A kind of extraction method and application of Atractylodes macrocephala polysaccharide

Abstract

The present invention provides an extraction method and application of Atractylodes macrocephala polysaccharide, which belongs to the technical field of traditional Chinese medicine. The present invention uses Atractylodes macrocephala as raw material, adopts ultrasonic synergistic subcritical water extraction and graded alcohol precipitation to extract Atractylodes macrocephala polysaccharide AMP-50, and purifies and separates AMP-50-I to III through DEAE-650M cellulose ion exchange resin. The extraction method of the present invention is not only easy to operate, time-saving and efficient, but also can improve the extraction rate and purity of Atractylodes macrocephala polysaccharide. Among them, the AMP-50 extraction rate can reach 12.13%, the sugar content is 84.31%, and the relative molecular weight is 7768 to 24628Da. And the Atractylodes macrocephala polysaccharide extracted by the present invention has the effect of effectively improving breast damage and breast hyperplasia.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, and more particularly to an extraction method and application of Atractylodes macrocephala polysaccharide. Background Art

[0002] Fibrocystic breast disease is a degenerative disease caused by the proliferation of mammary fibroids and epithelial tissue, resulting in progressive connective tissue growth. Modern medical research indicates that the occurrence and progression of fibrocystic breast disease is closely related to endocrine disorders. An imbalance in the ratio of estradiol to progesterone leads to excessive proliferation and retraction of the mammary gland parenchyma. Currently, the diagnosis and treatment of fibrocystic breast disease often involve biopsy or surgical resection of suspicious nodules detected by imaging studies. Oral medications primarily include oral sex hormones and non-sex hormones. However, surgical resection is invasive and can cause irreversible physiological and psychological harm to patients. Medication can cause a variety of side effects in women, including decreased menstrual bleeding, menstrual irregularities, dizziness, nausea, and vomiting. Some medications can also cause damage to the gastrointestinal tract and central nervous system. Therefore, the search for a non-invasive, highly effective treatment for fibrocystic breast disease has become a hot topic of research both domestically and internationally.

[0003] Atractylodes macrocephala (Atractylodes macrocephala Koidz.) is the dried rhizome of the Asteraceae plant. It has a bitter and sweet taste and warm properties. It enters the spleen and stomach meridians, benefiting from the effects of strengthening the spleen and replenishing qi. Ancient scholars have called it "the most important herb for replenishing qi and strengthening the spleen," with the saying "nine out of ten prescriptions contain Atractylodes macrocephala." The Shennong Bencao Jing lists Atractylodes macrocephala as a top-grade herb, citing its benefits of invigorating the spleen and replenishing qi, removing dampness and promoting diuresis, and arresting perspiration and stabilizing pregnancy. The Origin of Medicine states: "Atractylodes macrocephala can remove dampness and replenish dryness, harmonize the middle and replenish qi, warm the middle, remove dampness from the spleen and stomach, eliminate stomach heat, and strengthen the spleen and stomach." Atractylodes macrocephala polysaccharide is one of the main active ingredients in Atractylodes macrocephala and a key component in its pharmacological effects. It improves gastrointestinal function, protects the liver, and promotes cell apoptosis. Therefore, further rational development and utilization of Atractylodes macrocephala resources are warranted.

[0004] Therefore, how to provide a Atractylodes macrocephala polysaccharide with high extraction rate and purity and at the same time having the effect of regulating breast hyperplasia and its extraction method is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a method for extracting Atractylodes macrocephala polysaccharide and the use of Atractylodes macrocephala polysaccharide AMP-50 in the preparation of a drug for treating breast hyperplasia. Specifically, the present invention first heats Atractylodes macrocephala with petroleum ether for reflux, then adopts ultrasonic extraction, subcritical water extraction, centrifugal filtration, graded alcohol precipitation, and freeze-drying to obtain Atractylodes macrocephala polysaccharide AMP-50. The Atractylodes macrocephala polysaccharide AMP-50 obtained by this extraction method has a high extraction rate and purity. Compared with other extraction methods, the present invention overcomes a series of problems such as low extraction efficiency, low polysaccharide purity, cumbersome operation, and difficult post-processing during the extraction process of Atractylodes macrocephala polysaccharide. Therefore, the extraction method of Atractylodes macrocephala polysaccharide defined by the present invention is a green, environmentally friendly emerging extraction and separation technology, and the Atractylodes macrocephala polysaccharide of the present invention has the effect of effectively improving breast hyperplasia.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides a method for extracting Atractylodes macrocephala polysaccharide AMP-50, comprising the following steps:

[0008] (1) First, the Atractylodes macrocephala is dried, crushed, sieved, defatted, and air-dried to obtain Atractylodes macrocephala powder;

[0009] (2) Then the Atractylodes macrocephala powder was treated at a solid-liquid ratio of 1: (8-12) / g·mL -1 After adding distilled water and ultrasonic extraction to obtain the mixed solution, the mixture was diluted according to the ratio of 1: (20-30) / g·mL -1 The material-liquid ratio was adjusted to add corresponding distilled water for subcritical water extraction, and the supernatant was obtained by centrifugation;

[0010] (3) taking the supernatant and performing a second alcohol precipitation, and centrifuging to obtain a precipitate;

[0011] (4) Finally, the precipitate is redissolved, concentrated under reduced pressure, and freeze-dried to obtain Atractylodes macrocephala polysaccharide AMP-50.

[0012] The present invention, for the first time, employs a method combining ultrasonic-assisted subcritical water extraction with multi-stage alcohol precipitation extraction, resulting in a high extraction rate and high purity for the resulting Atractylodes macrocephala polysaccharide AMP-50. Furthermore, within the specified material-to-liquid ratio range for ultrasonic and subcritical water extraction, the present invention can improve the dissolution of the polysaccharide, thereby increasing the extraction rate of the Atractylodes macrocephala polysaccharide.

[0013] Preferably, the drying temperature in step (1) is 60° C. and the product is passed through a 40-mesh sieve.

[0014] The beneficial effect of adopting the above further scheme is that, on the basis of ensuring the biological activity of the Atractylodes macrocephala polysaccharide in the Atractylodes macrocephala, drying is carried out within this temperature range, which facilitates the subsequent crushing process.

[0015] Preferably, the degreasing treatment in step (1) is as follows: adding the sieved Atractylodes macrocephala to the solution at a material-liquid ratio of 1: (5-7) / g·mL -1 Petroleum ether was added and the mixture was heated under reflux at 70°C for 2 h.

[0016] The beneficial effect of adopting the above further scheme is that the petroleum ether heating reflux treatment before ultrasonic-subcritical water extraction can remove components with low polarity, such as fat-soluble components, to eliminate their influence on the extraction of Atractylodes macrocephala polysaccharides.

[0017] Preferably, the ultrasonic power in step (2) is 60-70 W, the ultrasonic time is 30-40 min, and the ultrasonic temperature is 50-60.

[0018] The beneficial effect of adopting the above further scheme is that the present invention changes the permeability of the cell wall of Atractylodes macrocephala through ultrasonic extraction, thereby effectively softening and swelling the cell wall and improving the extraction rate of polysaccharides.

[0019] Preferably, the subcritical water extraction temperature in step (2) is 90-120°C, the water extraction time is 15-25 min, the pH value of the water extraction solution is 5-6, and the centrifugal speed is 3500-4000 r·min -1 , the centrifugation time is 10 to 15 minutes.

[0020] Preferably, the two alcohol precipitations in step (3) are specifically as follows: in the first alcohol precipitation, the supernatant in step (2) is added with anhydrous ethanol until the ethanol concentration of the solution reaches 40%; in the second alcohol precipitation, the supernatant of the first alcohol precipitation is added with anhydrous ethanol until the ethanol concentration of the solution reaches 50%.

[0021] The beneficial effect of adopting the above further scheme is that the graded alcohol precipitation can fully precipitate the polysaccharide and avoid the loss of polysaccharide.

[0022] Preferably, the two alcohol precipitations were allowed to stand at 4°C for 48 hours, and then heated at 4000 r·min -1 Centrifuge at 1000rpm for 10 min.

[0023] Preferably, the extraction method further comprises separating and purifying the Atractylodes macrocephala polysaccharide AMP-50 through DEAE-650M cellulose ion exchange resin to obtain AMP-50-I to AMP-50-III, with weight percentages of 38.67%, 3.89% and 2.57% respectively.

[0024] The beneficial effect of adopting the above further scheme is that sugars with different solubilities can be separated through separation and purification by DEAE-650M, and impurities in polysaccharides can be stripped off, thereby achieving the purpose of obtaining uniform polysaccharides.

