Application of green tea acidic polysaccharide GTPS in improving metabolic syndrome

Abstract

This invention discloses the application of green tea acidic polysaccharide GTPS in improving metabolic syndrome, aiming to provide a new use for green tea acidic polysaccharide GTPS. The extraction process involves first grinding green tea into powder, boiling, filtering, deproteinizing using the Seval method, concentrating, adding ethanol, allowing it to stand overnight, centrifuging to collect the precipitate, dissolving it, removing the ethanol, and freeze-drying to obtain GTPSs. The GTPSs are then passed through a DEAE-agarose FF column, eluted with distilled water and 0.2, 0.5, and 1.0 mol / L sodium chloride, and the fractions are collected. The fraction eluted with 0.2 mol / L sodium chloride is dialyzed to desalt, yielding green tea polysaccharide GTPS. Applying this to the improvement of metabolic syndrome provides a solid foundation for revealing the key active components in green tea that promote metabolism and paves the way for the future utilization of tea polysaccharides in the pharmaceutical and food industries, offering a promising solution for the integration of tea resources and the application of natural products.

Description

Technical Field

[0001] This invention relates to the field of new applications of functional substances, and more specifically to the application of a green tea acidic polysaccharide GTPS in improving metabolic syndrome. Background Technology

[0002] Plant polysaccharides have attracted academic attention due to their low toxicity, high biocompatibility, biodegradability, and various biological benefits. Tea is a traditional beverage made from the fresh leaves of the tea plant. In recent years, various tea polysaccharides have been studied, and their extraction, purification, and functions have been reviewed. However, only a few studies have focused on revealing the structure, function, and bioactivity of tea polysaccharides and the relationships between them.

[0003] Metabolic syndrome is a prevalent metabolic disorder accounting for the largest proportion of non-communicable diseases worldwide, including obesity, hyperlipidemia, insulin resistance, and hepatic steatosis. Tea polysaccharides improve metabolic syndrome by modulating the gut microbiota, showing potential as a natural phytotherapy strategy for the prevention and clinical management of metabolic syndrome. Green tea is the most consumed of the six major tea categories, and its chemical composition is most similar to that of fresh tea leaves. Its unique fixation process can immediately inactivate polyphenol oxidase and peroxidase. Green tea polysaccharides possess antioxidant, antitumor, and antidiabetic effects. However, the structural characteristics, physical and chemical properties of structurally homogeneous green tea polysaccharides still require further characterization and research to reveal the relationship between their structure and function.

[0004] 'Longjing 43' is one of the main tea tree varieties cultivated, and also one of the representative tea tree varieties most suitable for making green tea. How to study and utilize it to improve metabolism is an urgent problem to be solved by those in the field. Summary of the Invention

[0005] In view of this, the present invention provides the application of green tea polysaccharide GTPS in improving metabolic syndrome.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] First, this invention defines the application of green tea acidic polysaccharide GTPS in the preparation of drugs to improve metabolic syndrome.

[0008] This invention also defines the application of green tea acidic polysaccharide GTPS in the preparation of foods that improve metabolic syndrome.

[0009] This invention also provides a method for preparing green tea acidic polysaccharide GTPS, comprising the following steps:

[0010] (1) Grind green tea into powder, then boil it in distilled water for 2-3 hours, filter and collect the filtrate, remove the protein, and then concentrate it to 1 / 10 of the original volume;

[0011] (2) Add 4 times the volume of ethanol to the concentrated mixture of step (1), let it stand overnight at 4°C, centrifuge to collect the precipitate, redissolve it in distilled water, remove the ethanol by rotary evaporation under reduced pressure at 50-70°C, and freeze-dry the remaining liquid to obtain polysaccharide GTPPSs.

[0012] (3) Pass the GTPSS obtained in step (2) through an elution column and elute with three portions of distilled water and 0.2, 0.5 and 1.0 mol sodium chloride solutions at a flow rate of 15 mL / min. The eluted fractions are automatically collected.

