New use of atractylodes rhizome polysaccharide

Abstract

The present application aims to disclose a new use of atractylodes rhizome polysaccharide, i.e. the application of atractylodes rhizome polysaccharide in the preparation of health care products or functional foods for reducing the ratio of the relative abundance of intestinal bacteria Firmicutes (F) and Bacteroidetes (B) in people with spleen deficiency, regulating the intestinal bacteria F / B balance, and preventing or treating obesity caused by spleen deficiency intestinal bacteria disorder, wherein the obesity also includes symptoms such as hyperuricemia. The atractylodes rhizome polysaccharide is a heteropolysaccharide composed of five monosaccharides of rhamnose (Rha), galacturonic acid (GalA), glucose (Glu), galactose (Gal) and arabinose (Ara) and has specific fingerprint characteristics. The present application provides a new weight loss product with wide application prospect, and the atractylodes rhizome polysaccharide is safe and effective for preventing and treating the above symptoms.

Description

TECHNICAL FIELD

[0001] The present application relates to a new use of atractylodes rhizome polysaccharide, in particular to regulating the intestinal flora structure homeostasis of spleen deficiency syndrome and its application, belonging to the technical field of traditional Chinese medicine. BACKGROUND

[0002] The intestinal flora is a huge ecosystem, and the number of normal adult human cells is 10 13 times, and the number of intestinal bacteria is about 10 14 times the total number of somatic cells, about 100-150 times the number of genes encoded by the human genome. Microorganisms can exchange information with each other, and can also contact host cells, while participating in the regulation of their metabolism, genes and protein expression. Intestinal flora antagonism, cooperation, maintain the balance between health and disease. Intestinal flora imbalance can increase the risk of chronic diseases such as obesity, metabolic syndrome, inflammatory bowel disease, Alzheimer's disease, and affect human health. The bacterial phylum in human intestinal contents is Firmicutes (F), Bacteroidetes (B), Actinobacteria, and Proteobacteria. The proportion of these four bacteria accounts for more than 90% of all intestinal flora. Most bacteria live in the intestinal tract in symbiosis with the human body, not only helping the human body to digest and absorb, providing energy and nutrients to the human body, but also promoting the secretion of short-chain fatty acids, bile acids, and building an immune system to prevent pathogen invasion. However, the growth of some pathogenic bacteria is inhibited, and once the intestinal microecology is imbalanced, they will rapidly reproduce and cause various chronic diseases such as obesity, cardiovascular disease, metabolic disorder, and Alzheimer's disease. Therefore, regulating intestinal flora and anti-obesity, high blood lipids, and high uric acid syndrome have become urgent problems in current medical research. In recent years, intestinal flora has been considered as a possible pathogenic factor and therapeutic target for many diseases.

[0003] Plant polysaccharides have a prebiotic effect, can promote the growth of beneficial bacteria in the intestine, improve the structure of the flora, and have a positive effect on the prevention and treatment of diseases. Plant polysaccharides from different sources have structural differences in molecular weight distribution, monosaccharide composition and molar ratio, so different polysaccharides have a nurturing effect on different types of intestinal bacteria. In other words, a specific polysaccharide has a regulatory effect on a specific intestinal bacterium, regulates the intestinal flora structure under different disease states, and has different preventive and therapeutic effects.

[0004] It is found through research that intestinal flora is widely involved in the processes of digestion, nutrition, metabolism and immunity of human body, almost all the functional changes of TCM's spleen, and highly related to TCM's spleen, and most of the patients with spleen deficiency syndrome have intestinal flora structure disorder, and modern research finds that the ratio (F / B) of the intestinal flora of the patients with spleen deficiency, especially obese people, is higher than that of normal people. Although research shows that some spleen-invigorating traditional Chinese medicines can regulate intestinal flora disorder, such as Shenling Baizhu Powder and Sijunzi Decoction, but the effect is still not ideal in clinical practice. Although there are reports that polysaccharides in Atractylodes rhizome can proliferate intestinal lactobacillus and bifidobacterium, but the effect of reducing the ratio (F / B) of the relative abundance of intestinal flora of the patients with spleen deficiency and improving the symptoms of obesity and hyperuricemia has not been reported.

[0005] Our research shows that after the intervention of Atractylodes rhizome polysaccharide, the relative abundance of intestinal flora of the mice in the spleen deficiency and obesity model group is obviously reduced, and the relative abundance of Bacteroidetes is obviously increased, and the F / B value tends to be stable, and Atractylodes rhizome polysaccharide has a positive effect on regulating the balance of intestinal flora F / B. Based on a large number of experimental researches on Atractylodes rhizome polysaccharide, it is proved that Atractylodes rhizome polysaccharide can regulate the intestinal flora homeostasis of the patients with spleen deficiency, improve the symptoms of spleen deficiency, obesity, hyperlipidemia and hyperuricemia, and the development of the medicine with the effects of weight loss, spleen invigoration, blood lipid reduction and uric acid reduction has great potential. SUMMARY

[0006] The purpose of the present application is to provide Atractylodes rhizome polysaccharide for regulating the intestinal flora structure homeostasis, i.e. regulating the balance of intestinal flora F / B, for preparing the application of the medicine for preventing or treating obesity and hyperuricemia caused by intestinal flora disorder.

[0007] The Atractylodes rhizome polysaccharide described in the present application is a heteropolysaccharide composed of five monosaccharides of rhamnose (Rha), galacturonic acid (GalA), glucose (Glu), galactose (Gal) and arabinose (Ara) and has specific fingerprint characteristics (see Figure 1 ).

[0008] The specific fingerprint characteristics of the atractylodes rhizome polysaccharide refer to the fact that the atractylodes rhizome polysaccharide is composed of five monosaccharides of rhamnose (Rha), galacturonic acid (GalA), glucose (Glu), galactose (Gal) and arabinose (Ara). The specific fingerprint information is that there are five characteristic peaks in the atractylodes rhizome polysaccharide HPLC fingerprint, wherein the peak No. 1 is rhamnose (Rha), the peak No. 2 is galacturonic acid (GalA), the peak No. 3 is glucose (Glu), the peak No. 4 is galactose (Gal), and the peak No. 5 is arabinose (Ara). These sugars are qualitatively determined according to the retention time of the PMP derivatives of the monosaccharide mixed standard solution measured under the same chromatographic conditions as the atractylodes rhizome polysaccharide sample, and the sum of the peak areas of the five sugars accounts for more than 90% of the total peak area. In the atractylodes rhizome polysaccharide HPLC fingerprint, the glucose chromatographic peak has good separation degree and accounts for the largest proportion in the total peak area of the chromatographic peak, and is used as an internal reference peak to calculate the relative peak area ratio of each main peak. The relative retention time (RT) of each main peak is calculated by taking the derivatization reagent PMP as the internal reference peak. That is, the average RT of the peak No. 1 (Rha) is 0.83, and the relative peak area is 0.15; the average RT of the peak No. 2 (GalA) is 0.79, and the relative peak area is 0.23; the average RT of the peak No. 3 (Glu) is 0.70, and the relative peak area is 1 (reference peak); the average RT of the peak No. 4 (Gal) is 0.66, and the relative peak area is 0.19; and the average RT of the peak No. 5 (Ara) is 0.60, and the relative peak area is 0.20. The relative peak area ratio and the relative standard deviation RSD of the relative retention time of the five characteristic peaks in the atractylodes rhizome polysaccharide HPLC fingerprint are both less than 2% (see Figure 2 ). The above description constitutes the atractylodes rhizome polysaccharide HPLC standard fingerprint characteristics (see Figure 1 ).

[0009] The atractylodes rhizome polysaccharide is extracted from the rhizome or flower of the plant atractylodes lancea (R. Koidz.). (Atractylenolides macrocephala The atractylodes rhizome polysaccharide HPLC fingerprint characteristics have a similarity of more than 60% to the standard fingerprint, preferably 70%, and more preferably 90%. The purity can be 1%-99%, preferably 80%, and more preferably 99%.

