Purified polysaccharide of polygonatum, its preparation method and application in preparing medicine for relieving mammary gland hyperplasia

Abstract

The present application belongs to the technical field of natural polymer materials, and particularly relates to a purified polysaccharide of Rhizoma Polygonati, a preparation method thereof and application of the purified polysaccharide in preparation of a medicine for relieving mammary gland hyperplasia. The purified polysaccharide of Rhizoma Polygonati comprises xylose, mannose, glucose and galactose, and the molar ratio of the xylose, mannose, glucose and galactose is 6.45:30.26:7.43:55.87. The molecular weight distribution of the purified polysaccharide of Rhizoma Polygonati is within 2000-30000 Da, and the molecular weight is concentrated at 22877 Da. The purified polysaccharide of Rhizoma Polygonati can reduce the toxic damage of estrogen and progesterone imbalance to HC11 mammary epithelial cells, and a low concentration of the purified polysaccharide of Rhizoma Polygonati can produce a protective effect on the cells. The purified polysaccharide of Rhizoma Polygonati is verified to have a significant inhibitory effect on mammary gland hyperplasia of mice through a mammary gland hyperplasia model intervention experiment, and therefore can be used for developing a medicine for treating mammary gland hyperplasia.

Description

Technical Field

[0001] This invention belongs to the field of natural polymer materials technology, specifically relating to a purified polysaccharide of Polygonatum odoratum, its preparation method, and its application in the preparation of drugs to alleviate breast hyperplasia. Background Technology

[0002] Breast hyperplasia is a common non-inflammatory breast disease, mainly caused by an imbalance of estrogen and progesterone in the body. It is common in premenopausal and perimenopausal women, with clinical symptoms primarily including cyclical breast tenderness, breast lumps, and thickening of the breast lobes. With the fast pace of modern life and changes in living environment, young people face immense social pressure, leading to a trend of breast hyperplasia occurring at younger ages. Although breast hyperplasia is a benign condition, it still carries the risk of becoming cancerous, especially breast cancer caused by atypical hyperplasia. Therefore, this disease should be taken seriously, and early detection and treatment are crucial.

[0003] Currently, the main medications used clinically to treat breast hyperplasia are hormone-based drugs, vitamin-based drugs, and iodine preparations. Hormone-based drugs, such as tamoxifen, inhibit the effects of estrogen by competing with its receptors. Vitamin-based drugs, such as vitamin E, primarily work by improving liver function to inactivate excess estrogen. Iodine preparations mainly promote the production of luteinizing hormone, repair the ovaries, and balance estrogen levels. Although they all have some efficacy, their effects are either short-lived or have significant side effects, making them unsuitable for long-term treatment of breast hyperplasia. Therefore, unless symptoms severely impact daily life, Western medicine is generally not the first choice for treating breast hyperplasia. Due to the non-invasive, low-toxicity, and significant efficacy characteristics of traditional Chinese medicine (TCM), its use in treating breast hyperplasia is gradually gaining acceptance.

[0004] Polygonatum rhizome is the dried rhizome of a plant belonging to the genus Polygonatum in the Liliaceae family. It has a sweet and neutral flavor and enters the spleen, lung, and kidney meridians. It is believed to tonify the middle energizer and replenish qi, nourish the kidneys and yin, strengthen the spleen and moisten the lungs, and dispel wind-dampness. Polygonatum contains various chemical components, including polysaccharides, saponins, flavonoids, and alkaloids, with polysaccharides being one of its main components. Further research is needed to explore the pharmacological effects and applications of purified polysaccharides from Polygonatum rhizome. Summary of the Invention

[0005] The first objective of this invention is to provide a purified polysaccharide from Polygonatum odoratum; the second objective of this invention is to provide a method for preparing the purified polysaccharide from Polygonatum odoratum; and the third objective of this invention is to provide an application of the purified polysaccharide from Polygonatum odoratum.

[0006] According to a first aspect of the present invention, a purified polysaccharide of Polygonatum odoratum is provided, the monosaccharide composition of which includes xylose, mannose, glucose and galactose, wherein the molar ratio of xylose, mannose, glucose and galactose is 6.45:30.26:7.43:55.87.

