Phyllanthus emblica polysaccharide with weight losing effect as well as efficient preparation method and application of phyllanthus emblica polysaccharide

Abstract

The invention provides emblic leafflower fruit polysaccharide with a weight losing effect and an efficient preparation method and application thereof, and belongs to the field of plant polysaccharide. The preparation method comprises the following steps: performing powdering, water extraction and concentration on a phyllanthus emblica raw material, adding an alcohol solvent for precipitation, and drying to obtain phyllanthus emblica crude polysaccharide; after the phyllanthus emblica crude polysaccharide is dissolved, impurities are removed through membrane filtration, protein substances are adsorbed through a reversed-phase chromatographic column, flow-through liquid and water leacheate are collected and combined, small-molecular-weight substances are removed through a membrane bag, intercepted liquid is collected and dried, and the phyllanthus emblica polysaccharide is obtained. The phyllanthus emblica polysaccharide has alpha-type and beta-type glycosidic bonds, and has linear polysaccharide chains and aggregates formed by a plurality of chains. The emblic leafflower fruit polysaccharide is used for preparing products for reducing fat and blood sugar, and can also be used for preparing medicines for preventing and treating abnormal glucose metabolism, abnormal lipid metabolism, related diseases, obesity and liver injury caused by obesity. The phyllanthus emblica polysaccharide extraction and purification preparation method is simple and efficient, and is convenient for industrial implementation.

Description

Technical Field

[0001] This invention belongs to the field of plant polysaccharide technology, and specifically relates to a Phyllanthus emblica polysaccharide with weight loss effects, its efficient preparation method, and its application. Background Technology

[0002] With the fast pace of modern life and changes in dietary structure, overweight and obesity have become global health problems. Obesity not only affects physical appearance but is also a risk factor for many chronic diseases, including diabetes, cardiovascular disease, hypertension, and certain types of cancer. Therefore, developing safe and effective weight-loss products has become a hot research topic in the medical and health food fields. Existing weight-loss methods mainly include exercise therapy, diet control, drug treatment, and surgical intervention. However, drug treatment often comes with certain side effects, such as nausea, palpitations, and liver and kidney damage; while surgical weight loss involves significant trauma, high risks, and long recovery periods. Therefore, finding natural, safe, and side-effect-free functional food ingredients has become one of the important directions of current weight-loss research.

[0003] Phyllanthus emblica ( Phyllanthus emblica Indian gooseberry (Phyllanthus emblica), also known as Indian gooseberry or amla, is a traditional Chinese medicine and functional fruit widely distributed in tropical and subtropical Asia. It is rich in various bioactive components, including polyphenols, flavonoids, organic acids, vitamin C, and polysaccharides. Polysaccharides are considered one of its main functional components, possessing various biological activities such as antioxidant, anti-inflammatory, immunomodulatory, and anti-tumor effects. Studies have shown that certain natural polysaccharides can regulate lipid metabolism, inhibit fat absorption, and promote energy expenditure, thus exhibiting potential weight-loss benefits.

[0004] Existing research has found that amla polysaccharides have blood sugar and lipid-lowering activities. However, amla polysaccharides prepared by conventional extraction and purification methods are not effective in preventing and alleviating obesity-related symptoms. For example, invention patent CN120463833A discloses an amla polysaccharide with blood sugar and lipid-lowering activities, its preparation method and application.

[0005] Therefore, there is an urgent need to develop a polysaccharide from Phyllanthus emblica with a clear and efficient weight-loss function. Phyllanthus emblica The study developed a highly efficient and controllable extraction and preparation process for polysaccharides, providing a new natural functional ingredient for the weight loss field, which has significant scientific value and promising industrial application prospects. Summary of the Invention

[0006] Therefore, the present invention aims to provide a Phyllanthus emblica polysaccharide with weight loss effects, its efficient preparation method and application, in order to solve at least one technical problem in the background art.

[0007] This invention is implemented as follows: The first aspect of this invention provides a highly efficient method for preparing Phyllanthus emblica polysaccharide with weight-loss effects, the preparation method comprising the following steps: S1. After the raw material of Phyllanthus emblica is powdered, extracted with water and concentrated, an alcohol solvent is added to precipitate it, the precipitate is collected and dried to obtain crude polysaccharide of Phyllanthus emblica. S2. After the crude polysaccharide of Phyllanthus emblica is dissolved, it is first filtered through a membrane to remove impurities, and then passed through a reversed-phase chromatography column to adsorb protein substances. The flow-through liquid and water eluent are collected, combined, and then small molecular weight substances are removed by membrane encapsulation. The retained liquid is collected and dried to obtain Phyllanthus emblica polysaccharide.

[0008] Furthermore, step S1 specifically includes: S11. Remove the pits from fresh amla fruits and crush them to obtain amla powder; S12. Mix amla powder with ultrapure water at a solid-liquid ratio of 1g:(8~12)mL, heat to 120℃~150℃ and keep warm for 1h~3h for water extraction to obtain the extract; the number of water extractions is 1~3 times. S13. Combine the extracts and concentrate them to obtain a concentrated solution; S14. Add anhydrous ethanol to the concentrate to make the final volume concentration of ethanol 75%~85%, and let it stand at 3℃~5℃ for 12h~20h. Centrifuge at 7000rpm~12000rpm for 5min~15min, discard the supernatant, filter the obtained precipitate and freeze dry to obtain crude polysaccharide of Phyllanthus emblica.

