CN114917245B - Application of raspberry polysaccharide R1 in the preparation of anti-tumor drugs and anti-inflammatory preparations - Google Patents

Abstract

The invention discloses application of raspberry polysaccharide R1 in preparing anti-tumor drugs and anti-inflammatory preparations, wherein the raspberry polysaccharide R1 is prepared by the following steps: pulverizing dry fructus Rubi, sieving, adding water, heating for leaching, concentrating the leaching solution, and filtering to obtain crude polysaccharide; deproteinizing crude raspberry polysaccharide, loading on DEAE agarose gel column, eluting at least three column volumes with deionized water, eluting with 0.2mol/L NaCl solution until the eluent does not contain polysaccharide, mixing 0.2mol/L NaCl solution eluents, and dialyzing to obtain raspberry polysaccharide R1; the tumor is cervical cancer, liver cancer and colon cancer. The extraction method of the invention does not affect the biological activity of the raspberry polysaccharide, the obtained polysaccharide pure product has remarkable effects in anti-tumor, anti-inflammatory and moisturizing aspects, and cytotoxicity tests prove that the polysaccharide is nontoxic in the test range, and can be further used for developing health care products, medicines and cosmetics.

Description

Application of raspberry polysaccharide R1 in the preparation of anti-tumor drugs and anti-inflammatory preparations

Technical field

The invention belongs to the field of plant active ingredients, and specifically relates to the application of raspberry polysaccharide R1 in the preparation of anti-tumor drugs and anti-inflammatory preparations.

Background technique

Raspberry palmatum is a vine-like shrub of the genus Rubus in the Rosaceae family. It is named after its leaves (mostly five- or seven-lobed) that resemble the shape of a palm. It is a medicinal and edible plant.

The 2020 version of the "Chinese Pharmacopoeia" stipulates: Raspberry (Rubus idaeus L.) is the dried fruit of the Rosaceae plant Raspberry raspberry (also known as raspberry palm leaf). It has the functions of nourishing the kidneys, strengthening sperm, shrinking urine, nourishing the liver and improving eyesight. It can be used It is used to treat spermatorrhea, enuresis and frequent urination, impotence and premature ejaculation, dim vision and other symptoms. Raspberries are rich in vitamin A, vitamin C, vitamin E, 17 kinds of amino acids, zinc, magnesium, calcium and other trace elements. The average soluble solids, total soluble sugar, and titratable acid contents of red ripe raspberries are 13.68%, 111.8mg/g, and 1.27% respectively. Not only do they have high sugar content, but they also have good flavor, and their Vc content is much higher than that of other products on the market during the same period. Fruits such as loquat and bayberry are also rich in active ingredients such as ellagic acid and kaempferol-3-O-rutinoside.

In my country, raspberries have been widely used in traditional Chinese medicine preparations and diet for a long time, and are precious medicinal and food homologous substances. There is a lack of research on raspberry plants in the records of the past, and the varieties are not the same, as well as geographical reasons, resulting in differences in raspberry ingredients and unstable efficacy, and there are few related studies. At present, there are few studies on the bioactive components of raspberries, and the relevant pharmacological effects and mechanisms are not sufficiently studied.

Contents of the invention

The object of the present invention is to provide the application of raspberry polysaccharide R1 in the preparation of anti-tumor drugs and anti-inflammatory preparations. The polysaccharide is extracted from raspberries by water extraction and alcohol precipitation, and the polysaccharide is separated and purified by ion exchange chromatography and dialysis. Freeze-drying, thereby extracting and separating polysaccharide components with higher purity and biological activity.

The object of the present invention is achieved through the following technical solutions:

Application of raspberry polysaccharide R1 in the preparation of anti-tumor drugs and anti-inflammatory preparations;

The raspberry polysaccharide R1 is prepared by the following steps:

(1) Crush and sieve the dried raspberry fruit, then add water to heat and extract, and concentrate and filter the extract to obtain raspberry crude polysaccharide;

(2) Deproteinize the raspberry crude polysaccharide, and then put it on the DEAE Sepharose column. First use deionized water to elute for at least three column volumes, and then use 0.2 mol/L NaCl solution to elute until the eluent is no longer Then containing polysaccharide, combined with 0.2mol/L NaCl solution eluate, and after dialysis, raspberry polysaccharide R1 was obtained;

The heating leaching described in step (1) is leaching at 80°C;

The filtration described in step (1) is to add anhydrous ethanol or an ethanol solution to the concentrated liquid, so that the polysaccharide is alcohol-precipitated to form a brown flocculent precipitate, which is left to stand and then filtered with suction. The resulting precipitate is raspberry crude polysaccharide;

