A method for preparing and applying crude and homogeneous polysaccharides of Bacteroides-regulating Mesona chinensis.

Abstract

This invention discloses a method for preparing and applying crude and homogeneous polysaccharides of *Mesona chinensis* that regulate *Bacteroides*. The method includes the following steps: pulverizing *Mesona chinensis* herb, extracting by reflux with deionized water, filtering out the residue, concentrating to 1 / 4 of the volume, adding ethanol to 80% by volume, stirring evenly, letting stand overnight, centrifuging the precipitate, washing three times with anhydrous ethanol, ethyl acetate, and acetone respectively, dissolving in an appropriate amount of deionized water, and freeze-drying to obtain crude *Mesona chinensis* polysaccharide. The obtained crude *Mesona chinensis* polysaccharide is diluted with deionized water and dialyzed using dialysis bags in the range of 7000D, 100000D, 300000D, and 500000D respectively. The solutions are combined, concentrated, and freeze-dried to obtain homogeneous polysaccharides of *Mesona chinensis* with different molecular weights. The prepared crude and homogeneous polysaccharides of *Mesona chinensis* have good antioxidant capacity and can promote the growth of *Bacteroides* in the gut. In vitro experiments verified that the metabolites of *Mesona chinensis* polysaccharide have good lipid-lowering activity.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine, specifically relating to the preparation method and application of polysaccharides and homogeneous polysaccharides of the medicinal and edible herb Meadowfoam, especially its applications in anti-oxidation and lipid-lowering. Background Technology

[0002] With the continuous improvement of living standards, people's lifestyles are undergoing tremendous changes. Obesity and related metabolic diseases caused by improper lifestyles and unreasonable dietary habits have become a major global public health problem threatening individuals, families, and even society. The incidence of hyperlipidemia in China is showing an increasing trend year by year, and its damage to human health is mainly manifested in damage to the cardiovascular system, especially the heart, brain, and kidney vessels.

[0003] Hyperlipidemia is a metabolic disease caused by lipid metabolism disorders, resulting in elevated blood lipid levels. Clinically, it is characterized by elevated serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and decreased serum high-density lipoprotein cholesterol (HDL-C). Hyperlipidemia is also one of the main causes of cardiovascular disease, and the number of people dying from cardiovascular disease worldwide is increasing year by year. Hyperlipidemia has become a serious public health problem that endangers people's health and social development.

[0004] Currently, statins are the primary lipid-lowering drugs used in clinical practice. They effectively inhibit cholesterol synthesis by competitively inhibiting 3-hydroxymethylglutaryl-CoA reductase (HMGCR) to block the intracellular hydroxymethylglutaryl acid metabolic pathway. However, with ongoing clinical research, researchers have discovered that statins also have a range of toxic side effects, including inducing liver damage, muscle toxicity, diabetes, and gastrointestinal reactions, which limits their application. Therefore, finding safe and effective lipid-lowering drugs has become a hot topic in medical research.

[0005] Mesona chinensis Benth., also known as fairy grass, immortal grass, divine grass, frozen grass, and firewood grass, is the dried whole herb of the plant. According to the *Chinese Materia Medica*, it is astringent, sweet, and cold in nature, possessing the effects of clearing heat and detoxifying, and can be used to treat diabetes, acute nephritis, and hypertension. It is mainly distributed in Fujian, Guangdong, Guangxi, and Jiangxi provinces. Its cool nature and sweet, bland taste allow it to promote salivation, clear heat, and relieve summer heat. It is used to treat heatstroke, fever, burns, thirst, edema, jaundice, diarrhea, general pain, toothache due to wind-heat, and syphilis.

[0006] Numerous studies have shown that an imbalance in the gut microbiota is a significant intrinsic factor contributing to metabolic diseases such as hyperglycemia, hyperlipidemia, and hypertension, as well as obesity. For instance, Bacteroides' main role in the gut is to degrade complex polysaccharides, providing energy and beneficial metabolites for itself, other bacteria, and the host. It also exhibits different immunomodulatory effects and functions on host cells and tissues.

[0007] Therefore, how to process the components of jelly grass into novel extracts and develop new applications or uses to give them new functions is a technical problem that urgently needs to be solved by technicians in this field. Summary of the Invention

[0008] The purpose of this invention is to provide a method for preparing crude polysaccharides and homogeneous polysaccharides from *Mesona chinensis*. The prepared homogeneous polysaccharides are homogeneous polysaccharides with four different molecular weights from *Mesona chinensis*. The homogeneous polysaccharides are homogeneous polysaccharides with molecular weights of less than 7000D, 7000-100000D, 100000-300000D, and 300000D-500000D.

[0009] Another objective of this invention is to provide the application of crude and homogeneous polysaccharides of Mesona chinensis in lowering blood lipids; and the application of crude and homogeneous polysaccharides of Mesona chinensis in regulating the structure of intestinal flora.

[0010] To achieve the above objectives, this invention discloses a method for preparing crude polysaccharide and homogeneous polysaccharide from *Gnaphalium affine*, comprising the following process steps:

[0011] 1) Crush and sieve the herbal powder, add 20 times the amount of deionized water, soak for 30 minutes, reflux extract for 90 minutes, remove and filter, concentrate the filtrate under reduced pressure to 1 / 4, add ethanol to the volume fraction of ethanol to 80%, stir well, and let stand overnight at 4℃.