[0009] Furthermore, step S2 specifically includes: S21. The crude polysaccharide of Phyllanthus emblica was dissolved in ultrapure water to prepare a crude polysaccharide solution of Phyllanthus emblica with a concentration of 1 mg / mL to 10 mg / mL. S22. Filter the crude polysaccharide solution of Phyllanthus emblica using a microporous membrane with a pore size of 0.4 to 0.5 µm, and collect the filtrate; S23. The filtrate was separated and purified by passing it through a C18-YE reversed-phase chromatography column at a flow rate of 30 mL / min to 50 mL / min, using ultrapure water as the eluent, and the flow-through and ultrapure water eluent were collected separately. S24, the combined solution of the flow-through liquid and the ultrapure water rinsing liquid is separated by a membrane with a molecular weight cutoff of ≥8kDa, and ultrapure water is used as the replacement liquid to obtain a high-purity amla polysaccharide solution. S25. Phyllanthus polysaccharide was obtained by freeze-drying the Phyllanthus polysaccharide solution.

[0010] The second aspect of this invention provides an efficient method for preparing Phyllanthus emblica polysaccharide with weight-loss effects, as described above.

[0011] Furthermore, the amla polysaccharide has α- and β-glycosidic bonds; the amla polysaccharide contains linear polysaccharide chains and aggregates formed by multiple chains.

[0012] Furthermore, in descending order of component content, the components of the amla polysaccharide include galactose, galacturonic acid, glucose, arabinose, mannose, rhamnose, xylose, and glucuronic acid.

[0013] A third aspect of the present invention provides the application of the aforementioned Phyllanthus emblica polysaccharide, the application comprising at least one of the following (1) to (6): (1) The application of the amla polysaccharide in the preparation of weight loss products, wherein the products are functional foods, health products or drugs; (2) The amla polysaccharide is used in the preparation of products that lower blood sugar, and the products are functional foods, health products or drugs; (3) The application of the above-mentioned amla polysaccharide in the preparation of drugs for the prevention and treatment of glucose metabolism disorders and related diseases; (4) The application of the above-mentioned amla polysaccharide in the preparation of drugs for the prevention and treatment of lipid metabolism disorders and related diseases; (5) The application of the above-mentioned amla polysaccharide in the preparation of drugs for the prevention and treatment of obesity; (6) The application of the above-mentioned Phyllanthus emblica polysaccharide in the preparation of drugs for preventing and treating liver damage caused by obesity.

[0014] Furthermore, the drug prepared from the amla polysaccharide significantly inhibits α-amylase, α-glucosidase, and lipase.

[0015] Furthermore, the drug prepared from the amla polysaccharide significantly inhibits fat deposition.

[0016] Furthermore, the drug prepared from the amla polysaccharide significantly regulates lipid accumulation in adipocytes and reduces inflammation.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention is the first to start with the extraction and purification of Phyllanthus emblica polysaccharide. It uses a C18-YE reversed-phase chromatography column to adsorb protein substances in crude polysaccharide, thereby achieving the purpose of deproteinization. Furthermore, it uses membrane separation method to efficiently remove small molecules from crude Phyllanthus emblica polysaccharide, and the efficiency is better than the commonly used Sevage method, trichloroacetic acid method and enzymatic hydrolysis method.

[0018] 2. This invention systematically studies the application of Phyllanthus emblica polysaccharide in weight loss and prevention of obesity-related side effects. Compared with the prior art, the Phyllanthus emblica polysaccharide involved in this invention has the advantages of being natural, safe, and having few side effects. Moreover, its mechanism of action in regulating glucose and lipid metabolism is unique, and it is expected to provide a new strategy for the prevention and treatment of obesity.

[0019] 3. The research results of this invention will provide a theoretical basis for the further development and application of Phyllanthus emblica polysaccharides, promote the application of Phyllanthus emblica, a substance that is both food and medicine, in functional foods, and have important scientific and social value. Attached Figure Description