[0007] In some embodiments, the main stretching vibration absorption peak of the infrared spectrum of purified Polygonatum polysaccharide is related to the attached... Figure 4 The infrared spectra shown are basically consistent.

[0008] In some embodiments, the molecular weight of the purified polysaccharide from Polygonatum odoratum is distributed in the range of 2000-30000 Da, with the molecular weight concentrated at 22877 Da.

[0009] According to a second aspect of the present invention, a method for preparing the above-mentioned purified polysaccharide from Polygonatum sibiricum is provided, comprising the following steps:

[0010] (1) Weigh out the Polygonatum powder, add water at a ratio of 1:8~15, stir evenly, soak for 20~30 minutes to obtain a Polygonatum water-foam mixture, then extract the Polygonatum water-foam mixture at 55~70℃ 2~4 times, each time for 2~4 hours, combine the extracts obtained from each extraction to obtain a Polygonatum water extract; then concentrate the Polygonatum water extract under reduced pressure to obtain a concentrate, then add anhydrous ethanol to the concentrate at a volume ratio of 1:3~5, let stand overnight at room temperature, then centrifuge and collect the precipitate;

[0011] (2) The precipitate obtained in step (1) is redissolved with water to obtain a polysaccharide redissolved solution. Then, Sevage reagent is added to the polysaccharide redissolved solution to remove protein. The solution is shaken and allowed to stand overnight. Then, the solution is centrifuged and the supernatant is collected. The supernatant is then concentrated under reduced pressure to obtain a concentrated solution. The concentrated solution is then added to a macroporous resin for decolorization. The solution is eluted with pure water and the eluent is collected. The eluent is then freeze-dried to obtain crude polysaccharide of Polygonatum odoratum.

[0012] (3) The crude polysaccharide of Polygonatum obtained in step (2) is prepared into a solution with a concentration of 40~60 mg / mL by adding water. After filtration through a 0.22μm filter membrane, the filtrate is obtained. The filtrate is then separated by a DEAE-52 cellulose ion exchange column with a sample loading amount of 1.2~1.8% of the column volume. Pure water is used for elution, and the eluent is collected manually. The eluent is then concentrated under reduced pressure to obtain a concentrate. The concentrate is then freeze-dried to obtain the water-washed polysaccharide of Polygonatum.

[0013] (4) The polysaccharide obtained in step (3) was diluted with water to prepare a solution with a concentration of 40~60 mg / mL. After filtration through a 0.22 μm filter membrane, the filtrate was obtained. The filtrate was then added to a Sephadex G-50 dextran gel column for separation and purification. The sample loading amount was 1.2~1.8% of the column volume. The solution was eluted with pure water. The eluent was collected manually, 2 mL per tube. The eluents from tubes 21 to 41 were collected and combined. The solution was concentrated under reduced pressure to obtain a concentrate. The concentrate was then dialyzed to obtain a dialysate. The dialysate was then freeze-dried to obtain purified polysaccharide from Polygonatum.

[0014] This invention first extracts Polygonatum polysaccharides from a complex mixture using a DEAE-52 cellulose ion exchange column, and then separates the different sugar components from the Polygonatum polysaccharides using a Sephadex G-50 dextran gel column. Since filtration through a membrane removes some impurities and improves sample purification before column loading, this invention uses membrane filtration before purification. Because the separated polysaccharide is water-soluble, pure water is used for elution.

[0015] In some embodiments, in step (1), the precipitate is collected by centrifugation at 6000 r / min for 15 min; in step (2), the supernatant is taken after centrifugation at 12000 r / min for 10 min.

[0016] In some embodiments, in step (2), elution is performed with 2 BV of pure water at a flow rate of 20 mL / min; in step (3), elution is performed with 2 BV of pure water at a flow rate of 10 mL / min; and in step (4), elution is performed with 2 BV of pure water at a flow rate of 1 mL / min.

[0017] In some embodiments, in step (2), the Sevage reagent is obtained by mixing chloroform and n-butanol at a volume ratio of 4:1, and the volume ratio of the Sevage reagent to the alcohol-precipitated polysaccharide reconstitution solution is 2:1.

[0018] In some embodiments, in step (4), the concentrate is dialyzed through a dialysis bag with a molecular weight cutoff of 2000 Da for 24 hours.