[0012] 2) Centrifugation was used to obtain a precipitate. The precipitate was washed three times with anhydrous ethanol, ethyl acetate and acetone respectively. The polysaccharide was dissolved in an appropriate amount of deionized water, filtered, and the filtrate was freeze-dried to obtain crude polysaccharide of Mesona chinensis.

[0013] 3) The obtained crude polysaccharide (MA) of *Gynostemma pentaphyllum* was diluted with deionized water and dialyzed using dialysis bags with molecular weights of 7000D, 100000D, 300000D, and 500000D respectively. The solutions were combined, concentrated, and freeze-dried to obtain four homogeneous polysaccharides of *Gynostemma pentaphyllum* with different molecular weights: MA1 (<7000D), MA2 (7000~100000D), MA3 (100000~300000D), and MA4 (300000D~500000D).

[0014] The crude and homogeneous polysaccharides of *Gynostemma pentaphyllum* can promote the growth of *Bacteroides intestinalis*. The homogeneous polysaccharides MA1 (<7000D) and MA2 (7000–100000D), determined through isolation and purification, can promote *Bacteroides intestinalis* proliferation. After degradation and utilization by *Bacteroides intestinalis*, they can produce active ingredients, and the metabolites have good lipid-lowering activity. The active ingredients are at least one of monosaccharides, oligosaccharides, and acetic acid or butyric acid.

[0015] The crude and homogeneous polysaccharides of *Gynostemma pentaphyllum* obtained by processing them using the method of this invention can be made into various preparations, including suppositories, tablets, pills, granules, films, microcapsules, drop pills, aerosols, tinctures, syrups, oral liquids, etc.

[0016] The beneficial effects of the crude and homogeneous polysaccharides of *Gnaphalium affine* and their applications disclosed in this invention are as follows:

[0017] 1) The crude polysaccharide and homogeneous polysaccharide of the grass extract of the present invention can promote the growth of intestinal Bacteroides. The preparation process is simple, the product is stable, and it is suitable for mass production. It has a good effect on regulating hyperlipidemic intestinal flora disorder and lowering blood lipids. It can be applied to the fields of drugs or health foods that lower blood lipids and regulate intestinal flora structure.

[0018] 2) The raw materials of this invention are abundant, the preparation method is simple, it is easy to produce and prepare, it is convenient to take, and it has good effects. It opens up a new way of treating hyperlipidemia, improves the medicinal value of licorice, and is an innovation in the treatment of hyperlipidemia with significant economic and social benefits. Attached Figure Description

[0019] Figure 1 The diagram shows the scavenging effect of mesona chinensis polysaccharide on ABTS free radicals (n=3).

[0020] Figure 2 The graph shows the results of FRAP determination of mesona chinensis polysaccharide (n=3).

[0021] Figure 3 The growth curves of Bacteroides were obtained by the action of different molecular weight Mesona chinensis polysaccharides.

[0022] Figure 4The supernatant after the interaction of BT, BO, and BC bacteria with MA1 was used for HPGPC detection.

[0023] Figure 5 This study aimed to detect the effects of BT, BO, and BC bacteria on the supernatant of *Gynostemma pentaphyllum* polysaccharides of different molecular weights using TLC.

[0024] Figure 6 To determine the content of SCFAs, metabolites of Gynostemma pentaphyllum polysaccharides of different molecular weights.

[0025] Figure 7 The effect of total lipid accumulation on the polysaccharide metabolites of Mesona chinensis.

[0026] Figure 8 The effect of TG levels on the polysaccharide metabolites of Mesona chinensis.

[0027] Figure 9 The effect of glucose consumption, a metabolite of mesona chinensis polysaccharide. Detailed Implementation

[0028] This invention provides a method for preparing and applying crude and homogeneous polysaccharides of *Bacteroides montana* from *Mesona chinensis*. The method for preparing the crude and homogeneous polysaccharides of *Bacteroides montana* from *Mesona chinensis* includes the following steps:

[0029] 1) The herbal medicine of jelly grass was crushed, extracted by reflux with deionized water, the residue was filtered out, the volume was concentrated to 1 / 4, ethanol was added to 80% by volume, the mixture was stirred evenly and left to stand overnight, the precipitate was centrifuged and washed three times with anhydrous ethanol, ethyl acetate and acetone respectively, dissolved in an appropriate amount of deionized water and freeze-dried to obtain crude polysaccharide of jelly grass.

[0030] 2) The crude polysaccharide obtained from *Gynostemma pentaphyllum* was diluted with deionized water and dialyzed using dialysis bags in the range of 7000D, 100000D, 300000D, and 500000D respectively. The solutions were combined, concentrated, and freeze-dried to obtain homogeneous polysaccharides of *Gynostemma pentaphyllum* with different molecular weights.

[0031] The preparation method described in this invention is simple to operate and easy to implement. The raw materials are natural products with low toxicity and side effects. The prepared crude polysaccharide and homogeneous polysaccharide of different molecular weights of grass have good antioxidant capacity and can promote the growth of intestinal Bacteroides. In vitro experiments have verified that the metabolites of grass polysaccharide have good lipid-lowering activity.

[0032] This invention further demonstrates the lipid-lowering and hypoglycemic activities of crude polysaccharides from *Mesona chinensis* through in vitro experiments using the HepG2 fat accumulation model and insulin resistance model. Intervention of *Mesona chinensis* ethanol extract (containing polysaccharides) in hyperlipidemic rats showed that *Mesona chinensis* polysaccharides are degraded and metabolized by intestinal flora, and the abundance of *Bacteroides* was significantly increased in the *Mesona chinensis*-treated group. *Bacteroides* is a major genus of bacteria in the human gut that degrades and metabolizes polysaccharides; therefore, exploring the bioactivity of *Bacteroides* and polysaccharides is an important approach to investigating the biological activity of *Mesona chinensis* polysaccharides.

