Rosa roxburghii polysaccharide and its preparation method and application

Abstract

The present invention discloses roxburghii polysaccharide and its preparation method and application. Wherein roxburghii crude polysaccharide is purified to obtain two polysaccharides RP1 and RP3, RP1 monosaccharide types include galacturonic acid, galactose, rhamnose and arabinose, and its molar ratio is 1.00:3.45:1.88:1.82, RP3 monosaccharide types include galactose, rhamnose and arabinose, galacturonic acid, galactose, rhamnose and arabinose, and its molar ratio is 1.00:0.37:0.37:0.25. It has been confirmed by in vitro experiments that the above-mentioned roxburghii polysaccharide has the ability to significantly inhibit the proliferation of MCF-7 breast cancer cells, as well as the effect of inducing apoptosis of MCF-7 breast cancer cells, and can be developed to prepare breast cancer therapeutic drugs; At the same time, the above-mentioned roxburghii polysaccharide has no effect on normal L929 cells. Therefore, the above-mentioned roxburghii polysaccharide has broad application prospects and has good social and economic benefits.

Description

Technical Field

[0001] The invention relates to the technical field of natural product development, and in particular to roxburghii polysaccharide and a preparation method and application thereof. Background Art

[0002] Rosa roxburghii is a fruit rich in a variety of natural active ingredients. It is concentrated in southwest my country and has abundant wild resources. Rosa roxburghii fruit tastes sour and astringent, has thorns on the surface and a hard texture, so it is more suitable for processing into products. Polysaccharides were discovered by researchers as one of the active ingredients of Rosa roxburghii, but the research on Rosa roxburghii polysaccharides is limited to the hot water extraction of crude polysaccharides and the study of their in vitro antioxidant, hypoglycemic and lipid-lowering activities. The selection and optimization of the extraction method, purification and fine structure of Rosa roxburghii polysaccharides, and their anti-breast cancer activity have not been reported. Therefore, it is of great significance to select an extraction method and its optimization that can improve the yield of Rosa roxburghii polysaccharides. At the same time, it is of great significance to purify Rosa roxburghii polysaccharides and analyze their fine structure, explore their biological activity, and provide a theoretical basis for their application in functional foods and medicine.

[0003] In view of this, the present invention is proposed. Summary of the invention

[0004] The purpose of the present invention is to provide roxburghii polysaccharide and a preparation method and application thereof, wherein the obtained roxburghii polysaccharide has an inhibitory effect on the growth of MCF-7 breast cancer cells.

[0005] The present invention is achieved in that:

[0006] In a first aspect, the present invention provides a roxburghii polysaccharide extract, comprising roxburghii polysaccharides RP1 and RP3;

[0007] The monosaccharide types in the roxburghii polysaccharide RP1 include galacturonic acid, galactose, rhamnose and arabinose, with a molar ratio of 1.00:3.45:1.88:1.82, and the monosaccharide composition of the roxburghii polysaccharide RP3 is galacturonic acid, galactose, rhamnose and arabinose, with a molar ratio of 1.00:0.37:0.37:0.25.

[0008] In an alternative embodiment, the monosaccharide species in the roxburghii polysaccharide include galacturonic acid, galactose, rhamnose, arabinose, xylose and glucose.

[0009] In an alternative embodiment, the glycosidic bonds of the polysaccharide RP1 include →4)-GalpA-(1→, T-Rhap-(1→, →2)-Rhap-(1→, →2,4)-Rhap-(1→, →4)-Rhap-(1→, T-Galp-(1→, →4)-Galp-(1→, →6)-Galp-(1→, →3,6)-Galp-(1→, →3)-β-Galp-(1→, T-Araf-(1→, and →3)-Araf-(1→.

[0010] In an optional embodiment, the molar ratio of the glycosidic bonds of the polysaccharide RP1 is 12.88, 5.35, 3.16, 17.95, 1.65, 19.33, 12.95, 11.21, 4.63, 3.68, 4.51 and 2.66.