The deproteinization described in step (2) includes the following steps:

Dissolve raspberry crude polysaccharide in ultrapure water, slowly add trichloroacetic acid solution in an ice bath, mix thoroughly and let it stand; then adjust the pH value to 7; centrifuge at 5000-10000r/min for 5-10 minutes to remove the colloidal protein precipitate , polysaccharide filtrate; after concentration, add 95% ethanol solution, stir continuously to uniformly precipitate the polysaccharide, then let it stand at 4°C for 8 to 24 hours, take it out, and vacuum filtrate to obtain the precipitate, to obtain the deproteinized raspberry polysaccharide;

The dialysis described in step (2) includes the following steps:

Concentrate the NaCl solution eluate and transfer it to a dialysis bag for dialysis at 4°C for 24 to 96 hours to remove small molecule impurities. The retention solution in the dialysis bag contains purified raspberry polysaccharide R1;

The molecular weight cutoff of the dialysis bag is 3000 Da.

The monosaccharide composition of the raspberry polysaccharide R1 is: arabinose (Ara): galactose (Gal): xylose (Xyl): glucose (Glc): mannose (Man): glucuronic acid (Glc-UA): Fucose (Fuc): Guluronic acid (Gul-UA): Galacturonic acid (Gal-UA): Ribose (Rib) = 31.15: 27.64: 13.61: 13.48: 10.60: 1.34: 0.76: 0.59: 0.54 :0.29 (mass percentage);

The raspberry is preferably Rubus idaeus L.;

The tumors are cervical cancer, liver cancer and colon cancer;

The anti-tumor drugs and anti-inflammatory preparations also contain other active ingredients and excipients (carriers);

The excipients (carriers) are preferably sustained release agents, excipients, fillers, adhesives, wetting agents, disintegrants, absorption accelerators, adsorption carriers, surfactants or lubricants, etc.;

The dosage forms of the anti-tumor drugs and anti-inflammatory preparations are aerosols, tablets, capsules, pills, pills, powders, solutions, suspensions, emulsions, granules, lipid agents, transdermal agents, oral Buffers, suppositories or freeze-dried powder injections, etc.

Compared with the existing technology, the present invention has the following advantages and effects:

(1) The method of the present invention can be used to complete large-throughput polysaccharide extraction operations, and is suitable for industrial large-scale production. The DEAE-Sepharose fast flow ion exchange chromatography column has good decolorization effects, good separation effects, good repeatability, and reduces additional costs. The polysaccharide loss caused by the decolorization step results in a polysaccharide component with very low impurity content and stable properties;

(2) The biological activity of raspberry polysaccharide is not affected by the extraction method of the present invention. The pure polysaccharide obtained has significant effects in anti-tumor, anti-inflammatory and moisturizing effects, and the cytotoxicity test proves that the polysaccharide is non-toxic within the test range. It can be further used in the development of health care products, pharmaceuticals and cosmetics.

(3) R1 is composed of 10 kinds of monosaccharides, the main components of which are arabinose and galactose, and does not contain rhamnose. It is significantly different from the monosaccharide composition of raspberries discovered at this stage and is novel.

Description of the drawings

Figure 1 is an ion chromatogram analyzing the composition of monosaccharides.

Figure 2 shows the survival rate of RAW264.7 cells.

Figure 3 shows the survival rate of HeLa cells.

Figure 4 shows the survival rate of HepG2 cells.

Figure 5 shows the survival rate of HCT116 cells.

Figure 6 is a diagram showing the effect of polysaccharides on IL-6 expression.

Figure 7 is a diagram showing the effect of polysaccharides on IL-1β expression.

Figure 8 is a diagram showing the effect of polysaccharides on TNF-α expression.

Figure 9 is the hygroscopicity curve of polysaccharides.

Figure 10 is the moisture retention curve of polysaccharides.

Detailed ways

The present invention will be described in further detail below with reference to the examples and drawings, but the implementation of the present invention is not limited thereto.

In the examples, the physical and chemical properties analysis of raspberry polysaccharide R1 was conducted by Hangzhou Yanqu Information Technology Co., Ltd., and the report number is YY220127001.

Example 1

The method for isolating and purifying raspberry polysaccharide R1 includes the following steps:

(1) Preparation of raspberry crude polysaccharide

Weigh 120g of dried raspberry palm fruit, crush it, pass through 60 mesh and 80 mesh sieves successively, weigh 100g of powder and add 2000 mL of deionized water, place in a water bath at 80°C, stir and extract at 200 rpm for 6 hours. Extract twice, combine the filtrate, and concentrate under reduced pressure at 60°C to 400 mL. After cooling to room temperature, slowly add 1600 mL of 95% ethanol to the water extract concentrate while stirring. After the polysaccharide is alcohol-precipitated, a brown flocculent precipitate is formed. It is left to stand in a refrigerator at 4°C for 12 hours and then filtered to obtain a crude polysaccharide precipitate.