[0033] Meanwhile, the antioxidant properties of polysaccharides were assessed using the ABTS free radical scavenging test and the FRAP antioxidant assay. The growth curves of three Bacteroides species (Bacteroides multiforme (BT), Bacteroides ovalis (BO), and Bacteroides fibrolyticus (BC)) that were negatively correlated with lipid-lowering activity were measured to evaluate the bioactivity of each polysaccharide component.

[0034] HPGPC (High-Performance Gel Permeation Chromatography) and TLC (Thin-Layer Chromatography) were used to determine the products of polysaccharides degraded by three Bacteroides species to evaluate whether each polysaccharide component could produce monosaccharides, oligosaccharides, and short-chain fatty acids, which are beneficial metabolites to the human body. Using an oleic acid-induced human hepatocellular carcinoma HepG2 cell lipid accumulation model and an insulin-induced human hepatocellular carcinoma HepG2 cell insulin resistance model, the contents of total lipids, triglycerides (TG), and glucose consumption were measured to verify the lipid-lowering and glucose-lowering activities of the polysaccharide metabolites.

[0035] The results showed that the prepared crude polysaccharide and homogeneous polysaccharides of *Mesona chinensis* with different molecular weights could promote the growth of *Bacteroides intestinalis*. Through isolation and purification, the homogeneous polysaccharides MA1 (<7000 Da) and MA2 (7000–100000 Da) were identified as bioactive polysaccharides that promote *Bacteroides intestinalis* proliferation. After degradation and utilization by *Bacteroides intestinalis*, they can produce monosaccharides, oligosaccharides, and active ingredients such as acetic acid and butyric acid. In vitro experiments verified that the metabolites of MA1 and MA2 (degraded by three types of *Bacteroides intestinalis*) have good lipid-lowering activity. This demonstrates that the crude polysaccharide and homogeneous polysaccharides (MA1 and MA2) of *Mesona chinensis* have excellent lipid-lowering activity.

[0036] Unless otherwise specified, all reagents involved in the embodiments of this invention are commercially available products and can be purchased through commercial channels.

[0037] The following specific examples and experiments demonstrate that the present invention has good antioxidant capacity and lipid-lowering effect.

[0038] The experimental material used in this invention is the above-ground part of the plant Mesona chinensis, belonging to the genus Mesona of the Lamiaceae family.

[0039] Example 1:

[0040] The preparation methods of crude polysaccharide and homogeneous polysaccharide from *Gnaphalium affine* are as follows:

[0041] The herbal extract of *Gnaphalium affine* was pulverized and sieved. It was then soaked in 20 times its volume of deionized water for 30 minutes, refluxed for 90 minutes, filtered, and the filtrate concentrated under reduced pressure to 1 / 4. Ethanol was added until the ethanol volume fraction reached 80%, and the mixture was stirred thoroughly and allowed to stand overnight at 4°C. The precipitate was obtained by centrifugation and washed three times with anhydrous ethanol, ethyl acetate, and acetone, respectively. The polysaccharide was dissolved in an appropriate amount of deionized water, filtered, and the filtrate was freeze-dried to obtain crude *Gnaphalium affine* polysaccharide. The obtained crude *Gnaphalium affine* polysaccharide was diluted with deionized water and dialyzed using dialysis bags in the ranges of 7000D, 100000D, 300000D, and 500000D, respectively. The solutions were combined, concentrated, and freeze-dried to obtain homogeneous *Gnaphalium affine* polysaccharides of different molecular weights.

[0042] Results: After separation by dialysis bag, MA polysaccharide was successively separated into components with different molecular weight ranges: MA1 (yield 36.8%), MA2 (yield 16.4%), MA3 (yield 4.1%), and MA4 (yield 37.3%).

[0043] Example 2:

[0044] The preparation methods of crude polysaccharide and homogeneous polysaccharide from *Gnaphalium affine* are as follows:

[0045] The herbal extract of *Gnaphalium affine* was pulverized and sieved. It was then soaked in 50 times its volume of deionized water for 60 minutes, refluxed for 120 minutes, filtered, and the filtrate concentrated under reduced pressure to 1 / 5. Ethanol was added until the ethanol volume fraction reached 80%, and the mixture was stirred thoroughly and allowed to stand overnight at 4°C. The precipitate was obtained by centrifugation and washed four times with anhydrous ethanol, ethyl acetate, and acetone, respectively. The polysaccharide was dissolved in an appropriate amount of deionized water, filtered, and the filtrate was freeze-dried to obtain crude *Gnaphalium affine* polysaccharide. The obtained crude *Gnaphalium affine* polysaccharide was diluted with deionized water and dialyzed using dialysis bags in the ranges of 7000D, 100000D, 300000D, and 500000D, respectively. The solutions were combined, concentrated, and freeze-dried to obtain homogeneous *Gnaphalium affine* polysaccharides of different molecular weights.

[0046] The following is the content of the efficacy test case.