[0010] The atractylodes rhizome polysaccharide has a purity of 1%-99%, preferably a purity of 50%-80%, and more preferably a purity of more than 90%.

[0011] The atractylodes rhizome polysaccharide has an ash content of ≤0.2%, and a neutral taste without sweetness.

[0012] The application provides the use of the above-mentioned atractylodes rhizome polysaccharide in the preparation of a medicine for regulating the structure homeostasis of the spleen deficiency intestinal flora, i.e., regulating the F / B balance of the intestinal flora.

[0013] The application provides application of the above-mentioned atractylodes rhizome polysaccharide in preparation of medicines for preventing and treating obesity caused by intestinal flora structure disorder due to spleen deficiency.

[0014] The application provides application of the above-mentioned atractylodes rhizome polysaccharide in preparation of medicines for preventing and treating obesity and hyperuricemia caused by intestinal flora structure disorder due to spleen deficiency.

[0015] The application of the atractylodes rhizome polysaccharide in preparation of medicines for reducing the ratio of the relative abundance of intestinal flora Firmicutes and Bacteroidetes and adjusting the F / B balance of intestinal flora is characterized in that the atractylodes rhizome polysaccharide is a probiotic molecule, can directionally regulate intestinal flora structure homeostasis, enhance the function of the spleen, enhance the water and dampness transport and transformation capacity, and significantly improve the effect of improving the symptoms of obesity and hyperuricemia due to spleen deficiency.

[0016] Preferably, the atractylodes rhizome polysaccharide directionally regulates the homeostasis of the relative abundance of intestinal flora Firmicutes and Bacteroidetes, reduces the increase of the F / B ratio of intestinal flora caused by obesity, and achieves the purpose of weight loss.

[0017] Preferably, the weight loss effect of the atractylodes rhizome polysaccharide has a tonifying effect, can significantly increase the relative abundance of intestinal flora Bacteroides and Parabacteroides, increase the spleen coefficient, and has the effect of invigorating the spleen, and more preferably refers to reducing the symptoms of obesity and hyperuricemia due to spleen deficiency.

[0018] The above-mentioned atractylodes rhizome polysaccharide is supplemented with common pharmaceutical excipients to prepare dosage forms such as aqueous preparations, granules, powders and capsules, and is used for preparing medicines for preventing or treating obesity and metabolic disorders caused by spleen deficiency.

[0019] The technical scheme for solving the above-mentioned problems is as follows.

[0020] The application of the atractylodes rhizome polysaccharide in preparation of medicines for reducing the ratio of the relative abundance of intestinal flora Firmicutes and Bacteroidetes and adjusting the F / B balance of intestinal flora is characterized in that the preparation method of the atractylodes rhizome polysaccharide comprises the following steps:

[0021] a. Collecting rhizome or flower of atractylodes (Atractylodes Koidz.) and crushing and sieving. Atractylenolides macrocephala koidz.) rhizome or flower, crushing and sieving.

[0022] b. Preparing atractylodes crude polysaccharide by using a common water extraction and alcohol precipitation method.

[0023] c. Adding distilled water to dissolve the atractylodes crude polysaccharide, adding mixed solvents trichloroacetic acid and n-butanol (volume ratio 1:8-10) with the same volume of the distilled water, violently shocking, standing, taking supernatant after layering, removing the upper n-butanol layer and the middle layer of impurities, adding a certain concentration of NaOH solution to neutralization, evaporating to dryness, and repeatedly performing the above steps multiple times to remove glycoprotein and decolorize to obtain pure atractylodes polysaccharide.

[0024] The crude atractylodes rhizome polysaccharide can also be purchased through market channels, and should have the fingerprint characteristics of the atractylodes rhizome polysaccharide HPLC as shown in Figure 1 , and the similarity compared with the standard fingerprint is greater than 60%, preferably 70%, and more preferably 90%. The purity can be 1%-99%, preferably 80%, and more preferably 99%, and can be prepared into a suitable concentration according to the actual situation.

[0025] The atractylodes rhizome polysaccharide has a new use for improving the symptoms of spleen deficiency obesity and hyperuricemia by regulating the intestinal flora homeostasis of the spleen deficiency population, and is characterized in that the monosaccharide composition is rhamnose (Rha), galacturonic acid (GalA), glucose (Glu), galactose (Gal), and arabinose (Ara) heteropolysaccharide; the weight average molecular weight Mw of the polysaccharide ranges from 1500 to 10000 Da, preferably from 2000 to 6000 Da, and more preferably from 3500 to 5000 Da; the infrared spectrum of the polysaccharide has characteristic peaks of polysaccharide, O-H stretching vibration absorption peak near 3342 or 3325 cm -1 , C-H stretching vibration peak near 2953 cm -1 , 2924 cm -1 , and 2854 cm -1 , C=O stretching vibration absorption peak near 1737 cm -1 , and larger absorption peaks at 1122 and 1095, indicating C-O and sugar ring stretching vibration signals, and preferably, the main stretching vibration absorption peaks of the infrared spectrum of the polysaccharide are basically the same as those in the infrared spectrum shown in Figure 3 .

[0026] In the present application, the atractylodes rhizome polysaccharide can be modified to further improve its performance or broaden its use without affecting its target application effect. The modification can be a conventional modification method for polysaccharides in the art, such as by a fluorescent labeling reaction, an etherification reaction, an esterification reaction, etc. The polysaccharide can be further modified into a fluorescently labeled product, a carboxymethylated product, a hydroxyethylated product, a hydroxypropylated product, a glycolated product, a propylene glycolated product, a polyethylene glycolated product, etc. of the above-mentioned polysaccharide according to the application target. It should be understood that the above-mentioned modified polysaccharides should also be within the scope of protection of the present application.

[0027] The beneficial effects of the present application are: spleen deficiency combined with obesity and hyperuricemia has become a common public health problem in the world. At present, the drugs used for weight loss purposes mainly include: hormone, appetite suppressants, metabolism promoters, fat absorption inhibitors and other weight loss drugs. These functional weight loss drugs affect the normal function of the human body and have safety hazards. It is of great significance to find safe and effective weight loss products. Traditional Chinese medicine believes that the spleen governs transportation and transformation, ascending clear and unifying blood. Obesity is caused by insufficient transportation and transformation of the spleen due to spleen deficiency, and the ingested food and water cannot be converted into fine substances needed by the body in time, forming evil qi such as phlegm and dampness, thereby causing obesity. Weight loss requires tonifying the spleen and invigorating qi to promote the transportation and transformation of water and dampness by the spleen and stomach. Only when the transportation and transformation function of the spleen is good, can the weight loss effect be achieved. Modern research has also confirmed that obesity is related to the change of the relative abundance of intestinal thick-walled bacteria and Bacteroidetes, and the relative proportion of Bacteroidetes in the intestinal tract of obese people is reduced, and the F / B ratio is increased. Our research found that the atractylodes rhizome polysaccharide with the above characteristics can reduce the intestinal F / B ratio, regulate the intestinal flora homeostasis of the spleen deficiency population, and improve the symptoms of obesity, hyperuricemia and the like. The present application provides a new weight loss and uric acid reducing drug with wide application prospect, which has extremely important significance. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 . HPLC standard fingerprint spectrum of atractylodes rhizome polysaccharide [No. 1 peak is rhamnose (Rha), No. 2 peak is galacturonic acid (GalA), No. 3 peak is glucose (Glu), No. 4 peak is galactose (Gal), and No. 5 peak is arabinose (Ara)].

[0029] Figure 2 . HPLC fingerprint spectrum of 38 batches of atractylodes rhizome polysaccharide.

[0030] Figure 3 . Infrared spectrum of atractylodes rhizome polysaccharide.

[0031] Figure 4 . Systematic diversity index of fecal flora of mice [ (a) alpha diversity analysis; (b) beta diversity analysis; CK: control group; A0: spleen deficiency and obesity mouse model group; PC: positive control group; AH: high-dose atractylodes rhizome polysaccharide intervention group (n = 5 in each group). Compared with the A0 group, * p < 0.05 and ** p < 0.01; compared with the CK group, # P < 0.05 and ## P < 0.01].