[0011] In an alternative embodiment, the glycosidic bonds of the polysaccharide RP3 include T-GalpA-(1→,→4)-GalpA-(1→,→3,4)-GalpA-(1→,T-Rhap-(1→,→2)-Rhap-(1→,→2,4)-Rhap-(1→,T-Galp-(1→,→6)-Galp-(1→,→3,6)-Galp-(1→,T-Araf-(1→,→5)-Araf-(1→,→4)-Glcp-(1→,T-Xylp-(1→.

[0012] In an optional embodiment, the molar ratios of the glycosidic bonds of the polysaccharide RP3, T-GalpA-(1→,→4)-GalpA-(1→,→3,4)-GalpA-(1→,T-Rhap-(1→,→2)-Rhap-(1→,→2,4)-Rhap-(1→,T-Galp-(1→,→6)-Galp-(1→,→3,6)-Galp-(1→,T-Araf-(1→,→5)-Araf-(1→,→4)-Glcp-(1→,T-Xylp-(1→,→4)-Glcp-(1→,T-Xylp-(1→,→6)-Galp-(1→,→3,6)-Galp-(1→,T-Araf-(1→,→5)-Araf-(1→,→4)-Glcp-(1→,T-Xylp-(1→,→4)-Glcp-(1→,T-Xylp-(1→,→4)-Glcp-(1→,T-Xylp-(1→,→4)-Glcp-(1→,T-Rhap-(1→,→2)-Rhap-(1→,→2,4)-Rhap-(1→,T-Galp-(1→,→6)-Galp-(1→,→3,6)-Galp-(1→,T-Araf-(1→,→5)-Araf-(1→,→4)-Glcp-(1→,T-

[0013] In an optional embodiment, the number average molecular weight of polysaccharide RP1 is 629.41 kDa; the number average molecular weight of polysaccharide RP3 is 74.70 kDa.

[0014] In a second aspect, the present invention provides a method for preparing the above-mentioned roxburghii extract, which comprises: extracting and purifying roxburghii dry powder to obtain a roxburghii extract containing polysaccharide RP1 and polysaccharide RP3.

[0015] In an optional embodiment, the extraction includes dissolving the roxburghii dry powder with a buffer solution and performing enzymolysis with pectinase, and then performing ultrasonic extraction on the enzymolysis solution.

[0016] In an optional embodiment, the conditions for enzymolysis are: the liquid ratio of buffer solution to roxburghii dry powder is 10:1-50:1 mL / g; the amount of pectinase added is 0.4%-4% of the mass of roxburghii dry powder; the pH value of the enzymolysis system is 3-6; the enzymolysis temperature is 35-55°C; and the enzymolysis time is 5-70 minutes.

[0017] More preferably, the liquid-to-buffer ratio of the buffer solution to the roxburghii dry powder is 1-30 mL / g; the amount of pectinase added is 2.5% of the mass of the roxburghii dry powder; the pH value of the enzymatic hydrolysis system is 4.0; the enzymatic hydrolysis temperature is 50° C.; and the enzymatic hydrolysis time is 40 minutes.

[0018] In an optional embodiment, the ultrasonic conditions are: ultrasonic power of 80-200 W; ultrasonic temperature of 40-90° C.; and time of 5-60 minutes.

[0019] More preferably, the ultrasonic power is 120 W; the ultrasonic temperature is 80° C.; and the time is 20 minutes.

[0020] In an optional embodiment, the purified Rosa roxburghii extract is obtained by separation through anion exchange column.

[0021] Specifically, the extract is decolorized by passing through AB-8 macroporous resin, trichloroacetic acid is added to remove protein, the protein precipitate is removed by centrifugation, the supernatant is taken and precipitated with alcohol, then redissolved, dialyzed and freeze-dried to obtain crude roxburghii polysaccharide; then the extracted crude roxburghii polysaccharide is dissolved in water, separated by DEAE Sepharose Fast Flow anion exchange chromatography column, and the roxburghii polysaccharide components are collected.

[0022] In a third aspect, the present invention also provides the use of the above-mentioned roxburghii extract in preparing anti-breast cancer drugs.

