Application of beautiful millettia root extract in resisting obesity

Abstract

The invention discloses application of a beautiful millettia root extract in preparation of an anti-obesity product. The beautiful millettia root extract comprises at least one of an extract A and an extract B, wherein the preparation process of the extract A comprises the following steps: taking a dispersion liquid containing crude beautiful millettia root polysaccharide, adding the dispersion liquid into a cellulose anion exchange column for purification, eluting with water, and collecting an eluent, so as to obtain the extract A; and the preparation process of the extract B comprises the following steps: taking a dispersion liquid containing crude beautiful millettia root polysaccharide, adding the dispersion liquid into a cellulose anion exchange column for purification, eluting the dispersion liquid with a 0.35-0.45 mol / L NaCl solution, and collecting the eluent. Compared with the conventional beautiful millettia root extract, the beautiful millettia root extract disclosed by the invention shows higher biological activity in the aspect of resisting obesity, and has a wide application prospect in research and development of anti-obesity related products.

Description

Technical Field

[0001] The invention relates to the technical field related to the application of natural active extracts, and in particular to the application of a Herba Lycopodii extract in anti-obesity. Background Art

[0002] There is a profound and subtle relationship between the complex structure of plant polysaccharides and their biological activity in obesity regulation. This relationship is not only reflected in the basic level of monosaccharide composition, but also profoundly affects the type of glycosidic bonds, the wide distribution of molecular weight, and the subtle characteristics of higher-level structures (such as conformation, interchain interactions, etc.), which together construct a unique biological function map of plant polysaccharides.

[0003] In the complex mechanism of obesity regulation, the structural characteristics of plant polysaccharides play a crucial role. They may directly affect energy balance and fat accumulation by regulating the digestion rate, absorption efficiency and subsequent metabolic pathways in the body. Specifically, the molecular weight, branching degree and higher-order structural characteristics of polysaccharides can affect their physical form in the gastrointestinal tract, thereby regulating their residence time in the intestine, fermentation pattern and interaction with digestive enzymes, thereby slowing down the release and absorption of carbohydrates, helping to control blood sugar levels and energy intake.

[0004] In addition, plant polysaccharides can indirectly regulate obesity-related metabolism by affecting the intestinal microecological system, that is, the composition and function of the intestinal flora. As a substrate for intestinal flora, polysaccharides have a specific structure that can selectively promote or inhibit the growth of certain flora, thereby affecting the production of metabolites such as short-chain fatty acids. These metabolites play an important role in regulating host energy metabolism, fat synthesis and decomposition.

[0005] In recent years, with the development of biotechnology and health sciences, plant polysaccharides, as valuable components of natural resources, have become a global research hotspot due to their diverse biological activities and relatively low toxic side effects. However, given the high complexity of the structure and composition of polysaccharides, the biological activities of plant polysaccharide compositions obtained by different types and extraction methods may vary greatly due to differences in polarity, purity, structural integrity and other factors. Therefore, its regulatory effect on obesity is also difficult to predict. Summary of the invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes an application of a Herba Streptococci extract in anti-obesity, and the composition has a better effect in anti-obesity.

[0007] According to one aspect of the present invention, a use of a Herba Lycopodii extract in the preparation of an anti-obesity product is provided, wherein the Herba Lycopodii extract comprises at least one of extract A or extract B;

[0008] The preparation process of extract A comprises the following steps: adding a dispersion containing crude polysaccharide of Radix Codonopsis pilosulae to a cellulose anion exchange column for purification, eluting with water, and collecting the eluate to obtain the extract;

[0009] The preparation process of extract B comprises the following steps: taking a dispersion containing crude polysaccharide of Eleutherococcus argyi and adding it into a cellulose anion exchange column for purification, eluting it with a 0.35-0.45 mol / L NaCl solution, and collecting the eluate to obtain the extract.

[0010] According to a preferred embodiment of the present invention, at least the following beneficial effects are achieved: the extract of the present invention shows stronger biological activity in anti-obesity than the conventional extract of the present invention, which is specifically embodied in the following aspects: first, it can more effectively reduce the accumulation of lipids in fat cells, thereby reducing the accumulation of fat in the body; secondly, it can also significantly reduce the efficiency of energy conversion into fat after intake, which helps to control weight gain; furthermore, it has a positive effect on improving the health of liver cells, and can effectively alleviate liver cell damage and excessive lipid deposition in liver cells, thereby better protecting liver function; preferably, it can significantly reduce the volume and provide a new strategy for obesity management. Therefore, the extract of the present invention has broad application prospects in the research and development of anti-obesity related products. Furthermore, its preparation process is simple and has good industrial application prospects.

[0011] In some embodiments of the present invention, the cellulose anion exchange column is a DEAE-52 cellulose anion exchange column. Other cellulose anion exchange columns may also be used.

[0012] In some embodiments of the present invention, the weight average molecular weight of the crude polysaccharide of Radix Glehniae is 26.5-29.0 kDa, and the number average molecular weight is 20.5-22.5 kDa. For example, the weight average molecular weight (Mw) is 26.7, 27, 27.2, 27.5, 27.7, 28.0, 28.5, 28.7 kDa, etc. The number average molecular weight (Mn) is 20.7, 21.2, 21.5, 21.7 kDa, etc.

[0013] In some embodiments of the present invention, the monosaccharide composition of the crude polysaccharide of Radix Eupatorii includes glucose, arabinose, galactose, xylose, mannose, and glucosamine hydrochloride.

[0014] In some embodiments of the present invention, the crude polysaccharide of Radix Glehniae contains glucose, arabinose and galactose, and the molar percentage of the glucose, arabinose and galactose in the monosaccharide of the crude polysaccharide of Radix Glehniae is 60-63%, 14-16% and 12-14% respectively. The crude polysaccharide of Radix Glehniae is mainly composed of glucose, arabinose and galactose.

[0015] In some embodiments of the present invention, the monosaccharide composition of the crude polysaccharide of Eleutherococcus chinensis further includes at least one of glucuronic acid, fucose, rhamnose or galacturonic acid.

[0016] In some embodiments of the present invention, the extract A contains Eleutherodactyl polysaccharide I, which is a neutral polysaccharide rich in glucose (preferably, rich in glucose means that the molar percentage of glucose is more than 70% of the monosaccharide composition in the polysaccharide, preferably more than 71.3%).

[0017] In some embodiments of the present invention, the monosaccharide composition of the Eleutherodactyl polysaccharide I is glucose, galactose, arabinose, mannose, xylose, and glucosamine hydrochloride.

[0018] In some embodiments of the present invention, the molar percentage ratio of glucose, galactose, arabinose, mannose, xylose and glucosamine hydrochloride in the monosaccharide of the Herba Polysaccharides I is 71-72:13-14:11-12:2-3:1-2:0.4-0.6.

[0019] In some embodiments of the present invention, the molar percentage ratio of glucose, galactose, arabinose, mannose, xylose, and glucosamine hydrochloride in the Eleutherodactyl polysaccharide I is 71.3:13.0:11.5:2.4:1.3:0.5.

[0020] In some embodiments of the present invention, the extract B contains Eleutherodactyl polysaccharide II, which is an acidic polysaccharide rich in galacturonic acid (preferably, the rich in galacturonic acid means that the molar percentage of galacturonic acid is more than 27% of the monosaccharide composition in the polysaccharide, preferably more than 37.9%).

[0021] In some embodiments of the present invention, the monosaccharide composition of the Eleutherodactyl polysaccharide II is galacturonic acid, glucose, rhamnose, galactose, arabinose, fucose, glucuronic acid, mannose, xylose, and glucosamine hydrochloride.

[0022] In some embodiments of the present invention, the molar percentage ratio of galacturonic acid, glucose, rhamnose, galactose, arabinose, fucose, glucuronic acid, mannose, xylose, and glucosamine hydrochloride in the monosaccharides of the Eleutherodactyl polysaccharide II is 27.5-28.5:16.5-17.5:16-17:8-10:6-7:2.5-3.5:0.5-1:0.5-1:0.2-0.4.