[0019] According to a third aspect of the present invention, the use of the above-described purified Polygonatum polysaccharide in the preparation of a medicament for relieving / treating breast hyperplasia is provided.

[0020] According to a fourth aspect of the present invention, a medicament for relieving / treating breast hyperplasia is provided, which is prepared from the above-described purified polysaccharide of Polygonatum odoratum and pharmaceutically acceptable excipients.

[0021] The beneficial effects of this invention include:

[0022] (1) The purified polysaccharide of Polygonatum sibiricum of the present invention can reduce the toxic damage of estrogen-progesterone imbalance to HC11 mammary epithelial cells. A low concentration (5 μg / mL) of purified polysaccharide of Polygonatum sibiricum can protect the cells. When the concentration of purified polysaccharide of Polygonatum sibiricum reaches 500 μg / mL, it can completely protect HC11 mammary epithelial cells from cytotoxic damage caused by estrogen-progesterone imbalance.

[0023] (2) The present invention verified through an intervention experiment using a mouse mammary hyperplasia model that the purified polysaccharide of Polygonatum sibiricum in the present invention has a significant inhibitory effect on mouse mammary hyperplasia, which suggests that it can be used to develop drugs for the treatment of mammary hyperplasia. Attached Figure Description

[0024] Figure 1 This is a flowchart illustrating the preparation process of the purified polysaccharide HJ01 from Polygonatum sibiricum in Example 1 of the present invention.

[0025] Figure 2 The elution curve of the purified polysaccharide HJ01 from Polygonatum sibiricum in Example 1 of this invention is obtained by Sephadex G-50 gel chromatography.

[0026] Figure 3 The results of GC-MS determination of the purified polysaccharide HJ01 from Polygonatum sibiricum in Example 1 of this invention are shown.

[0027] Figure 4 The infrared spectrophotometer of the purified polysaccharide HJ01 from Polygonatum sibiricum in Example 1 of this invention is shown.

[0028] Figure 5 The results of the MTT cell activity experiment of the purified polysaccharide HJ01 from Polygonatum sibiricum in Example 1 of this invention are shown.

[0029] Figure 6 The results show the effect of purified polysaccharide HJ01 from Polygonatum sibiricum in Example 1 of this invention on hormone-induced cell viability.

[0030] Figure 7 This is a comparative diagram showing the effect of purified polysaccharide HJ01 from Polygonatum sibiricum on histological changes in mammary gland tissue in mice with mammary hyperplasia, according to Example 1 of the present invention. In the diagram, A is the blank control group, B is the model group, C is the Rupixiao tablet group, and D is the purified polysaccharide HJ01 group from Polygonatum sibiricum.

[0031] Figure 8 The results of the effect of purified polysaccharide HJ01 from Polygonatum sibiricum in Example 1 of this invention on the expression of estrogen receptor (ERα) in mouse mammary tissue, wherein DAPI is a live cell staining agent.

[0032] Figure 9 The results of the effect of purified polysaccharide HJ01 from Polygonatum sibiricum in Example 1 of this invention on the expression of progesterone receptor (PR) in mouse mammary tissue, wherein DAPI is a live cell staining agent. Detailed Implementation

[0033] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. The experimental materials and reagents involved in the following embodiments are all commercially available. Experimental methods not specifically described in the embodiments are generally performed under conventional conditions or according to the manufacturer's recommendations.

[0034] Example 1

[0035] The process flow diagram for preparing purified polysaccharides from Polygonatum sibiricum in this embodiment is as follows: Figure 1 As shown. Specifically, the preparation method of purified polysaccharide from Polygonatum sibiricum in this embodiment includes the following steps:

[0036] (1) Clean the Polygonatum rhizome, crush it into powder, add water at a ratio of 1:10 and pour it into a mixer to stir evenly. Soak for 20 min to obtain a mixture of Polygonatum rhizome water and foam. Then extract the mixture of Polygonatum rhizome water and foam three times at 60℃ for 3 h each time. Combine the extracts obtained from each extraction to obtain a water extract of Polygonatum rhizome. Then concentrate the water extract of Polygonatum rhizome under reduced pressure at 60℃ to a concentration of 1 g / mL to obtain a concentrated solution. Then add anhydrous ethanol to the concentrated solution at a volume ratio of 1:3, let it stand overnight at room temperature, and then centrifuge at 6000 r / min for 15 min to collect the precipitate and obtain Polygonatum rhizome precipitate.