[0025] Preferably, the AMP-50-I to III all present single symmetrical peaks, and the molecular weights are 7768 Da, 17232 Da and 24628 Da, respectively.

[0026] To achieve the above object, the present invention also provides another technical solution: use of the Atractylodes macrocephala polysaccharide AMP-50 extracted according to the above extraction method in the preparation of a drug for treating breast hyperplasia.

[0027] In Traditional Chinese Medicine, breast hyperplasia is classified as a "breast disease" and other symptom categories. Its onset originates from Qi and is blamed on the liver. Liver depression and qi stagnation are the pathological basis of breast hyperplasia. The underlying deficiency is caused by disorders of the Chong and Ren meridians, spleen, liver, and kidneys, while the symptoms are phlegm coagulation, qi stagnation, and blood stasis. Therefore, Traditional Chinese Medicine often uses traditional Chinese medicines such as soothing the liver and regulating Qi, replenishing Qi and strengthening the spleen, regulating Chong and Ren meridians, and dispersing stagnation and removing blood stasis to treat breast hyperplasia through long-term conditioning. Atractylodes macrocephala is bitter, sweet, and warm in nature, replenishes Qi and strengthens the spleen, and eliminates dampness and promotes diuresis. The Atractylodes macrocephala polysaccharide AMP-50 extracted by the present invention can reduce serum estradiol levels and regulate progesterone levels by antagonizing estrogen, achieving the effect of alleviating breast hyperplasia.

[0028] It can be seen from the above technical solutions that, compared with the prior art, the present invention discloses a method for extracting and applying Atractylodes macrocephala polysaccharide, which has the following excellent effects:

[0029] 1) The present invention adopts the method of ultrasonic assisted subcritical water extraction, which not only solves the problems of low extraction efficiency and purity of Atractylodes macrocephala polysaccharide, but also provides a reference for subcritical water extraction in the extraction of natural products.

[0030] 2) The extraction method disclosed in the present invention has the advantages of easy operation, time-saving, high efficiency and no pollution.

[0031] 3) The Atractylodes macrocephala polysaccharide AMP-50 extracted by the present invention has the effect of improving breast hyperplasia. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0033] Figure 1 This is the HPGPC spectrum of AMP-50-Ⅰ~Ⅲ extracted in the present invention.

[0034] Figure 2 This is the effect of AMP-50 of the present invention on the levels of E2, P and PRL in the serum of rats with mammary hyperplasia.

[0035] Note: Compared with the Con group, #P<0.05, ##P<0.01; compared with the Mod group, *P<0.05, **P<0.01.

[0036] Figure 3 This is a graph showing the results of the mammary tissue pathological observation of rats with mammary hyperplasia by AMP-50 of the present invention. DETAILED DESCRIPTION

[0037] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0038] The present invention discloses a method for extracting Atractylodes macrocephala polysaccharide (AMP-50), which specifically comprises the following steps:

[0039] Weigh the Atractylodes macrocephala, crush it, pass it through a 40-mesh sieve, and calculate the concentration of the mixture at a ratio of 1: (5-7) g·mL. -1 Add petroleum ether at a material-liquid ratio of 1: (8-12) / g·mL at 70-80°C, heat under reflux for 1.5-2h, and dry in the shade. -1 Distilled water was added at a material-liquid ratio of 1:(20-30) / g·mL, and ultrasonic extraction was performed at 50-60°C for 30-40 min. -1 The material-liquid ratio was supplemented with corresponding distilled water, and the pH was adjusted to 5-6. Subcritical water extraction was performed at 90-120°C for 15-25 minutes and at 3500-4000 r·min -1 Centrifuge for 10-15 minutes to obtain the filtrate. Add anhydrous ethanol to the filtrate until the ethanol concentration in the solution reaches 40%, stir evenly, and let it stand at 4℃ for 48 hours at 4000 r·min. -1 Centrifuge for 10 minutes, take the supernatant and add anhydrous ethanol until the ethanol concentration in the solution reaches 50%, stir well, and let it stand at 4℃ for 48 hours at 4000 r·min. -1 After centrifugation for 10 minutes, the precipitate was taken and added into distilled water for redissolution, concentrated under reduced pressure, and freeze-dried to obtain Atractylodes macrocephala polysaccharide AMP-50.