[0023] In some embodiments of the present invention, the molar percentage ratio of galacturonic acid, glucose, rhamnose, galactose, arabinose, fucose, glucuronic acid, mannose, xylose, and glucosamine hydrochloride in the monosaccharides of the Eleutherodactyl polysaccharide II is 27.9:18.2:17.1:16.3:9:6.4:3.1:0.9:0.8:0.3.

[0024] In some embodiments of the present invention, the crude polysaccharide of Herba Lycopodii is prepared by the following steps: taking Herba Lycopodii raw materials, defatting by soaking, water extraction, alcohol precipitation, protein removal, and dialysis, and collecting components with a molecular weight range of 3500-14000Da.

[0025] In some embodiments of the present invention, the Herba Dioscoreae raw material is in powder form; preferably, the Herba Dioscoreae raw material can pass through a 40-mesh sieve.

[0026] In some embodiments of the present invention, the soaking and degreasing is performed by soaking and degreasing with ethanol-water solution I; wherein the mass volume ratio of ethanol-water solution I to Herba Glehniae is 6.3-7L:1.5kg.

[0027] In some embodiments of the present invention, the volume proportion of ethanol in the ethanol-water solution I is 90-99%.

[0028] In some embodiments of the present invention, the volume proportion of ethanol in the ethanol-water solution I is 95-99%, such as 98%.

[0029] In some embodiments of the present invention, the water extraction is specifically performed by adding 5.5 to 6.5 L of water per 500 grams of the solid phase after soaking and defatting, and boiling.

[0030] In some embodiments of the present invention, the alcohol precipitation is to concentrate the water-extracted extract to 1 / 12 of the original volume, and then mix the concentrate with the ethanol-water solution II, wherein the concentrate and the ethanol-water solution II are mixed at a volume ratio of 1:3.5 to 4.5, such as a volume ratio of 1:4.

[0031] In some embodiments of the present invention, the volume proportion of ethanol in the ethanol-water solution II is 75-85%, such as 80%.

[0032] In some embodiments of the present invention, the Sevag method is used to remove protein. The filter residue obtained by alcohol precipitation is added to water to dissolve the polysaccharide solution, and then the organic reagent is added in a ratio of 3.5 to 4.5:1 (such as 4:1) of chloroform to n-butanol. The polysaccharide solution and the organic reagent are mixed in a ratio of 4.5 to 5.5:1 (such as 5:1), shaken for 30 minutes, and allowed to stand until stratification.

[0033] According to another aspect of the present invention, the use of a Herba Lycopodii extract in preparing a product is provided, wherein the product has at least one of the following functions:

[0034] 1) Protect liver function;

[0035] 2) Reduce fat accumulation;

[0036] 3) Improve blood sugar metabolism disorders;

[0037] 4) Improve blood lipid metabolism disorders;

[0038] 5) Reduce oxidative stress levels;

[0039] 6) Lose weight;

[0040] The Herba Lysimachiae extract includes at least one of extract A or extract B;

[0041] The preparation process of extract A comprises the following steps: adding a dispersion containing crude polysaccharide of Radix Codonopsis pilosulae to a cellulose anion exchange column for purification, eluting with water, and collecting the eluate to obtain the extract;

[0042] The preparation process of extract B comprises the following steps: taking a dispersion containing crude polysaccharide of Eleutherococcus argyi and adding it into a cellulose anion exchange column for purification, eluting it with a 0.35-0.45 mol / L NaCl solution, and collecting the eluate to obtain the extract.

[0043] In some embodiments of the present invention, the protecting liver function comprises at least one of reducing liver hypertrophy, reducing liver cell damage, and reducing liver lipid accumulation.

[0044] In some embodiments of the present invention, the reducing fat accumulation is reducing liver fat accumulation.

[0045] In some embodiments of the present invention, the improvement of blood sugar metabolism disorder includes at least one of improving glucose tolerance and / or improving insulin sensitivity.

[0046] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0048] Figure 1 It is the elution curve of the embodiment of the present invention and comparative example 1.

[0049] Figure 2It is the ultraviolet spectra of the extracts in the examples of the present invention and comparative examples 1-2.

[0050] Figure 3 It is the infrared spectra of the extracts in the examples of the present invention and comparative examples 1-2.

[0051] Figure 4 1 is a molecular weight distribution diagram of a) NSP1 and b) NSP3 in the examples of the present invention.

[0052] Figure 5 It is the chromatogram of the standard product detected in the test example of the present invention.

[0053] Figure 6 This is a chromatogram when NSP1 is detected in the test example of the present invention.

[0054] Figure 7 This is a chromatogram when NSP3 is detected in the test example of the present invention.

[0055] Figure 8 It is a schematic diagram of the animal experiment grouping and drug administration design in the test examples of the present invention.

[0056] Fig. 9 Graph showing changes in mouse body weight in the test examples of the present invention.

[0057] Fig.10 It is a bar graph of the average feed intake and average energy intake of mice in the test examples of the present invention.

[0058] Fig.11 It is the organ weight and index of each group of mice in the test example of the present invention.

[0059] Fig.12 1 is a graph showing the test results of liver tissue of each group of mice in the test examples of the present invention.

[0060] Fig.13 1 is a graph showing the fat weight of mice in each group and the H&E staining results in the test examples of the present invention.

[0061] Fig.14 1 is a graph showing the blood glucose test results of each group of mice in the test example of the present invention.

[0062] Fig.15 It is a graph showing the glucose tolerance and insulin tolerance test results of each group of mice in the test example of the present invention.

[0063] Fig.16 It is a graph showing the test results of blood lipid levels of each group of mice in the test example of the present invention.

[0064] Fig.17 This is a graph showing the test results of AST and ALT levels in the liver and serum of each group of mice in the test examples of the present invention.

[0065] Fig.18It is a graph showing the test results of oxidative stress indexes of each group of mice in the test examples of the present invention. DETAILED DESCRIPTION

[0066] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of them. Based on the embodiments of the present invention, other embodiments obtained by technicians in this field without creative work are all within the scope of protection of the present invention. The test methods used in the embodiments are conventional methods unless otherwise specified; the materials, reagents, etc. used, unless otherwise specified, can be reagents and materials obtained from commercial channels. Unless otherwise specified, the same parameter in each embodiment has the same value. The embodiments described below are exemplary and are only used to explain the present invention, and cannot be understood as limitations on the present invention.

[0067] In the description of the present invention, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0068] The sources of the main reagents used in the following examples are shown in Table 1 below, and the information of the instruments and equipment used is shown in Table 2 below.

[0069] Table 1

[0070]

[0071]

[0072] Note: All reagents were of analytical grade.

[0073] Table 2

[0074]

[0075]

[0076] Example

[0077] This example provides an application of a Herba Lycopodii extract in anti-obesity.

[0078] (I) The specific process of extracting the herb is as follows:

[0079] Extraction of crude polysaccharide from S1

[0080] ① Grinding of Niu Da Li: Take an appropriate amount of Niu Da Li (purchased from Fengkai County Market), dry it, grind it with a grinder, and sieve it through a 40-mesh sieve. Then, place the sieved powder in a dryer and dry it at 40°C for 3 to 4 hours.

[0081] ② Soaking and defatting: Mix 7kg of Radix Codonopsis powder with 98% alcohol in a ratio of 7L alcohol to 1.5kg of powder, and soak for 24 hours. Filter through gauze to obtain the filter residue, and evaporate the alcohol in the filter residue. ③ Water extraction: Add 6L water to one pound of filter residue and boil for 2 hours. Repeat this process twice, filter through gauze after each boiling, and centrifuge. Combine the two water extracts and concentrate to 1000mL.

[0082] ④ Alcohol precipitation: Mix the concentrate with ethanol (80 vol%) in a ratio of 1:4, place in a 4°C refrigerator and let stand for 48 hours, then filter to obtain the residue.