[0023] In a fourth aspect, the present invention further provides a pharmaceutical composition, which comprises the above-mentioned polysaccharide RP1 and / or polysaccharide RP3, and a pharmaceutically acceptable carrier.

[0024] The present invention has the following beneficial effects:

[0025] The invention separates roxburghii polysaccharide from roxburghii, and in vitro experiments confirm that the roxburghii polysaccharide has the ability to significantly inhibit the proliferation of MCF-7 breast cancer cells and the effect of inducing apoptosis of MCF-7 breast cancer cells, and can be further developed to prepare breast cancer therapeutic drugs; meanwhile, the roxburghii polysaccharide has no effect on normal L929 cells. Therefore, the roxburghii polysaccharide of the invention has broad application prospects and good social and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0027] Figure 1 The elution diagram of the DEAE Sepharose Fast Flow anion exchange chromatography column of the roxburghii polysaccharide provided by the present invention;

[0028] Figure 2 The effects of different extraction conditions on the extraction yield of roxburghii polysaccharides in the present invention are as follows;

[0029] Figure 3 The monosaccharide composition of the pure roxburghii polysaccharides RP1 and RP3 provided by the present invention;

[0030] Figure 4 The infrared spectra of the pure roxburghii polysaccharides RP1 and RP3 provided by the present invention;

[0031] Figure 5 The one-dimensional nuclear magnetic spectrum of the pure roxburghii polysaccharides RP1 and RP3 provided by the present invention;

[0032] Figure 6 The effect of the pure roxburghii polysaccharide RP1 provided by the present invention on the survival rate of MCF-7 cells;

[0033] Figure 7 The invention discloses an effect of the pure roxburghii polysaccharide RP1 provided by the present invention on the survival rate of L929 cells. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0035] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0036] Example 1

[0037] This example is the preparation of roxburghii polysaccharide, and the specific steps are as follows:

[0038] (1) grinding and sieving the dried roxburghii fruit purchased from Liupanshui, Guizhou Province, adding 95% ethanol and reflux in a 70° C. water bath for 3 h, repeating twice for decolorization and defatting, filtering and drying the residue in an oven at 50° C. overnight to obtain roxburghii dry powder;

[0039] (2) putting the roxburghii dry powder obtained in step (1) into a round-bottom flask, adding 2.5% pectinase, adding 0.1 mol / L sodium citrate-sodium citrate buffer with a pH of 4.0 (the ratio of liquid to raw material is 30:1 mL / g), mixing, and placing the solution in a 50° C. water bath for enzymolysis for 40 min;

[0040] (3) placing the liquid obtained in step (2) in an ultrasonic reactor at a power of 120 W and a temperature of 80° C. for 20 min;

[0041] (4) filtering the solution obtained in step (3) to obtain a filtrate;

[0042] (5) adding AB-8 macroporous resin to the filtrate obtained in step (4) for decolorization;

[0043] (6) adding trichloroacetic acid with a final concentration of 4% to the filtrate obtained in step (5), stirring at room temperature for half an hour, centrifuging, and collecting the supernatant;

[0044] (7) adding four times the volume of anhydrous ethanol to the supernatant obtained in step (6), subjecting the mixture to alcohol precipitation, centrifuging and collecting the precipitate;

[0045] (8) dissolving the precipitate obtained in step (7) with water, dialyzing, and vacuum freeze-drying to obtain crude roxburghii polysaccharide;

[0046] (9) dissolving the crude roxburghii polysaccharide obtained in step (8) in water to obtain a crude roxburghii polysaccharide solution;

[0047] (10) passing the crude roxburghii polysaccharide solution obtained in step (9) through a DEAE-Sepharose Fast Flow anion exchange chromatography column to obtain RP1 and RP3 components;

[0048] (11) The roxburghii polysaccharide solution obtained in step (10) is dialyzed and vacuum freeze-dried to obtain purified roxburghii polysaccharide.

[0049] The yield of roxburghii polysaccharide obtained by the preparation method of this example is 4.78±0.10%.

[0050] Figure 1 The figure is the elution diagram of the DEAE Sepharose Fast Flow anion exchange chromatography column of the roxburghii polysaccharide of the present invention.