[0032] Figure 5 . Relative abundance of bacterial phylum in feces of mice (CK: control group; A0: spleen deficiency and obesity model group; PC: positive control group; AH: high-dose atractylodes rhizome polysaccharide intervention group (n = 5)).

[0033] Figure 6. The relative abundance of Firmicutes and Bacteroidetes in the intestinal contents of patients before and after Baizhu polysaccharide intervention Figure 6 a) and the ratio of Firmicutes and Bacteroidetes in the intestinal contents Figure 6 b).

[0034] Figure 7 . Comparison of the relative abundance of the genera of Streptococcus ( Ruminococcaceae ), Akkermansia ( Akkermansia ), and Lachnospira ( Lachnospiraceae ) in the intestinal contents of patients before and after Baizhu polysaccharide intervention.

[0035] The following experimental examples are used to further illustrate but are not limited to the present application.

[0036] Experimental Example 1: Establishment of Baizhu polysaccharide standard fingerprint

[0037] 1.1 Instruments Waters high-performance liquid system (2695 Separations Module, 2996 Photodiode Array Detector, Empower chromatography workstation)

[0038] 1.2 Reagents and samples

[0039] Monosaccharide standards: Rhamnose (Rha, ≥99%), Galacturonic acid (GalA, ≥99%), Glucose (Glul, ≥99%), Galactose (Gal, ≥99%), Xylose (Xyl, ≥99%), and Arabinose (Ara, ≥99%) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (China, Shanghai); Derivatization reagents: 1-phenyl-3-methyl-5-pyrazolium (PMP), purity 99% (Acros Organics Company, USA); trifluoroacetic acid (TFA), purity not less than 99% (National Pharmaceutical Group Chemical Reagent Co., Ltd.). Acetonitrile, HPLC grade (Tedia Company, USA); water, ultrapure grade; other reagents were of analytical purity.

[0040] 1.3 Test materials

[0041] Thirty-eight batches of Baizhu medicinal material samples were collected from farmers in the place of origin or from medicinal material markets, and the specific information is shown in Table 1. Part of the medicinal material samples from each production area were crushed and placed in sealed bags for use. The collected Baizhu powder was passed through a 60-mesh sieve, and the obtained Baizhu coarse powder was used.

[0042] Table 1. Information on the production area and collection time of Baizhu medicinal materials

[0043]

[0044] 1.4 Preparation of the test solution

[0045] The crude powder of Atractylodes macrocephala Koidz was used to obtain polysaccharides by water extraction and alcohol precipitation. The polysaccharides were hydrolyzed by trifluoroacetic acid (TFA), and the monosaccharide derivatives were obtained after PMP derivatization and chloroform extraction purification. The test solution was prepared after 0.45 μm microporous membrane filtration. The PMP derivatization product solution of the mixed standard was prepared under the same conditions and analyzed by HPLC.

[0046] 1.5 HPLC chromatographic conditions

[0047] A Waters high-performance liquid chromatograph was used for analysis. A Sun Fire C18 column (250 mm*4.6 mm, 5 μm) from Waters was used, the column temperature was 30 °C, the injection volume was 20 μL, the flow rate was 1 mL / min, the injection time was 50 min, and the monosaccharide detection wavelength was 245 nm. The mobile phase A was phosphate buffer (0.1 mol / L KH2PO4-0.2 mol / L Na2OH, pH=6.0), and the mobile phase B was acetonitrile, which was gradient eluted at the proportions shown in Table 2. System suitability tests were performed, and the results showed that the precision, repeatability, and stability met the requirements.

[0048]

[0049] 1.6 PMP derivatization product fingerprint analysis by reversed-phase high-performance liquid chromatography:

[0050] Under these conditions, the PMP derivatization products of the test sample and the monosaccharide mixed standard were analyzed by injection, and the chromatographic fingerprints of Atractylodes macrocephala Koidz polysaccharides and the mixed standard were obtained. By comparing the HPLC spectra of the PMP derivatization samples of the five monosaccharides, the characteristics of the peaks corresponding to each Atractylodes macrocephala Koidz polysaccharide test solution were determined. In the comparison of the chromatograms of the control and the sample, glucose was found to have good separation and to account for the largest proportion of the peak area in the chromatographic peak, so it was used as an internal reference peak to calculate the relative peak area ratio of each main peak; the PMP derivatization reagent was used as an internal reference peak to calculate the relative retention time of each main peak.

[0051] 1.7 Establishment of standard fingerprint

[0052] The 38 batches of Atractylodes macrocephala Koidz polysaccharide test solutions were analyzed by high-performance liquid chromatography under the same chromatographic conditions, and the high-performance liquid chromatograms of the 38 batches of Atractylodes macrocephala Koidz polysaccharides were obtained, as shown in Figure 1 The measured data of the 38 batches were imported and analyzed using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2004A" software of the National Pharmacopoeia Committee. The chromatographic peaks were automatically matched to generate a control chromatogram. After multi-point correction, a common mode control fingerprint was generated, and the HPLC standard fingerprint of Atractylodes macrocephala Koidz polysaccharides was established, as shown in Figure 2 .

[0053] The average RT of peak 1 (Rha) was 0.83 (RSD 0.05%), and the relative peak area was 0.15 (RSD 1.9%).

[0054] The average RT for peak 2 (GalA) was 0.79 (RSD of 0.08%), and the relative peak area was 0.23 (RSD of 2.0%).

[0055] The average RT for peak 3 (Glu) was 0.7 (RSD 0.70%), and the relative peak area was 1 (reference peak) (RSD 0).

[0056] The average RT for peak 4 (Gal) was 0.66 (RSD 1.68%), and the relative peak area was 0.19 (RSD 0.58%).

[0057] The average RT of peak 5 (Ara) is 0.60 (RSD is 0.90%), and the relative peak area is 0.20 (RSD is 1.4%).

[0058] The ratio of the relative peak areas and the relative standard deviation (RSD) of the relative retention times of the five characteristic peaks in the HPLC fingerprint of Atractylodes macrocephala polysaccharide were both less than 2.0% (e.g., Figure 2 (As shown).

[0059] Experiment Example 2: Effects of Atractylodes macrocephala polysaccharide on spleen deficiency and obesity symptoms in mice

[0060] 2.1 Medicinal Material: Atractylodes macrocephala polysaccharide, obtained from Atractylodes macrocephala (… Atractylenolides macrocephala (koidz.) After extraction and purification, the polysaccharide has a purity of 99%. The fingerprint spectrum of the Atractylodes macrocephala polysaccharide was analyzed using the HPLC method of this invention. The fingerprint spectrum of the Atractylodes macrocephala polysaccharide showed five characteristic peaks: peak 1 (Rha) with a relative peak area of ​​0.15; peak 2 (GalA) with a relative peak area of ​​0.23; peak 3 (Glu) as a reference peak with a relative peak area of ​​1; peak 4 (Gal) with a relative peak area of ​​0.19; and peak 5 (Ara) with a relative peak area of ​​0.20. The similarity to the HPLC standard fingerprint spectrum of Atractylodes macrocephala polysaccharide provided in Experimental Example 1 was greater than 90%. The main stretching vibration absorption peak of the infrared spectrum of the Atractylodes macrocephala polysaccharide was similar to... Figure 2 The infrared spectra shown are basically consistent, and the weight-average molecular weight (Mw) ranges from 3500 to 5000 Da.

[0061] 2.2 Laboratory Animals and Materials

[0062] 2.2.1 Experimental animals: 70 SPF KM male mice (18-20 g) were purchased from Zhejiang Provincial Academy of Medical Sciences, and were raised in a SPF barrier system with an ambient temperature of 20-23°C and a relative humidity of 45-65%, and were allowed to drink water and eat freely.