[0037] (2) The Polygonatum precipitate obtained in step (1) was diluted with water to prepare a solution with a concentration of 50 mg / mL to obtain a polysaccharide redissolved solution. Then, Sevage reagent was added to the polysaccharide redissolved solution to remove protein. Sevage reagent is obtained by mixing chloroform and n-butanol at a volume ratio of 4:1. The volume ratio of Sevage reagent to polysaccharide redissolved solution is 2:1. The solution was shaken and allowed to stand overnight. Then, it was centrifuged at 12000 r / min for 10 min and the supernatant was taken. The supernatant was then concentrated under reduced pressure at 60℃ to a concentration of 0.1 g / mL to obtain a concentrate. The concentrate was then added to a macroporous resin (column height 80 cm, inner diameter 10 cm, and filled with a 1:1 mixture of D101 and AB-8 resin) for decolorization. After standing for 12 h of adsorption, it was eluted with 2 BV pure water at a flow rate of 20 mL / min. The eluent was collected and freeze-dried to obtain crude Polygonatum precipitate.

[0038] (3) The crude polysaccharide of Polygonatum obtained in step (2) was prepared into a solution with a concentration of 50 mg / mL by adding water. After filtration through a 0.22 μm filter membrane, the filtrate was obtained. The filtrate was then separated by a DEAE-52 cellulose ion exchange column with a sample loading amount of 1.5% of the column volume. The solution was eluted with 2 BV pure water at a flow rate of 10 mL / min. The eluent was collected manually and then concentrated under reduced pressure at 60 °C to a concentration of 1 g / mL to obtain a concentrated solution. The concentrated solution was then freeze-dried to obtain the water-washed polysaccharide of Polygonatum.

[0039] (4) The Polygonatum polysaccharide obtained in step (3) was diluted with water to prepare a solution with a concentration of 50 mg / mL. After filtration through a 0.22 μm filter membrane, the filtrate was obtained. The filtrate was then slowly added to a Sephadex G-50 dextran gel column for separation and purification. The sample loading volume was 1.5% of the column volume. Elution was performed with 2 BV of pure water. The eluent was collected manually at a flow rate of 1 mL / min for 2 min per tube. The polysaccharide content of each tube of eluent was tracked and determined using the phenol-sulfuric acid method. The elution curve of the Sephadex G-50 dextran gel column is shown below. Figure 2 As shown, the eluent from tubes 21 to 41 was collected, combined, and concentrated under reduced pressure at 60°C to a concentration of 0.5 g / mL to obtain a concentrated solution. The concentrated solution was then dialyzed with running water for 24 hours using a dialysis bag with a molecular weight cutoff of 2000 Da to obtain a dialysate. The dialysate was then freeze-dried to obtain purified polysaccharide from Polygonatum rhizome, which was named purified polysaccharide from Polygonatum rhizome HJ01.

[0040] Below, the average relative molecular weight of the purified polysaccharide HJ01 prepared by Polygonatum sibiricum in Example 1 was determined by experiment, its monosaccharide composition was analyzed, and its efficacy in relieving breast hyperplasia and its uses were further verified.

[0041] I. Chemical Structure Identification of the Purified Polysaccharide HJ01 from Polygonatum sibiricum

[0042] 1. Average relative molecular weight of purified polysaccharide HJ01 from Polygonatum sibiricum

[0043] The average relative molecular weight of purified polysaccharide HJ01 from Polygonatum sibiricum was determined by gel permeation chromatography (GPC) using a SEC-RI-MALLS system. The purified polysaccharide HJ01 was dissolved in a 1 mg / mL solution using 0.1 M Na₂SO₄ solution as the mobile phase. Chromatographic conditions were as follows: HQSB 802.5 and HQSB 804 columns in series; 0.1 M Na₂SO₄ solution as the mobile phase; flow rate of 0.6 mL / min; column temperature of 35 ℃; injection volume of 100 μL; system pre-calibration; and a curve was plotted using the logarithm of the average molecular weight of PEG standard. The weight-average molecular weight (Mw) was calculated using the calibration formula Mw = ∑(RIiMi) / ∑RIi. The average relative molecular weight of purified polysaccharide HJ01 from Polygonatum sibiricum was determined to be 22877 Da.