[0051] Example 2

[0052] This example adopts the preparation method of Example 1, and screens the best preparation scheme by single factor variation, wherein the single factor variation factors are enzymolysis temperature, enzymolysis time, material-liquid ratio, system pH, enzyme amount, ultrasonic temperature, ultrasonic time and ultrasonic power.

[0053] Test results such as Figure 2 As shown, under the conditions that the material-liquid ratio of the buffer solution to the roxburghii dry powder is 10:1-50:1 mL / g, the addition amount of pectinase is 0.4%-4% of the mass of the roxburghii dry powder, the pH value of the enzymatic hydrolysis system is 3-6, the enzymatic hydrolysis temperature is 35-55°C, the enzymatic hydrolysis time is 5-70 minutes, the ultrasonic power is 80-200W, the ultrasonic temperature is 40-90°C and the ultrasonic time is 5-60 minutes, the preparation method of the present invention can be used to prepare roxburghii polysaccharides, but the best conditions are: the addition amount of pectinase is 2.5% of the mass of the roxburghii dry powder, the enzymatic hydrolysis temperature is 50°C, the enzymatic hydrolysis time is 40min, the enzymatic hydrolysis pH is 4.0, the reaction liquid-material ratio of the buffer solution to the roxburghii dry powder is 1:30, the ultrasonic power is 120W, the ultrasonic extraction temperature is 80°C and the ultrasonic extraction time is 20min.

[0054] Example 3

[0055] This example is the identification of the roxburghii polysaccharide obtained in Example 1

[0056] 3.1 Molecular weight determination

[0057] The specific steps are as follows:

[0058] (1) RP1 and RP3 obtained in Example 1 were dissolved in 0.1 M NaNO3 aqueous solution to a final concentration of 1 mg / mL, and filtered through a filter with a pore size of 0.45 μm;

[0059] (2) The filtrate (100 μL) obtained in step (1) was injected into a gel exclusion chromatography column (Ohpak SB 805HQ (300×8 mm), Ohpak SB 804HQ (300×8 mm), Ohpak SB 803HQ (300×8 mm)), and eluted with NaNO3 solution (0.1 M) at a flow rate of 0.4 mL / min, and the column temperature was 45°C to obtain response value data;

[0060] (3) The obtained data were collected and processed, and the molecular weight was calculated according to the Mark Houwink equation. The results showed that the average molecular weights of RP1 and RP3 were 629.41 kDa and 74.70 kDa, respectively.

[0061] 3.2 Determination of monosaccharide composition

[0062] The specific steps are as follows:

[0063] (1) 5 mg of RP1 and RP3 obtained in Example 1 were hydrolyzed with trifluoroacetic acid (TFA) at 121° C. for 2 hours;

[0064] (2) Pass nitrogen gas, blow dry, add methanol to clean, blow dry again, and repeat the methanol cleaning 2 to 3 times;

[0065] (3) Add sterile water to dissolve and transfer to a chromatographic bottle for testing;

[0066] (4) The standard fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, guluronic acid, and mannuronic acid were used as comparisons by high performance anion exchange chromatography (HPAEC) with pulsed amperometric detection and Dionex TM CarboPac TM The monosaccharide composition and monosaccharide content of the hydrolysate were determined using a PA20 (150 mm × 1.0 mm, 10 μm) liquid chromatography column;

[0067] Among them, the HPAEC operating conditions are as follows:

[0068] Mobile phase A: 0.1 M NaOH; Mobile phase B: 0.1 M NaOH, 0.2 M NaAc; Flow rate: 0.5 mL / min.

[0069] The gradient elution conditions were as follows: 0 min, 95% A, 5% B; 30 min, 80% A, 20% B; 30.1 min, 60% A, 40% B; 45 min, 60% A, 40% B; 45.1 min, 95% A, 5% B; 60 min, 95% A, 5% B.