[0083] ⑤ Sevag method for protein removal: Add the filter residue to 300 mL of pure water and then add an organic reagent in a volume ratio of chloroform to n-butanol of 4:1. Mix the filter residue-water mixture with the organic reagent in a volume ratio of 5:1, shake in a conical flask for 30 minutes, and let stand until the layers are separated. Repeat this process several times until there is no intermediate layer of protein.

[0084] ⑥ Dialysis: The protein-free solution was placed in a dialysis bag (molecular weight range 3500-14000Da) and dialyzed in ultrapure water. Stir with a magnetic stirrer and dialyze for 48 hours, during which the ultrapure water was replaced every six hours.

[0085] ⑦ Centrifuge the polysaccharide solution at 8000 rpm / min for 5 min, freeze it in a -20°C refrigerator, and freeze-dry it in a freeze dryer to obtain crude polysaccharide.

[0086] S2 DEAE-52 cellulose resin purification

[0087] (1) DEAE-52 cellulose pretreatment

[0088] Weigh 100g DEAE-52 fiber resin in 500mL deionized water, stir gently until foaming, and remove the suspended particles with a pipette. After 5 hours, remove the water with a sand core filter. Activate the resin with 0.5mol / L sodium hydroxide, 0.5mol / L hydrochloric acid, and 0.5mol / L sodium hydroxide.

[0089] (2) Column Packing and Balancing

[0090] Fix the chromatography column and add DEAE-52 into the column slowly and evenly along the glass rod until the resin is 4 cm to 5 cm away from the upper end of the column. Stop filling the column and balance it with deionized water (1 mL / min) for 12 hours.

[0091] (3) Sample loading

[0092] Weigh an appropriate amount of crude polysaccharide from Radix Codonopsis pilosulae to prepare a 50 mg / mL solution, centrifuge (4500 rpm / min) for 10 min, take the supernatant and filter it through a 0.45 μm microporous filter membrane, slowly add the crude polysaccharide solution from Radix Codonopsis pilosulae to the DEAE-52 cellulose chromatography column, elute with water or 0.4 mol / L sodium chloride solution, collect the solution with an automatic collector, and draw an elution curve.

[0093] The above operation was repeated several times, and the corresponding components were collected and combined, and freeze-dried.

[0094] Among them, the component collected after elution with water is the Herba Lycopodii Extract A (hereinafter referred to as "NSP1"), and the component collected after elution with 0.4 mol / L sodium chloride solution is the Herba Lycopodii Extract B (hereinafter referred to as "NSP3").

[0095] Comparative Example 1

[0096] This example provides an application of a Herba Lycopodii extract in anti-obesity. The Herba Lycopodii extract is an extract obtained by eluting with a DEAE-52 cellulose chromatography column using an eluent of 0.2 mol / L sodium chloride solution (hereinafter referred to as "NSP2") in the example.

[0097] Comparative Example 2

[0098] This example provides an application of a Herba Lycopodii extract in anti-obesity treatment, which differs from the embodiment in that it only includes a crude polysaccharide extraction process (hereinafter referred to as "NSP").

[0099] Test example

[0100] This example tests the components and contents of the extracts of Herba Lycopodii in the above examples and comparative examples and their biological activities in the field of anti-obesity.

[0101] (I) Composition and content test

[0102] 1. Determination of total sugar content of polysaccharides

[0103] Prepare 1.00 mg / mL glucose mother solution, and prepare the mother solution into solutions with concentrations of 0.02 mg / mL, 0.04 mg / mL, 0.06 mg / mL, 0.08 mg / mL, 0.10 mg / mL, 0.12 mg / mL, and 0.14 mg / mL. Take 200 μL of each dilution of the above concentrations, add 100 μL of 6% phenol and 500 μL of concentrated sulfuric acid. Mix well and return to room temperature. Measure the absorbance with an enzyme marker (490 nm) and draw a standard curve.

[0104] Total sugar content of polysaccharide sample: Weigh 40 mg of crude polysaccharide sample and dilute to 40 mL with distilled water to obtain a 10 mg / mL solution. Take 40 μL of it and add 3960 μL of distilled water to dilute to 0.1 mg / mL. Then, measure the absorbance of the solution according to the operating steps of the sulfuric acid-phenol method.

[0105] When NSP was purified by DEAE-52, the elution curve was as follows: Figure 1 As shown. Three elution peaks were obtained: water elution component (NSP1), 0.2mol / LNaCl elution component (NSP2) and 0.4mol / LNaCl elution component (NSP3). The components were collected and vacuum freeze-dried to obtain the polysaccharides of each component. The yields of NSP1, NSP2 and NSP3 were 21.9%, 13.09% and 9.1% of NSP, respectively; the polysaccharide contents of NSP, NSP1, NSP2 and NSP3 were measured by the sulfuric acid phenol method and were 42.6%, 70.3%, 65.51% and 69.31%, respectively, indicating that after purification by DEAE-52 cellulose, the polysaccharide contents of NSP1, NSP2 and NSP3 were increased.

[0106] 2. Ultraviolet spectrum analysis

[0107] The extract of Herba Lysimachiae was dissolved in ultrapure water to prepare a polysaccharide solution with a concentration of 1 mg / mL, and the ultraviolet absorption spectrum of the polysaccharide solution was scanned (190-600 nm) by a UV-visible spectrophotometer.

[0108] The UV spectra of NSP, NSP1, NSP2 and NSP3 are as follows Figure 2 As shown in the figure, it can be seen that aromatic amino acids (such as tryptophan, tyrosine, and phenylalanine) in proteins have characteristic absorption peaks near 280nm, and NSP has an absorption peak near 280nm, indicating that it contains protein; NSP1, NSP2, and NSP3 do not have obvious absorption peaks near 280nm, indicating that these two components do not contain protein or the protein content is extremely low after purification. Chlorophyll, carotenoids, etc. usually have characteristic absorption peaks near 380nm, and NSP, NSP1, NSP2, and NSP3 have no absorption peaks at 380nm, indicating that these pigments are not contained or the pigment content is extremely low.

[0109] 3. Fourier transform infrared spectroscopy (FT-IR) analysis

[0110] The infrared absorption spectrum of the polysaccharide samples of Radix Codonopsis pilosulae was determined by KBr tablet method.

[0111] The results of infrared spectrometry of NSP, NSP1, NSP2 and NSP3 are as follows Figure 3 As shown in the figure, after analyzing the infrared spectra of polysaccharides, it was found that NSP, NSP1, NSP2, and NSP3 had a peak at 3400 cm -1 Nearby and 2920cm -1 There are peaks near the -OH and CH stretching vibration peaks, 1650cm -1 The sharp peak near the position is attributed to the OH stretching vibration, which is likely caused by the binding of polysaccharides to water molecules. -1 The peaks near 1545cm may be caused by the angular vibration of CH. These two peaks together with the other two characteristic peaks constitute the typical absorption characteristic peaks of polysaccharides. -1 The peaks on the left and right indicate the in-plane bending vibration of NH. NSP, NSP1, NSP2, and NSP3 have peaks at 1240 cm -1 The peaks around are assigned to the CO vibrations of the O-acetyl group; the peaks at 1200-1000 cm -1 The peaks in the region may be attributed to COH and COC stretching vibrations and 700–500 cm -1 The peaks in the wavelength range all indicate the characteristic absorption peaks of the pyranose ring. NSP1 is at 930cm -1 The peaks around 1740 cm-1 are probably due to the antisymmetric ring vibration absorption peaks of D-glucose isomers. -1 The absorption peak corresponds to the carbonyl (C=O) vibration peak, indicating that NSP3 may contain uronic acid.

[0112] 4. Molecular weight determination

[0113] (1) Reagent preparation

[0114] Preparation of 0.05mol / L NaCl mobile phase solution: weigh 5.844g NaCl, add pure water to dissolve, transfer to a 2L volumetric flask, ultrasonicate for 10min, and filter through a 0.22μm filter membrane.