[0013] (4) The fraction eluted by 0.2 moles of sodium chloride was desalted by dialysis and collected to obtain green tea polysaccharide GTPS.

[0014] Preferably, the powder particle size in step (1) is 40-80 mesh, and the mass concentration of green tea powder in distilled water is 10%.

[0015] Preferably, the specific operation of deproteinization in step (1) is as follows:

[0016] Prepare a mixture with a volume ratio of chloroform:n-butanol = 4-5:1. Mix this mixture with the filtrate at 1 / 4-1 / 3 of the filtrate volume, shake vigorously for 15-30 minutes, and then let it stand or centrifuge at 4000 rpm for 10-15 minutes. Collect the upper aqueous phase. Repeat the operation several times until the white, flocculent protein precipitate disappears. Remove the residual organic solvent by rotary evaporation and vacuum drying to obtain the deproteinized polysaccharide solution.

[0017] Preferably, the centrifugation speed in step (2) is 8000 rpm and the time is 10 minutes.

[0018] Preferably, the elution column in step (3) is a DEAE-agarose FF column with a diameter of 26 mm and a length of 30 cm.

[0019] Preferably, the dialysis desalination in step (4) specifically includes:

[0020] The elution fraction was injected into a 3500 Dalton dialysis bag. The sealed dialysis bag was placed in a container containing a large amount of ultrapure water dialysis solution. The container was placed on a magnetic stirrer with a stir bar and stirred at 4°C. The dialysis solution was changed several times until the conductivity of the dialysis solution was close to the background value of the ultrapure water used. The dialysis bag was then removed and the sample was recovered.

[0021] As can be seen from the above technical solution, compared with the prior art, the present invention discloses an application of green tea polysaccharide GTPS in improving metabolic syndrome, which has the following beneficial effects:

[0022] In this study, a homogeneous green tea polysaccharide (named GTPS) isolated from the tea variety 'Longjing 43' was found to improve metabolism in vivo. Its application in the preparation and research of metabolic syndrome-related products provides a solid foundation for revealing the key active components in green tea that promote metabolism, and paves the way for the future use of tea polysaccharides in the pharmaceutical and food industries. It also provides a promising solution for the integration of tea resources and the application of natural products. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0024] Figure 1 Purification and identification of GTPS prepared in Example 1; wherein: (A) elution curve of GTPS on DEAE-SepHaroseFast Flow column, (B) elution curve of GTPS on Superdex 200 column, (C) HPGPC curve of GTPS, (D) FT-IR spectrum of GTPS.

[0025] Figure 2 The UV-Vis spectrum (A) and monosaccharide composition (orange) (B) of the GTPS prepared in Example 1;

[0026] Figure 3 The nuclear magnetic resonance spectrum of GTPS prepared in Example 1, wherein: (A) 1 H spectrum, (B) 13 C spectrum, (C) 13 C DEPT-135 spectrum, (D) 1 H- 13 C HSQC spectrum, (E) 1 H- 1 H COSY spectrum, (F) 1 H- 13 C HMBC spectrum, (G) 1 H- 1 H NOESY spectrum, (H)GTPS predicted structure;

[0027] Figure 4Scanning electron microscope images of GTPS prepared in Example 1, wherein: (A) 200x, (B) 300x, (C) 10000x, (D) 50000x;

[0028] Figure 5 Physicochemical properties of GTPS prepared in Example 1, wherein: (A) DSC (dashed line) and TGA (solid line) thermograms of GTPS, (B) viscosity of 6% (blue) and 12% (purple) concentrations of GTPS, (C) average zeta potential value, (D) particle size of GTPS as a function of pH value, and error bars represent the standard deviation of a set of triple measurements;

[0029] Figure 6 The emulsifying properties of GTPS prepared in Example 1 are shown in: (A) appearance changes at 24 hours and (B) 168 hours and (C) histogram of EC, with error bars representing the standard deviation of a set of triple measurements.