[0070] The results of the assay were compared with those of the standard and the results showed that: RP1 contained galacturonic acid, galactose, rhamnose and arabinose as well as a small amount of xylose and glucose; the molar ratio of galacturonic acid, galactose, rhamnose and arabinose was 1.00:3.45:1.88:1.82. RP3 contained galacturonic acid, galactose, rhamnose and arabinose as well as a small amount of xylose and glucose; the molar ratio of galacturonic acid, galactose, rhamnose and arabinose was 1.00:0.37:0.37:0.25.

[0071] Figure 3 It is the monosaccharide composition of the roxburghii polysaccharides RP1 and RP3 of the present invention.

[0072] 3.3 FT IR spectroscopy

[0073] The specific steps are as follows:

[0074] RP1 and RP3 obtained in Example 1 were respectively mixed with KBr at a ratio of 1:100 and ground, and then pressed into thin sheets by vacuuming; the pressed thin sheets of RP1 and RP3 were heated at 4000-400 cm 1 The results were measured on an IR Tracer 100 Fourier transform infrared spectrometer.

[0075] The results of the test are as follows Figure 4 As shown, at approximately 3403.41cm -1 (RP1) and 3422.55cm -1 The broad stretching peak at (RP-3) represents the -OH stretching vibration peak at about 2927.65 cm -1 (RP1) and 2931.86cm -1 The small peak observed at (RP3) is attributed to the stretching vibration of CH. -1 The absorption band centered at about 1615.96 cm is caused by the asymmetric stretching vibration of C=O. -1 (RP1) and 1616.08cm -1 The peak at (RP3) represents the asymmetric stretching vibration of the non-esterified carbonyl C=O, which indicates the presence of uronic acid. -1 (RP1) and 1740.09cm -1 Valence vibration at (RP3), 1249 cm -1 The symmetric CH bending vibration at 1200-1000 cm -1 The region is a characteristic absorption band of polysaccharides, which is related to the COC glycosidic bond and COH link in the pyranose unit. In addition, 893.26cm -1 The characteristic absorption peak at indicated that RP1 had β-type glycosidic bonds between sugar units.

[0076] 3.4 Methylation detection

[0077] The specific steps are as follows:

[0078] (1) Weigh 10 mg of RP1 and RP3, dissolve each in 1 ml of primary water, add 1 ml of 100 mg / mL carbodiimide, and react for 2 h.

[0079] (2) Add 1 ml of 2 M imidazole, divide the sample into two equal parts, add 1 ml of 30 mg / mL NaBH4 and 1 ml of 30 mg / mL NaBD4 respectively, and react for 3 h.

[0080] (3) Add 100 μL of glacial acetic acid to terminate the reaction. Dialyze the sample for 48 h. After dialysis is complete, freeze-dry the sample and perform methylation treatment.

[0081] (4) Add 500 μL DMSO to the freeze-dried sample to dissolve.

[0082] (5) Add 1 mg NaOH and incubate for 30 min.

[0083] (6) Add 50 μL of iodomethane solution and react for 1 h.

[0084] (7) Add 1 mL of water and 2 mL of dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. Repeat the water wash three times.

[0085] (8) Take the lower dichloromethane phase and evaporate to dryness.

[0086] (9) Add 100 μL 2 M TFA, react at 121 °C for 90 min, and evaporate to dryness at 30 °C.

[0087] (10) Add 50 μL 2 M ammonia water and 50 μL 1 M NaBD4, mix well, and react at room temperature for 2.5 h.

[0088] (11) 20 μL of acetic acid was added to terminate the reaction, the mixture was dried under nitrogen, and the mixture was washed twice with 250 μL of methanol and dried under nitrogen.

[0089] (12) Add 250 μL of acetic anhydride, vortex to mix, and react at 100 °C for 2.5 h.

[0090] (13) Add 1 mL of water and let stand for 10 min.

[0091] (14) Add 500 μL of dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. Repeat the water washing process three times.

[0092] (15) The lower dichloromethane phase was removed and the acetylated product was qualitatively analyzed by GC-MS (Agilent 7890A-5977B; Agilent Technologies, Inc., USA) using a BPX70 capillary column (25 m × 0.22 mm × 0.25 μm).

