, 10 mM MgCl2, 1 mM PMSF, and 2% β-mercaptoethanol) was added. The mixture was sonicated for 5 s, placed on ice for 30 min, and centrifuged at 15000 ×g for 15 min. The supernatant was collected and loaded onto an affinity chromatography column containing 2 mL of ConA-agarose gel 4B. 6 mL of deionized water was added for elution, followed by 6 mL of eluent. 1 mL of eluent was collected from each tube. Protein detection was performed at 280 nm using a UV spectrophotometer. The tubes containing protein were combined and loaded onto a dextran gel G-75 affinity chromatography column with a 2 cm packing height. 6 μL of protein was collected and centrifuged. Elute with 6 mL of deionized water, collect the eluent into centrifuge tubes, 500 µL per tube, and detect protein using a UV spectrophotometer at 280 nm. Combine the protein-containing tubes and load them into a dextran gel G-50 affinity chromatography column with a 2 cm packing height. Elute with 6 mL of deionized water, collect the eluent into centrifuge tubes, 200 µL per tube, and detect protein using a UV spectrophotometer at 280 nm. Combine the protein-containing solutions and incubate at -20 ℃ overnight. Freeze-dry at -69 ℃ for 72 h using a vacuum freeze dryer to obtain a white yam glycoprotein solid powder.

[0041] 1.2 Wash and peel the fresh Chinese yam tubers, then cut them into 1 cm pieces. 3Small pieces of yam were freeze-dried at -69℃ for 48 h using a vacuum freeze dryer. 10 g of the freeze-dried yam powder was weighed and placed in a 50 mL centrifuge tube. 2 mL of protein extraction buffer (0.5 M Tris-HCl (pH = 8), 5 mM EDTA (pH = 8), 150 mM NaCl, 10 mM MgCl2, 1 mM PMSF, and 2% β-mercaptoethanol) was added. The mixture was sonicated for 1 min, placed on ice for 30 min, and centrifuged at 15000 ×g for 30 min. The supernatant was collected and loaded onto an affinity chromatography column containing 10 mL of ConA-agarose gel 4B. 30 mL of deionized water was added for elution, followed by 30 mL of eluent. 1.5 mL of eluent was collected from each tube. Protein detection was performed at 280 nm using a UV spectrophotometer. The tubes containing protein were combined and loaded onto a centrifuge column containing 10 mL of ConA-agarose gel 4B. A dextran gel G-75 affinity chromatography column with a 10 cm packing height was used for elution with 30 mL of deionized water. The eluent was collected into centrifuge tubes, 1 mL per tube. Protein detection was performed using a UV spectrophotometer at a wavelength of 280 nm. The collected tubes containing protein were combined and loaded into a dextran gel G-50 affinity chromatography column with a 10 cm packing height. Elution was performed with 30 mL of deionized water. The eluent was collected into centrifuge tubes, 1 mL per tube. Protein detection was performed using a UV spectrophotometer at a wavelength of 280 nm. The solutions from the collected tubes containing protein were combined and incubated at -20 ℃ overnight. Then, the column was freeze-dried at -69 ℃ for 72 h using a vacuum freeze dryer to obtain a white yam glycoprotein solid powder.

[0042] Example 2: Effects of yam glycoprotein on body weight

[0043] Fifteen 8-week-old male C57BL / 6J mice (Speford (Beijing) Biotechnology Co., Ltd.) weighing 20 ± 2 g were randomly divided into three groups of five mice each: a control group, a high-fat group, and a yam glycoprotein group. After 7 days of acclimatization feeding with a normal diet (D12450B, Speford, Beijing, China), the diets of the high-fat and yam glycoprotein groups were replaced with a 60% high-fat diet (D12492, Speford, Beijing, China), and the mice were administered medication. The control and high-fat groups were administered 200 µL of distilled water by gavage, while the yam glycoprotein group was administered 200 µL of yam glycoprotein solution (400 mg / kg) by gavage for 36 consecutive days. The yam glycoprotein solution was prepared by dissolving the yam glycoprotein powder obtained in Example 1 in an appropriate amount of distilled water. Mice were housed at 22 ± 2 ℃ under a 12-hour light / dark cycle. Body weight changes were recorded every 3 days during the administration period. Figure 1As shown, the experimental results indicate that, compared with the control group, the weight of mice in the high-fat group increased significantly, while yam glycoprotein can significantly reduce the weight gain induced by the high-fat diet in mice.

[0044] Example 3: Effect of yam glycoprotein on white fat deposition

[0045] On day 37, the mice in Example 2 were weighed and euthanized. Inguinal white adipose tissue was collected, and its weight recorded. The white adipose tissue was fixed in 4% paraformaldehyde at room temperature for 12 hours. It was then dehydrated and cleared sequentially with 70%, 80%, 90%, 95%, and 100% ethanol and xylene, and embedded in paraffin. Serial sections (6 µm) were then hydrated with xylene and a gradient of ethanol (100%, 95%, 90%, 80%, and 70%). Hematoxylin staining was added for 3 minutes; after blue reversal, the cell nuclei turned blue. Eosin staining was then added for 1 minute. After dehydration with a gradient of ethanol (98% and 100%), the sections were cleared in xylene, mounted with neutral resin, and air-dried at room temperature. Ten areas were randomly captured under a microscope to observe morphological changes in the adipose tissue and count the number of adipocytes. Figure 2 As shown, the experimental results indicate that, compared with the control group, the high-fat group showed a significant increase in the weight of white adipose tissue and a significant increase in the area of ​​adipocytes. Yam glycoprotein significantly reduced white adipose tissue deposition induced by a high-fat diet and decreased the area of ​​adipocytes. In summary, the embodiments of this invention demonstrate that yam glycoprotein improves the weight increase and histological morphology of inguinal white adipose tissue induced by a high-fat diet.