[0020] Figure 1 This is a flowchart of the efficient preparation of Phyllanthus emblica polysaccharide in Example 1 of the present invention; Figure 2 This is a molecular weight distribution diagram of amla polysaccharides measured in Example 2 of the present invention; Figure 3 This is a compositional distribution diagram of the monosaccharides of Phyllanthus emblica polysaccharides detected in Example 2 of the present invention; Figure 4 This is the Fourier transform infrared spectrum of amla polysaccharide detected in Example 2 of the present invention; Figure 5 This is a scanning electron microscope image of the polysaccharides detected in Phyllanthus emblica in Example 2 of the present invention; Figure 6 This is a comparison chart of the inhibitory effects of Phyllanthus emblica polysaccharide and acarbose on α-amylase and α-glucosidase in Example 3 of the present invention. Figure 7 This is a comparison chart of the inhibitory effects of Phyllanthus emblica polysaccharide and acarbose on lipase in Example 3 of the present invention; Figure 8 This is a comparison chart of the body weight and fasting blood glucose of mice in each group in Example 4 of the present invention; Figure 9 This is a comparison chart of blood glucose and area under the GTT curve in each group of mice in the GTT experiment of Example 4 of the present invention; Figure 10 This is a comparison chart of serum TC, serum TG, serum AST, and serum ALT in each group of mice in Example 4 of the present invention. Figure 11 This is a comparison chart of subcutaneous fat weight and epididymal fat weight in each group of mice in Example 4 of the present invention; Figure 12 The images show H&E staining of mouse adipose tissue in each group in Example 4 of this invention. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0022] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods and techniques used in the following examples are the same as those used conventionally. All reagents / instruments used in the following examples are commercially available. Any modifications or substitutions made without departing from the spirit and essence of the invention should be within the scope of protection of the invention.

[0023] The invention will now be described in detail with reference to specific embodiments.

[0024] Example 1 This embodiment describes a highly efficient method for preparing Phyllanthus emblica polysaccharide with weight-loss effects. High-purity Phyllanthus emblica polysaccharide is obtained from fresh Phyllanthus emblica fruit through extraction and purification steps including water extraction and alcohol precipitation, membrane filtration, C18-YE column chromatography, and membrane encapsulation. The process is as follows: Figure 1 As shown, it specifically includes steps S1 and S2.

[0025] S1. Wash and pit fresh Phyllanthus emblica fruits, dry, pulverize, and pass through a 40-mesh sieve to obtain Phyllanthus emblica powder. Accurately weigh the Phyllanthus emblica powder and add pure water at a material-to-liquid ratio of 1:10. Heat to boiling using an electric heating mantle and maintain for 1 hour. Then, centrifuge at 7000 rpm for 10 minutes, collect the supernatant, and filter through qualitative filter paper to remove any residual precipitate. Add the centrifuged residue to the same volume of pure water as before and extract again. Repeat this step three times. Combine the supernatants obtained from the three extractions and concentrate them to 1 / 3 of the original volume by rotary evaporation. Then, add anhydrous ethanol to achieve a final ethanol concentration of 80% (v / v) and let stand overnight at 4°C. Finally, centrifuge at 7000 rpm for 3 minutes, collect the precipitate, reconstitute the precipitate with pure water, and freeze-dry to obtain crude Phyllanthus emblica polysaccharide.

[0026] S2. The crude polysaccharide of Phyllanthus emblica was dissolved in ultrapure water to prepare a solution with a concentration of 5 mg / mL. Subsequently, the solution was filtered using a microporous membrane with a pore size of 0.45 µm to remove any possible particulate impurities and ensure the clarity and homogeneity of the solution. The filtered solution was then purified by separation using a C18-YE reversed-phase chromatography column at a flow rate of 30 mL / min. Ultrapure water was used as the eluent, and the flow-through and ultrapure water eluents were collected separately for subsequent analysis and application. The combined solution of the flow-through and ultrapure water eluents was then separated by a membrane with a molecular weight cutoff ≥8 kDa to separate substances of different molecular weights. Ultrapure water was used as the replacement fluid to effectively remove small molecule impurities from the solution, thereby obtaining a high-purity Phyllanthus emblica polysaccharide solution. After freeze-drying, the Phyllanthus emblica polysaccharide was obtained.

[0027] Example 2 This embodiment describes the property testing and structural characterization analysis of the amla polysaccharide prepared in Example 1.

[0028] 1. Molecular weight experiment; The molecular weight of Phyllanthus emblica polysaccharides was determined using an Alliance E2695 high-performance liquid chromatograph. First, 10 mg of dextran standards with different relative molecular weights were dissolved in 1 mL of 150 mM NaCl initial mobile phase to prepare dextran solutions of different molecular weights. Then, the polysaccharide sample to be tested was accurately weighed and dissolved in the initial mobile phase to prepare a 10 mg / mL solution. A TSKgel G5000PWXL column (300 mm × 7.8 mm) and a G7162A differential refractive index (RI) detector were used to detect the polysaccharide samples. The flow rate was set to 0.6 mL / min, column temperature to 30℃, injection volume to 10 µL, and RI detector temperature to 35℃. Standard regression equations for the molecular weight (MW) and refractive index (TR) of dextran were established to calculate the molecular weight of the Phyllanthus emblica polysaccharides to be tested. The results are as follows: Figure 2 As shown.

[0029] Figure 2 The results showed that the molecular weight of Phyllanthus emblica polysaccharide was 464 kDa.