[0030] Figure 7 Images of metabolic disorders in mice with high-fat diet-induced metabolic syndrome, improved by GTPS drinking water, including: (A) body weight, (B) plasma triglycerides, (C) plasma total cholesterol, (D) plasma low-density lipoprotein cholesterol / high-density lipoprotein cholesterol ratio, and (E) epididymal fat images of mice in different groups.

[0031] Figure 8 The effect of GTPS on lipid and glucose metabolism disorders induced by a high-fat diet in mice with metabolic syndrome includes: (A) weight gain, (B) epididymal fat, (C) representative image of epididymal fat, (D) percentage of lean meat and fat by mass, (E) obesity index, (F) oral glucose tolerance test and area under the curve of oral glucose tolerance test, and (G) insulin resistance test and area under the curve of insulin resistance test.

[0032] Figure 9 The image shows the effects of GTPS on lipid accumulation and liver damage in mice with metabolic syndrome caused by high-fat diet-induced metabolic disorders, where: (A) plasma triglycerides, (B) plasma total cholesterol, (C) LDL cholesterol / HDL cholesterol ratio, (D) liver image, HE staining and Oil Red O staining, and (E) food intake. Detailed Implementation

[0033] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0034] Example 1

[0035] Extraction and purification of GTPS

[0036] Grind green tea into powder, pass it through a 60-mesh sieve, and then boil it in distilled water (10% by mass) for 3 hours. Filter the mixture, collect the filtrate, and deproteinize it using the Seval method (prepare a mixture of chloroform and n-butanol at a volume ratio of 4-5:1, mix this mixture with 1 / 4-1 / 3 of the filtrate volume, shake vigorously for 15-30 minutes, then let it stand or centrifuge at 4000 rpm for 10-15 minutes, collect the upper aqueous phase, repeat the operation several times until the white, flocculent protein precipitate disappears, then remove residual organic solvent by rotary evaporation and vacuum drying to obtain a deproteinized polysaccharide solution), concentrate it 10 times, add 4 times the volume of ethanol to the mixture, and then let it stand overnight at 4°C to precipitate the protein.

[0037] Centrifuge at 8000 rpm for 10 minutes at 4°C, collect the precipitate and redissolve it in distilled water, then remove the ethanol by rotary evaporation under reduced pressure at 60°C, and freeze-dry the remaining liquid to obtain polysaccharides, which are named GTPSSs.

[0038] Next, GTPSS were loaded into a DEAE-agarose FF column (26 mm diameter × 30 cm length, Amsgarm Biosciences, Uppsala, Sweden) and eluted with three portions of distilled water and 0.2, 0.5, and 1.0 mol sodium chloride solutions at a flow rate of 15 mL / min. The eluted fractions were automatically collected and the carbohydrate content was measured using the phenol-sulfuric acid method.

[0039] The fraction eluted with 0.2 mol of sodium chloride was then desalted by dialysis (the eluted fraction was injected into a pre-treated 3500 Dalton dialysis bag, the sealed dialysis bag was placed in a container containing a large amount of ultrapure water dialysis solution, the container was placed on a magnetic stirrer, a stir bar was added, and the mixture was stirred at 4°C. The dialysis solution was changed several times until the conductivity of the dialysis solution was close to the background value of the ultrapure water used. The dialysis bag was then removed, and the sample was recovered), and collected and named GTPS.

[0040] Experimental Example 1

[0041] The following studies were conducted using the GTPS prepared in Example 1.

[0042] I. Purification of GTPS

[0043] GTPS were purified using a DEAE-SepHarose fast flow column. Four distinct fractions were collected as a result (e.g., Figure 1A). The fraction obtained by elution with 0.2 M NaCl solution (which had the highest yield) was selected and named GTPS. Subsequently, GTPS was freeze-dried and separated. Figure 1 As shown in Figure B, the labeled components corresponding to the peak center region of the online monitoring chromatogram (22 to 44 minutes) were collected. The purity of GTPS was confirmed by the single peak and symmetrical characteristics displayed in the HPGPC chromatogram at 40 minutes. UV scanning ( Figure 2 A) This indicates that there is almost no protein or nucleic acid contamination in this component, as there is no absorption peak at 260 / 280 nm.