[0093] The measurement results are shown in Table 1:

[0094] Table 1 Methylation results of roxburghii polysaccharides

[0095]

[0096] According to the methylation results of RP1 and RP3 listed in Table 1, it can be concluded that the important glycosidic bond connection modes obtained by methylation of RP1 are →4)-GalpA-(1→, T-Rhap-(1→, →2)-Rhap-(1→, →2,4)-Rhap-(1→, →4)-Rhap-(1→, T-Galp-(1→, →4)-Galp-(1→, →6)-Galp-(1→, →3,6)-Galp-(1→, →3)-β-Galp-(1→, T-Araf-(1→, and →3)-Araf-(1→, with molar ratios of 12.88, 5.35, 3.16, 17.95, 1.65, 19.33, 12.95, 11.21, 4.63, 3.68, 4.51 and 2.66, respectively.

[0097] The important glycosidic bond connection modes of RP3 obtained through methylation are T-GalpA-(1→,→4)-GalpA-(1→,→3,4)-GalpA-(1→,T-Rhap-(1→,→2)-Rhap-(1→,→2,4)-Rhap-(1→,T-Galp-(1→,→6)-Galp-(1→,→3,6)-Galp-(1→,T-Araf-(1→,→5)-Araf-(1→,→4)-Glcp-(1→,T-Xylp-(1→), with molar ratios of 7.80, 43.22, 1.79, 3.68, 4.06, 5.29, 7.86, 4.84, 3.78, 9.15, 1.79, 2.71 and 1.97, respectively.

[0098] 3.5NMR detection

[0099] The specific steps are as follows:

[0100] About 30 mg of dried sample powder was dissolved in D2O (0.5 mL) and freeze-dried three times for NMR analysis. The residue was redissolved in D2O with added 2,2,3,3-d4-3-(trimethylsilyl) propionate (TMSP) as an internal standard and analyzed by 1D NMR spectroscopy. Spectral analysis was performed at 298K on a Bruker 800MHz NMR spectrometer (Avance III-800MHz, Bruker, Switzerland).

[0101] The results of the test are as follows Figure 5As shown: RP1 was confirmed to be rhamnosyl lactobionic acid, with a main chain consisting of 1,4-linked α-GalpA and 1,2,4-linked α-Rap, and rich in arabinogalactan (AG-I) and Gal side chains. RP3 was confirmed to be a pectin polysaccharide with a longer HG backbone and RG-I domain, and side chains composed of Gal and Ara, etc.

[0102] Example 4

[0103] This example is a functional verification of the roxburghii polysaccharide obtained in Example 1

[0104] 4.1 Effects of roxburghii polysaccharides RP1 and RP3 on the proliferation of MCF-7 breast cancer cells

[0105] The CCK-8 method was used to evaluate the inhibitory effects of RP1 and RP3 on MCF-7 cancer cells and L929 cells cultured in vitro.

[0106] The specific steps are as follows: Take cells in the logarithmic growth phase and add 5×10 3 The cells were seeded into 96-well plates at a density of 100 μL of culture medium in each well and incubated at 37°C and 5% CO2 for 24 hours. 200 μL of RP1 and RP3 solutions at different concentrations of 0, 10, 20, 50, 100, 500, 1000 and 2000 μg / mL were added to the wells and incubated for 24, 48 and 72 hours, respectively. After the incubation, 100 μL of fresh culture medium containing 10% CCK-8 was added to each well and incubated at 37°C for 1.5 hours. The absorbance at 450 nm was measured using a Synergy-H1 microplate reader.

[0107] The results of the test are as follows Figure 6 and Figure 7 As shown: RP1 and RP3 showed time- and dose-dependent inhibitory effects on the growth of MCF-7 cells after co-incubation with polysaccharides at concentrations of 10, 20, 50, 100, 500, 1000, and 2000 μg / mL for 24, 48, and 72 hours. When the concentration was below 100 μg / mL, RP1 and RP-3 had little cytotoxicity to MCF-7 cells, with cell viabilities of 73.9% and 79.9% at 48 hours, respectively. At a concentration of 1000 μg / mL, the survival rates of RP1 and RP3 on MCF-7 cells at 48 hours were 42.5% and 74.6%, respectively, indicating that the inhibitory effect of RP1 was more significant than that of RP3. Also within a certain concentration range, RP1 and RP3 had no significant effect on the growth of L929 cells. The results showed that RP1 and RP3 could reduce the viability of MCF-7 cells in a dose-dependent manner without affecting the growth of normal cells. Therefore, this water-soluble polysaccharide from the fruit of Rosa roxburghii may be a potential anti-breast cancer substance.