[0063] 2.2.2 Experimental materials: 16S rDNA sequencing analysis was performed by Jiangsu Jinweizhi Biotechnology Co., Ltd.; MetaVx™ library construction kit was from GENEWIZ; Illumina MiSeq sequencing platform was from GENEWIZ; Qubit 2.0 Fluorometer was from Invitrogen Carlsbad; VSEARCH (1.9.6) was from GENEWIZ; Beckman AU5400 automatic biochemical analyzer was from the United States; electronic balance (AUY120 Shimadzu analytical balance) was from Shimadzu Corporation; triglyceride assay kit (Changchun Hui Li, batch number 2019007); total cholesterol assay kit (Changchun Hui Li, batch number 2019004); high-density lipoprotein assay kit (Changchun Hui Li, batch number 2019006); low-density lipoprotein assay kit (Changchun Hui Li, batch number 2019008).

[0064] 2.3 Test method

[0065] 2.3.1 Model establishment

[0066] ① Animal grouping: 70 male mice were adaptively raised and quarantined for one week, and were randomly divided into 2 groups, of which 10 were normal group and 60 were modeling group for the previous experiment, and five mice were raised in each cage.

[0067] ② Modeling: 79% Hubei Wanqianjiaxing basic feed (protein content 30%) was used as the basis, and 10% high-cholesterol animal oil, 10% cholesterol, and 1% cholate were added to form the modeling feed. After one week of feeding with high-fat feed, the mice were simultaneously fed with high-fat feed and swum once a day until the endurance limit was reached, and then the mice were returned to the cage. The swimming duration was 21 days. The mice were continuously fed with high-fat feed for seven weeks, and the modeling was completed. The mice with body weight > normal group and average body weight + 2SD were selected as successfully modeled obese mice.

[0068] 2.3.2 Determination of related indexes in each group after intervention:

[0069] A experimental group (30) and B experimental group (30), each experimental group was divided into blank control group, model group, high-dose group (AH), medium-dose group (AM), low-dose group (AL) and positive control group (liraglutide). A experimental group was used for general behavior test and body weight test; B experimental group was used for determination of physiological indexes (organ coefficient, triglyceride, total cholesterol, low-density lipoprotein), fecal DNA extraction.

[0070] ① High-throughput sequencing of intestinal flora: The total DNA of the feces of mice in each group on day 7 after treatment was extracted according to the Upgraded Version of the Fecal Genomic DNA Rapid Extraction Kit (Centrifugal Column Type) of Beijing Baitaike Biotechnology Co., Ltd. The amplification and sequencing library construction of the V3-V4 region of 16S rDNA and the bioinformatics analysis were completed by Jiangsu Jinweizhi Biotechnology Co., Ltd. based on the Illumina MiSeq sequencing platform. The sequencing library was constructed using the MetaVx™ Library Construction Kit (GENEWIZ, Inc. South Plainfield, NJ, US), and the sequence results were optimized and analyzed using Bcl2fastq (v2.17.1.14), VSEARCH (1.9.6), Qiime (1.9.1), and R language software.

[0071] ② Detection of blood lipid components in mice: After the end of treatment, the mice were fasted for 12 h, and the blood was taken from the eyes and centrifuged to separate the serum. The content of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), and uric acid (UA) in the serum was detected using a full-wavelength multifunctional enzyme label instrument according to the detailed operation steps in the mouse ELISA kit instruction manual; and the spleen coefficient was observed by taking the spleen.

[0072]

[0073] ③ Body weight and general behavior test: The body weight of mice in the blank control and model groups was weighed and recorded before and after modeling, and the behavior was observed; the mental state, body characteristics, and other changes of mice in each group were observed and recorded during the treatment.

[0074] 2.4 Test results

[0075] 2.4.1 Effect on the structure of intestinal flora in mice

[0076] At the end of the experiment, the feces of mice were taken under sterile conditions, and the intestinal flora of the feces of mice was sequenced and analyzed using 16S rRNA to compare the changes in the composition of intestinal flora between groups. The V3-V4 region of the 16S rDNA gene sequence was subjected to a diversity analysis. As shown in Figure 4 The community diversity index (Shannon index, Figure 4 a), the community diversity of the A0 and AH groups was significantly lower than that of the CK group (P<0.01). In addition, in order to evaluate the similarity and distance of the ecological complexity between all group samples (β diversity), the OUT distance was subjected to principal coordinate analysis (PCoA) based on the Brary-Curtis method. As shown in Figure 4b shows that the intestinal flora is similar between A0 and PC groups, while there is a significant difference between the intestinal flora of A0 and AH groups and that of CK group. These results indicate that the intervention of Atractylodes Rhizome Polysaccharide significantly changes the intestinal flora structure of the spleen deficiency type obese mice.

[0077] Figure 5 As shown, compared with the CK group, the relative abundance of Bacteroidetes in the intestinal feces of the model group (A0) of spleen deficiency type obesity mice is significantly reduced, while the relative abundance of Firmicutes is significantly increased. After the intervention of Atractylodes Rhizome Polysaccharide (AH), the relative abundance of Bacteroidetes in the feces of mice is increased, the relative abundance of Firmicutes is reduced, and the F / B ratio tends to be stable, while there is no significant change in the positive drug intervention group (PC).

[0078] 2.4.2 Behavioral observation of mice:

[0079] The mice in the blank control group are active and lively, have lustrous hair, and normal defecation. The mice in the model group have limb weakness, lethargy, debilitation, back arching, and reduced feces. After drug treatment, the mice have more lustrous hair, are lively, and have normal fecal form and defecation particle number.

[0080] 2.4.3 Changes in body weight of mice and spleen coefficient:

[0081] After 8 weeks of feeding, the body mass index of the model group is significantly higher than that of the normal group (P<0.05), indicating that the model mice meet the obesity conditions. The fecal water content of the model group is significantly higher than that of the normal group (P<0.05), which is consistent with the TCM syndrome type of TCM diarrhea, indicating that the model mice begin to change to the spleen deficiency obesity type. After 2 weeks of intervention, the mice in the high-dose Atractylodes Rhizome Polysaccharide group have a decreased body weight, which is maintained at a low level for 4 weeks after treatment, showing a significant difference from the model group and showing a trend of.

[0082] The spleen coefficients of the Atractylodes Rhizome Polysaccharide groups are higher than those of the negative control and positive control groups, among which the middle-dose group shows a significant difference from the model group (p<0.05), and there is no significant difference between the other groups.

[0083] Table 3. Comparison of body weight and spleen coefficient between the normal group and the model group after 4 weeks of intervention of Atractylodes Rhizome Polysaccharide

[0084]

[0085] Note: Compared with the normal group: *P<0.05, **p<0.01; compared with the model group: #P<0.05, ##p<0.01.

[0086] 2.4.4 Effect on blood lipids and uric acid of obese mice, the results are shown in Table 4.

[0087] Table 4. Effects of Atractylodes macrocephala polysaccharide on blood lipid and uric acid levels in mice (n=5)

[0088]

[0089] Note: Compared with the normal group: *P<0.05, **p<0.01; Compared with the model group: #P<0.05, ##p<0.01.

[0090] The results showed that compared with the normal group, the levels of blood lipid components TG, TC, LDL, and UA in the model group were significantly increased. Compared with the model group, the levels of blood lipids TG, TC, LDL, and UA in the positive control group, high-dose group, and medium-dose group of Atractylodes macrocephala polysaccharide were significantly decreased. The low-dose group of Atractylodes macrocephala polysaccharide also showed a significant decrease in TG, TC, and UA levels. The Atractylodes macrocephala polysaccharide intervention group had a significant reducing effect on hyperuricemia in obese mice, exhibiting a dose-response relationship.

[0091] Experimental Example 3: Clinical observation of the treatment of spleen-deficiency type obesity with Atractylodes macrocephala polysaccharide of the present invention.