[0030] 2. Experimental methods for monosaccharide composition; The monosaccharide composition of Phyllanthus emblica polysaccharide was determined using a combination of 3-methyl-1-phenyl-5-pyrazolone (PMP) pre-column derivatization high-performance liquid chromatography (HPLC) and a non-derivatization method. The PMP derivatization method involved adding 2 mg of Phyllanthus emblica polysaccharide sample to 1 mL of 2M trifluoroacetic acid (TFA), hydrolyzing at 120°C for 2 h, freeze-drying to neutral pH, and then reacting with monosaccharide standards and mixed standards in NaOH and PMP solution, followed by water bath reaction, centrifugation, and extraction. Finally, HPLC analysis was performed using a Homemade-C18ME column and other conditions. The non-derivatization method involved adding 2 mg of the desired Phyllanthus emblica polysaccharide sample to 1 mL of 0.1M trifluoroacetic acid (TFA). TFA was hydrolyzed at 80℃ for 30 min and then treated to neutral pH. Analysis was performed using a CYS column and other conditions. Since the PMP derivatization method cannot detect fructose (Fru), a non-derivatization method was used to determine the ratio of the total content of the three monosaccharides (Man, Glc, and Gal) to Fru. This ratio was then substituted into the total content of the three monosaccharides (Man, Glc, and Gal) from the PMP derivatization results to calculate the Fru content. The results are as follows: Figure 3 As shown.

[0031] Figure 3The results showed that the main components of the amla polysaccharide prepared in Example 1 were galactose (Gal, 37.59%), galacturonic acid (GalA, 27.13%), and glucose (Glc, 18.18%), which together accounted for 82.9%. In addition, small amounts of arabinose (Ara, 6.9%), mannose (Man, 5.1%), rhamnose (Rha, 2.9%), xylose (Xyl, 1.48%), and glucuronic acid (GlcA, 0.75%) were also present.

[0032] 3. Fourier transform infrared spectroscopy detection experimental method; In Fourier Transform Infrared Spectrometer (FTIR) tests, 3 mg of Phyllanthus emblica polysaccharide sample and 100 mg of dried potassium bromide powder were mixed, ground evenly, compressed into tablets, and tested at 4000-400 cm⁻¹. -1 FTIR analysis was performed internally, with an infrared spectral resolution of 1 cm⁻¹. -1 The result is as follows Figure 4 As shown.

[0033] Figure 4 The FTIR spectrum of Phyllanthus emblica polysaccharide showed that at 3384.25 cm⁻¹... -1 The presence of a broad and strong absorption peak nearby is typically associated with the stretching vibration of hydroxyl groups (-OH), indicating the presence of numerous hydroxyl groups in the polysaccharide structure. At 2920.12 cm⁻¹ -1 The absorption peak at 1737.03 cm⁻¹ is typically due to the stretching vibration of the CH bond, further confirming the carbon skeleton structure of the polysaccharide. -1 The absorption peak at 1627.07 cm⁻¹ further supports the presence of ester groups. -1 The strong absorption peak at 1440.30 cm⁻¹ may be related to the stretching vibration of the C=O bond, suggesting that the polysaccharide structure may contain a carboxyl group (-COOH) or an ester group (-COOR). -1 and 1016.06cm -1 The absorption peak at 913.23 cm⁻¹ may be related to the stretching vibration of the CO bond and the bending vibration of the C-OH group. -1 and 830.87cm -1 The absorption peak at the specified location indicates that the polysaccharide has a complex cyclic structure, confirming that Phyllanthus emblica polysaccharide has α- and β-glycosidic bonds.

[0034] 4. Scanning electron microscopy (SEM) detection method; The amla polysaccharide sample was adhered to adhesive tape on a copper sample stage, and a layer of gold was vacuum-sprayed onto it. The microstructure of the amla polysaccharide was observed using a scanning electron microscope (SEM) under conditions of an acceleration voltage of 5.00 kV, a magnification factor of 600, and working distances of 8.1 and 8.2 mm. Figure 5 As shown.

[0035] The microstructure of *Ligusticum striatum* polysaccharide was observed using SEM. Figure 5 The results showed that Phyllanthus emblica polysaccharide exhibited a dense, sheet-like structure with curled and folded edges. Furthermore, the structure contained filamentous networks and randomly distributed coiled regions. These morphological features revealed the presence of linear polysaccharide chains and aggregates formed by multiple chains within the polysaccharide sample.

[0036] Example 3 This embodiment tests the inhibition rates of α-amylase, α-glucosidase and lipase in the amla polysaccharide prepared in Example 1.

[0037] 1. Detection experiment of α-amylase inhibition rate; (1) Preparation of reagents Weigh the amla polysaccharide sample prepared in Example 1, dissolve it using ultrapure water as solvent by vortexing, and bring the volume to a mother liquor concentration of 5 mg / mL. Perform a serial dilution using the proportional dilution method, mixing the mother liquor with ultrapure water at a 1:1 (v / v) ratio. Through continuous dilution steps, five concentration gradients of amla polysaccharide solutions were finally obtained: 0.3125 mg / mL, 0.625 mg / mL, 1.25 mg / mL, 2.5 mg / mL, and 5 mg / mL.