[0044] II. Structural Identification of GTPS

[0045] (1) Molecular weight and monosaccharide composition of GTPS

[0046] According to the standard calibration curve, the weight-average molecular weight (Mw) of GTPS is 41,864 Daltons, and the mean molecular weight (Mn) is 28,561 Daltons. Therefore, the polydispersity index (PDI), which reflects the Mw distribution range and peak sharpness (i.e., PDI = Mw / Mn) and affects emulsion properties, is 1.47, indicating that GTPS has a narrow Mw range. Figure 1 C). As for the monosaccharide composition, GTPS is mainly composed of RHa, Ara, Gal, Glu, GalA and GluA, with a ratio of 5.3:10.3:9.3:0.9:71.3:2.8 (Figure 2B).

[0047] (2) Fourier transform infrared spectroscopy analysis of GTPS

[0048] Fourier transform infrared spectrum of GTPS ( Figure 1 D) shows 4000 to 400 cm -1 The absorption spectrum between [values]. Approximately 3359.39 cm⁻¹ -1 and 2960.20 cm -1 The strong peak at 1741.41 cm⁻¹ is attributed to the stretching vibrations of the OH and CH bonds in the monosaccharide unit. -1 The peak at approximately 1427.07 cm⁻¹ likely represents the C=O stretching vibration in the methylated compound. -1 and 1099.23 cm -1 The peak at 1637.27 cm⁻¹ typically represents the stretching vibration of the CO bond. -1 The peak at 1330.64 cm⁻¹ is attributed to related water molecules. -1 The peak at that point corresponds to a symmetrical C=O stretching vibration. The intensity is between 1000-1200 cm⁻¹. -1The bands between these points originate from the stretching vibrations of C-OH, COC, and CC, indicating the presence of a pyranose ring. Located at 1238.08 cm⁻¹ -1 and 1014.37 cm -1 The peaks correspond to the rocking vibration of the terminal methyl group and the stretching vibration of the asymmetric ring of the pyranose ring, respectively. Finally, 892.88 cm⁻¹ -1 The peak at this point indicates the rocking vibration of the β-isomer of CH in GTPS.

[0049] (3) Methylation analysis

[0050] To investigate the glycosidic bond structure of GTPS, the galacturonic acid units were first reduced and methylated, followed by hydrolysis, reduction, and acetylation, and then analyzed by gas chromatography-mass spectrometry. Based on the fragment ion mass spectra (Table 1), the glycosidic bond structure between the monosaccharide residues of GTPS was determined.

[0051] Table 1. Methylation analysis of GTPS

[0052] RT MetHylated sugar Mass fragments (m / z) Molar ratio Type of linkage 16.546 2,3,5-Me3-Araf 43,71,87,101,117,129,145,161 0.317 Araf-(1→ 20.402 3-Me1-RHap 43,87,101,117,129,143,159,189 0.044 →2,4)-RHap-(1→ 21.969 2,3-Me2-Araf 43,71,87,99,101,117,129,161,189 0.229 →5)-Araf-(1→ 25.332 2,3,4,6-Me4-Galp 43,71,87,101,117,129,145,161,205 0.051 Galp-(1→ 26.094 2-Me1-Araf 43,58,85,99,117,127,159,201 0.094 →3,5)-Araf-(1→ 29.597 2,3,6-Me3-Galp 43,87,99,101,113,117,129,131,161,173,233 0.102 →4)-Galp-(1→ 30.587 2,4,6-Me3-Galp 43,87,99,101,117,129,161,173,233 0.061 →3)-Galp-(1→ 33.191 2,3,4-Me3-Galp 43,87,99,101,117,129,161,189,233 0.034 →6)-Galp-(1→ 39.399 2,4-Me2-Galp 43,87,117,129,159,189,233 0.067 →3,6)-Galp-(1→

[0053] (4) Nuclear magnetic resonance spectroscopy analysis

[0054] One-dimensional nuclear magnetic resonance analysis

[0055] The C / H chemical shift values ​​of the GTPS monosaccharide residues were determined based on nuclear magnetic resonance spectroscopy and literature data (Table 2).