[0092] 3.1 Medicines

[0093] Atractylodes macrocephala polysaccharide is used to extract Atractylodes macrocephala polysaccharide. Atractylenolides macrocephala The polysaccharide obtained by extraction and purification (koidz.) with a purity of 99% was prepared into capsules. The fingerprint spectrum of the Atractylodes macrocephala polysaccharide was analyzed using the HPLC method of this invention. Five characteristic peaks were found in the fingerprint spectrum: peak 1 (Rha) with a relative peak area of ​​0.15; peak 2 (GalA) with a relative peak area of ​​0.23; peak 3 (Glu) as a reference peak with a relative peak area of ​​1; peak 4 (Gal) with a relative peak area of ​​0.19; and peak 5 (Ara) with a relative peak area of ​​0.20. The similarity to the HPLC standard fingerprint spectrum of Atractylodes macrocephala polysaccharide provided in Experimental Example 1 was greater than 90%. The main stretching vibration absorption peak of the infrared spectrum of the Atractylodes macrocephala polysaccharide was similar to... Figure 2 The infrared spectra shown are basically consistent, and the weight-average molecular weight (Mw) ranges from 3500 to 5000 Da.

[0094] 3.2 Case Data

[0095] Referring to the "Guidelines for Surgical Treatment of Obesity in China" and the "Guiding Principles for Clinical Research of New Traditional Chinese Medicine" published by the Chinese Medical Association in 2007, 50 patients with simple obesity of spleen deficiency and dampness obstruction type were selected; according to the body mass index (BMI) method (kg / m²), the following criteria were applied: 2), overweight BMI = 24-29.9, mild obesity BMI = 30-34.9, moderate obesity BMI = 35-39.9, severe obesity BMI = 40, abdominal obesity is defined as waist circumference greater than or equal to 85 cm in men and greater than or equal to 80 cm in women. The diagnosis of spleen deficiency and dampness type refers to the symptoms of spleen deficiency and dampness type, such as obesity, overweight, abdominal fullness and softness, more facial oil, easy to be tired, teeth marks on the edge of the tongue, fatigue, heavy limbs, abdominal distension, poor appetite, small amount of urine, loose and thin stool, mild edema in lower limbs, pale and obese tongue with teeth marks, and white and greasy tongue coating.

[0096] Randomly divided into treatment group and control group. Among them, 25 cases in the treatment group, 25 cases in the control group. There was no significant difference in gender, age, disease distribution and other general data between the two groups, which was comparable.

[0097] 3.3 Usage

[0098] Treatment group: take the medicine for treatment, take the capsule prepared in Example 1, each Atractylodes rhizome polysaccharide (purity 99%) 0.4 g, 4-5 capsules once a day, half an hour after meal, warm water, 3 months as a course of treatment.

[0099] Control group: take oral Qingshen Xiaobang pills for treatment, GMP code Z11020496, 240s*3 bottles, 30 pills at a time, 3 times a day.

[0100] The curative effects of the two groups were evaluated after 2 months of treatment.

[0101] 3.4 Curative effect evaluation standard

[0102] Clinical efficacy evaluation of Western medicine (1) clinical cure: clinical symptoms disappear or basically disappear, waist circumference reduction > 10.0 cm, BMI reduction > 2.5 to normal or overweight range (BMI < 25); (2) significant effect: most clinical symptoms disappear or basically disappear, waist circumference reduction > 7.0 cm, BMI reduction > 1.8, body weight reduction > 5.0 kg; (3) effective: clinical symptoms are obviously relieved, waist circumference reduction > 4.0 cm, BMI reduction > 1.0, body weight reduction > 3.0 kg; (4) ineffective: no obvious improvement in clinical symptoms, waist circumference reduction < 4.0 cm, BMI reduction < 1.0, body weight reduction < 3.0 kg.

[0103] TCM syndrome score: The main symptoms of obesity of spleen deficiency and dampness obstruction type include obesity, body fatigue, lazy speech, tasteless food, abdominal distension after eating, and heavy head and body. According to the patient's condition, normal score 0 points, mild score 2 points, moderate score 4 points, and severe score 6 points. Tongue and pulse symptoms include tongue body obesity, edge with tooth marks, white and greasy fur, and thin and smooth pulse. Among them, tongue and fur are scored according to symptoms 0, 2, 4, and 6 points; pulse is soft and smooth, and is scored 2 points. The total score is 0-44 points, and the higher the score, the more severe the symptoms. The TCM syndrome efficacy evaluation refers to the "Guiding Principles for Clinical Research of New Drugs of Traditional Chinese Medicine". The efficacy of TCM is determined by the TCM syndrome score, and the efficacy index = (pre-treatment score - post-treatment score) / pre-treatment score x 100.0%. (1) Clinical cure: TCM clinical symptoms and signs disappear or basically disappear, and the efficacy index is ≥90.0%; (2) Marked effect: TCM clinical symptoms and signs are significantly improved, and the efficacy index is 70.0%≤efficacy index<90.0%; (3) Effective: TCM clinical symptoms and signs are improved, and the efficacy index is 30.0%≤efficacy index<70.0%; (4) Ineffective: TCM clinical symptoms and signs have no obvious improvement, and even aggravate, and the efficacy index is <30.0%.

[0104] 3.5 Laboratory index detection: The patient's venous blood was collected before and after treatment, and the total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and uric acid (UA) were detected by spectrophotometric determination and potentiometric determination using Beckman AU5400 automatic biochemical analyzer.

[0105] 3.6 Statistical method The data were analyzed by SPSS 22.0 statistical software. P<0.05 was considered statistically significant.

[0106] 3.7 Treatment effect:

[0107] 3.7.1 Comparison of physical examination indexes of patients in two groups before and after treatment

[0108] As shown in Table 5, the physical examination indexes of patients in two groups before and after treatment were compared: before treatment, the height, body mass, waist circumference, hip circumference, and BMI of patients in two groups had no statistically significant difference (p>0.05). After treatment, the body mass, waist circumference, and BMI of patients in two groups had statistically significant difference (p<0.05); the hip circumference had no statistically significant difference (p>0.05). After treatment, the body mass, waist circumference, hip circumference, and BMI of patients in the control group had no statistically significant difference compared with those before treatment (p>0.05). After treatment, the body mass, waist circumference, hip circumference, and BMI of patients in the observation group had statistically significant difference compared with those before treatment (p<0.05).

[0109] Table 5 Comparison of examination indexes of patients in two groups before and after treatment

[0110]

[0111] Note: compared with the control group, *p<0.05.

[0112] 2.4.2 Comparison of TCM syndrome score between the two groups before and after treatment

[0113] As shown in Table 6: before treatment, the TCM syndrome scores of the control group and the treatment group were (21.3±0.5) and (22.3±4.2) points, respectively, and the difference between the two groups was not statistically significant (p>0.05); after treatment, the TCM syndrome scores of the two groups were (12.4±0.5) and (9.8±2.9) points, respectively, and the difference between the two groups was statistically significant (p<0.05). After treatment, the TCM syndrome scores of the two groups were significantly different from those before treatment (p<0.05). After treatment, the TCM syndrome scores of the control group and the treatment group were 2 cases of marked effectiveness, 22 cases of effectiveness, and 10 cases of ineffectiveness in the control group, and 5 cases of marked effectiveness, 26 cases of effectiveness, and 2 cases of ineffectiveness in the treatment group.

[0114] Table 6 Comparison of clinical efficacy between the two groups (cases)

[0115]

[0116] Note: compared with the control group, #P<0.05; compared with before treatment, *P<0.05.

[0117] 2.4.3 Comparison of blood lipid indexes between the two groups before treatment TC, TG, LDL-C, and HDL-C had no statistically significant difference (P>0.05). After treatment, TC and HDL-C of the two groups had no statistically significant difference (P>0.05); TG and LDL-C had statistically significant difference (P<0.05). After treatment, TC of the two groups had no statistically significant difference from that before treatment (P>0.05); TG, LDL-C, and HDL-C had statistically significant difference from those before treatment (P<0.05), see Table 7.

[0118] Table 7 Comparison of blood lipid indexes between the two groups before and after treatment (mmol / L)

[0119]

[0120] Note: compared with the control group, #P<0.05; compared with before treatment, *P<0.05.