[0040] Example 1

[0041] A method for preparing Atractylodes macrocephala polysaccharide, comprising the following steps:

[0042] Weigh 1 kg of Atractylodes macrocephala, crush it, pass it through a 40-mesh sieve, and calculate the mass fraction at a ratio of 1:6 g·mL -1 Add petroleum ether at a material-liquid ratio of 1:10 g·mL, heat under reflux at 70°C for 1.5 h, and dry in the shade.-1 Distilled water was added at a solid-liquid ratio of 1:25 g·mL and ultrasonic extraction was performed at 55°C for 35 min. -1 The material-liquid ratio was supplemented with corresponding distilled water, and the pH was adjusted to 5.5. Subcritical water extraction was performed at 110 ° C for 20 min and the speed was 4000 r·min -1 Centrifuge for 10 minutes to obtain the filtrate. Add anhydrous ethanol to the filtrate until the ethanol concentration of the solution reaches 40%, stir well, and let it stand at 4℃ for 48 hours at 4000 r·min. -1 Centrifuge for 10 minutes, take the supernatant and add anhydrous ethanol until the ethanol concentration of the solution reaches 50%, stir evenly, and let it stand at 4℃ for 48 hours at 4000 r·min. -1 After centrifugation for 10 minutes, the precipitate was taken and added into distilled water for redissolution, concentrated under reduced pressure, and freeze-dried to obtain 0.1213 kg of Atractylodes macrocephala polysaccharide (AMP-50).

[0043] The extraction rate of AMP-50 was 12.13% and the sugar content was 84.31%.

[0044] Note: 1. The extraction rate formula is: Extraction rate (%) = (m / M) × 100%

[0045] Where: m——polysaccharide sample mass;

[0046] M——Quality of Atractylodes macrocephala powder.

[0047] 2. Sugar content is determined by measuring the proportion of glucose in AMP using glucose as an indicator. It is calculated using the phenol-concentrated sulfuric acid method: prepare 0.1 mg mL -1 Glucose standard solution: pipette 0.0, 0.2, 0.4, 0.6, 0.8 and 1.0 mL into 10 mL stoppered test tubes, add distilled water to make up to 1.0 mL, add 1 mL of 5% phenol solution and 5 mL of concentrated sulfuric acid in sequence, shake well, let stand for 5 min, boil in water bath for 15 min, cool to room temperature, measure absorbance A at 490 nm, and use glucose concentration (0.1 mg mL -1 ) as the x-axis and absorbance (A) as the y-axis to draw a standard curve.

[0048] Prepare 0.1 mg mL -1 Take 2.0 mL of AMP-50 sample solution and perform three parallel tests according to the above method. Take the average value and calculate the polysaccharide content according to the standard curve.

[0049] In addition, to further illustrate the non-obviousness of the process parameter conditions in the technical solution disclosed in the present application, the inventors optimized the power and material-liquid ratio of Atractylodes macrocephala and ultrasonic extraction, and the material-liquid ratio and extraction temperature of Atractylodes macrocephala and subcritical water extraction, and the specific contents are as follows:

[0050] Examples 2-4

[0051] A method for extracting Atractylodes macrocephala polysaccharide, comprising the following steps:

[0052] Weigh 3 portions of 50g of Atractylodes macrocephala, crush them, pass through a 40-mesh sieve, and adjust the ratio to 1:6 / g·mL. -1 Add petroleum ether at a material-liquid ratio of 1:10 / g·mL, heat under reflux at 70°C for 1.5h, and dry in the shade. -1 Distilled water was added at a material-liquid ratio of 1:25 / g·mL and ultrasonic extraction was performed at 55°C for 35 min. -1 The material-liquid ratio was supplemented with corresponding distilled water, and the pH was adjusted to 5.5. Subcritical water extraction was performed at 110 ° C for 20 min and the speed was 4000 r·min -1 Centrifuge for 10 minutes to obtain the filtrate. Add anhydrous ethanol to the filtrate until the ethanol concentration of the solution reaches 40%, stir well, and let it stand at 4℃ for 48 hours at 4000 r·min. -1 Centrifuge for 10 minutes, take the supernatant and add anhydrous ethanol until the ethanol concentration of the solution reaches 50%, stir evenly, and let it stand at 4℃ for 48 hours at 4000 r·min. -1 The mixture was centrifuged for 10 min, and the precipitate was re-dissolved in distilled water, concentrated under reduced pressure, and freeze-dried to obtain Atractylodes macrocephala polysaccharides 2, 3, and 4.