[0038] Preparation of phosphate buffer: Mix 9 mL of 0.1 mol / L sodium dihydrogen phosphate aqueous solution with 11 mL of 0.1 mol / L disodium hydrogen phosphate aqueous solution, and add ultrapure water to a final volume of 40 mL to prepare phosphate buffer. Prepare an α-amylase solution with a concentration of 10 U / mL. Prepare a soluble starch solution with a mass concentration of 1%. Preparation of DNS reagent: Add 63 mg of 3,5-dinitrosalicylic acid, 1.82 g of potassium sodium tartrate, 200 mg of sodium hydroxide, 50 mg of phenol, and 50 mg of anhydrous sodium bisulfite to ultrapure water, stir well, and bring the volume to 10 mL.

[0039] (2) Experimental procedure 50 µL of Phyllanthus emblica polysaccharide solution and 50 µL of α-amylase solution were respectively transferred to 1.5 mL test tubes, thoroughly mixed, and heated in a 37 °C water bath for 10 min. 50 µL of 1% soluble starch solution was added, and the mixture was further mixed and heated in a 37 °C water bath for another 10 min. Subsequently, 100 µL of DNS reagent was added, and the mixture was heated in a boiling water bath for 5 min. After the reaction was complete, the reaction system was quickly transferred to an ice bath to rapidly cool to room temperature, thus effectively terminating the reaction. Ultrapure water was added to a total volume of 1 mL, and its absorbance at 540 nm was measured. Acarbose was used as a positive control reagent, and the inhibition rate of α-amylase in the sample was calculated according to formula (1).

[0040] (1) In formula (1), ultrapure water is used instead of the compound in the control well. The absorbance of the sample solution and enzyme mixture; The absorbance of the mixture of sample solution and phosphate buffer; A represents the absorbance when ultrapure water is used to replace the sample and enzyme; A represents the absorbance when ultrapure water is used to replace the sample and phosphate buffer mixture.

[0041] (3) Results Analysis Starch chains can be broken down into small amounts of glucose and maltose by α-amylase. Inhibiting α-amylase can lower blood sugar levels. Some medications, such as acarbose, reduce glucose uptake by inhibiting α-amylase activity, thereby lowering blood sugar.

[0042] The effects of different concentrations of acarbose and the amla polysaccharide prepared in Example 1 on the inhibition rate of α-amylase are as follows: Figure 6 As shown in Figure A, the results indicated a positive correlation between the concentrations of acarbose and Phyllanthus emblica polysaccharide and their inhibitory rates on α-amylase. Within the concentration range of 0.3125 mg / mL to 5 mg / mL, the inhibitory activity of acarbose was in the order of > Phyllanthus emblica polysaccharide. At a concentration of 5 mg / mL, the inhibitory rates of acarbose and Phyllanthus emblica polysaccharide on α-amylase reached their maximum, at 24.9% and 66.7%, respectively. These results demonstrate that at the same concentration, Phyllanthus emblica polysaccharide has a significantly higher inhibitory effect on α-amylase than acarbose (p < 0.0001). Invention patent CN120463833A discloses a polysaccharide from Phyllanthus emblica with blood sugar and lipid-lowering activity. Results show that to achieve the same α-amylase inhibition effect as the Phyllanthus emblica polysaccharide (5 mg / mL) in Example 1 of this invention, the required concentration of the remaining Phyllanthus emblica polysaccharide is 10 mg / mL. Therefore, it can be seen that the blood sugar-lowering effect of the Phyllanthus emblica polysaccharide extracted by this invention is significantly better than that obtained by existing extraction methods.

[0043] 2. α-Glucosidase inhibition rate detection experiment; (1) Preparation of reagents Weigh the amla polysaccharide sample prepared in Example 1, dissolve it using ultrapure water as solvent by vortexing, and bring the volume to a mother liquor concentration of 5 mg / mL. Perform a serial dilution using the proportional dilution method, mixing the mother liquor with ultrapure water at a 1:1 (v / v) ratio. Through continuous dilution steps, five concentration gradients of amla polysaccharide solutions were finally obtained: 0.3125 mg / mL, 0.625 mg / mL, 1.25 mg / mL, 2.5 mg / mL, and 5 mg / mL.

[0044] Preparation of phosphate buffer: Mix 9 mL of 0.1 mol / L sodium dihydrogen phosphate aqueous solution with 11 mL of 0.1 mol / L disodium hydrogen phosphate aqueous solution, and add ultrapure water to a final volume of 40 mL to prepare phosphate buffer. Preparation of 0.48 U / mL α-glucosidase solution. Preparation of PNPG solution: Prepare a 1 mg / mL PNPB (p-nitrophenyl butyrate) solution with acetonitrile, taking care to avoid light; weigh PNPG (p-nitrophenyl-β-D-galactopyranoside) powder, dissolve it in a small amount of DMSO (p-nitrophenyl butyrate), and then dilute with the PNPB solution to obtain a 2 mmol / L PNPG solution.

[0045] (2) Experimental procedure Mix 10 µL of Phyllanthus emblica polysaccharide solution with 49 µL of α-glucosidase and incubate at 37 °C for 5 min. Then add 50 µL of LPNPG solution and incubate at 37 °C for 30 min. After the reaction is complete, add 100 µL of Na2CO3 solution to terminate the enzymatic reaction and measure its absorbance at 405 nm. Using acarbose as a positive control, calculate the inhibition rate of α-glucosidase in the sample according to formula (2).