[0044] 2. Monosaccharide composition of purified polysaccharide HJ01 from Polygonatum sibiricum

[0045] The monosaccharide composition of purified polysaccharide HJ01 from Polygonatum sibiricum was analyzed according to the following method, with the specific steps as follows: 5 mg of purified polysaccharide HJ01 from Polygonatum sibiricum was accurately weighed and placed in a stoppered test tube. 1.0 mL of 2 mol / L trifluoroacetic acid (TFA) was added, shaken, sealed, and hydrolyzed in an oil bath at 110 ℃ for 6 h. After cooling, the residual TFA was dried with N2, dissolved in a small amount of methanol, mixed, shaken, and dried with N2. This process was repeated 3 times until all TFA was removed. Then, the product was evaporated under reduced pressure at 40 ℃ to obtain the polysaccharide hydrolysis product.

[0046] Add 10 mg of hydroxylamine hydrochloride and 0.5 mL of pyridine sequentially to the above polysaccharide hydrolysis product, shake to mix, seal, and then heat in a 90 ℃ water bath for 30 min with continuous shaking. After cooling, add 0.5 mL of acetic anhydride, seal, and continue heating in a 90 ℃ water bath for 30 min with occasional shaking. After the reaction stops, dry the reaction product with N2 in a 70 ℃ water bath, add 1 mL of chloroform to redissolve, and obtain the polysaccharide acetylated sample. Take 0.5 μL of the polysaccharide acetylated sample for gas chromatography analysis.

[0047] Take 2 mg of each standard monosaccharide (rhamnose, arabinose, xylose, mannose, glucose, galactose) into a round-bottom flask, add 10 mg of hydroxylamine hydrochloride and 0.5 mL of pyridine, shake to mix, seal, and perform acetylation treatment of the monosaccharide sample according to the above steps for acetylation of polysaccharide hydrolysis products to obtain monosaccharide acetylated sample.

[0048] The monosaccharide composition of the above acetylated samples was analyzed using a DB-17 column. The operating conditions were as follows: column: DB-17 (30 m × 0.32 mm × 0.5 μm); detector: flame ionization detector (FID); carrier gas: N2; injection port temperature: 280℃; detector temperature: 280℃; flow rate: 1 mL / min; column temperature: 190℃.

[0049] The GC-MS analysis results of purified polysaccharide HJ01 from Polygonatum sibiricum are shown in Table 1 and 2. Figure 3 As shown in Table 1 and Figure 3 It can be concluded that the purified polysaccharide HJ01 of Polygonatum sibiricum is mainly composed of xylose, mannose, glucose and galactose, and the molar ratio of the four is 6.45:30.26:7.43:55.87.

[0050] Table 1. GC-MS analysis of purified polysaccharide HJ01 from Polygonatum sibiricum.

[0051]

[0052] 3. Infrared spectral analysis of purified polysaccharide HJ01 from Polygonatum sibiricum

[0053] Weigh 1 mg of purified Polysaccharide HJ01 from Polygonatum sibiricum and 200 mg of dried KBr, place them in a mortar, mix and grind them evenly, then press them into transparent tablets using a tablet press. Use blank KBr tablets as a blank control. Scan the polysaccharide sample using a Fourier transform infrared spectroscopy (FTIR) spectrometer with a scanning wavelength range of 400–4000 cm⁻¹. -1 Resolution: 4 cm -1 Scan count: 16. The infrared spectrum of purified polysaccharide HJ01 from Polygonatum sibiricum was obtained as follows: Figure 4 As shown.

[0054] Depend on Figure 4 It can be seen that the purified polysaccharide HJ01 from Polygonatum sibiricum is at a wavelength of 2934.23 cm⁻¹ -1 A C-H stretching vibration absorption peak was generated at 1633.80 cm⁻¹. -1 There is an absorption peak for CO stretching vibration at 1417.50 cm⁻¹. -1 A CH-angle vibrational absorption peak is present at all locations; 1137.08 cm⁻¹ -1 932.65 cm -1 and 817.83 cm -1 This is one of the characteristic absorptions of pyranose, indicating that this polysaccharide is a pyranose, of which 932.65 cm⁻¹ -1 It is β-D-galactopyranose, 817.83 cm -1 It is α-D-galactopyranose; 686.84 cm -1The weak, sharp peak is the β-pyran ring bending vibration peak. These results indicate that this polysaccharide exists in both α- and β-type pyranose forms.