[0053] According to the optimization of different ultrasonic powers, the extraction rate of Atractylodes macrocephala polysaccharide 2 was 8.56% and the sugar content was 76.31%.

[0054] The extraction rate of Atractylodes macrocephala polysaccharide 3 was 12.03% and the sugar content was 81.03%;

[0055] The extraction rate of Atractylodes macrocephala polysaccharide 4 was 9.73% and the sugar content was 78.14%.

[0056] Therefore, it is preferred that the ultrasonic extraction power of Atractylodes macrocephala polysaccharide be 65W for extraction.

[0057] Examples 5-7

[0058] A method for extracting Atractylodes macrocephala polysaccharide, comprising the following steps:

[0059] Weigh 3 portions of 50g of Atractylodes macrocephala, crush them, pass through a 40-mesh sieve, and adjust the ratio to 1:6 / g·mL. -1 Add petroleum ether at a material-liquid ratio of 1:10 / g·mL, heat under reflux at 70°C for 1.5h, and dry in the shade. -1 Distilled water was added at a solid-liquid ratio of 1:20, 1:25, and 1:30 / g·mL, and ultrasonic extraction was performed at 55°C for 35 min. -1The material-liquid ratio was supplemented with corresponding distilled water, and the pH was adjusted to 5.5. Subcritical water extraction was performed at 110 ° C for 20 min and the speed was 4000 r·min -1 Centrifuge for 10 minutes to obtain the filtrate. Add anhydrous ethanol to the filtrate until the ethanol concentration of the solution reaches 40%, stir well, and let it stand at 4℃ for 48 hours at 4000 r·min. -1 Centrifuge for 10 minutes, take the supernatant and add anhydrous ethanol until the ethanol concentration of the solution reaches 50%, stir evenly, and let it stand at 4℃ for 48 hours at 4000 r·min. -1 The mixture was centrifuged for 10 min, and the precipitate was re-dissolved in distilled water, concentrated under reduced pressure, and freeze-dried to obtain Atractylodes macrocephala polysaccharide 5, Atractylodes macrocephala polysaccharide 6, and Atractylodes macrocephala polysaccharide 7.

[0060] According to the different material-liquid ratios of subcritical water extraction, the extraction rate of Atractylodes macrocephala polysaccharide 5 was 9.11% and the sugar content was 77.14%.

[0061] The extraction rate of Atractylodes macrocephala polysaccharide 6 was 11.67% and the sugar content was 81.36%;

[0062] The extraction rate of Atractylodes macrocephala polysaccharide 7 was 9.06% and the sugar content was 80.06%.

[0063] Therefore, the preferred material-liquid ratio for subcritical water extraction is 1:25 g·mL -1 Perform extraction.

[0064] Examples 8-10

[0065] A method for extracting Atractylodes macrocephala polysaccharide, comprising the following steps:

[0066] Weigh 3 portions of 50g of Atractylodes macrocephala, crush them, pass through a 40-mesh sieve, and adjust the ratio to 1:6 / g·mL. -1 Add petroleum ether at a material-liquid ratio of 1:10 / g·mL, heat under reflux at 70°C for 1.5h, and dry in the shade. -1 Distilled water was added at a material-liquid ratio of 1:25 / g·mL and ultrasonic extraction was performed at 55°C for 35 min. -1 The material-liquid ratio was supplemented with corresponding distilled water, and the pH was adjusted to 5.5. Subcritical water extraction was carried out at 100, 110, and 120 °C for 20 min and at 4000 r·min -1 Centrifuge for 10 minutes to obtain the filtrate. Add anhydrous ethanol to the filtrate until the ethanol concentration of the solution reaches 40%, stir well, and let it stand at 4℃ for 48 hours at 4000 r·min. -1 Centrifuge for 10 minutes, take the supernatant and add anhydrous ethanol until the ethanol concentration of the solution reaches 50%, stir evenly, and let it stand at 4℃ for 48 hours at 4000 r·min. -1After centrifugation for 10 minutes, the precipitate was taken and added into distilled water for redissolution, concentrated under reduced pressure, and freeze-dried to obtain Atractylodes macrocephala polysaccharide 8, Atractylodes macrocephala polysaccharide 9, and Atractylodes macrocephala polysaccharide 10.

[0067] By optimizing the subcritical water extraction temperature, the extraction rate of Atractylodes macrocephala polysaccharide 8 was 9.07% and the sugar content was 76.56%.