[0115] Preparation of standard solution: Accurately weigh 5 mg of each standard (dextran), dissolve in 1 mL of mobile phase solution to prepare a 5 mg / mL solution, and transfer the sample to a 1.8 mL injection vial.

[0116] Preparation of sample solution: Accurately weigh 5 mg of sample and dissolve it in 1 mL of mobile phase solution to prepare a 5 mg / mL solution. After vortexing to dissolve, centrifuge for 10 min (12000 rpm / min) and pass the supernatant through a water-based microporous filter membrane (0.22 μm).

[0117] (2) Chromatographic conditions

[0118] Mobile phase: 0.05 mol / L NaCl solution; Chromatographic column: BRT105-103-101 series gel column (8×300 mm); Flow rate: 0.7 mL / min; Column temperature: 40°C; Injection volume: 25 μL; Detector: Differential detector RID-20A; Analysis time: 70 min

[0119] (3) Analysis steps

[0120] Place the standard in the sample injection plate, select the above-mentioned chromatographic method for analysis, obtain the retention time, draw the lgMp-RT (Mp peak molecular weight), lgMw-RT (Mw weight average molecular weight), lgMn-RT (Mn number average molecular weight) standard curves, and obtain the molecular weight calculation formula.

[0121] Place the sample in the sample injection tray, select the above-mentioned chromatographic method for analysis, obtain the chromatogram and retention time, and substitute the retention time into the formula to obtain the molecular weight (Mp, Mw, Mw).

[0122] The equation of the lgMp-RT calibration curve is: y = -0.203x + 11.86, R 2 =0.993; lgMw-RT calibration curve equation is: y = -0.205x + 11.91, R 2 =0.993; lgMn-RT calibration curve equation: y = -0.205x + 11.90, R 2 =0.993; Based on the standard curve, the calculation formula was obtained to calculate the molecular weight of each sample, and the results are shown in Table 3.

[0123] Table 3

[0124]

[0125] The number average molecular weight (Mn) is the sum of the molecular weights of all polymers divided by the total number of polymer molecules, and the weight average molecular weight (Mw) is the sum of the product of the mass of each polymer molecule and the total mass divided by the total mass of the polymer molecules. It places more emphasis on the contribution of macromolecules to the overall molecular weight. The molecular weight information of NSP1 and NSP3 is shown in Table 3. Figure 4As shown. The molecular weight distribution of NSP1 polysaccharide components showed two peaks. The peak area of ​​NSP1-1 accounted for 54.63%, the number average molecular weight (Mn) and weight average molecular weight (Mw) were 31.5 kDa and 35.2 kDa, respectively, and the polydispersity coefficient was 1.1179 (Mw / Mn); the peak area of ​​NSP1-2 was 45.37%, the number average molecular weight (Mn) and weight average molecular weight (Mw) were 31.5 kDa and 35.2 kDa, respectively, and the polydispersity coefficient was 1.1179 (Mw / Mn). W ) were 8.0 kDa and 10.0 kDa respectively, and the polydispersity coefficient was 1.2588.

[0126] The peak area of ​​NSP3-1 is 14.45%, the number average molecular weight (Mn) and weight average molecular weight (Mw) are 586.2kDa and 599.8kDa respectively, and the polydispersity coefficient is 1.0232; the peak area of ​​NSP3-2 accounts for 85.55%, the number average molecular weight (Mn) and weight average molecular weight (Mw) are 29.1kDa and 29.8kDa respectively, and the polydispersity coefficient is 1.0240. The polydispersity coefficient is used to measure the width of the molecular weight distribution of polysaccharides. When the polydispersity coefficient of polysaccharides is closer to 1, it means that the molecular weight distribution of polysaccharides is more concentrated, that is, the molecular size of polysaccharides in the sample is relatively consistent. NSP1-1, NSP1-2, NSP3-1, and NSP3-2 are all close to 1, indicating that the molecular weight distribution of polysaccharides is more concentrated after purification by DEAE-52. The weight average and number average molecular weights (Mw and Mn) of crude polysaccharide of Radix Codonopsis pilosulae are 27.7 kDa and 21.2 kDa, respectively, and the polydispersity coefficient is 1.3066.

[0127] 5. Determination of monosaccharide composition

[0128] (1) Preparation and calculation methods of standards and samples

[0129] Take 15 kinds of commercially available monosaccharide standards (the main parameters are shown in Table 4), take appropriate amounts of monosaccharide standards, add 2 mL of 3 mol / L trifluoroacetic acid (TFA), hydrolyze at 120 °C for 3 h, blow dry with nitrogen, add deionized water and vortex mix to prepare standard mother solution.

[0130] Take each monosaccharide standard solution and accurately prepare the concentration standard as the mixed standard. According to the absolute quantitative method, the mass of different monosaccharides is measured, and the molar ratio is calculated according to the molar mass of the monosaccharides. The test results are as follows Figure 5 The calculated results are shown in Table 4.

[0131] Table 4 Standard product information

[0132]

[0133] (2) Sample preparation

[0134] 5 mg of sample was accurately weighed and placed in an ampoule. The hydrolysis method was the same as that of the standard. 100 μL of the sample solution after hydrolysis was taken and added to 900 μL of deionized water. Centrifuged for 5 min (12000 rpm / min). The supernatant was taken and analyzed by ion chromatography for the monosaccharide components of NSP1 and NSP3.

[0135] (3) Chromatographic method

[0136] Column: Dionex Carbopac TM PA20 (3*150mm); mobile phase: A: H2O; B: 15mM NaOH; C: 15mMNaOH&100mM NaAc; flow rate: 0.3mL / min; injection volume: 25μL; column temperature: 30℃.

[0137] Elution gradient: 0-18 min phase A / phase B / phase C (98.8:1.2:0, V / V), 20-30 min phase A / phase B / phase C (50:50:0, V / V), 30.1-46 min phase A / phase B / phase C (0:0:100, V / V), 46.1-50 min phase A / phase B / phase C (0:100:0, V / V), 50.1-80 min phase A / phase B / phase C (98.8:1.2:0, V / V).

[0138] Detector: Electrochemical detector.

[0139] The test results of NSP1 are shown in Table 5 and Figure 6 shown.

[0140] Table 5

[0141]

[0142] The test results of NSP3 are shown in Table 6 and Figure 7 shown.

[0143] Table 6

[0144]

[0145]

[0146] From Table 5 and Figure 6 It can be seen that the monosaccharide composition of NSP1 was determined by comparing the peak time of each monosaccharide with that of the standard. The results showed that the main monosaccharide components of NSP1 were glucose, galactose, arabinose, mannose, xylose and glucosamine hydrochloride, and their contents (molar percentage) were 71.3%, 13.0%, 11.5%, 2.4%, 1.3% and 0.5%, respectively, indicating that NSP1 is a neutral polysaccharide rich in glucose. From Tables 6 and Figure 7It can be seen that by comparing the peak time of each monosaccharide of NSP3 with the standard, the monosaccharide composition results show that NSP3 is composed of galacturonic acid, glucose, rhamnose, galactose, arabinose, fucose, glucuronic acid, mannose, xylose, and glucosamine hydrochloride, and their contents (molar percentage) are 27.9%, 18.2%, 17.1%, 16.3%, 9%, 6.4%, 3.1%, 0.9%, 0.8%, and 0.3%, respectively, indicating that NSP3 is an acidic polysaccharide rich in galacturonic acid. The crude polysaccharide of the Herba Lysimachiae used in the embodiment of the scheme of the present invention is mainly composed of glucose (62.6%), arabinose (15.3%), galactose (13.4%), rhamnose (2.9%), and galacturonic acid (2.8%).

[0147] According to existing research, changes in the physical properties of polysaccharides may affect the bioavailability of polysaccharides by the human intestinal flora. The molecular size, solubility, and monosaccharide composition of polysaccharides will significantly affect the speed and extent of their fermentation in the intestine.