[0056] Table 2. Chemical shifts of GTPS

[0057]

[0058] 1 H nuclear magnetic resonance spectrum ( Figure 3 A) shows that the main peaks are located in the range of 3.0–6.0 ppm. The peaks at 5.18, 4.97, 5.02, 5.05, 4.98, 4.4, 5.18, 4.55, 4.47, and 4.44 ppm correspond to the H-1 of the AJ residues, respectively. The peaks between 3.3 and 4.0 ppm correspond to the protons of the sugar ring. 13 C nuclear magnetic resonance spectrum ( Figure 3B) shows peaks from 60 to 120 ppm. Key isomeric carbon signals appear at 99.81, 100.39, 104.48, 104.69, 104.9, 105.39, 108.78, 108.89, 108.91, and 110.63 ppm, corresponding to C-1 of G, E, F, I, J, H, B, C, D, and A residues, respectively. Furthermore, non-isomeric carbon signals (60–180 ppm) are classified as C-2, C-3, or C-4. Next, 13 C DEPT-135 spectrum ( Figure 3 C) shows negative peaks in the 60-72 ppm range, which is due to chemical shifts in C-5 or C-6. The peaks at 62.65, 62.34, and 68.28 ppm correspond to A-C5, B-C5, and C-C5, respectively.

[0059] Two-dimensional nuclear magnetic resonance analysis

[0060] 2D NMR experiments were conducted, including 1 H- 13 C HSQC, 1 H- 1 H COSY、 1 H- 13 C HMBC and 1 H- 1 H NOESY, to further clarify the structure of GTPS. (Through...) 1 H- 13 The H / C correlation peaks at 5.18 / 110.63 ppm in the C HSQC spectrum (Figure 3D) identified α-L-Araf-(1→ residues, which is consistent with the monosaccharide composition and glycosidic bond. 1 H- 1 H COSY analysis assigned the signal to residues H1 (5.18 ppm), H2 (4.14 ppm), H3 (3.88 ppm), H4 (4.07 ppm), H5a (3.77 ppm), and H6a (3.65 ppm). Figure 3 E). The corresponding carbon signal of this residue is in 1 H- 13 The C HSQC spectrum detected residues at 110.63, 82.63, 77.98, 85.23, and 62.65 ppm. The (3,6)-β-D-Galp-(1→) residues were identified by H / C correlation peaks at 4.47 / 104.69 ppm. 1 H- 13 Heterogeneous regions of C HSQC spectrum ( Figure 3In D), the cross peak at 5.02 / 108.89 ppm was assigned to the H1 / C1 of the →5)-α-L-Araf-(1→ residue. The remaining residues were determined based on published literature.

[0061] The G→E connection was confirmed by HMBC cross-peaks. Figure 3 F), meaning there is a cross-peak between the anomeric proton of G and the C4 of E. Furthermore, there is a strong cross-peak between the anomeric proton of E and its own C4, and the same is true between the anomeric carbon of E and its own H4, indicating the existence of an E→E connection. Figure 3 G). Furthermore, the coupling between H1 at site E and H6 at site I indicates the presence of an E→I link. A strong cross-peak exists between the anomeric proton at site I and C6 and H6 at site J, indicating an I→J link. Taken together, these results suggest that the GTPS backbone is composed of G→E→E→I→J links. The D→G link is confirmed by the interaction between H1 of residue D and C4 of residue G. This indicates that the O-4 peptide chain of the side chain 3,5)-α-L-Araf-(1→ is linked to residue 2,4)-α-L-RHa-(1→). Furthermore, the presence of B→C→D→G and A→D can be inferred from the correlations between H1 of residue B and C5 of residue C, H1 of residue C and C5 of residue D, and H1 of residue A and C3 of residue D. Similarly, the F→H→I link can also be inferred. Overall, the structure of GTPS has been predicted ( Figure 3 H).