[0068] The extraction rate of Atractylodes macrocephala polysaccharide 9 was 12.06% and the sugar content was 82.17%;

[0069] The extraction rate of Atractylodes macrocephala polysaccharide 10 was 9.17% and the sugar content was 80.61%.

[0070] Therefore, the subcritical water extraction temperature is preferably 110°C.

[0071] Examples 11-13

[0072] A method for extracting Atractylodes macrocephala polysaccharide, comprising the following steps:

[0073] Weigh 3 portions of 50g of Atractylodes macrocephala, crush them, pass through a 40-mesh sieve, and adjust the ratio to 1:6 / g·mL. -1 Add petroleum ether at a material-liquid ratio of 1:8, 1:10, and 1:12 / g·mL, respectively, at 70°C, reflux for 1.5h, and dry in the shade. -1 Distilled water was added at a material-liquid ratio of 1:25 / g·mL and ultrasonic extraction was performed at 55°C for 35 min. -1 The material-liquid ratio was supplemented with corresponding distilled water, and the pH was adjusted to 5.5. Subcritical water extraction was performed at 110 ° C for 20 min and the speed was 4000 r·min -1 Centrifuge for 10 minutes to obtain the filtrate. Add anhydrous ethanol to the filtrate until the ethanol concentration of the solution reaches 40%, stir well, and let it stand at 4℃ for 48 hours at 4000 r·min. -1 Centrifuge for 10 minutes, take the supernatant and add anhydrous ethanol until the ethanol concentration of the solution reaches 50%, stir evenly, and let it stand at 4℃ for 48 hours at 4000 r·min. -1 The mixture was centrifuged for 10 minutes, and the precipitate was taken and re-dissolved in distilled water. The mixture was concentrated under reduced pressure and freeze-dried to obtain Atractylodes macrocephala polysaccharide 11, Atractylodes macrocephala polysaccharide 12, and Atractylodes macrocephala polysaccharide 13.

[0074] By optimizing the subcritical water extraction temperature, the extraction rate of Atractylodes macrocephala polysaccharide 11 was 9.43% and the sugar content was 78.56%.

[0075] The extraction rate of Atractylodes macrocephala polysaccharide 12 was 11.89% and the sugar content was 80.13%;

[0076] The extraction rate of Atractylodes macrocephala polysaccharide 13 was 8.96% and the sugar content was 77.76%.

[0077] Comparative Example 1:

[0078] Atractylodes macrocephala polysaccharide was extracted according to the method of Example 1, except that Atractylodes macrocephala was not subjected to ultrasonic extraction before subcritical water extraction. Other steps were the same as in Example 1 to extract Atractylodes macrocephala polysaccharide (AMP-50-A).

[0079] Using the method of Comparative Example 1, the extraction rate of AMP-50-A was 9.67% and the sugar content was 70.56%. It can be seen that the extraction rate and sugar content of the Atractylodes macrocephala polysaccharide extracted by ultrasound were significantly higher than those of the Atractylodes macrocephala polysaccharide extracted without ultrasound. Therefore, ultrasonic extraction changes the permeability of the cell wall, thereby effectively softening and swelling the cell wall and improving the extraction rate of polysaccharides. However, the control of power and time during the ultrasonic extraction of Atractylodes macrocephala is a technical difficulty in this extraction method. The power should not be too large, and it is only necessary to destroy the plant cell wall.

[0080] Comparative Example 2:

[0081] Atractylodes macrocephala polysaccharide was extracted according to the method of Example 1, except that only ultrasonic wave was used for extraction. Other steps were the same as in Example 1 to extract Atractylodes macrocephala polysaccharide (AMP-50-B).

[0082] The extraction method of Comparative Example 2 yielded AMP-50-B at a yield of 4.05% and a sugar content of 58.04%. Ultrasonic extraction alone cannot completely remove polysaccharides from the medicinal material, negatively impacting the extraction yield. Therefore, increasing the solid-liquid ratio appropriately can increase the polysaccharide dissolution rate in the solution. This extraction process is simple and highly efficient.

[0083] Comparative Example 3:

[0084] Atractylodes macrocephala polysaccharide was extracted according to the method of Example 1, except that distilled water was not added for subcritical water extraction after ultrasonic extraction. Other steps were the same as in Example 1 to extract Atractylodes macrocephala (AMP-50-C).