[0046] (2) In formula (2), The absorbance of the sample solution and enzyme mixture; The absorbance is the value of the mixture of sample solution and phosphate buffer.

[0047] (3) Results Analysis α-Glucosidase is one of the important hydrolytic enzymes in the human digestive system. α-Glucosidase hydrolyzes glucosidic bonds into glucose. Inhibiting α-glucosidase can lower blood glucose levels and protect the pancreas. The effects of different concentrations of acarbose and the *Phyllanthus emblica* polysaccharide prepared in Example 1 on the inhibition rate of α-glucosidase activity are shown below. Figure 6As shown in Figure B, the results indicate that within the concentration range of 0.3125 mg / mL to 5 mg / mL, the inhibition rates of both Phyllanthus emblica polysaccharide and acarbose on α-glucosidase exhibited a concentration-dependent effect, with the inhibition rates decreasing in the order of acarbose > Phyllanthus emblica polysaccharide. At a concentration of 5 mg / mL, the inhibition rates of Phyllanthus emblica polysaccharide and acarbose on α-glucosidase were 65.9% and 87.9%, respectively.

[0048] Invention patent CN120463833A discloses a polysaccharide from Phyllanthus emblica with blood sugar and lipid-lowering activity. The results show that the inhibition rate of Phyllanthus emblica polysaccharide (10 mg / mL) on α-glucosidase is only about 10%. It can be seen that the blood sugar-lowering effect of the Phyllanthus emblica polysaccharide extracted by the present invention is significantly better than that of Phyllanthus emblica polysaccharide obtained by existing extraction methods.

[0049] 3. Experimental method for detecting lipase inhibition rate: (1) Preparation of reagents Weigh the amla polysaccharide sample prepared in Example 1, dissolve it using ultrapure water with vortexing, and bring the volume to a mother liquor concentration of 5 mg / mL. Perform a serial dilution using a proportional dilution method, mixing the mother liquor with ultrapure water at a 1:1 (v / v) ratio. Through continuous dilution steps, five concentration gradients of amla polysaccharide solutions were finally obtained: 0.3125 mg / mL, 0.625 mg / mL, 1.25 mg / mL, 2.5 mg / mL, and 5 mg / mL. All solutions were prepared fresh and stored at 4°C in the dark to prevent polysaccharide degradation.

[0050] Preparation of lipase solution: Weigh pancreatic lipase and add it to PBS (pH=7.4) to obtain a lipase solution with an activity of 150 U / mL. Preparation of PNPG solution: Prepare a 1 mg / mL PNPB (p-nitrophenyl-β-D-glucopyranoside) solution using chilled acetonitrile, taking care to avoid light. Weigh PNPG powder, dissolve it in a small amount of DMSO, and then dilute it with PNPB solution to a concentration of 2 mM to obtain the PNPG solution.

[0051] (2) Experimental procedure Take 50 µL of Phyllanthus emblica polysaccharide solution and 50 µL of lipase solution separately, mix them thoroughly, and incubate at 37 °C for 30 min. Then add 50 µL of PNPG solution, mix well, and shake in the dark for 20 min. Measure the absorbance at 405 nm. Using orlistat as a positive control, the inhibition rate of the sample against pancreatic lipase is calculated as shown in equation (3).

[0052] (3) In equation (3), A 1 represents the absorbance of the sample solution with added lipase solution; A2 represents the absorbance of the sample solution with added ultrapure water; A 3 represents the absorbance of ultrapure water with added lipase; A 4 represents the absorbance of ultrapure water.

[0053] (3) Results Analysis The inhibitory effect of amla polysaccharides on lipase, such as Figure 7 As shown, within the concentration range of 0.3125 mg / mL to 5 mg / mL, the inhibition rates of both Phyllanthus emblica polysaccharide and orlistat on lipase exhibited a concentration-dependent effect, with the inhibition rates decreasing in the order of orlistat > Phyllanthus emblica polysaccharide. At a concentration of 5 mg / mL, the inhibition rates of Phyllanthus emblica polysaccharide and orlistat on lipase were 76% and 90.7%, respectively.

[0054] Example 4 This embodiment evaluates the weight-loss efficacy of Phyllanthus emblica polysaccharide by constructing an animal obesity model.

[0055] 1. Establishment of an obesity model; Experimental Methods: Thirty-two healthy 8-week-old SPF-grade male C57 mice were housed at the Ganjiang Traditional Chinese Medicine Innovation Center - Experimental Animal Center. Mice were only allowed to proceed to the next stage of the experiment after observing normal growth and development, normal activity, glossy fur, and the absence of diarrhea or other adverse conditions. After one week of acclimatization, the mice were divided into two groups: a control group (n=8) and a high-fat diet group (n=24). The high-fat diet group was used to establish an obese mouse model. Feed was changed every two days, and water bottles, bedding, and cages were changed weekly, with weight recorded. After 8 weeks of feeding, the success of the model was determined by indicators such as a 20% weight gain, fasting blood glucose greater than 7 mmol / L, and a glucose tolerance test (GTT).