[0046] Example 4: Effect of yam glycoprotein on glucose tolerance

[0047] On day 30 of the drug administration, the mice in Example 2 were transferred to clean cages in the afternoon and fasted for 16 hours without restriction of water intake. They were then orally administered glucose (2 g / kg body weight). Blood glucose concentrations were measured at 0, 15, 30, 60, and 120 minutes after administration via the tail vein. Figure 3 As shown, oral glucose tolerance tests revealed that the high-fat group exhibited significantly impaired glucose tolerance compared to the control group, while the yam glycoprotein group significantly improved glucose tolerance in mice. In summary, these embodiments demonstrate that yam glycoprotein helps alleviate high-fat diet-induced glucose metabolism abnormalities in mice.

[0048] Example 5: Effects of yam glycoprotein on the expression of key factors in adipose tissue development

[0049] RNA and proteins were extracted from the white adipose tissue of the mouse groin collected in Example 3. The expression levels of PPARγ, GLUT4, and AdipoQ were detected using quantitative PCR (qPCR) and Western blotting. Figure 4 As shown, the results indicate that, compared with the high-fat diet group, the expression levels of the aforementioned genes and proteins in the yam glycoprotein treatment group were significantly regulated, suggesting that yam glycoprotein can improve the functional state of adipose tissue. In summary, the embodiments of this invention demonstrate that yam glycoprotein can regulate the expression levels of PPARγ, GLUT4, and AdipoQ in adipose tissue, and can reduce the expression of PPARγ while increasing the expression of GLUT4 and AdipoQ.

[0050] The present invention has been described through the above embodiments. However, it should be understood that the above embodiments are for illustrative purposes only and are not intended to limit the invention to the scope of the described embodiments. Furthermore, those skilled in the art will understand that the present invention is not limited to the above embodiments, and many variations and modifications can be made based on the teachings of the present invention, all of which fall within the scope of protection claimed by the present invention. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. The use of a yam glycoprotein in the preparation of a drug for treating obesity, characterized in that... The yam glycoprotein mentioned above is derived from Dioscorea opposita, a plant belonging to the Dioscoreaceae family and the Dioscorea genus. The yam glycoprotein is prepared according to the following steps: (1) Peel the fresh yam, cut it into small pieces of about 1 cubic centimeter, and freeze-dry it under vacuum at -69 ℃ for about 48 h; (2) Place the freeze-dried sample from step (1) into a mortar, add a small amount of liquid nitrogen to the mortar, and grind it into powder; (3) Collect the powder from step (2) into a centrifuge tube, add protein extraction solution to the centrifuge tube, the protein extraction solution contains 0.5 M Tris-HCl, 5 mM EDTA, 150 mM NaCl, 10 mM MgCl2, 1 mM PMSF and 2% β-mercaptoethanol, pH 8, use an ultrasonic homogenizer to break up the solution in the centrifuge tube, and place it on ice for 30 min. (4) Centrifuge the sample from step (3) at 15000 ×g for 15 min and collect the supernatant. (5) Load the supernatant from step (4) into a ConA-agarose gel 4B affinity chromatography column, and elute with deionized water and elution buffer (10 M Tris-HCl (pH = 4), 0.5 M NaCl) in sequence, and collect the eluent. (6) Load the eluent collected in step (5) into a dextran gel G-75 affinity chromatography column, elute with deionized water, and collect the eluent. (7) Load the eluent collected in step (6) into a dextran gel G-50 affinity chromatography column, elute with deionized water, and collect the eluent. (8) Place the eluent collected in step (7) at -20 °C overnight, freeze-dry at -69 °C for 72 h using a vacuum freeze dryer, collect the freeze-dried sample, and obtain yam glycoprotein.

2. The use according to claim 1, wherein the yam glycoprotein is derived from Dioscorea opposita, a species of yam belonging to the genus Dioscorea in the family Dioscoreaceae.

3. The use according to claim 1, wherein the obesity is caused by a high-fat diet.

4. The use according to any one of claims 1-3, characterized in that... The yam glycoprotein described herein can regulate the expression of adipose tissue genes in subjects in need: it can reduce the expression of PPARγ in subjects in need, increase the expression of GLUT4 in subjects in need, and / or increase the expression of AdipoQ in subjects in need.

5. The use according to any one of claims 1-3, characterized in that... The yam glycoprotein mentioned above can reduce the area of ​​fat cells and the weight of adipose tissue.

6. The use according to any one of claims 1-3, characterized in that... The drug is a pharmaceutical preparation made with yam glycoprotein as the active ingredient, plus pharmaceutical excipients or auxiliary ingredients.

7. The use according to claim 6, characterized in that... The pharmaceutical preparations mentioned are oral liquids, suspensions, granules, tablets, or capsules.

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