[0047] Experimental Example 1: Antioxidant Capacity Test of Mesona chinensis Polysaccharides

[0048] 1) ABTS free radical scavenging test

[0049] Preparation of ABTS working solution: Mix 1 ml each of ABTS stock solution (8.16 mg ABTS added to 2 ml distilled water to obtain ABTS stock solution) and K2S2O8 stock solution (1.4 mg K2S2O8 added to 2 ml distilled water to obtain K2S2O8 stock solution) thoroughly and place in a dark place for 12 hours; dilute with 95% ethanol before use so that its absorbance value at 734 nm is 0.700±0.005.

[0050] Take 40 μl of polysaccharide sample solutions of different concentrations and 160 μl of ABTS working solution, place them in a 96-well plate, shake well, and react in the dark for 6 min. Measure the absorbance at 734 nm using a microplate reader. Use vitamin C (VC) as a positive control and purified water as a blank to zero the plate.

[0051] Each sample was measured in triplicate, and the ABTS free radical scavenging rate and IC50 value of the sample were calculated. Scavenging rate (%) = [1 - (A1 - A2) / A3] × 100%.

[0052] A1 represents the absorbance value of the mixture of ABTS solution and sample solution, A2 represents the absorbance value of the mixture of sample solution and blank solvent, and A3 represents the absorbance value of the mixture of ABTS solution and blank solvent.

[0053] Table 1. ABTS free radical scavenging test reaction system

[0054]

[0055] 2) FRAP method for determining antioxidant activity

[0056] FRAP working solution preparation: Prepare 20 mmol / L FeCl3 solution, 10 mmol / L TPTZ solution, and 0.3 mol / L sodium acetate buffer solution, and mix them in a ratio of 1:1:10 before use.

[0057] FeSO4 solutions with concentrations of 80, 120, 160, 200, 240, and 280 μg / mL were prepared. Using FeSO4 as a standard, a standard curve was plotted with concentration on the x-axis and absorbance on the y-axis: y = 0.00003x + 0.00378, R0. 2 =0.9953.

[0058] Extracts from each fraction of the sample were prepared to the same and appropriate concentration. 20 μl of the sample solution and 180 μl of the FRAP working solution were placed in a 96-well plate, shaken thoroughly, and incubated at 37°C for 20 min. The absorbance was measured at 740 nm. Each sample was measured in triplicate, and the FRAP value was calculated. Its antioxidant capacity is expressed as the FRAP value, i.e., Fe... 2+ Equivalent, n=3.

[0059] 3) Results as follows Figure 1 As shown, the ABTS free radical scavenging abilities of polysaccharides of different molecular weights from *Gynostemma pentaphyllum* were compared as follows: MA > MA1 > MA4 > MA2 > MA3. The ABTS free radical scavenging ability of MA decreased after separation and purification, but MA (IC50 = 25.52 μg / ml) showed significantly higher scavenging ability than MA1 (IC50 = 23.35 μg / ml) and MA4.

[0060] The scavenging ability of MA2 (IC50 = 20.83 μg / ml) against ABTS free radicals was higher than that of the positive control VC (IC50 = 11.23 μg / ml).

[0061] 4) Results are as follows Figure 2 As shown, the FRAP value indicates that the in vitro antioxidant capacity of polysaccharides of different molecular weights from *Gynostemma pentaphyllum* is best for MA4, followed by MA, MA3, MA2, and MA1.

[0062] Experimental Example 2: Activity Screening Experiment of Three Bacteroides on Polysaccharide Components

[0063] 1) Test samples: B. thetaiotaomicron GDMCC 1.1104 (BT) and B. ovatus GDMCC 1.1702 (BO) were ordered from the Guangzhou Culture Collection Center in China; B. cellulosilyticus CCUG 44979 (BC) was ordered from the CCUG Culture Collection Center in Sweden.

[0064] 2) Bacteroidetes growth curve determination: Three types of Bacteroides, BT, BO, and BC, were anaerobic in BHI medium at 37°C for 12-24 hours to reach the logarithmic growth phase. MM medium formulation: 100mM KH₂PO₄ (pH 7.2), 15mM NaCl, 8.5mM (NH₄)₂SO₄, 4mM L-cysteine, 1.9μM hematin, 200μM L-histidine, 100nM MgCl₂, 1.4nM FeSO₄·7H₂O, 50μM CaCl₂, 1μg / ml Vitamin K₃, 5ng / ml Vitamin B12. Take 1ml of bacterial suspension, centrifuge at 3000rpm for 5min, discard the supernatant, filter MM through a 0.22μm filter, add 1ml of resuspended (gently pipet) bacterial cells, centrifuge at 3000rpm for 5min, discard the supernatant, and collect the bacterial cells.

[0065] The bacterial cells were diluted with MM to an OD600 value of approximately 0.2-0.3. 100 μL of the diluted bacterial solution and 100 μL of polysaccharide filtered through a 0.22 μm filter were added to each well of a 96-well plate. Autoclaved ultrapure water served as a negative control, and a glucose solution with the same concentration as the polysaccharide served as a positive control. Three replicates were set for each well. OD600 was measured and recorded using a microplate reader at 0 h, 12 h, 16 h, 20 h, 26 h, 32 h, 38 h, 42 h, 48 h, 60 h, 64 h, 68 h, and 72 h. Finally, a growth curve was plotted.

[0066] 3) High-performance gel permeation chromatography (HPGPC) detection of Bacteroides degradation polysaccharide products: BT, BO and BC three types of Bacteroides were cultured in MM containing 5 mg / ml polysaccharide in an anaerobic environment at 37℃ for 48 h. After centrifugation at 3000 rpm for 5 minutes, the supernatant was collected. The supernatant was then desalted three times using a desalting column. The desalted solution was freeze-dried and dissolved in deionized water.