(2) Deproteinization of raspberry crude polysaccharide

Dissolve the crude polysaccharide precipitate in 200 mL of ultrapure water, slowly add 200 mL of 10% trichloroacetic acid solution under ice bath conditions, shake thoroughly, and let stand in a 4°C refrigerator for 2 hours. Neutralize the filtrate with 5% NaOH and adjust the pH to 7. Centrifuge at 8000 r/min for 5 minutes to remove the colloidal protein precipitate and obtain polysaccharide filtrate. Concentrate the filtrate under reduced pressure at 60°C to 200 mL. Slowly add 800 mL of 95% ethanol to the polysaccharide solution, while stirring continuously with a glass rod to uniformly precipitate the polysaccharide, and then let it stand at 4°C for 12 hours. Then take it out, vacuum filtrate to obtain the precipitate, and obtain the deproteinized raspberry polysaccharide.

(3) Isolation and purification of raspberry polysaccharides

Weigh 2.5g of deproteinized raspberry polysaccharide and dissolve it in 50mL of deionized water. After suction filtration through a 0.45μm microporous filter membrane, put it on a DEAE agarose gel column. First, use deionized water to elute three times at a flow rate of 0.5mL/min. The column bed volume was then eluted with a NaCl solution with a concentration of 0.2 mol/L at a flow rate of 0.5 mL/min. One bottle was collected for every 50 mL, and the polysaccharide content was determined bottle by bottle using the phenol sulfuric acid method. The polysaccharide solution eluted with sodium chloride was combined, concentrated under reduced pressure at 60°C, and then transferred to a dialysis bag (3000Da) for dialysis at 4°C for 72 hours to remove small molecule impurities. The remaining liquid in the dialysis bag was concentrated under reduced pressure at 50°C and then freeze-dried to obtain the purified component R1 of raspberry polysaccharide.

Specific operations for detecting polysaccharide content by phenol-sulfuric acid method: Precisely weigh 0.1g of anhydrous glucose standard dried at 105°C to constant weight, place it in a 100ml volumetric flask, add distilled water to dissolve and adjust to volume, shake well, and prepare a solution of 1mg/ml The standard solution is ready for use. Dilute this solution into standard solutions with different concentrations of 0, 20, 40, 60, 80, and 100 μg/ml. Take 1 ml of each of the above solutions into a test tube, add 1 ml of 6% phenol solution and mix well, then add 5 ml of concentrated sulfuric acid and mix well, let it stand at room temperature for 20 minutes, use distilled water as the blank control, measure the absorbance at 490 nm, and use the glucose concentration as The abscissa is, the OD value is the ordinate, and a standard curve is drawn. The polysaccharide content of unknown samples was determined using the standard curve method.

Example 2

The monosaccharide composition analysis was performed on the raspberry polysaccharide R1 obtained in Example 1. The specific experimental method is as follows:

An ion chromatography system is used to analyze and detect monosaccharide components using an electrochemical detector. Take a clean chromatography bottle, accurately weigh 5 mg (±0.05 mg) of the polysaccharide sample, add 1 mL of 2M TFA acid solution, and heat at 121°C for 2 hours. Blow through nitrogen and blow dry. Add methanol to clean, blow dry, and repeat methanol cleaning 2-3 times. Add sterile water to dissolve and transfer to chromatography bottle for testing.

Dionex ^TM CarboPac ^TM PA20 (150*3.0mm, 10um) liquid chromatography column was used; the injection volume was 5uL. Mobile phase A (0.1M NaOH), mobile phase B (0.1M NaOH, 0.2M NaAc), flow rate 0.5mL/min; column temperature is 30°C; elution gradient: 0min Phase A/Phase B (95:5V/V ), 30min Phase A/Phase B (80: 20V/V), 30.1min Phase A/Phase B (60: 40V/V), 45min Phase A/Phase B (60: 40V/V), 45.1min Phase A/ Phase B (95: 5V/V), 60min Phase A/Phase B (95: 5V/V).

13 monosaccharide standards (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, mannuronic acid, gulose Aldehydic acid) was tested according to the same steps, and the processed standard monosaccharide was analyzed by gas chromatography according to the same detection level. The results are shown in Figure 1.