[0148] In summary, the component analysis shows that:

[0149] (1) NSP was separated into three components, NSP1, NSP2 and NSP3, by DEAE-52, with yields of 21.9%, 13.09% and 9.1%, respectively; the polysaccharide contents of NSP, NSP1, NSP2 and NSP3 were 42.6%, 70.3%, 65.51% and 69.31%, respectively. NSP contained a small amount of impurities such as proteins and nucleic acids; NSP1, NSP2 and NSP3 were free of proteins and nucleic acids. All components of polysaccharides were at 3400cm -1 、2920cm -1 、1650cm -1 、1410cm -1 Nearby, typical polysaccharide absorption peaks appear, corresponding to the vibration of -OH and CH. NSP, NSP1, NSP2, and NSP3 have absorption peaks of 1200-1000 cm -1 Area and 700-500cm -1 The peaks in the wavelength range indicate that pyranose rings are very likely to exist in NSP, NSP1, NSP2, and NSP3. -1 The peaks around 1740 cm-1 are probably due to the antisymmetric ring vibration absorption peaks of D-glucose isomers. -1 The absorption peak corresponds to the carbonyl (C=O) vibration peak, indicating that NSP3 may contain uronic acid.

[0150] (2) The molecular weight distribution of NSP1 showed two main peaks. The peak area of ​​NSP1-1 accounted for 54.63%, the weight average molecular weight (Mw) was 35.26 kDa, and the polydispersity coefficient was 1.12; the peak area of ​​NSP1-2 accounted for 45.37%, the weight average molecular weight (Mw) was 10.08 kDa, and the polydispersity coefficient was 1.26; NSP1 was mainly composed of glucose (71.3%). The molecular weight distribution of NSP3 mainly showed one main peak, with a peak area of ​​85.55%, a weight average molecular weight (Mw) of 29.17 kDa and 29.85 kDa, and a molecular weight distribution of 1.02; NSP3 was mainly composed of galacturonic acid (27.9%), glucose (18.2%), rhamnose (18.2%), and galactose (16.3%).

[0151] (II) Anti-obesity effect test

[0152] A high-fat diet-induced obese mouse model was used to evaluate the effects of Rhizoma Dioscoreae extract on obesity.

[0153] Sixty C57BL / 6 male mice (6 weeks old) weighing 15 ± 0.5 g were housed in Jiangmen Big Health International Innovation Institute in strict accordance with animal husbandry regulations. During the experiment, the mice were housed in standard cages with adequate food and water ad libitum, while maintaining a 12-hour light and 12-hour dark day-night cycle to simulate the lighting conditions in the natural environment.

[0154] (1) High-fat diet-induced obesity model

[0155] The animals were randomly divided into groups, with 10 animals in each group, and were given normal diet or high-fat diet and gavage with 100 mg / kg / d NSP, NSP1, NSP2, and NSP3 for 13 weeks. Figure 8 As shown:

[0156] CHOW: water / normal diet (10% fat, 3.5 kcal / g); HFD: water / high-fat diet (45% fat, 4.73 kcal / g); NSP: water / high-fat diet (45% fat, 4.73 kcal / g) / 100 mg / kg / dNSP; NSP1: water / high-fat diet (45% fat, 4.73 kcal / g) / 100 mg / kg / dNSP1; NSP2: water / high-fat diet (45% fat, 4.73 kcal / g) / 100 mg / kg / dNSP2; NSP3: water / high-fat diet (45% fat, 4.73 kcal / g) / 100 mg / kg / dNSP3.

[0157] The body weight of mice was monitored weekly. Fig. 9As shown (Figure, N = 10. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001. ns indicates no statistical significance). At the beginning of the experiment, the initial body weight of mice in each group was roughly the same, and showed a gradual increase trend as time went by. Before the 7th week of gavage, except for the CHOW group, there was no significant difference in body weight gain between the other groups and the HFD group. However, starting from the 7th week, the weight gain rate of mice in each drug-treated group began to slow down compared with that in the HFD group. When the experiment reached the 13th week, the weight of mice in the HFD group increased significantly compared with that in the normal diet group (P<0.0001), indicating that the obese mouse model was successfully established. Compared with the HFD group, the weight of mice in each group gavaged with Botrytis cava polysaccharide decreased, among which the weight of NSP, NSP1, and NSP3 groups decreased significantly (P<0.01), indicating that the NSP, NSP1, and NSP3 polysaccharide components can reduce the weight of mice induced by a high-fat diet.

[0158] The food intake of each group of mice was recorded weekly. Fig.10 As shown in the figure (N=10. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001. ns indicates no statistical significance). Compared with the CHOW group, after being given a high-fat diet, the food intake of mice in both the HFD group and the drug-treated group was significantly reduced (P<0.0001). Compared with the HFD group, the food intake of mice in the NSP group and the NSP3 group decreased significantly (P<0.01). From the energy intake chart, it can be seen that compared with the CHOW group, the energy intake of the HFD group was higher (P<0.01); compared with the HFD group, the energy intake of the NSP and NSP3 groups decreased (P<0.01). This shows that the polysaccharides in the NSP and NSP3 groups may reduce the weight of mice by reducing the energy intake of mice.

[0159] (2) Anatomical sampling

[0160] One week before the end of the experiment, fresh feces of mice were collected. At the end of the experiment, mice were dislocated from the neck, blood was collected from the ocular artery, centrifuged statically, serum was packaged, and relevant indicators were measured; the liver, heart, spleen, and kidney were separated and weighed, and the liver and colon were collected, part of which was frozen in liquid nitrogen and the other part was fixed in 4% paraformaldehyde for subsequent experiments.

[0161] Intake of high-calorie foods will cause the body to accumulate more energy than it needs, causing fat to accumulate in the body. When fat accumulates in the liver, it may cause fatty liver. Therefore, the weight of the heart, liver, kidney, and spleen can reflect the degree of obesity in mice. Fig.11 As shown in the figure (N=10. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001. ns indicates no statistical significance). Organ weight and organ index showed that compared with the CHOW group, the high-fat diet had the greatest effect on the mouse liver compared with other organs (P<0.0001). Different components of Rhizoma Dioscoreae polysaccharides had different degrees of relief effects on the liver index and liver weight increase of obese mice induced by high-fat diet. Among them, the effect of alleviating the increase of liver weight in mice in NSP1 and NSP3 groups was better (P<0.0001). Compared with the HFD group, the liver index of NSP1 group was extremely significantly reduced (P<0.001), and the liver index of NSP3 group was significantly reduced (P<0.05). It can be seen that NSP1 and NSP3 polysaccharide components can significantly improve liver hypertrophy induced by a high-fat diet.

[0162] A high-fat diet can cause structural disorder in liver cells, with loose and crowded hepatocytes. At the same time, due to the accumulation of fat in the liver, H&E staining will show transparent or vacuolar structures in the hepatocytes. Fig.12 As shown (in the figure, N = 6. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001. ns indicates no statistical significance). Fig.12 As shown in a), the liver of the CHOW group was moderate in size and bright red in color, while the liver of the HFD group was significantly larger and light brown in color. The size and color depth of the liver of each drug-treated group were between the CHOW group and the HFD group. Fig.12bc) H&E staining of the liver and semi-quantitative analysis of hepatocyte nuclei. The liver cells of the CHOW group mice were neatly arranged, with intact morphology, clear boundaries, and no vacuolar structures. However, in the HFD group, more and larger vacuoles appeared, which was the result of lipid accumulation in the liver. At the same time, the boundaries between hepatocytes were unclear, and the hepatocyte nuclei were reduced and crowded. The high-fat diet led to obvious changes in liver morphology. After the administration of NSP, NSP1 and NSP3, the situation of liver vacuoles and the reduction of hepatocyte nuclei (NSP1, P<0.0001; NSP3, P<0.01) were alleviated to varying degrees. The tissue morphology and number of nuclei in the NSP2 group were no different from those in the HFD group. These results show that the polysaccharide components of NSP1 and NSP3 have a certain improvement effect on liver cell damage and lipid accumulation caused by a high-fat diet.