[0085] The extraction method of Comparative Example 2 yielded an AMP-50-C extraction rate of 8.05% and a sugar content of 71.31%. Because polysaccharides cannot be completely dissolved when the liquid-to-solid ratio is too low, this adversely affects the extraction rate. Therefore, increasing the liquid-to-solid ratio appropriately can increase the polysaccharide dissolution rate in the solution. This extraction process is simple and efficient.

[0086] Comparative Example 4:

[0087] Atractylodes macrocephala polysaccharide was extracted according to the method of Example 1, except that after ultrasonic extraction, traditional water extraction, microwave extraction and high-pressure extraction were used for extraction. Other steps were the same as in Example 1 to extract Atractylodes macrocephala polysaccharide (AMP-50-D~F).

[0088] The method of Comparative Example 3 was adopted, and the results are shown in Table 1 below.

[0089] Table 1 Extraction rate and sugar content of AMP-50 and AMP-50-C~E

[0090]

[0091] Example 14:

[0092] The pretreated DEAE-650M cellulose ion exchange resin was loaded into the column using a wet method to prepare 5 mg mL -1 AMP-50 solution extracted from Example 1, 4000 r·min -1 After centrifugation for 10 min, the supernatant was taken and loaded with distilled water, 0.1, 0.2, 0.3, 0.4, and 0.5 mol·L -1 NaCl was used for elution at a flow rate of 1 mL min -1 The eluate was collected, the absorbance was detected by phenol-sulfuric acid method, the single peak elution components were collected and combined, and after concentration, a dialysis bag with a molecular weight cutoff of 3500Da was selected for dialysis desalination and freeze-dried to obtain three polysaccharides, which were recorded as AMP-50-Ⅰ, AMP-50-Ⅱ, and AMP-50-Ⅲ.

[0093] The relative molecular weight of the obtained AMP-50 was distributed between 7768 and 24628 Da. AMP-50-Ⅰ to -Ⅲ all showed single symmetrical peaks with molecular weights of 7768 Da, 17232 Da and 24628 Da, respectively.

[0094] In order to further illustrate the excellent effect of the Atractylodes macrocephala polysaccharide extracted by the present invention in improving breast hyperplasia, the inventors also conducted the following biological effect verification experiments

[0095] Effect test:

[0096] Thirty-two SD rats, SPF grade, female, 200±20 g, were selected and adapted to feeding for 7 days. The rats were randomly divided into 4 groups: blank group (Con group), model group (Mod group), positive drug group (Pos group), and Atractylodes macrocephala polysaccharide group (AMP-50 group) extracted from Example 1, with 8 rats in each group. Among them, the positive drug group: Tamoxifen citrate dosage conversion: According to the maximum daily dosage of a 70 kg adult on the instructions, the dosage of rats is 1.8 mg kg -1 ·d -1 AMP-50 dosage conversion: According to the Chinese Pharmacopoeia (2020 edition), the maximum daily dosage of Atractylodes macrocephala for a 70kg adult is 12g. Based on the dosage of Atractylodes macrocephala and the yield of AMP-50, the maximum dosage of AMP-50 is 131mg·kg -1 ·d-1 Except for the blank group, rats in other groups were injected intramuscularly with 0.5 mg·kg -1 ·D -1 Estradiol benzoate, 25 days, followed by intramuscular injection of 5 mg kg -1 ·D -1 Progesterone was administered for 5 consecutive days; the blank group received an intramuscular injection of the corresponding saline. Rats in each group were fasted for 24 hours before the end of the experiment, with free access to water. Blood was collected after anesthesia, and serum was assayed for E2, PRL, and P levels. The results are as follows:

[0097] (1) Impact on E2 content:

[0098] Compared with the Con group, the E2 content of rats in the Mod group was extremely significantly increased (P<0.01); compared with the Mod group, the E2 content of the Pos group was extremely significantly decreased (P<0.01), and the E2 content of the AMP-50 group was significantly decreased (P<0.05). The results showed that AMP-50 could reduce the E2 content in the serum of MGH rats. Figure 2 .

[0099] (2) Effect on PRL content:

[0100] Compared with the Con group, the PRL content of rats in the Mod group was extremely significantly increased (P<0.01); compared with the Mod group, the PRL content of the Pos group was extremely significantly decreased (P<0.01), and the PRL content of the AMP-50 group was significantly decreased (P<0.05). The experimental results show that AMP-50 can reduce the PRL content in the serum of MGH rats. Figure 2 .