[0067] Detection was performed using a Waters e2695 pump and a 2424 ELSD evaporative light detector on an Ultrahydrogel 2000 column. The sample was eluted with 100% ultrapure water at a flow rate of 1 ml / min. The drift tube temperature was 90 °C, the gain was 500, the injection volume was 20 μL, and the column temperature was 30 °C.

[0068] 4) Thin-layer chromatography (TLC) detection of Bacteroides-degraded polysaccharide products: The obtained bacterial solution was analyzed by TLC using a Siliconegel 60F254 (Merck) instrument. The mobile phase was n-butanol:ethanol:water (5:3:2), and the chromogenic reagent was concentrated sulfuric acid:anhydrous ethanol (1.9:40). 2 μl of the product was spotted onto a silica gel plate, and 10 mg / ml of L-rhamnose, D-arabinose, D-xylose, D-mannose, D-anhydrous glucose, and D-galactose were used as standards. The sample was developed in the mobile phase, and then the silica gel plate was dried with a hairdryer. The chromogenic reagent was evenly sprayed onto the silica gel plate, and then the plate was dried at high temperature with a hot air gun for chromogenic development.

[0069] 5) Determination of short-chain fatty acids: BO and BC were cultured in MM medium containing 5 mg / ml for 60 h. 1 ml of the bacterial culture was centrifuged at 3000 rpm for 5 min. The supernatant was collected, and 100 μL of 50% sulfuric acid was added, followed by 1 ml of diethyl ether. The mixture was shaken for 30 s and centrifuged at 10000 rpm for 5 min. 200 μL of the supernatant was injected for analysis. Acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid were used as standards. The standards were diluted to 50, 100, 200, 400, and 800 μg / ml. An InertCap FFA fused silica capillary column (30 m × 0.25 mm × 0.25 μm) from Shimadzu Corporation, Japan was used. High-purity (greater than 99.999%) helium was used as the carrier gas at a flow rate of 1.0 ml / min.

[0070] A gradient temperature program was used for sample separation and analysis. The gradient temperature program was as follows: the initial temperature was 80℃ and held for 1 min, then increased to 150℃ at a rate of 15℃ / min, and then further increased to 220℃ at a rate of 20℃ / min. 1 μl of sample was injected in a 10:1 split mode. The solvent delay time was set to 3.0 min. The transfer line and electron collision (EI) ionization source temperatures were set to 220℃ and 230℃, respectively.

[0071] 6) Results as follows Figure 3 As shown, the promoting effect of *Mesona chinensis* polysaccharides on the proliferation of *Bacteroides* was as follows: MA1 polysaccharide at concentrations of 2.5 mg / ml and 5.0 mg / ml exhibited a strong growth-promoting effect, enabling the three *Bacteroides* species to rapidly reach the growth plateau phase in approximately 48 hours. This indicates that low molecular weight (≤100 kDa) polysaccharides can effectively promote the growth of *Bacteroides*. MA2 at a concentration of 2.5 mg / ml showed a strong promoting effect on the three *Bacteroides* species, and the promoting effect was also good at a concentration of 5 mg / ml. MA3 and MA4, at concentrations of 2.5 mg / ml and 5.0 mg / ml, did not significantly promote the growth of *Bacteroides BT*, *Bacteroides BO*, and *Bacteroides BC*, meaning that *Mesona chinensis* polysaccharides with a molecular weight greater than 10 kDa may not be utilized by the three *Bacteroides* species. The effect of MA at a concentration of 5.0 mg / ml on the three *Bacteroides* species was relatively weak, possibly because the proportion of polysaccharides with a molecular weight greater than 10 kDa was large, representing an ineffective component, while polysaccharides with a molecular weight less than 10 kDa were fully utilized, causing the *Bacteroides* to stop proliferating after reaching a certain growth level.

[0072] The results showed that the isolated and purified polysaccharide components MA1 (<7000 Da) and MA2 (7000~100000 Da) had high activity in promoting the growth and reproduction of Bacteroides, and were significantly better than D-glucose and other molecular weight polysaccharide components as carbon sources for the three Bacteroides species.

[0073] 7) Results as follows Figure 4 As shown, the polysaccharide analysis of Bacteroides metabolites revealed that after digestion and decomposition of the herbicide MA1 by BT, BO, and BC, the retention time of the polysaccharide peak in the supernatant of the bacteria detected in HPGPC was higher than that of the MA1 peak. This indicates that BT, BO, and BC can digest and decompose MA1 to produce polysaccharides with smaller molecular weights, which may be related to their binding with proteins produced by Bacteroides. The similarity of the chromatographic peaks of BT, BO, and BC may be due to the presence of the same or similar enzymes to degrade the polysaccharide, thus yielding the same metabolites. Further research is needed.

[0074] 8) Results as follows Figure 5 The image shows TLC images of the supernatant containing different molecular weight *Gynostemma pentaphyllum* polysaccharides, analyzed by BT, BO, and BC bacteria. 1: L-Rha; 2: D-Ara; 3: D-Xyl; 4: D-Man; 5: D-Glc; 6: D-Gal; 7: AJ; 8: AY; 9: BJ; 10: BY; 11: CJ; 12: CY

[0075] Analysis of monosaccharides and oligosaccharides in metabolites: Analysis of bacterial supernatants containing polysaccharide components MA1, MA2, MA3, MA4, and MA polysaccharides of different molecular weights revealed that the oligosaccharide bands were lighter than those of the crude polysaccharides isolated from the stems and leaves of *Mesona chinensis*. For example... Figure 4-8BT bacteria produced fewer oligosaccharide fragments from MA1, MA3, MA4 and MA, while no oligosaccharide fragments were detected in MA2, and no monosaccharides were detected in any of them.