[0108] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A roxburghii polysaccharide extract, characterized in that The roxburghii polysaccharide extract comprises roxburghii polysaccharide; The monosaccharide types in the roxburghii polysaccharide include galacturonic acid, galactose, rhamnose and arabinose, and the molar ratio of galacturonic acid, galactose, rhamnose and arabinose is 1.00:3.45:1.88:1.82 or 1.00:0.37:0.37:0.25; The monosaccharide types in the roxburghii polysaccharide include galacturonic acid, galactose, rhamnose, arabinose, and a small amount of xylose and glucose; The roxburghii polysaccharide includes polysaccharide RP1, whose glycosidic bonds include →4)-GalpA-(1→, T-Rhap-(1→, →2)-Rhap-(1→, →2,4)-Rhap-(1→, →4)-Rhap-(1→, T-Galp-(1→, →4)-Galp-(1→, →6)-Galp-(1→, →3,6)-Galp-(1→, →3)-β-Galp-(1→, T-Araf-(1→, and →3)-Araf-(1→; the corresponding molar ratios are 12.88, 5.35, 3.16, 17.95, 1.65, 19.33, 12.95, 11.21, 4.63, 3.68, 4.51 and 2.66; The roxburghii polysaccharide includes polysaccharide RP3, whose glycosidic bonds include T-GalpA-(1→,→4)-GalpA-(1→,→3,4)-GalpA-(1→,T-Rhap-(1→,→2)-Rhap-(1→,→2,4)-Rhap-(1→,T-Galp-(1→,→6)-Galp-(1→,→3,6)-Galp-(1→,T-Araf-(1→,→5)-Araf-(1→,→4)-Glcp-(1→,T-Xylp-(1→; the corresponding molar ratios are 7.80, 43.22, 1.79, 3.68, 4.06, 5.29, 7.86, 4.84, 3.78, 9.15, 1.79, 2.71 and 1.97; The number average molecular weight of the polysaccharide RP1 is 629.41 kDa; the number average molecular weight of the polysaccharide RP3 is 74.70 kDa.

2. The method for preparing the roxburghii polysaccharide extract according to claim 1, wherein Extracting and purifying the roxburghii dry powder to obtain a roxburghii polysaccharide extract containing the polysaccharide RP1 and the polysaccharide RP3; The extraction comprises dissolving the roxburghii dry powder with a buffer solution and performing enzymolysis with pectinase, and then performing ultrasonic extraction on the enzymolysis solution; The conditions for the enzymolysis are: the liquid ratio of the buffer solution to the roxburghii dry powder is 10:1-50:1 mL / g; the amount of pectinase added is 0.4%-4% of the mass of the roxburghii dry powder; the pH value of the enzymolysis system is 3-6; the enzymolysis temperature is 35-55° C.; and the enzymolysis time is 5-70 minutes; The ultrasonic conditions are: ultrasonic power of 80-200W; ultrasonic temperature of 40-90°C; time of 5-60 minutes; The purification comprises subjecting the extract obtained after ultrasound to gradient elution with 0, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L and 0.5 mol / L sodium chloride solutions through a DEAE Sepharose Fast Flow anion exchange chromatography column, collecting the eluates of 0 and 0.2 mol / L sodium chloride solutions, dialyzing and freeze-drying to obtain the purified polysaccharides RP1 and RP3.

3. Use of the roxburghii polysaccharide extract according to claim 1 in the preparation of anti-breast cancer drugs.

4. A drug combination, characterized in that The drug combination comprises the polysaccharide RP1 and / or polysaccharide RP3 according to claim 1, and a pharmaceutically acceptable carrier.

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