[0163] White adipose tissue is the main part of the body responsible for storing excess energy. It stores chemical energy in the form of triglycerides. These adipose tissues are widely distributed in the subcutaneous layer and around certain internal organs (such as the epididymis and kidneys). The weight and index of white fat can reflect the obesity level of mice. In order to evaluate the effects of various polysaccharide components of Botrytis cinerea on white fat in mice, subcutaneous, epididymal and perirenal fat were separated and weighed, and the fat coefficient (i.e., the percentage of body weight) was calculated. The results are as follows: Fig.13 As shown (in the figure, N = 8. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001. ns indicates no statistical significance). Fig.13 af), compared with the CHOW group, the weights of perirenal fat, subcutaneous fat and epididymal fat in the HFD group were significantly increased (P<0.0001), and the fat index was also significantly increased (P<0.0001), indicating that a high-fat diet would increase fat accumulation in multiple parts of the body of mice; compared with the HFD group, the weights and indexes of perirenal and epididymal fat of each polysaccharide component were significantly different, the weights and indexes of subcutaneous fat in the NSP and NSP1 groups were also significantly different (P<0.01, P<0.05), while the weights of subcutaneous fat in the NSP2 and NSP3 groups were significantly different, but the indexes were not statistically different. Fig.13gh) H&E staining of epididymal fat found that after feeding with various polysaccharide components of Rhizoma Coptidis, the increase in fat cell volume was reduced to varying degrees compared with the HFD group, among which NSP, NSP1 and NSP3 had the most obvious reduction effects. In summary, various polysaccharide components of Rhizoma Coptidis can reduce the accumulation of lipids in fat cells to varying degrees. Studies have shown that increased body fat is closely related to adipocyte dysfunction, which manifests as metabolic dysfunctions such as decreased insulin sensitivity. Each polysaccharide component can reduce lipid accumulation to varying degrees, which may indicate that each polysaccharide component will also increase the insulin sensitivity of mice to varying degrees.

[0164] (3) Oral glucose tolerance test (OGTT)

[0165] Two weeks before the end of the experiment. The mice were fasted for 12 hours but not water. A 200 mg / mL D-glucose solution was prepared and intraperitoneally injected with 0.1 mL / 10 g of glucose solution. Blood was collected from the tail vein of the mice at 0, 15, 30, 60, and 120 min after injection. The blood glucose levels were measured with a Roche blood glucose meter and recorded in a table. The area of ​​the glucose tolerance curve was calculated using GraphPad Prism 9.0 software. Fig.14 a)-c) (in the figure, N=10. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001. ns indicates no statistical significance).

[0166] (4) Insulin tolerance test (ITT)

[0167] One week before the end of the experiment, an insulin tolerance test was performed. The mice were fasted but not watered for 6 hours, and 300U / mL of protamine recombinant human insulin was diluted to 0.03U / mL using normal saline. The insulin solution was injected intraperitoneally into the mouse at a volume ratio of 0.1mL / 10g. Blood was collected from the tail vein of the mice at 0, 15, 30, 60, 90 and 120min, and the blood glucose level was measured with a Roche blood glucose meter and recorded in a table. These data were analyzed using GraphPad Prism 9.0 software, and the results are shown below. Fig.14 de) (in the figure, N=10. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001. ns indicates no statistical significance).

[0168] In obese individuals, elevated blood sugar and insulin resistance usually occur. Due to insulin resistance, the risk of diabetes increases, and fasting blood sugar level is one of the key parameters for diagnosing diabetes. In order to explore the effect of radix sutchuenensis polysaccharides on the glucose metabolism of mice fed a high-fat diet, the mice were gavaged with glucose solution, and the blood sugar levels at different time points were tested to evaluate the potential effect of radix sutchuenensis polysaccharides on improving glucose metabolism. Fig.14 ac). The fasting blood glucose values ​​of mice in the CHOW group were normal, while those of mice in the HFD group increased significantly (P<0.0001), and the area under the glucose tolerance curve also increased significantly (P<0.0001). However, the fasting blood glucose values ​​of mice in the NSP1 and NSP3 groups decreased significantly (P<0.0001). There was no statistical difference in fasting blood glucose between the NSP and NSP2 groups, but the area under the glucose tolerance curve decreased significantly (P<0.01, P<0.05). It can be seen from the area under the curve that, compared with the HFD group, different polysaccharide components of Rhizoma Dioscoreae can increase the body's sensitivity to glucose to varying degrees. The NSP1 and NSP3 components have the best effect.

[0169] The insulin tolerance test (ITT) is used to evaluate the insulin sensitivity of an individual. Blood is collected from the tail tip of the mouse, and a blood glucose meter is used to monitor the changes in blood glucose over time after the mouse receives intraperitoneal insulin injection. Fig.14 de), at 15 minutes, except for the CHOW group, the blood glucose levels of all groups of mice dropped to the lowest point, and then slowly recovered. The area under the insulin tolerance curve showed that compared with the HFD group, all polysaccharide treatment groups showed significant differences (P<0.001, P<0.0001). Comprehensive analysis showed that each component of the polysaccharide from Rhizoma Dioscoreae could improve the insulin sensitivity of obese mice to varying degrees, among which the effects of the NSP group, NSP1 group and NSP3 group were better than those of the NSP2 group.

[0170] Glucose tolerance reflects the body's ability to process glucose. Increased glucose tolerance means that the body can effectively convert glucose into energy or store it after ingesting it, which means that insulin sensitivity is improved, allowing the body to convert glucose into energy or store it as glycogen more quickly after ingesting it, rather than allowing blood sugar levels to continue to rise. Decreased insulin tolerance usually means that the body's responsiveness to insulin is enhanced, that is, less insulin can produce the same blood sugar-lowering effect. This section found that different polysaccharide components of Botrytis cinerea can increase the body's sensitivity to glucose and improve insulin sensitivity to varying degrees. The experimental results are similar to the fact that each polysaccharide component of Botrytis cinerea can reduce the accumulation of lipids in fat cells to varying degrees. It may be that each polysaccharide component of Botrytis cinerea reduces the accumulation of lipids in fat cells, thereby increasing insulin sensitivity.

[0171] (5) Serum biochemical index detection

[0172] The mouse blood was placed in an EP tube, allowed to stand for 3 hours, and then centrifuged (4°C, 1500rpm / 15min). The supernatant was taken, packaged, and frozen at -80°C. The corresponding kits were used to measure the levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and superoxide dismutase (SOD) in the serum. The results are as follows: Fig.15 As shown (N=8. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001. ns indicates no statistical significance).

[0173] Dyslipidemia is usually accompanied by abnormal increase of TC, TG, and LDL-C in the blood, while HLD-C decreases. Obesity caused by a high-fat diet is a major risk factor for hyperlipidemia and other related diseases. Persistent hyperlipidemia is positively correlated with abnormal lipid accumulation in the liver and may lead to non-alcoholic fatty liver disease. Fig.15 As shown in the figure, in order to evaluate the effect of edible polysaccharide components of Radix Codonopsis pilosulae on blood lipid metabolism in mice, the TC, TG, LDL-C and HDL-C levels in the serum of each experimental group were measured. Compared with the CHOW group, the concentrations of serum TC, TG and LDL-C in the HFD group were significantly increased (P<0.001, P<0.0001); compared with the HFD group, the serum TG content in the NSP group was significantly decreased (P<0.01), the serum TC and TG content in the NSP1 group was extremely significantly decreased (P<0.001, P<0.0001), the changes in serum TC, TG and LDL-C content in the NSP2 group were not statistically significant, and the serum TC, TG and LDL-C content in the NSP3 group was extremely significantly decreased (P<0.0001, P<0.01). There was no statistical difference in serum HDL-C content between the HFD group and the CHOW group and between the drug-treated groups. In summary, the NSP, NSP1 and NSP3 fractions of Eleutherococcus polysaccharides showed a certain effect in improving lipid metabolism disorders, among which the lipid-lowering effects of the NSP1 and NSP3 groups were the most significant.