[0055] II. Experiment on the effect of purified polysaccharide HJ01 from Polygonatum sibiricum on relieving mammary hyperplasia in living cells

[0056] 1. Main materials and reagents: Polysaccharide HJ01 purified from Polygonatum sibiricum (prepared in Example 1), embryonic bovine serum (Sigma), MTT (Sigma), estradiol benzoate (Ningbo General Pharmaceutical Co., Ltd.), progesterone (Zhejiang Xianju Pharmaceutical Co., Ltd.), microplate reader (Tecan, Austria), CO2 incubator (Thermo Scientific, USA), optical inverted microscope (Thermo Scientific, USA).

[0057] The PBS solution was prepared as follows: First, prepare 800 mL of distilled water in a container. Then, add 8 g NaCl, 200 mg KCl, 1.44 g Na2HPO4, and 240 mg KH2PO4 to the distilled water in sequence to adjust the pH of the solution to 7.4. Then, add distilled water until the solution volume is 1 L.

[0058] 2. Cytotoxic effect of purified polysaccharide HJ01 from Polygonatum sibiricum in HC11 cells

[0059] With 1×10 3 Mouse mammary epithelial cells (HC11 cells) were seeded into 96-well plates at a concentration of 100 μL of cell culture medium per well. After culturing for 24 h at 37 °C with 5% CO2, the old culture medium was removed, and the cells were divided into 7 groups. 200 μL of culture medium containing 0, 5, 25, 125, 250, 500, and 1000 μg / mL of purified polysaccharide HJ01 from Polygonatum sibiricum was added to each well, with three parallel controls. The cells were cultured for 24 h. After removing the culture medium, 200 μL of 0.5 mg / mL MTT solution was added in the dark, and the cells were cultured at 37 °C for 4 h. After removing the MTT solution, 200 μL of DMSO was added, and the mixture was shaken for 15 min. The absorbance was measured at 490 nm, and the cell viability was calculated. The results are shown below. Figure 5 As shown.

[0060] from Figure 5 It can be seen that the purified polysaccharide HJ01 from Polygonatum rhizome can significantly improve the cell viability of HC11 mammary epithelial cells. After treatment with the purified polysaccharide HJ01, the activity of HC11 mammary epithelial cells was enhanced in a concentration-dependent manner. Even a low concentration (5 μg / mL) of the purified polysaccharide HJ01 could promote cell growth, and the growth-promoting effect on HC11 cells was greatest when the polysaccharide concentration reached 1000 μg / mL.

[0061] 3. Effects of purified polysaccharide HJ01 from Polygonatum sibiricum on the proliferation of mammary epithelial cells after hormone-induced modeling.

[0062] With 1×10 3 HC11 cells were seeded into 96-well plates at a concentration of 100 μL of cell culture medium per well. After culturing for 24 h, the old culture medium was aspirated, and the cells were divided into 8 groups: a control group (200 μL of culture medium), a model group (200 μL of culture medium containing 250 μg / mL estradiol benzoate (E2) and 25 μg / mL progesterone (P), and experimental groups (5, 25, 125, 250, 500, and 1000 μg / mL of purified polysaccharide HJ01 from Polygonatum sibiricum). After 24 h, the old culture medium was aspirated, and the cells were washed once with PBS and aspirated. 200 μL of 0.5 mg / mL MTT solution was added under light-protected conditions, and the cells were incubated for 4 h. After 4 h, the liquid was aspirated, 200 μL of DMSO was added, and the mixture was shaken well for 10 min. Cell viability was then detected at 490 nm. The results are as follows: Figure 6 As shown.

[0063] from Figure 6 It can be seen that estrogen-progesterone imbalance causes significant damage to HC11 mammary epithelial cells, exhibiting strong cytotoxicity and significantly reducing cell viability. After treatment with purified polysaccharide HJ01 from Polygonatum rhizome, the cytotoxicity decreased in a concentration-dependent manner. Even a low concentration (5 μg / mL) of purified polysaccharide HJ01 could protect the cells, and a concentration of 500 μg / mL completely protected HC11 mammary epithelial cells from the cytotoxic damage caused by estrogen-progesterone imbalance. This indicates that purified polysaccharide HJ01 from Polygonatum rhizome can inhibit the effects of hormones on the proliferation of HC11 mammary epithelial cells and protect cells from hormone damage, suggesting that purified polysaccharide HJ01 from Polygonatum rhizome has a good effect in alleviating mammary hyperplasia.