[0076] BO bacteria only detected large oligosaccharide fragments in MA, while no oligosaccharide fragments were detected in MA1, MA2, MA3, and MA4, and no monosaccharide fragments were detected in any of them. BC bacteria TLC analysis only showed oligosaccharide bands in MA4; no oligosaccharide fragments or monosaccharides were detected in the supernatants of the other polysaccharide samples. BO, BT, and BC may have also produced glucose when utilizing Mesona chinensis polysaccharides, but glucose was not detected in the TLC results. Glucose can be rapidly utilized by Bacteroides as a growth-required resource, which may explain the lack of glucose detection; further investigation is needed.

[0077] 9) Results are as follows Figure 6 As shown, Bacteroides utilize polysaccharides to produce short-chain fatty acids: different molecular weight polysaccharide components MA1, MA2, MA3, MA4 and MA polysaccharide can also be detected in the supernatant by three types of Bacteroides BT, BC and BO.

[0078] The acetic acid produced by MA4 and MA in the BT supernatant was significantly higher than that of other samples, at 217.46 mg / ml and 228.76 mg / ml, respectively. MA1 and MA2 also showed high acetic acid levels in the BO bacteria supernatant, at 100.03 mg / ml and 72.56 mg / ml, respectively. The acetic acid levels detected by MA1 and MA2 in the BC bacteria supernatant were higher than those of MA3, MA4, and MA, at 84.90 mg / ml and 66.55 mg / ml, respectively. A small amount of butyric acid was detected in the D-glucose positive control group.

[0079] Experimental Example 3: Verification of the lipid-lowering activity of mesona chinensis polysaccharide metabolites

[0080] 1) Test sample: Metabolites of human hepatocellular carcinoma HepG2 cell line.

[0081] 2) MTT cytotoxicity assay: Human hepatocellular carcinoma HepG2 cells in logarithmic growth phase were seeded at a density of 5000 cells / well in 96-well plates and cultured in an incubator (37℃, 5% CO2) for 24 h. After cell adhesion and growth, a control group and a drug-treated group were set up, with 3 replicates in each group. The drug-treated group was given polysaccharide metabolites at final concentrations of 5, 10, 50, 100, 200, 500, 1000, and 2000 μg / ml, respectively. The control group was given an equal volume of complete culture medium and cultured for another 24 h. The supernatant was then carefully aspirated and washed three times with a small amount of PBS. 180 μl of PBS and 20 μl of 5 mg / ml MTT solution were added to each well and cultured in the dark for 4 h. The supernatant was carefully aspirated, avoiding the removal of purple crystals. 200 μl of dimethyl sulfoxide was added to each well and the plates were shaken for 15 min. The absorbance was measured at 570 nm and the IC50 value of cell viability was calculated.

[0082] 3) Intervention effect of oleic acid (OA) induced fat accumulation model in HepG2 cells: Preparation of Oil Red O staining solution: Take 5g of Oil Red O powder, dissolve it in 100ml of isopropanol, store it at 4℃ in the dark as a stock solution; before use, dilute it according to the stock solution: water (4:6), filter it until clear and transparent, and use it as Oil Red O staining solution.

[0083] Human HepG2 liver cancer cells in logarithmic growth phase were harvested at a concentration of 1×10⁻⁶. 5 The cells were seeded at a density of cells / ml in 24-well or 6-well plates and incubated in an incubator (37°C, 5% CO2). The culture medium was changed every two days. When the cells grew to cover about 80% of the field of view, the drug intervention was carried out for 24 hours according to the table below. Each group was set up with 3 replicates.

[0084] Table 2. Grouping and Dosing Regimens for the HepG2 Cell Lipid Accumulation Model

[0085]

[0086] Total lipids were determined by Oil Red O staining: After the drug intervention, the upper culture medium was aspirated, and 500 μl of 4% paraformaldehyde was added to each well to fix the cells for 30 min. The 4% paraformaldehyde was aspirated, and the cells were washed three times with PBS. 500 μl of Oil Red O staining solution was added to each well, and the cells were stained by shaking at low speed on a shaker for 30 min. The staining solution was aspirated, and the cells were washed three times with water. Finally, 500 μl of isopropanol was added to each well, and the cells were shaken for 30 min. The absorbance A was measured at 510 nm.

[0087] Triglyceride (TG) level determination: After the drug administration intervention, the upper culture medium was aspirated and the cells were washed once with PBS. 200 μl of cell lysis buffer was added to each well and the cells were lysed on ice for 10 min. The cell pellet was carefully scraped off with a cell scraper and collected in a 1.5 ml EP tube. The tube was placed on ice and lysed for another 20 min. During this period, the cells were vortexed multiple times to ensure complete lysis. The cell lysis buffer was measured according to the instructions of the triglyceride (TG) test kit.