[0062] The core structure of GTPS comprises a backbone of 2,4-linked α-L-rhamnose, 4-linked α-galactomannan, 3,6-linked β-D-galactose, and 6-linked β-galactose residues, with branches at positions O-4 and O-3 of the α-L-rhamnose and β-D-galactose residues, respectively. α-L-alofose and β-D-galactose form the inner side chains. The side chains have reducing ends of α-L-alofose and β-D-galactose. Figure 3 As shown in H, the side chain of GTPS consists of α-L-arabinose linked to rhamnose residues and β-D-galactose linked to β-D-galactose residues.

[0063] III. Physicochemical Properties Analysis of GTPS

[0064] Scanning electron microscopy (SEM) analysis: The surface morphology of GTPS was characterized using scanning electron microscopy, including its shape and distribution. Figure 4 GTPS particles are flaky and consist of broken flakes.

[0065] Thermal properties: Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) indicate that GTPS exhibits good thermal stability, with relatively low weight loss (18.58%) when heated to 220°C. Figure 5 A), with a melting point of 104.9℃ and an enthalpy change of 354.1 J / g. These properties indicate that GTPS can withstand typical food processing conditions without significant degradation, making it a promising candidate for inclusion in a variety of food products.

[0066] Rheological properties: Flow curves of GTPS at different concentrations (6% and 12%) were analyzed under stable shear conditions. Figure 5 B). The results show that the viscosity of GTPS increases with increasing concentration and exhibits typical shear-thinning behavior, which is a typical characteristic of non-Newtonian fluids, indicating that GTPS can improve the stability of liquids by reducing fluidity and preventing droplet aggregation.

[0067] ζ-potential and particle size analysis: such as Figure 5 As shown in Figure C, the zeta potential of the GTPS solution gradually becomes negative from -0.26 mV to -17.95 mV as the solution pH increases. Within the pH range of 2.0 to 10.0, the zeta potential of GTPS remains negative, pK... a The estimated value of ~2.0 indicates that GTPS is acidic, which is due to its high glucuronic acid content. The zeta potential of GTPS, measured to be -15.7 mV without the influence of ionic strength, confirms its anionic nature. These results, along with the increase in particle size with increasing pH ( Figure 5 (D) This confirms that GTPS is rich in carboxyl groups, which contribute to its zeta potential through dissociation, forming a negatively charged layer, thereby enhancing emulsion stability.

[0068] Emulsifying performance: To verify the emulsifying performance of GTPS, it was evaluated by observing its behavior during storage. Specifically, this involved monitoring its appearance and measuring its emulsifying capacity. Figure 6 As shown in A and 6B, visual observations indicate that higher concentrations of GTPS exhibit stronger emulsion stability. The stability behavior was similar between 2.0% GTPS and 0.1% pectin. After 24 hours of storage, the cream layer stabilized by GTPS (0.5% to 2.0%) was almost as stable as that stabilized by pectin. After 168 hours of storage, the emulsion stabilized by 2.0% GTPS was as stable as that stabilized by 0.1% pectin, while emulsions stabilized by GTPS at concentrations of 0.5%, 1.0%, and 1.5%, as well as the emulsion stabilized by 0.5% pectin, became unstable. Figure 6As shown in Figure C, the emulsifying capacity (EC24) of 2.0% GTPS was calculated to be 0.76 after 24 hours and 0.74 after 168 hours. There was no significant difference between EC24 and EC168 for 2.0% GTPS, both being almost identical to the values ​​for 0.1% pectin. These results indicate that emulsions stabilized by GTPS exhibit excellent long-term stability.

[0069] Experimental Example 2

[0070] The following experiments were conducted using the GTPS prepared in Example 1.