[0064] III. Experiment on the inhibition of mouse mammary gland hyperplasia by purified polysaccharide HJ01 from Polygonatum sibiricum

[0065] 1. Experimental animals: KM, SPF grade female mice, weighing 35±2g, purchased from Zhuhai Beston Co., Ltd., Experimental animal production license number SCXK (Guangdong) 2020-0051.

[0066] 2. Main reagents: Polysaccharide HJ01 purified from Polygonatum sibiricum (prepared in Example 1), estradiol benzoate (Ningbo General Pharmaceutical Co., Ltd.), progesterone (Zhejiang Xianju Pharmaceutical Co., Ltd.).

[0067] 3. Establishment and administration of a mouse model of mammary hyperplasia

[0068] Twenty-four female nonpregnant KM mice were randomly divided into four groups: a blank control group, a model group, a Rupixiao tablet group, and a Huangjing purified polysaccharide HJ01 group, with six mice in each group. The blank control group received an intramuscular injection of 0.1 mL of physiological saline per mouse once daily for 30 days. The other groups received an intramuscular injection of estradiol benzoate (0.5 mg / kg) once daily for the first 25 days, followed by an intramuscular injection of progesterone (4 mg / kg) once daily for the next 5 days. The blank control group and model group received 0.2 mL of physiological saline by gavage before injection; the Rupixiao tablet group received 0.2 mL of Rupixiao tablet solution (0.25 g / kg / d) by gavage before injection; and the Huangjing purified polysaccharide HJ01 group received 0.2 mL of Huangjing purified polysaccharide HJ01 solution (0.67 mg / kg / d) by gavage before injection.

[0069] 4. Effects of purified polysaccharide HJ01 from Polygonatum sibiricum on pathological changes in mammary tissue of a mouse model of mammary hyperplasia.

[0070] After 30 days of modeling, the third pair of mammary gland tissues from mice were collected, fixed by immersion in 4% paraformaldehyde, and prepared for later use. After fixation, the tissues were embedded in paraffin, sectioned, and stained with H&E. Morphological observation of the mammary gland tissues from each group of mice was performed using an optical microscope. The results are as follows: Figure 7 As shown in the figure, A represents the blank control group, B represents the model group, C represents the Rupixiao tablet group, and D represents the Huangjing purified polysaccharide HJ01 group.

[0071] from Figure 7 The results showed that, compared with the blank control group, the model group exhibited proliferative lesions in the mammary epithelial cell tissue, and the number of acini and ducts in the mammary lobules increased. Compared with the model group, both the Rupixiao tablet group and the Huangjing purified polysaccharide HJ01 group significantly alleviated the symptoms of hormone-induced mammary hyperplasia in mice, with Huangjing purified polysaccharide HJ01 showing better efficacy in improving mammary hyperplasia lesions.

[0072] 5. Effects of purified polysaccharide HJ01 from Polygonatum sibiricum on the expression of ERα and PR in mammary tissue of mice with mammary hyperplasia.

[0073] To investigate the effects of purified polysaccharide HJ01 from Polygonatum sibiricum on estrogen and progesterone receptors, immunofluorescence was used to detect the levels of estrogen receptor α (ERα) and progesterone receptor (PR). Pre-fixed and dehydrated embedded breast tissue was sectioned, and the expression of ERα and PR in the breast tissue was measured using immunofluorescence. The results are shown below. Figure 8 and Figure 9 As shown.

[0074] from Figure 8 and Figure 9As can be seen, the fluorescence intensity of ERα and PR was significantly increased in the model group compared with the blank control group. Compared with the model group, the fluorescence intensity of ERα and PR was significantly decreased in the Rupixiao tablet group and the purified polysaccharide HJ01 group from Polygonatum rhizome. This result indicates that the purified polysaccharide HJ01 from Polygonatum rhizome has a regulatory effect on estrogen and progesterone receptors in mice, suggesting that the purified polysaccharide HJ01 from Polygonatum rhizome has a significant inhibitory effect on mammary gland hyperplasia in mice.