[0121] Experiment 4 Effect of atractylodes rhizome polysaccharide on human intestinal microbial flora

[0122] 4.1 Medicines

[0123] Atractylodes Rhizome Polysaccharide, which is polysaccharide extracted and purified from Atractylodes Rhizome with a purity of 99%, is prepared into capsules. The Atractylodes Rhizome Polysaccharide has 5 characteristic peaks in the fingerprint spectrum detected by the HPLC method of the present application, the relative peak area of peak No. 1 (Rha) is 0.15; the relative peak area of peak No. 2 (GalA) is 0.23; peak No. 3 (Glu) is a reference peak with a relative peak area of 1; the relative peak area of peak No. 4 (Gal) is 0.19; and the relative peak area of peak No. 5 (Ara) is 0.20. The similarity of the Atractylodes Rhizome Polysaccharide HPLC standard fingerprint spectrum provided in Experimental Example 1 is greater than 98% compared with the Atractylodes Rhizome Polysaccharide. The main stretching vibration absorption peaks of the infrared spectrum of the Atractylodes Rhizome Polysaccharide are basically consistent with those in the infrared spectrum shown in Fig. 1, and the weight average molecular weight Mw ranges from 3500 to 5000 Da. Figure 2

[0124] 4.2 Fecal sample microbial flora analysis: Fecal samples of 5 obese volunteers in Experimental Example 3 before taking the Atractylodes Rhizome Polysaccharide and after taking it for 90 days were randomly collected, total DNA was extracted according to the Fecal Genomic DNA Rapid Extraction Kit Upgrade Version (Centrifugal Column Type) of Beijing Baitaike Biotechnology Co., Ltd., sequencing was performed on the high variable region of 16s rDNA using the Illumina MiSeq platform, Reads were spliced and filtered, OUTs were clustered, and species annotation and abundance analysis were performed. According to the analysis of the Alpha diversity (Alpha diversity) index, the ACE index and the Chao1 index represent the change in the abundance of intestinal flora.

[0125] 4.3 Results:

[0126] 4.3.1 As can be seen from the ACE index, Chao1 index, Shannon index and Simpson index of each group in Table 8, the intestinal flora structure of obese patients changed after taking the Atractylodes Rhizome Polysaccharide, and the relative abundance and diversity of intestinal flora after taking the Atractylodes Rhizome Polysaccharide for 90 days were significantly up-regulated compared with before taking it, and the Chao1, Shannon and ACE values were significantly different (P<0.05) compared with before taking it. The results show that the Chao1 index and Shannon index of the intestinal flora of patients after intervention with the Atractylodes Rhizome Polysaccharide increase, the Simpson index decreases, the diversity of the fecal flora increases, the species in the sample tend to be rich, and the ability to resist external interference is enhanced.

[0127] Table 8. Changes in the relative abundance and diversity of intestinal flora before and after taking the Atractylodes Rhizome Polysaccharide

[0128]

[0129] 4.3.2 As Figure 6 ​As shown, after taking atractylodes rhizome polysaccharide for 90 days, the relative abundance of the phylum Firmicutes decreased, the relative abundance of the phylum Bacteroidetes increased, and the F / B ratio significantly decreased (P<0.05); at the genus level, the relative abundance of Ruminococcaceae decreased, and the relative abundance of Akkermansia and Lachnospiraceae rebounded to varying degrees.

[0130] From the analysis of the intestinal flora structure of the intestinal contents of the volunteers before and after taking the atractylodes rhizome polysaccharide, it can be known that the atractylodes rhizome polysaccharide improves the intestinal flora abundance and species diversity of obese patients. It significantly affects the relative abundance ratio of the phylum Firmicutes and the phylum Bacteroidetes in the intestinal flora of obese patients with spleen deficiency, and plays a role in regulating the intestinal flora structure homeostasis of obese patients with spleen deficiency.

[0131] Experimental Example 5 Acute oral toxicity test

[0132] Reagents: atractylodes rhizome polysaccharide, the atractylodes rhizome polysaccharide detected by the HPLC method of the present application has 5 characteristic peaks in the atractylodes rhizome polysaccharide fingerprint, the relative peak area of peak No. 1 (Rha) is 0.15; the relative peak area of peak No. 2 (GalA) is 0.23; peak No. 3 (Glu) is a reference peak, and the relative peak area is 1; the relative peak area of peak No. 4 (Gal) is 0.19; and the relative peak area of peak No. 5 (Ara) is 0.20. The similarity of the atractylodes rhizome polysaccharide HPLC standard fingerprint provided in Experimental Example 1 is greater than 90% compared with the atractylodes rhizome polysaccharide HPLC standard fingerprint provided in Experimental Example 1. The main stretching vibration absorption peaks of the infrared spectrum of the atractylodes rhizome polysaccharide are basically consistent with those in the infrared spectrum shown in the present application. Figure 2 The weight average molecular weight Mw is in the range of 3500-5000 Da.

[0133] Experimental results: according to the maximum gavage concentration and the maximum gavage dose, the maximum gavage concentration is 0.33 g / ml, the gavage amount is 0.3 ml / 10 g, and the gavage is performed 3 times in 24 hours. After 7 days of observation, no mice died and the mice were active, and no obvious toxic symptoms were observed. Therefore, the total dosage of the mice is 0.59 g / 20 g (29.7 g / kg.bw), and since no mice died, the maximum tolerance dose is greater than 29.7 g / kg, which is equivalent to 445 times the human intake. After the mice were sacrificed, visual observation showed that there was no abnormal fluid in the chest and abdominal cavity of the mice; the color and shape of the heart, liver, spleen, stomach, kidney and other important organs were normal, and there were no bleeding points or other abnormal pathological changes. It can be determined that the median lethal dose LD 50 >29.7 g / kg, according to the acute toxicity dose classification evaluation standard, it is non-toxic level, so the atractylodes rhizome polysaccharide belongs to non-toxic level and has high safety.

[0134] The following examples can achieve the effects of the experimental examples: DETAILED DESCRIPTION

[0135] Example 1

[0136] Main drug: commercially available crude atractylodes rhizome polysaccharide with a content of 35%. The crude polysaccharide is subjected to HPLC fingerprint analysis according to the analysis method provided in Experimental Example 1, and there are 5 characteristic peaks in the atractylodes rhizome polysaccharide fingerprint, the relative peak area of No. 1 peak (Rha) is 0.18; the relative peak area of No. 2 peak (GalA) is 0.26; No. 3 peak (Glu) is a reference peak, and the relative peak area is 1; the relative peak area of No. 4 peak (Gal) is 0.18; the relative peak area of No. 5 peak (Ara) is 0.25, and the similarity is greater than 88% compared with the atractylodes rhizome polysaccharide HPLC standard fingerprint provided in Experimental Example 1. The commercially available atractylodes rhizome polysaccharide is added with 75-80 times the volume of distilled water, and the same volume of trichloroacetic acid:n-butanol (volume ratio of 1:8-10) is added, and the mixture is shaken vigorously for 20-25 min, and then allowed to stand until the layers are separated. The supernatant is taken, the upper n-butanol layer and the middle layer of impurities are removed, and a certain concentration of NaOH is added to neutralize it. The mixture is evaporated to dryness, and the above steps are repeated several times to remove glycoprotein, and then H2O2 is used to remove pigment to obtain pure atractylodes rhizome polysaccharide (content of 99%). After drying in a 60°C oven, it is ready for use. The main stretching vibration absorption peaks of the infrared spectrum of the purified atractylodes rhizome polysaccharide are basically consistent with those in the infrared spectrum shown in Figure 2 The weight average molecular weight Mw ranges from 1500 to 10000 Da.

[0137] Auxiliary material: starch

[0138] The above atractylodes rhizome polysaccharide is added with 30% of its weight of lactose, and a powder is prepared by a conventional method.