[0056] 2. Evaluation of the weight loss efficacy of Phyllanthus emblica polysaccharides; The high-fat diet group was divided into an obesity model group (saline gavage), a low-dose polysaccharide group (100 mg / kg), and a high-dose polysaccharide group (200 mg / kg) and administered the drug via gavage. The control group and the high-fat diet group were given 200 μl of the solution via gavage once a day. Body weight, fasting blood glucose, and food intake were measured weekly for 4 weeks. Results are as follows: Figure 8 As shown; the glucose tolerance test (GTT) was conducted in mice during the last week, and the changes in blood glucose and GTT curves during the experiment are shown in the figure. Figure 9 As shown. After 4 weeks of drug treatment, mice were fasted for 12 hours before dissection, anesthetized with isoflurane, and blood was drawn from the aorta via the heart. The blood collection vessels were centrifuged to separate the serum, and the mice were then euthanized by cervical dislocation. Subcutaneous and epididymal fat samples were weighed after euthanasia. The results are shown below. Figure 11 As shown, a portion of the tissue was fixed in 4% paraformaldehyde for paraffin sectioning, and the remaining tissue was flash-frozen in liquid nitrogen and immediately placed in a -80°C freezer for subsequent experiments.

[0057] 3. Indicator evaluation; Serum assay: Blood was centrifuged at 12000 rpm for 10 min to obtain serum. Triglycerides (TG), total cholesterol (TC), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were measured using a Nanjing Jiancheng triglyceride (TG), total cholesterol (TC), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) assay kit. Specific procedures followed the manufacturer's instructions. Results are as follows: Figure 10 As shown.

[0058] Histological analysis: Freshly isolated adipose tissue was immediately soaked in 4% paraformaldehyde and subsequently stained with eosin (H&E). Fixed liver tissue was also stained with Oil Red O. All sections were observed under a light microscope. Figure 12 As shown.

[0059] 4. Results Analysis; like Figure 8 As shown, by Figure 8 As can be seen from the body weight of mice, by week 6 of administration, the body weight of both the low-dose polysaccharide group and the high-dose polysaccharide group was significantly lower than that of the model group. p< 0.05). Figure 8 In mice B, blood glucose levels were significantly lower in both the low-dose and high-dose polysaccharide groups compared to the obesity model group. p< The concentration of 0.05 g / L indicates that Phyllanthus emblica polysaccharide has the effect of reducing body weight and blood sugar in obese mice.

[0060] like Figure 9 As shown, compared with the control group, the blood glucose levels of the obese model mice were significantly increased at all time points after intraperitoneal injection of glucose solution. Their blood glucose-time response curves shifted upwards overall, and the area under the curve increased significantly, fully confirming the significant glucose metabolism disorder and insulin resistance characteristics of the model group mice. After intervention with high-dose Phyllanthus emblica polysaccharide, the blood glucose response curve and the corresponding area under the curve were significantly lower than those of the model group (…). p< 0.05). This data clearly shows that Phyllanthus emblica polysaccharide can effectively improve glucose metabolism dysfunction in obese mice, suggesting that it has a certain regulatory effect on glucose metabolism homeostasis.

[0061] like Figure 10 As shown, compared with the blank group, the serum TC ( ) in the obese model group mice was higher. p <0.0001), TG ( p <0.0001), AST ( p <0.001) and ALT ( pThe levels of <0.0001 were significantly increased, indicating that the obese model mice had obvious lipid metabolism abnormalities and liver damage. After treatment with different doses of Phyllanthus emblica polysaccharide, the serum TC, TG, AST, and ALT levels of mice in each dose group were significantly lower than those in the model group. p <0.05). These results indicate that Phyllanthus emblica polysaccharide can effectively improve lipid metabolism disorders and alleviate liver damage caused by obesity in obese mice.

[0062] according to Figure 11 As shown in Figure A, the subcutaneous fat weight of obese mice treated with Phyllanthus emblica polysaccharide was significantly lower than that of the model group mice. p The value <0.05 indicates that Phyllanthus emblica polysaccharide can effectively reduce the accumulation of subcutaneous fat. Figure 11 In B, the epididymal fat weight of obese mice treated with high-dose Phyllanthus emblica polysaccharide was also significantly reduced. p The result of <0.001 further supports the role of Phyllanthus emblica polysaccharide in inhibiting fat deposition.

[0063] from Figure 12 H&E staining results of mouse adipose tissue showed that the adipocytes in the blank group were regular in shape and moderate in size, with vacuolated lipid droplets visible in the cytoplasm and no obvious inflammation; the adipocytes in the obesity model group were significantly enlarged and plump, with large and numerous lipid droplets, accompanied by inflammatory cell infiltration; while the adipocytes in the low- and high-dose treatment groups of Phyllanthus emblica polysaccharide were smaller in size, with significantly reduced number and size of lipid droplets, and significantly improved inflammatory response, indicating that Phyllanthus emblica polysaccharide can play a therapeutic role in obesity by regulating lipid accumulation in adipocytes and reducing inflammation.