[0101] (3) Impact on P content:

[0102] Compared with the Con group, the P content of rats in the Mod group was significantly increased (P<0.05); compared with the Mod group, the P content of the Pos group was significantly decreased (P<0.05), and the P content of the AMP-50 group was significantly decreased (P<0.05). The experimental results show that AMP-50 can reduce the P content in the serum of MGH rats. Figure 2 .

[0103] Hematoxylin-eosin (HE) staining was used to evaluate the pathological changes in breast tissue. The results are as follows:

[0104] The mammary tissue cell morphology and structure of rats in the Con group were normal, with a small number of mammary lobules and elongated ducts, and no proliferative lesions or secretions. Compared with the Con group, rats in the Mod group had obvious ductal hyperplasia and dilatation, with dense acinar cells in the lobules, varying in size and irregular in shape, and containing secretions. Compared with the Mod group, the acinar cells in the lobules of the Pos group had regular shapes, elongated ducts, and no secretions. The number of acinar cells in the lobules of rats in the AMP-50 group was reduced, with a small amount of secretions in the cavity, and the degree of duct dilatation was reduced. The experimental results show that AMP-50 can improve the degree of mammary gland lesions in MGH rats. Figure 3 .

[0105] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. Reference can be made to the common and similar parts between the various embodiments. For the devices disclosed in the embodiments, since they correspond to the methods disclosed in the embodiments, the description is relatively simple, and the relevant parts can be referred to the method description.

[0106] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of Atractylodes macrocephala polysaccharide AMP-50 in the preparation of drugs for the treatment of breast hyperplasia.

2. The use according to claim 1, characterized in that The extraction method of the Atractylodes macrocephala polysaccharide AMP-50 comprises: (1) drying, crushing, sieving, and defatting the Atractylodes macrocephala to obtain Atractylodes macrocephala powder; (2) The Atractylodes macrocephala powder was mixed with water at a solid-liquid ratio of 1: (8-12) / g·mL. -1 Distilled water was added for ultrasonic extraction to obtain a mixed solution, and then the mixture was concentrated at a ratio of 1: (20-30) / g·mL -1 The material-liquid ratio was adjusted to add corresponding distilled water for subcritical water extraction, and the supernatant was obtained by centrifugation; (3) taking the supernatant and performing secondary alcohol precipitation, and centrifuging to obtain a precipitate; (4) The precipitate is redissolved, concentrated under reduced pressure, and freeze-dried to obtain Atractylodes macrocephala polysaccharide AMP-50.

3. The use according to claim 2, characterized in that The drying temperature in step (1) is 60° C. and the product is passed through a 40-mesh sieve.

4. The use according to claim 2, characterized in that The degreasing step (1) is as follows: adding the sieved Atractylodes macrocephala to the sieved Atractylodes macrocephala according to a material-liquid ratio of 1: (5-7) / g·mL -1 Add petroleum ether and heat under reflux at 70-80°C for 1.5-2h.

5. The use according to claim 2, characterized in that The ultrasonic extraction power in step (2) is 60-70W, the ultrasonic extraction time is 30-40min, and the ultrasonic extraction temperature is 50-60°C.

6. The use according to claim 2, characterized in that The subcritical water extraction temperature in step (2) is 90-120°C, the water extraction time is 15-25 minutes, the pH value of the water extraction solution is 5-6, and the centrifugal speed is 3500-4000 r·min -1 , the centrifugation time is 10 to 15 minutes.

7. The use according to claim 2, characterized in that The two alcohol precipitations in step (3) are specifically as follows: the first alcohol precipitation is to take the supernatant of step (2) and add anhydrous ethanol until the ethanol concentration in the solution reaches 40%; the second alcohol precipitation is to take the supernatant of the first alcohol precipitation and add anhydrous ethanol until the ethanol concentration in the solution reaches 50%.

8. The use according to claim 2, characterized in that The two alcohol precipitations were allowed to stand at 4 ° C for 48 h, and then the mixture was stirred at 4000 r min -1 Centrifuge at 1000rpm for 10 min.

9. The use according to claim 2, characterized in that The extraction method further comprises separating and purifying the Atractylodes macrocephala polysaccharide AMP-50 through DEAE-650M cellulose ion exchange resin.

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