[0088] 4) Intervention effect of insulin-induced insulin resistance in HepG2 cells:

[0089] Human HepG2 liver cancer cells in logarithmic growth phase were harvested at a concentration of 1×10⁻⁶. 5 Cells were seeded at a density of cells / ml in 24-well or 6-well plates and cultured in an incubator (37℃, 5% CO2). The culture medium was changed every two days. When the cells grew to cover about 80% of the field of view, the culture medium was replaced with FBS-free DMEM basal medium and starved for 12 hours. Then, the drug intervention was carried out for 24 hours according to the table below. Each group was set up with 3 replicates. After the intervention, the cell culture supernatant was collected, the glucose concentration was measured, and the glucose consumption was calculated: Glucose consumption (mmol / L) = glucose consumption in blank medium - glucose consumption in experimental medium.

[0090] 5) Results as follows Figure 7 As shown:

[0091] Note: NC: Control group; Mo: Model group; S: Positive drug group; L: Low-dose group; M: Medium-dose group; H: High-dose group; MA1: ≤7KD; MA2: 7~100KD; MA3: 100~300KD; MA4≥300KD; Glu: Glucose; W: Water blank group; Statistical data are shown in Appendix Table 2. "*" indicates that compared with the NC group, P<0.05, which is statistically significant; "#" and "##" indicate that compared with the Mo group, P<0.05 and P<0.01, respectively, which are statistically significant.

[0092] Accumulation of total lipids from polysaccharide metabolites: HepG2 cells in the model group and the drug treatment group were induced to accumulate lipids using oleic acid (OA). Total lipids were measured by Oil Red O staining. The absorbance of the model group was greater than that of the control group (P<0.05), indicating that the HepG2 cell lipid accumulation model was successfully established. The absorbance of the positive control simvastatin was significantly reduced after intervention (P<0.01).

[0093] The intervention effect of BT (Bacteroides polymorpha) metabolites of MA1, MA2, MA3, MA4, and MA polysaccharides on total lipids in HepG2 cells, such as... Figure 7As shown, the absorbance values ​​of polysaccharides of different molecular weights in MA1, MA2, MA3, and MA4, as well as crude polysaccharide of MA (low, medium, and high doses), were all lower than those in the model group (P<0.01). This indicates that the metabolites of polysaccharides from BT bacteria can significantly reduce the total lipid accumulation in HepG2 cells. The absorbance values ​​of Glu (glucose group) and W (water blank group) (low, medium, and high doses) were also lower than those in the model group (P<0.01). Among them, the absorbance value of the low-dose MA3 group was the lowest (P<0.01), followed by the low-dose MA2 (low and high dose groups) and MA1 (low dose group) (P<0.01). The absorbance values ​​of MA4 (low, medium, and high dose groups) and Glu (medium dose group) were higher than those of other treatment groups (P<0.01). The absorbance values ​​of MA1, MA2, MA3, MA4, and MA (low, medium, and high doses) in the intervention effect of BO (Bacteroides ovatus) polysaccharide metabolites on total lipids in HepG2 cells were all lower than those in the model group (P<0.05). Among them, the absorbance value of the medium dose group of MA2 was the lowest (P<0.05). The intervention effects of MA4 (low and medium dose groups) and Glu and water (low, medium, and high dose groups) were relatively weaker than those of other treatment groups, i.e., the absorbance values ​​were higher (P<0.05). The results of the intervention effect of BC (Bacteroides fibrolyticus) polysaccharide metabolites on total lipids in HepG2 cells showed that the absorbance values ​​of MA1 (medium and high dose groups), MA2 (low and high dose groups), MA3 (low, medium, and high dose groups), MA (low, medium, and high dose groups), and Glu (low and high dose groups) were all lower than those in the model group (P<0.05).

[0094] 7) Results as follows Figure 8 As shown:

[0095] Note: NC: Control group; Mo: Model group; S: Positive drug group; L: Low-dose group; M: Medium-dose group; H: High-dose group; MA1: ≤7KD; MA2: 7~100KD; MA3: 100~300KD; MA4≥300KD; Glu: Glucose; W: Water blank group; Statistical data are shown in Appendix Table 2. "*" indicates that compared with the NC group, P<0.05, which is statistically significant; "#" and "##" indicate that compared with the Mo group, P<0.05 and P<0.01, respectively, which are statistically significant.

[0096] Effect of polysaccharide metabolite TG level: By measuring the intracellular TG level, it was found that the intracellular TG level in the model group was higher than that in the control group (P<0.05), indicating that the HepG2 cell lipid accumulation model was successfully established. Figure 8 The study showed the effect of Bacillus subtilis polysaccharide metabolites on intracellular TG levels. The TG levels decreased significantly after intervention in the low- and medium-dose groups of Mesona chinensis polysaccharide MA1, the low-dose group of MA2, and the low-, medium-, and high-dose groups of MA4 (P<0.05). The TG levels also decreased significantly after intervention in the low-dose glucose group (P<0.01).

[0097] The effects of Bacillus bronchiseptica polysaccharide metabolites on intracellular TG levels: In the positive control group, simvastatin intervention significantly decreased TG levels (P<0.01). The most significant decreases in intracellular TG levels were observed in the low, medium, and high dose groups of MA1 and MA4 (P<0.01), but the high dose group of Glu showed an increase in intracellular TG levels (P<0.01). The effects of Bacillus bronchiseptica polysaccharide metabolites on intracellular TG levels: The low, medium, and high dose groups of MA1 and MA2 showed significant decreases in intracellular TG levels (P<0.05), while the medium and high dose groups of MA3 showed significant increases in intracellular TG levels (P<0.01).