[0174] (6) Determination of liver biochemical indicators

[0175] 1) Determination of liver AST and ALT content: Weigh an appropriate amount of liver tissue, add 9 times normal saline (according to the weight-to-volume ratio), homogenize at low temperature, centrifuge for 10 minutes (2500r / min), take the supernatant and dilute it with normal saline to an appropriate concentration; take the supernatant and use the BCA protein quantitative kit to determine its protein concentration. The content determination is performed according to the steps of Nanjing Jiancheng AST and ALT kit.

[0176] 2) Determination of liver TC and TG content: Accurately weigh an appropriate amount of liver tissue, add 9 times normal saline (based on the weight-to-volume ratio), homogenize at low temperature, centrifuge for 10 minutes (2500r / min), and take the supernatant for testing. The content determination is performed according to the steps of Nanjing Jiancheng TC and TG kits.

[0177] 3) Determination of liver MDA, GSH, and GSH-PX content: ① Sample preparation for determination of MDA content in liver tissue: Weigh an appropriate amount of liver tissue, add the extract in the MDA kit at a ratio of weight (g): volume (mL) = 1:10, homogenize in an ice bath, centrifuge at 8000r / min, 4℃ for 10min, take the supernatant and operate according to the steps of Nanjing Jiancheng MDA kit. ② Sample preparation for determination of GSH content in liver tissue: Weigh an appropriate amount of liver tissue, add reagent 1 in the GSH kit at a ratio of weight (g): volume (mL) = 1:9, mechanically homogenize, centrifuge at 3500r / min, 10min, take the supernatant and operate according to the steps of Nanjing Jiancheng GSH kit. ③ Sample preparation for determination of GSH-PX content in liver tissue: Weigh liver tissue, add 9 times physiological saline (according to the weight to volume ratio), homogenize at low temperature, centrifuge for 10min (3000r / min), take the supernatant and operate according to the steps of Nanjing Jiancheng GSH-PX kit.

[0178] 4) Hematoxylin-eosin (H&E) staining

[0179] Tissue processing: Take the liver tissue out of the fixative and trim the target tissue with a scalpel in a fume hood. Place the trimmed tissue and the corresponding label in the embedding frame, and place all the embedding frames in a dehydration box. The steps for making paraffin sections and H&E staining refer to the conventional steps. The sections after staining are examined under a microscope and image acquisition and analysis is performed. Image result interpretation: The nucleus is blue and the cytoplasm is red.

[0180] 5) Oil Red O staining

[0181] 5.1 Tissue fixation: Take out the liver tissue from the fixative and trim the target area with a scalpel.

[0182] 5.2 Dehydration: Place the trimmed and fixed liver tissue in a 15% sucrose solution in a 4°C refrigerator for dehydration and sink to the bottom. Then transfer it to a 30% sucrose solution in a 4°C refrigerator for dehydration and sink to the bottom.

[0183] 5.3 OCT embedding: Take out the dehydrated liver tissue, dry the surface water with filter paper, and place it on the embedding table with the cut surface facing up. Drop OCT embedding agent around the tissue, and place the embedding table on the quick-freezing table for rapid freezing and embedding. After the OCT turns white and hard, it can be sectioned.

[0184] 5.4 Frozen section: Fix the embedding table on the slicer, cut roughly to make the tissue surface smooth before starting to section, the thickness of the section is 8-10μm, place a clean glass slide flat on the cut tissue slice and stick the tissue on the slide. After labeling the slide, store it at -20℃ for later use.

[0185] 5.5 Frozen section fixation: Take the frozen sections out of the -20℃ refrigerator and restore them to room temperature, fix them with tissue fixative for 15 minutes, wash them with tap water, and dry them.

[0186] 5.6 Oil red staining: 6 parts of saturated Oil Red O dye solution and 4 parts of distilled water are fully mixed and placed in a 60-70℃ water bath for 30 minutes. After natural cooling, filter with qualitative filter paper to obtain Oil Red O working solution, which is ready for use. Slices are immersed in the Oil Red dye solution for 8-10 minutes (cover and protect from light).

[0187] 5.7 Background differentiation: Take out the slices, let them stay for 3 seconds, then immerse them in two cylinders of 60% isopropanol for differentiation, 3 seconds and 5 seconds respectively. Then immerse the slices in two cylinders of pure water for 10 seconds each.

[0188] 5.8 Hematoxylin staining: Take out the slices, leave for 3 seconds, then immerse in hematoxylin for 3-5 minutes, rinse in 3 cylinders of pure water for 5 seconds, 10 seconds, and 30 seconds each. Differentiate in differentiation solution for 8 seconds, wash in 2 cylinders of distilled water for 10 seconds each, and blue in blue solution for 1 second. Gently immerse the slices in 2 cylinders of tap water for 5 seconds and 10 seconds each, and examine the staining effect under a microscope.

[0189] 5.9 Sealing: Seal the slides with glycerol-gelatin mounting medium.

[0190] 5.10 Microscope examination, image acquisition and analysis.

[0191] 5.11 Interpretation of results: Lipid droplets are orange to bright red, and cell nuclei are blue.

[0192] 6) Data analysis

[0193] All data analyses were performed using GraphPad Prism 9.0 software. The differences between different groups were analyzed using one-way ANOVA. * represents the comparison between the drug-treated group and the model group, and # represents the comparison between the model group and the normal group. The stained images were processed using imageJ.

[0194] From the above results, we can see that the liver weight, liver index, liver original state, and H&E stained liver pathological structure in the NSP1 and NSP3 groups showed obvious improvement.

[0195] The liver is a key organ for fat metabolism. When lipid intake is excessive, it increases the metabolic burden on the liver and may cause liver damage. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are important biochemical indicators for detecting whether liver function is impaired. In an organism with normal liver function, most of the ALT in its liver cells is in the cytoplasm, and AST is in the mitochondria, with low levels in the blood. When the liver is damaged or inflamed, the permeability of the cell membrane increases, allowing AST and ALT to enter the blood. Therefore, the potential effects of a high-fat diet on liver function were evaluated by measuring the AST and ALT levels in the liver and serum of mice. The results are as follows: Fig.16 As shown (N=8. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001; #P≤0.05, ##P≤0.01, ###P≤0.001, ####P≤0.0001. ns indicates no statistical significance). Fig.16 As shown in the figure, compared with the CHOW group, the AST and ALT levels in the liver and serum of mice in the HFD group were significantly increased, indicating that the high-fat diet had an adverse effect on liver function. Compared with the HFD group, the AST and ALT levels in the liver and serum of mice fed NSP1 were significantly reduced, indicating that NSP1 helps alleviate liver damage caused by a high-fat diet. Similarly, the AST and ALT levels in the serum of mice in the NSP3 group were also significantly lower than those in the HFD group (P<0.01, P<0.0001), indicating that NSP3 is also beneficial for protecting liver function. The inconsistency in the statistical difference analysis of the AST and ALT levels in the liver and serum of the NSP3 group may be caused by factors such as liver cell repair and metabolic changes. In summary, NSP1 and NSP3 can improve abnormal liver function induced by a high-fat diet.

[0196] The abnormal liver function of mice fed a high-fat diet is due to the inability of newly synthesized TG in the liver to be transported to adipose tissue in various parts of the body in a timely manner, resulting in lipid accumulation in the liver, damage to liver cells, and affect liver function. To evaluate the effect of polysaccharides on liver lipid accumulation caused by a high-fat diet, TG and TC in liver tissues of CHOW, HFD, NSP1, and NSP3 groups were measured, and Oil Red O staining was performed. The results are shown in Fig.17 ab) As shown in Figure 2, compared with the CHOW group, the TG and TC contents in the liver of the HFD group were significantly increased (P<0.001, P<0.0001), and a large amount of lipid accumulation appeared in the liver tissue (P<0.0001). After the intervention of NSP1 and NSP3, the TG and TC contents in the liver were significantly reduced (TG: P<0.001, P<0.05; TC: P<0.01). Fig.17cd) Oil red O staining and semi-quantitative analysis showed that the lipid contents of NSP1 and NSP3 were significantly reduced compared with those in the HFD group (P<0.001, P<0.01). Fig.17 N = 8. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001. ns indicates no statistical significance.