[0071] I. Experiment on the improvement of metabolic syndrome in mice by GTPS (free drinking water experiment)

[0072] GTPS was prepared into an aqueous solution to study its alleviating effect on metabolic syndrome in mice on a high-fat diet.

[0073] The experiment used C57BL / 6 mice as experimental animals. After the acclimatization period, the mice were randomly divided into 6 groups (n=5 per group):

[0074] (1) Normal diet group (ND group): fed with normal feed (ND, 10% kcal fat, D12450B, produced by ResearchDiets) and drinking distilled water;

[0075] (2) High-fat diet group (HFD group): fed with high-fat diet (HFD, 45% kcal fat, D12451, produced by ResearchDiets) and drinking distilled water;

[0076] (3) Low-dose group of crude polysaccharide (containing 50% GTPS) (GTPS-L group): On the basis of high-fat diet, drink 200 mg / kg crude polysaccharide (containing 50% GTPS) aqueous solution freely;

[0077] (4) Medium-dose group of crude polysaccharide (containing 50% GTPS) (GTPS-M group): On the basis of high-fat diet, the patient was given free access to a 400 mg / kg crude polysaccharide (containing 50% GTPS) aqueous solution;

[0078] (5) High-dose group of crude polysaccharide (containing 50% GTPS) (GTPS-H group): On the basis of high-fat diet, the patient was given free access to an aqueous solution of crude polysaccharide (containing 50% GTPS);

[0079] (6) Metformin group (MET group): On the basis of high-fat diet, patients were given free access to 250 mg / kg metformin aqueous solution.

[0080] Eight weeks after intervention, mice were fasted for 12 hours, and their body composition was assessed before they were anesthetized and euthanized. Liver, epididymal fat pad, and plasma samples were collected and stored at -80°C for subsequent analysis.

[0081] The results showed that GTPS administration significantly reduced body weight and improved plasma lipid profiles (triglycerides, total cholesterol, and LDL cholesterol / HDL cholesterol ratio) compared to mice on a high-fat diet. Figure 7 AD). Meanwhile, the increase in epididymal fat volume caused by a high-fat diet was reversed after treatment with GTPS and metformin. Figure 7 All these results indicate that GTPS infusion has considerable potential in improving high-fat diet-induced metabolic syndrome in mice.

[0082] II. Experiment on the improvement of metabolic syndrome in mice by GTPS (gavage intervention experiment)

[0083] After the acclimatization period, the mice were randomly divided into 7 groups (n=5 per group). All test compounds were administered daily by gavage, while the control groups (ND and HFD) were administered an equal volume of distilled water by gavage.

[0084] (1) Control group (ND): fed with ordinary feed and gavage with distilled water;

[0085] (2) NDGTPS group: fed with ordinary feed and administered 400 mg / kg crude polysaccharide (containing 50% GTPS) by gavage daily.

[0086] (3) HFD group: fed with high-fat diet and gavaged with distilled water;

[0087] (4) GTPS-M group: fed with a high-fat diet and administered 400 mg / kg crude polysaccharide (containing 50% GTPS) by gavage daily.

[0088] (5) GTPS-H group: fed with a high-fat diet and administered 800 mg / kg crude polysaccharide (containing 50% GTPS) by gavage daily.

[0089] (6) GTPS group: fed a high-fat diet and administered 400 mg / kg GTPS by gavage daily;

[0090] (7) MET group: fed with high-fat diet and administered 250 mg / kg metformin by gavage daily.

[0091] The intervention lasted for 9 weeks. After the intervention period, the mice were fasted for 12 hours, their body composition was assessed, and they were then euthanized under anesthesia. Liver, epididymal fat pad, and plasma samples were collected and stored at -80°C for subsequent analysis.