[0075] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. Application of purified polysaccharides from Polygonatum sibiricum in the preparation of drugs for relieving / treating breast hyperplasia, among which, The monosaccharide composition of the purified polysaccharide from Polygonatum includes xylose, mannose, glucose, and galactose, and the molar ratio of xylose, mannose, glucose, and galactose is 6.45:30.26:7.43:55.

87.

2. The application according to claim 1, characterized in that, The molecular weight of the purified polysaccharide from Polygonatum odoratum ranges from 2000 to 30000 Da, with the molecular weight concentrated at 22877 Da.

3. The application according to claim 1, characterized in that, The preparation method of the purified polysaccharide from Polygonatum includes the following steps: (1) Weigh out the Polygonatum powder, add water at a ratio of 1:8~15, stir evenly, soak for 20~30 minutes to obtain a Polygonatum water-foam mixture, then extract the Polygonatum water-foam mixture at 55~70℃ 2~4 times, each time for 2~4 hours, combine the extracts obtained from each extraction to obtain Polygonatum water extract. Then, the aqueous extract of Polygonatum was concentrated under reduced pressure to obtain a concentrated solution. Anhydrous ethanol was added to the concentrated solution at a volume ratio of 1:3~5. The solution was left to stand overnight at room temperature, and then centrifuged to collect the precipitate. (2) The precipitate obtained in step (1) is redissolved with water to obtain a polysaccharide redissolved solution. Then, Sevage reagent is added to the polysaccharide redissolved solution to remove protein. The solution is shaken and allowed to stand overnight. Then, the solution is centrifuged and the supernatant is collected. The supernatant is then concentrated under reduced pressure to obtain a concentrated solution. The concentrated solution is then added to a macroporous resin for decolorization. The solution is eluted with pure water and the eluent is collected. The eluent is then freeze-dried to obtain crude polysaccharide of Polygonatum odoratum. (3) The crude polysaccharide of Polygonatum obtained in step (2) is prepared into a solution with a concentration of 40~60 mg / mL by adding water. After filtration through a 0.22 μm filter membrane, the filtrate is obtained. The filtrate is then separated by a DEAE-52 cellulose ion exchange column with a sample loading amount of 1.2~1.8% of the column volume. Pure water is used for elution, and the eluent is collected manually. The eluent is then concentrated under reduced pressure to obtain a concentrate. The concentrate is then freeze-dried to obtain the water-washed polysaccharide of Polygonatum. (4) The polysaccharide obtained in step (3) was diluted with water to prepare a solution with a concentration of 40~60 mg / mL. After filtration through a 0.22 μm filter membrane, the filtrate was obtained. The filtrate was then added to a Sephadex G-50 dextran gel column for separation and purification. The sample loading amount was 1.2~1.8% of the column volume. The solution was eluted with pure water. The eluent was collected manually, 2 mL per tube. The eluents from tubes 21 to 41 were collected and combined. The solution was concentrated under reduced pressure to obtain a concentrate. The concentrate was then dialyzed to obtain a dialysate. The dialysate was then freeze-dried to obtain purified polysaccharide from Polygonatum.

4. The application according to claim 3, characterized in that, In step (1), the precipitate was collected by centrifugation at 6000 r / min for 15 min; in step (2), the supernatant was collected after centrifugation at 12000 r / min for 10 min.

5. The application according to claim 3 or 4, characterized in that, In step (2), 2 BV of pure water is used for elution at a flow rate of 20 mL / min; in step (3), 2 BV of pure water is used for elution at a flow rate of 10 mL / min; in step (4), 2 BV of pure water is used for elution at a flow rate of 1 mL / min.

6. The application according to claim 3 or 4, characterized in that, In step (2), the Sevage reagent is obtained by mixing chloroform and n-butanol at a volume ratio of 4:1, and the volume ratio of the Sevage reagent to the alcohol-precipitated polysaccharide reconstitution solution is 2:

1.

7. The application according to claim 3 or 4, characterized in that, In step (4), the concentrate is dialyzed through a dialysis bag with a molecular weight cutoff of 2000 Da for 24 hours.

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