[0098] 8) Results as follows Figure 9 As shown:

[0099] Note: NC: Control group; Mo: Model group; S: Positive drug group; L: Low-dose group; M: Medium-dose group; H: High-dose group; MA1: ≤7KD; MA2: 7~100KD; MA3: 100~300KD; MA4≥300KD; Glu: Glucose; W: Water blank group; Statistical data are shown in Appendix Table 2. "*" indicates that compared with the NC group, P<0.05, which is statistically significant; "#" and "##" indicate that compared with the Mo group, P<0.05 and P<0.01, respectively, which are statistically significant.

[0100] Effect of polysaccharide metabolite glucose consumption:

[0101] After high-concentration insulin intervention, the glucose consumption in the model group (Mo) was significantly lower than that in the control group (NC) (P<0.05), indicating that the IR-HepG model was successfully established; after metformin intervention in the positive control group, the cellular glucose consumption was significantly increased compared with the model group (P<0.01). Figure 9 The high-dose group of BT bacteria polysaccharide metabolites MA1, the low-dose group of MA3, and the low-dose group of MA all significantly increased cellular glucose consumption (P<0.05).

[0102] The effects of Bacillus bronchiseptica polysaccharide metabolites on cellular glucose consumption were investigated. Low and medium doses of MA1 and medium doses of MA3 significantly increased cellular glucose consumption (P<0.05). Low and medium doses of MA4, MA, and Glu also significantly increased cellular glucose consumption (P<0.05). Furthermore, the effects of Bacillus bronchiseptica polysaccharide metabolites on cellular glucose consumption were also examined. Low doses of MA1 and MA3, and high doses of MA4 and Glu significantly increased cellular glucose consumption.

[0103] The above results indicate that Mesona chinensis polysaccharides and homogeneous polysaccharides have good antioxidant capacity. The homogeneous polysaccharides MA1 (<7000 Da) and MA2 (7000-100000 Da) identified by isolation and purification are bioactive polysaccharides that promote the proliferation of Bacteroides. After being degraded and utilized by intestinal Bacteroides, they can produce monosaccharides, oligosaccharides, and active ingredients such as acetic acid and butyric acid. In vitro experiments have verified that the metabolites of MA1 and MA2 (degraded by three types of Bacteroides) have good lipid-lowering activity.

[0104] The above description is merely a preferred embodiment of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the original materials of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention. For example, those skilled in the art can directly or indirectly add the mesona chinensis extract to various pharmaceutically acceptable commonly used excipients required for preparing different dosage forms, such as fillers, disintegrants, lubricants, binders, etc., using conventional pharmaceutical preparation methods to prepare commonly used oral or injectable formulations. These improvements and modifications should also be considered within the scope of protection of the present invention.

[0105] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Therefore, any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. The application of crude polysaccharide and homogeneous polysaccharide from *Mesona chinensis* in the preparation of pharmaceutical formulations for regulating intestinal flora structure, characterized in that... The crude polysaccharide and homogeneous polysaccharide of *Gelugula styracifolium* were prepared by the following steps: 1) Crush and sieve the herbal powder, add 20 times the amount of deionized water, soak for 30 minutes, reflux extract for 90 minutes, remove and filter, concentrate the filtrate under reduced pressure to 1 / 4, add anhydrous ethanol to 80% ethanol volume fraction, stir well, and let stand overnight at 4℃. 2) Centrifugation was used to obtain a precipitate. The precipitate was washed three times with anhydrous ethanol, ethyl acetate and acetone respectively. The polysaccharide was dissolved in an appropriate amount of deionized water, filtered, and the filtrate was freeze-dried to obtain crude polysaccharide of Mesona chinensis. 3) The obtained crude polysaccharide of Mesona chinensis was diluted with deionized water and dialyzed using dialysis bags with molecular weights of 7000 D, 100000 D, 300000 D and 500000 D respectively. The solutions were combined, concentrated and freeze-dried to obtain homogeneous polysaccharides of Mesona chinensis with different molecular weights. The crude polysaccharide and homogeneous polysaccharide of the herb Mesona chinensis can promote the growth of intestinal Bacteroides. The homogeneous polysaccharide with a molecular weight of <7000 D and determined by separation and purification is MA1 and MA2 with a molecular weight of 7000~100000 D. It can promote the proliferation of Bacteroides and produce active ingredients after being degraded and utilized by intestinal Bacteroides. The metabolites have lipid-lowering activity. The active ingredient is at least one of monosaccharides, oligosaccharides, and acetic acid or butyric acid.

2. The application of the crude polysaccharide and homogeneous polysaccharide of *Gnaphalium affine* as described in claim 1, characterized in that: The extraction method was heating and reflux, and the heating and reflux extraction time was 90 minutes.

3. The application of the crude polysaccharide and homogeneous polysaccharide of *Gnaphalium affine* as described in claim 1, characterized in that: The concentration method is rotary evaporation, and the concentrated volume is 1 / 4 of the original.

4. The application of the crude polysaccharide and homogeneous polysaccharide of *Gnaphalium affine* as described in claim 1, characterized in that: Add anhydrous ethanol until the ethanol volume fraction is 80%, and stir while adding.

5. The application as described in any one of claims 1-4, characterized in that: The crude and homogeneous polysaccharides of the herb *Gynostemma pentaphyllum* can be made into various preparations, including suppositories, tablets, pills, granules, films, microcapsules, drop pills, aerosols, tinctures, and syrups.

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