[0064] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for efficient preparation of Phyllanthus emblica polysaccharides having weight loss efficacy, characterized by, The preparation method comprises the following steps: S1, the raw material of Phyllanthus emblica is powdered, water extracted, concentrated, and then precipitated with an alcohol solvent. The precipitate is collected and dried to obtain crude Phyllanthus emblica polysaccharide; S2, after the crude Phyllanthus emblica polysaccharide is dissolved, impurities are removed by membrane filtration, and then protein substances are adsorbed by a reverse phase chromatography column. The flow-through and water eluate are collected, combined, and then small molecular weight substances are removed by a membrane package. The cut-off liquid is collected, dried, and then Phyllanthus emblica polysaccharide is obtained.

2. The method of claim 1, wherein the method is characterized by, The step S1 specifically comprises: S11, fresh Phyllanthus emblica is cored and crushed to obtain Phyllanthus emblica powder; S12, the Phyllanthus emblica powder is mixed with ultrapure water at a solid-liquid ratio of 1g: (8-12) mL, heated to 120-150 DEG C and kept for 1-3 hours for water extraction, and then an extraction solution is obtained. The number of water extraction is 1-3 times; S13, the extraction solutions are combined and concentrated to obtain a concentrated solution; S14, anhydrous ethanol is added to the concentrated solution to make the final concentration of ethanol volume 75%-85%, and then the solution is placed at 3-5 DEG C for 12-20 hours; S15, centrifugation is performed at a speed of 7000-12000 rpm for 5-15 minutes, the supernatant is discarded, and the obtained precipitate is suction filtered and freeze-dried to obtain crude Phyllanthus emblica polysaccharide.

3. The method of claim 1, wherein the method is characterized by the steps of: The step S2 specifically comprises: S21, the crude Phyllanthus emblica polysaccharide is dissolved in ultrapure water to prepare a crude Phyllanthus emblica polysaccharide solution with a concentration of 1-10 mg / mL; S22, the crude Phyllanthus emblica polysaccharide solution is filtered by using a microporous filter membrane with a pore size of 0.4-0.5 µm to collect the filtrate; S23, the filtrate is separated and purified by a C18-YE reverse phase chromatography column at a flow rate of 30-50 mL / min, and ultrapure water is used as an eluent to collect the flow-through and the ultrapure water eluate, respectively; S24, the solution obtained by combining the flow-through and the ultrapure water eluate is separated by a membrane package with a molecular weight cut-off of ≥8 kDa, and ultrapure water is used as a displacement liquid to obtain a high-purity Phyllanthus emblica polysaccharide solution; S25, the Phyllanthus emblica polysaccharide solution is freeze-dried to obtain Phyllanthus emblica polysaccharide.

4. The Phyllanthus emblica polysaccharide obtained by the high-efficiency preparation method of Phyllanthus emblica polysaccharide with weight loss effect according to any one of claims 1-3.

5. The polysaccharide of Phyllanthus emblica as claimed in claim 4, wherein, The Phyllanthus emblica polysaccharide has α-type and β-type glycosidic bonds, linear polysaccharide chains, and aggregates formed by multiple chains.

6. The Phyllanthus emblica polysaccharide according to claim 4 or 5, characterized in that: According to the component content from large to small, the components of the Phyllanthus emblica polysaccharide include galactose, galacturonic acid, glucose, arabinose, mannose, rhamnose, xylose, and glucuronic acid.

7. Use of the Phyllanthus emblica polysaccharides according to any one of claims 4 to 6, characterized in that, The application comprises at least one of the following (1) to (6): (1) the application of the Phyllanthus emblica polysaccharide in preparing a weight loss product, which is a functional food, a health product, or a drug; (2) the application of the Phyllanthus emblica polysaccharide in preparing a product for reducing blood sugar, which is a functional food, a health product, or a drug; (3) the application of the Phyllanthus emblica polysaccharide in preparing a drug for preventing and treating abnormal glucose metabolism and related diseases; (4) the application of the Phyllanthus emblica polysaccharide in preparing a drug for preventing and treating abnormal lipid metabolism and related diseases; (5) the application of the Phyllanthus emblica polysaccharide in preparing a drug for preventing and treating obesity; (6) The use of the Phyllanthus emblica polysaccharide in the preparation of a medicine for preventing and treating liver damage caused by obesity.

8. The use of the Phyllanthus emblica polysaccharide according to claim 7, characterized in that, The medicine prepared from the Phyllanthus emblica polysaccharide significantly inhibits alpha-amylase, alpha-glucosidase and lipase.

9. The use of the Phyllanthus emblica polysaccharide according to claim 7, characterized in that, The medicine prepared from the Phyllanthus emblica polysaccharide significantly inhibits fat deposition.

10. The use of the Phyllanthus emblica polysaccharide according to claim 7, characterized in that, The medicine prepared from the Phyllanthus emblica polysaccharide significantly regulates fat cell lipid accumulation and reduces inflammation.

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