[0139] Example 2

[0140] Main drug: atractylodes rhizome polysaccharide with a content of 35% obtained from atractylodes rhizome (Rhizoma Atractylodis Macrocephalae) according to the method provided in Experimental Example 1. Atractylenolides macrocephalaAtractylodes lancea (Roxb.) Koidz. root and rhizome powder. The specific extraction method is as follows: take Atractylodes lancea (Roxb.) Koidz. root and rhizome powder, add water (w / v, 1:10), extract at 90°C for 3h, repeat twice, combine the filtrate, after standing, collect the filtrate by vacuum filtration, concentrate to extract. After cooling to room temperature, add 4-5 times the volume of 95% ethanol to precipitate polysaccharides, after 12h, filter to obtain Atractylodes lancea (Roxb.) Koidz. crude polysaccharides, then add 2 times the volume of water to dissolve, precipitate polysaccharides again, filter to obtain crude polysaccharides with a purity of 50%, dry in an oven at 60°C, and reserve. The crude polysaccharides are subjected to HPLC fingerprint analysis according to the analysis method provided in Experimental Example 1. There are 5 characteristic peaks in the Atractylodes lancea (Roxb.) Koidz. polysaccharide fingerprint, the relative peak area of peak 1 (Rha) is 0.13, the relative peak area of peak 2 (GalA) is 0.20, peak 3 (Glu) is the reference peak with a relative peak area of 1, the relative peak area of peak 4 (Gal) is 0.21, and the relative peak area of peak 5 (Ara) is 0.19, and the similarity is greater than 90% compared with the Atractylodes lancea (Roxb.) Koidz. polysaccharide HPLC standard fingerprint provided in Experimental Example 1. The crude polysaccharides are further refined to remove protein and pigment to obtain pure Atractylodes lancea (Roxb.) Koidz. polysaccharides with a purity of 99%, and the main stretching vibration absorption peaks of the infrared spectrum of the Atractylodes lancea (Roxb.) Koidz. polysaccharides are basically consistent with those in the infrared spectrum shown in Experimental Example 1, and the weight average molecular weight Mw ranges from 2000 to 5000 Da. Figure 2 The above Atractylodes lancea (Roxb.) Koidz. crude polysaccharides are added with 30% by weight of lactose, and granules are prepared by a conventional method.

[0141] Excipient: lactose

[0142] The above Atractylodes lancea (Roxb.) Koidz. crude polysaccharides are added with 30% by weight of lactose, and granules are prepared by a conventional method.

[0143] Example 3

[0144] Active ingredient: Atractylodes lancea (Roxb.) Koidz. polysaccharides Atractylenolides macrocephalaRhizoma Atractylodis Macrocephalae Crude Polysaccharide (purity 30%) was extracted from the rootstock powder of Atractylodes Macrocephala Koidz. The rootstock powder of Atractylodes Macrocephala Koidz. was taken in an appropriate amount, and water (w / v, 1:10) was added to extract at 90°C for 3 hours. The extraction was repeated twice, and the filtrates were combined. After standing, the filtrate was collected by vacuum filtration and concentrated into an extract. After the extract was cooled to room temperature, 4-5 times the volume of 95% ethanol was added to precipitate the polysaccharide. After 12 hours, the polysaccharide was collected by filtration. The Rhizoma Atractylodis Macrocephalae Crude Polysaccharide had a purity of 30%, and was dried in an oven at 60°C. The Rhizoma Atractylodis Macrocephalae Crude Polysaccharide was subjected to HPLC fingerprint analysis according to the analysis method provided in Experimental Example 1. In the Rhizoma Atractylodis Macrocephalae Polysaccharide Fingerprint, there were 5 characteristic peaks. The relative peak area of Peak 1 (Rha) was 0.17; the relative peak area of Peak 2 (GalA) was 0.24; Peak 3 (Glu) was the reference peak, and the relative peak area was 1; the relative peak area of Peak 4 (Gal) was 0.19; and the relative peak area of Peak 5 (Ara) was 0.22. The Rhizoma Atractylodis Macrocephalae Crude Polysaccharide was similar to the Rhizoma Atractylodis Macrocephalae Polysaccharide HPLC Standard Fingerprint provided in Experimental Example 1, with a similarity of greater than 90%. The Rhizoma Atractylodis Macrocephalae Crude Polysaccharide was further refined to remove protein and pigment to obtain pure Rhizoma Atractylodis Macrocephalae Polysaccharide with a purity of 99%. The main stretching vibration absorption peaks of the Rhizoma Atractylodis Macrocephalae Polysaccharide were basically consistent with those in the infrared spectrum shown in Figure 2 The weight average molecular weight Mw of the Rhizoma Atractylodis Macrocephalae Polysaccharide was in the range of 4400-9000 Da.

[0145] Excipient: lactose (starch, dextrin, sugar powder, lactose, mannitol, sorbitol, etc. are all good for molding)

[0146] The above Rhizoma Atractylodis Macrocephalae Polysaccharide was added with lactose at 14% by weight, and magnesium stearate was added to prepare tablets by a conventional method.

[0147] Example 4

[0148] Active ingredient: Rhizoma Atractylodis Macrocephalae Crude Polysaccharide was prepared by the method of Example 1, and was dried in an oven at 60°C. The Rhizoma Atractylodis Macrocephalae Crude Polysaccharide was subjected to HPLC fingerprint analysis according to the analysis method provided in Experimental Example 1, and the similarity was greater than 88% compared with the standard fingerprint provided in Experimental Example 1. The Rhizoma Atractylodis Macrocephalae Crude Polysaccharide was further refined to remove protein and pigment to obtain pure Rhizoma Atractylodis Macrocephalae Polysaccharide with a purity of 99%. The main stretching vibration absorption peaks of the Rhizoma Atractylodis Macrocephalae Polysaccharide were basically consistent with those in the infrared spectrum shown in Figure 2 The weight average molecular weight Mw of the Rhizoma Atractylodis Macrocephalae Polysaccharide was in the range of 4400-9000 Da.

[0149] Excipient: mannitol

[0150] The above Rhizoma Atractylodis Macrocephalae Polysaccharide was added with mannitol at 10% by weight, and Rhizoma Atractylodis Macrocephalae Polysaccharide capsules were prepared by a conventional method.

[0151] Example 5

[0152] Main ingredient: Commercially available Atractylodes macrocephala polysaccharide (80% purity). HPLC fingerprint analysis was performed using the analytical method provided in Example 1, and the similarity was greater than 80% when compared with the standard fingerprint spectrum of Atractylodes macrocephala polysaccharide provided in Example 1. After drying in a 60℃ oven, it was stored for later use. This crude polysaccharide was further purified by removing proteins and pigments to obtain pure Atractylodes macrocephala polysaccharide with a purity of 99%. The main stretching vibration absorption peak of this pure Atractylodes macrocephala polysaccharide was similar to that of the Atractylodes macrocephala polysaccharide described in the example. Figure 2 The infrared spectra shown are basically consistent, and the weight-average molecular weight (Mw) ranges from 4500 to 8000 Da.

[0153] Excipient: Sorbitol

[0154] The above-mentioned Atractylodes macrocephala polysaccharide was mixed with sorbitol at a ratio of 10% of its weight, and Atractylodes macrocephala polysaccharide capsules were prepared by conventional methods.

[0155] Example 6

[0156] Main ingredient: Atractylodes macrocephala ( Atractylenolides macrocephala Crude polysaccharide (purity 25%) was extracted from the powder of *Atractylodes macrocephala* flowers (*Koidz.*). The crude polysaccharide was analyzed by HPLC fingerprinting according to the analytical method provided in Example 1. The fingerprint chromatogram showed five characteristic peaks: peak 1 (Rha) with a relative peak area of ​​0.10; peak 2 (GalA) with a relative peak area of ​​0.25; peak 3 (Glu), a reference peak with a relative peak area of ​​1; peak 4 (Gal) with a relative peak area of ​​0.17; and peak 5 (Ara) with a relative peak area of ​​0.25. The similarity to the HPLC standard fingerprint chromatogram of *Atractylodes macrocephala* polysaccharide provided in Example 1 was greater than 60%. The Atractylodes macrocephala polysaccharide was added to 75-80 times its volume of distilled water, along with an equal volume of trichloroacetic acid:n-butanol (volume ratio 1:8-10). The mixture was vigorously shaken for 20-25 minutes, allowed to stand, and allowed to separate into layers. The supernatant was collected, and the upper n-butanol layer and middle layer of impurities were removed. NaOH of a certain concentration was added until neutral, and the mixture was evaporated to dryness. This process was repeated several times to remove glycoproteins. Finally, H2O2 was used to remove pigments, yielding pure Atractylodes macrocephala polysaccharide (99% purity). The main stretching vibration absorption peak in the infrared spectrum of the Atractylodes macrocephala polysaccharide described in this experimental example, after purification, is similar to... Figure 2 The infrared spectra shown are basically consistent, with a weight-average molecular weight (Mw) ranging from 5500 to 9000 Da. After drying in a 60°C oven, they are ready for use.