[0197] Continuous increase in blood sugar can cause lipid autooxidation in the body, triggering chronic oxidation reactions, and placing the body in a state of high oxidative stress, leading to excessive production of free radicals, which in turn damage cell membranes and DNA. Superoxide dismutase (SOD) is a key antioxidant enzyme that catalyzes the conversion of superoxide anion free radicals and reduces their damage to cells. Under the influence of a high-fat diet, the SOD content in mouse serum decreased, thereby weakening the body's antioxidant capacity. Fig.18 As shown in a), compared with the CHOW group, the SOD content in the serum of the mice in the HFD group was significantly decreased (P<0.01), while the SOD content in the serum of the mice in the NSP1 and NSP3 groups was significantly increased (P<0.0001, P<0.01), indicating that NSP1 and NSP3 help to increase the SOD content in the blood. Reduced glutathione (GSH) and glutathione peroxidase (GSH-PX) play an important role in maintaining the redox balance of the liver. GSH-PX catalyzes the reaction of GSH with peroxide to remove peroxide. Under a high-fat diet, the decrease in the levels of GSH and GSH-PX will aggravate the redox imbalance of the liver and lipid metabolism disorders. Fig.18 b) Although the HFD group showed a decrease in GSH and GSH-PX contents compared with the CHOW group, the decrease was not statistically significant. However, after treatment with NSP1 and NSP3, the contents of GSH and GSH-PX increased (P<0.01, P<0.05, P<0.0001), and the effect of NSP3 was better than that of NSP1.

[0198] Malondialdehyde (MDA) is the end product of lipid peroxidation, which can cause cross-linking and polymerization of proteins and nucleic acids and is cytotoxic. The detection of MDA levels can reflect the degree of lipid peroxidation and liver cell damage. Compared with the CHOW group, the MDA level in the HFD group was significantly increased (P<0.001), and the MDA content decreased after treatment with NSP1 and NSP3 (P<0.05). The above results show that these two polysaccharide components can effectively reduce liver lipid peroxidation and oxidative stress induced by a high-fat diet. The results are similar to those of Ganoderma lucidum polysaccharides, which can reduce the serum malondialdehyde content of mice and increase the activities of glutathione peroxidase and liver superoxidase. Fig.18N = 8. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001. ns indicates no statistical significance.

[0199] In summary, NSP1 and NSP3 can effectively reduce the weight of mice induced by high-fat diet (HFD), and NSP and NSP3 can reduce the energy intake of mice. The reason why these two can reduce the weight of mice may be that they can achieve the effect of weight loss by reducing energy intake. NSP, NSP1, NSP2 and NSP3 can significantly inhibit the increase of fat weight in various parts of mice, hypertrophy of adipocytes, and improve glucose tolerance and insulin sensitivity to varying degrees. Although NSP, NSP1, NSP2 and NSP3 may have certain potential in inhibiting lipid accumulation in adipose tissue and increasing insulin sensitivity, based on the comprehensive test results, NSP1 and NSP3 can more significantly reduce liver weight and liver index, protect liver tissue pathological morphology and liver function, improve blood lipid metabolism, reduce liver lipid accumulation, and reduce oxidative stress levels in mice. Therefore, NSP1 and NSP3 can simultaneously reduce adipose tissue and liver tissue lesions, thereby achieving the effect of significantly inhibiting the weight gain of mice.

[0200] The embodiments of the present invention are described in detail above, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. The use of a Herba Lycopodii extract in the preparation of an anti-obesity product, characterized in that: The Herba Lysimachiae extract includes at least one of extract A or extract B; The preparation process of extract A comprises the following steps: adding a dispersion containing crude polysaccharide of Radix Codonopsis pilosulae to a cellulose anion exchange column for purification, eluting with water, and collecting the eluate to obtain the extract; The preparation process of extract B comprises the following steps: taking a dispersion containing crude polysaccharide of Eleutherococcus argyi and adding it into a cellulose anion exchange column for purification, eluting it with a 0.35-0.45 mol / L NaCl solution, and collecting the eluate to obtain the extract.

2. The use of the extract of Radix Codonopsis pilosulae in preparing products, characterized in that: The product has at least one of the following functions: 1) Protect liver function; 2) Reduce fat accumulation; 3) Improve blood sugar metabolism disorders; 4) Improve blood lipid metabolism disorders; 5) Reduce oxidative stress levels; 6) Lose weight; The Herba Lysimachiae extract includes at least one of extract A or extract B; The preparation process of extract A comprises the following steps: adding a dispersion containing crude polysaccharide of Radix Codonopsis pilosulae to a cellulose anion exchange column for purification, eluting with water, and collecting the eluate to obtain the extract; The preparation process of extract B comprises the following steps: taking a dispersion containing crude polysaccharide of Eleutherococcus argyi and adding it into a cellulose anion exchange column for purification, eluting it with a 0.35-0.45 mol / L NaCl solution, and collecting the eluate to obtain the extract.

3. The use according to claim 1 or the use according to claim 2, characterized in that: The extract A contains Eleutherodactyl polysaccharide I, which is a neutral polysaccharide rich in glucose.

4. The use according to claim 1 or the use according to claim 2, characterized in that: The extract A contains Eleutherodactyl polysaccharide I, which is a neutral polysaccharide rich in glucose. The rich in glucose means that the molar percentage of glucose is more than 70% of the monosaccharide composition in the polysaccharide, and more preferably more than 71.3%.

5. The use according to claim 1 or the use according to claim 2, characterized in that: The extract A contains Eleutherodactyl polysaccharide I, and the monosaccharide composition of Eleutherodactyl polysaccharide I is glucose, galactose, arabinose, mannose, xylose, and glucosamine hydrochloride.

6. The use according to claim 1 or the use according to claim 2, characterized in that: The extract A contains Eleutherodactyl polysaccharide I, and the monosaccharides of Eleutherodactyl polysaccharide I are composed of glucose, galactose, arabinose, mannose, xylose, and glucosamine hydrochloride in the following molar percentage ratios: 71-72: 13-14: 11-12: 2-3: 1-2: 0.4-0.

6.

7. The use according to claim 1 or the use according to claim 2, characterized in that: The extract B contains Eleutherodactyl polysaccharide II, which is an acidic polysaccharide rich in galacturonic acid.

8. The use according to claim 1 or the use according to claim 2, characterized in that: The extract B contains Eleutherodactyl polysaccharide II, which is an acidic polysaccharide rich in galacturonic acid. The rich in galacturonic acid means that the molar percentage of galacturonic acid is more than 27% of the monosaccharide composition in the polysaccharide, and more preferably more than 27.9%.

9. The use according to claim 1 or the use according to claim 2, characterized in that: The extract B contains Eleutherodactyl polysaccharide II, and the monosaccharide composition of Eleutherodactyl polysaccharide II is galacturonic acid, glucose, rhamnose, galactose, arabinose, fucose, glucuronic acid, mannose, xylose, and glucosamine hydrochloride.

10. The use according to claim 1 or the use according to claim 2, characterized in that: The extract B contains Eleutherodactyl polysaccharide II, and the monosaccharides of Eleutherodactyl polysaccharide II are composed of galacturonic acid, glucose, rhamnose, galactose, arabinose, fucose, glucuronic acid, mannose, xylose, and glucosamine hydrochloride in the following molar percentage ratios: 27.5-28.5: 16.5-17.5: 16-17: 8-10: 6-7: 2.5-3.5: 0.5-1: 0.5-1: 0.2-0.4.

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