[0092] like Figure 8-9As shown, the results indicated that mice on a high-fat diet (HFD) had greater body weight gain, higher fat content, and lower food intake, while mice on a normal diet (ND) did not exhibit these characteristics. The HFD group also showed significantly higher energy utilization efficiency. Figure 8 A, B, D and Figure 9 E), increased epididymal fat ( Figure 8 C and E), and blood glucose levels were also higher in the HFD group than in the ND group. Similarly, the AUC of the OGTT and ITT curves in HFD mice was larger ( Figure 8 FG). GTPS alleviated the weight gain, fat gain, and increased epididymal fat mass caused by HFD (FG). Figure 8 AE). Mice treated with GTPS showed better blood parameters and improved glycemic homeostasis in OGTT and ITT tests compared to HFD mice (Figure 8F-G). GTPS intervention also significantly reduced plasma total cholesterol (TC), triglycerides (TG), and the LDL-c / HDL-c ratio (ALC / HDL-c). Figure 9 AC). Compared with previous experiments that simulated daily tea intake by administering the drug via drinking water, the effects of GTPS administered via gavage were more significant.

[0093] Furthermore, liver analysis using photographic images and tissue staining revealed that the livers of mice on a high-fat diet were deep yellow and contained granular protrusions. Figure 9 D), and GTPS can restore it to normal. More importantly, these livers showed significant lipid accumulation, vacuolar degeneration, ballooning degeneration, and inflammatory infiltration, which were also improved by GTPS, with effects comparable to MET. This indicates that GTPS alleviates metabolic disorders in mice with metabolic syndrome.

[0094] The various embodiments described in this specification are presented in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not 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 a green tea acidic polysaccharide GTPS in the preparation of a drug for improving metabolic syndrome, characterized in that, The preparation method of the green tea acidic polysaccharide GTPS includes the following steps: (1) Grind Longjing 43 into powder, then boil it in distilled water for 2-3 hours, filter and collect the filtrate, remove the protein, and then concentrate it to 1 / 10 of the original volume; (2) Add 4 times the volume of ethanol to the concentrated mixture of step (1), let it stand overnight at 4°C, centrifuge to collect the precipitate, redissolve it in distilled water, remove the ethanol by rotary evaporation under reduced pressure at 50-70°C, and freeze-dry the remaining liquid to obtain polysaccharide GTPPSs. (3) Pass the GTPSS obtained in step (2) through an elution column, the elution column being a DEAE-agarose FF column with a diameter of 26 mm × length of 30 cm, and elute with three portions of distilled water and 0.2, 0.5, and 1.0 M sodium chloride solutions at a flow rate of 15 mL / min. The eluted fractions are automatically collected. (4) The fraction eluted by 0.2M sodium chloride was desalted by dialysis and collected to obtain green tea acid polysaccharide GTPS.

2. The application according to claim 1, characterized in that, The powder particle size mentioned in step (1) is 40-80 mesh, and the mass concentration of Longjing 43 powder in distilled water is 10%.

3. The application according to claim 1, characterized in that, The specific steps for deproteinization in step (1) are as follows: Prepare a mixture with a volume ratio of chloroform:n-butanol = 4-5:

1. Mix this mixture with the filtrate at 1 / 4-1 / 3 of the filtrate volume, shake vigorously for 15-30 minutes, and then let it stand or centrifuge at 4000 rpm for 10-15 minutes. Collect the upper aqueous phase. Repeat the operation several times until the white, flocculent protein precipitate disappears. Remove the residual organic solvent by rotary evaporation and vacuum drying to obtain the deproteinized polysaccharide solution.

4. The application according to claim 1, characterized in that, The centrifugation speed in step (2) is 8000 rpm and the time is 10 minutes.

5. The application according to claim 1, characterized in that, The dialysis desalination in step (4) specifically involves: The elution fraction was injected into a 3500 Dalton dialysis bag. The sealed dialysis bag was placed in a container containing a large amount of ultrapure water dialysis solution. The container was placed on a magnetic stirrer with a stir bar and stirred at 4°C. The dialysis solution was changed several times until the conductivity of the dialysis solution was close to the background value of the ultrapure water used. The dialysis bag was then removed and the sample was recovered.

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