[0157] Excipients: Starch, Atractylodes macrocephala polysaccharide, and 30% lactose by weight were added to prepare a powder using conventional methods.

[0158] Example 7

[0159] Main ingredient: Atractylodes macrocephala ( Atractylenolides macrocephalaAtractylodes Rhizome Crude Polysaccharide (purity 25%) extracted from the rhizome of Atractylodes macrocephala Koidz. The specific extraction method is as follows: an appropriate amount of Atractylodes macrocephala Koidz. rhizome powder is weighed and added with water (w / v, 1:10), and then extracted at 90°C for 3 h, and the extraction is repeated twice. The filtrates are combined, and after standing, the filtrate is collected by vacuum filtration and concentrated into an extract. After the extract is cooled to room temperature, 4-5 times the volume of 95% ethanol is added to precipitate the polysaccharide, and after 12 h, the filtrate is collected by filtration and dried at low temperature to obtain Atractylodes macrocephala Koidz. crude polysaccharide, which is ready for use. The polysaccharide content is 39% as determined by the sulfuric acid-phenol method, and the similarity is greater than 92% as compared with the Atractylodes macrocephala Koidz. polysaccharide HPLC standard fingerprint provided in Experimental Example 1. The crude polysaccharide is further refined by removing protein and pigment to obtain pure Atractylodes macrocephala Koidz. polysaccharide with a purity of 99%. The main stretching vibration absorption peaks of the infrared spectrum of the Atractylodes macrocephala Koidz. polysaccharide are basically consistent with those in the infrared spectrum shown in Figure 2

[0160] Excipient: lactose

[0161] The above Atractylodes macrocephala Koidz. polysaccharide is added with sorbitol equivalent to 30% of the weight of the polysaccharide, and then CMC-Na is added to 2‰-5‰ of the total volume of the liquid to prepare Atractylodes macrocephala Koidz. polysaccharide oral solution by a conventional method.

[0162] Example 8

[0163] Active ingredient: sulfated modified Atractylodes macrocephala Koidz. polysaccharide. The sulfated modified Atractylodes macrocephala Koidz. polysaccharide is obtained by the following method: a certain amount of Atractylodes macrocephala Koidz. polysaccharide provided in Experimental Example 2 is accurately weighed, pre-dissolved with dimethyl sulfoxide, stirred in an ice water bath for 30 min, and then added with a sulfation reagent at a certain temperature for a period of time. After the reaction is completed, the heat source is removed, and the reaction liquid is cooled to below 0°C in an ice water bath. Then, 20% NaOH solution is added to adjust the pH value to 7.8-8.0. The reaction liquid is transferred to a dialysis bag, and then dialyzed with running water for 72 h. After being concentrated under reduced pressure, washed with acetone and precipitated with ethanol, the sulfated modified Atractylodes macrocephala Koidz. polysaccharide is obtained by freeze-drying.

[0164] Excipient: lactose

[0165] The above sulfated modified Atractylodes macrocephala Koidz. polysaccharide is added with an appropriate amount of 30% sorbitol, and then CMC-Na is added to 2‰-5‰ of the total volume of the liquid to prepare sulfated modified Atractylodes macrocephala Koidz. polysaccharide oral solution by a conventional method.

[0166] Example 9

[0167] ​Main drug: carboxymethyl atractylodes rhizome polysaccharide. The carboxymethyl atractylodes rhizome polysaccharide is obtained by the following method: a certain amount of atractylodes rhizome polysaccharide of experimental example 2 and a certain amount of 75% (volume fraction) isopropyl alcohol are weighed accurately and placed in a 100 mL three-necked flask, sodium hydroxide is added, and stirring is uniformly carried out under constant temperature conditions. After pretreatment for 30-75 min, a certain amount of chloroacetic acid solution is added, temperature control is carried out at 50-60 DEG C, reaction is carried out for 2.5-3.5 h, filtration is carried out, the obtained filter cake is washed with 60% (volume fraction) ethanol solution. After freeze-drying, the carboxymethyl atractylodes rhizome polysaccharide is obtained.

[0168] Auxiliary material: starch, carboxymethyl atractylodes rhizome polysaccharide is added with 30% of the weight of lactose, and a powder is prepared by a conventional method.

Claims

1. The application of Atractylodes macrocephala polysaccharide in the preparation of drugs for reducing the relative abundance ratio of Firmicutes and Bacteroidetes in intestinal flora of spleen deficiency syndrome and for the prevention and treatment of obesity. The Atractylodes macrocephala polysaccharide is composed of five monosaccharides: rhamnose (Rha), galacturonic acid (GalA), glucose (Glu), galactose (Gal), and arabinose (Ara), and possesses unique fingerprint information. The main stretching vibration absorption peak of the Atractylodes macrocephala polysaccharide infrared spectrum is 3342 cm⁻¹. -1 Or 3325cm -1 There is an absorption peak of OH stretching vibration near 2953 cm⁻¹. -1 2924cm -1 and 2854cm -1 It has a stretching vibration peak of CH, at 1737 cm⁻¹. -1 There is an absorption peak for the stretching vibration of C=O near the location, at 1122 cm⁻¹. -1 and 1095cm -1 An absorption peak is observed at the specified location; the weight-average molecular weight (Mw) of the Atractylodes macrocephala polysaccharide ranges from 3500 to 5000 Da.

2. The application as described in claim 1, characterized in that, The Atractylodes macrocephala polysaccharide is formulated into an oral preparation with the aid of pharmaceutical excipients.

3. The application as described in claim 1, further characterized in that: The specific fingerprint information of Atractylodes macrocephala polysaccharide refers to the following: There are five characteristic peaks in the HPLC fingerprint chromatogram of Atractylodes macrocephala polysaccharide, namely, peak 1 is rhamnose (Rha), peak 2 is galacturonic acid (GalA), peak 3 is glucose (Glu), peak 4 is galactose (Gal), and peak 5 is arabinose (Ara); the sum of the peak areas of the five characteristic peaks accounts for more than 90% of the total peak area; in the HPLC fingerprint chromatogram of Atractylodes macrocephala polysaccharide, glucose occupies the largest proportion of the total peak area, and is used as an internal reference peak to calculate the ratio of the relative peak areas of each major peak; the relative retention time (RT) of each major peak is calculated using the derivatization reagent 1-phenyl-3-methyl-5-pyrazolone as an internal reference peak; the relative standard deviation (RSD) of the ratio of the relative peak areas and the relative retention time of the five characteristic peaks in the HPLC fingerprint chromatogram of Atractylodes macrocephala polysaccharide is less than 2.0%; The average RT of peak 1 is 0.83, and the relative peak area is 0.

15. The average RT of peak 2 is 0.79, and the relative peak area is 0.

23. The average RT of peak 3 is 0.70, and the relative peak area is 1. The average RT of peak 4 is 0.66, and the relative peak area is 0.

19. The average RT of peak 5 is 0.60, and the relative peak area is 0.

20.

4. The application as described in claim 1, further characterized in that: The aforementioned Atractylodes macrocephala polysaccharide is extracted from the rhizome or flowers of the plant Atractylodes macrocephala koidz.

Images