The measured monosaccharide composition of R1 is as follows: arabinose (Ara): galactose (Gal): xylose (Xyl): glucose (Glc): mannose (Man): glucuronic acid (Glc-UA): fucose (Fuc): Guluronic acid (Gul-UA): Galacturonic acid (Gal-UA): Ribose (Rib) = 31.15: 27.64: 13.61: 13.48: 10.60: 1.34: 0.76: 0.59: 0.54: 0.29 ( mass percentage);

Example 3

Cytotoxicity was measured on the raspberry polysaccharide R1 obtained in Example 1. The specific experimental method is as follows:

Polysaccharide cytotoxicity was tested by the effect of raspberry polysaccharide on RAW264.7 cell viability.

1. Cell culture

Pipette the RAW264.7 cells in the logarithmic growth phase into a single cell suspension, centrifuge to collect the precipitate, add an appropriate amount of fresh DMEM complete medium (10% FBS+DMEM) to resuspend the cells, sample and count; in a 96-well plate Inoculate cells (1.5×10 ⁴ cells/100 μL/well) and culture them in a cell culture incubator for at least 24 hours.

2.Polysaccharide treatment

(1) After plating and culturing for 24 hours, observe and record the confluence and morphology of the cells;

(2) Prepare loading medium with different R1 concentrations (1, 5, 10, 50, 100, 200, 400, 600 μg/mL) in advance (added to DMEM complete medium), and the control is containing 10% sterile water culture medium.

(3) Discard the culture medium in the original well, add 100 μL of loading medium to each well according to groups, and continue culturing in the cell culture incubator for 24 hours.

3. Activity detection

(1) After adding the drug and culturing for 24 hours, observe and record the confluence and morphological changes of the cells under different sample concentrations;

(2) Shake off the culture medium in the 96-well plate, add 50 μL of methylene blue dye solution to each well, and incubate in a cell culture incubator for 1 hour.

(3) After incubating for 1 hour, take it out, wash away the excess dye with water, pat the well plate dry, add 100 μL of eluent to each well, and add 100 μL of eluent to 3 blank holes as blank controls (zero adjustment holes);

(4) The well plate was shaken for 15 minutes, then placed in a microplate reader and continued to shake for 1 minute to measure OD595.

(5) To analyze the data (after zero adjustment), you need to first get the average value of the control group, and then get the cell survival rate of each group (experimental data/control average × 100), and then calculate the deviation between each value (that is, each group deviation between the three wells), draw a graph to analyze the toxicity of the drug to cells.

Figure 2 is a graph showing the cytotoxicity results of polysaccharides. The data showed that the cell survival rate after R1 treatment was the lowest at 98.48%, indicating that raspberry polysaccharide has no toxic effect on RAW264.7 cells in the concentration range of 0-400 μg/mL.

Example 4

The anti-tumor activity of the raspberry polysaccharide obtained in Example 1 was determined. The specific experimental method is as follows:

The anti-tumor activity of polysaccharides was detected by CCK-8 method.

1. Cell culture

After digestion and centrifugation of three cancer cell lines (Hela, HCT116, and HepG2) in the logarithmic growth phase, add an appropriate amount of fresh complete culture medium to resuspend the cells (Hela: 10% FBS+MEM; HepG2: 10% FBS+DMEM; HCT116 : 10% FBS + McCoy's 5A), sample and count; inoculate cells in a 96-well plate at 3000 cells/100 μL/well, and culture in a cell culture incubator (37°C, 5% CO ₂ ) for 24 hours.

2.Polysaccharide treatment

After plating and culturing for 24 hours, observe and record the degree of confluence and morphology of the cells; prepare loading media with different R1 concentrations (0.2, 0.4, 0.6, 0.8, 1 mg/mL) in advance (added on the basis of the corresponding complete culture medium), The control was medium containing 10% sterile water. Discard the culture medium in the original well, add 100 μL of loading medium to each well according to groups, and continue culturing in the cell culture incubator for 24 hours.

3. Result determination

After adding the drug and culturing for 24 hours, add 10 μL CCK8 to each well, mix well, incubate in the incubator in the dark for 1 hour, and then measure OD450 with a microplate reader.

Experimental results show that R1 has the ability to inhibit cancer cell proliferation in a concentration-dependent manner.

Figure 3 is a diagram of the inhibitory effect of polysaccharides on human cervical cancer cells HeLa cells. The data in the figure shows that raspberry polysaccharide R1 has a significant inhibitory effect on the proliferation of HeLa cells and is positively correlated with concentration. When the polysaccharide concentration reached 1 mg/mL, the survival rate of Hela cells after R1 treatment was 67.04%.

Figure 4 is a diagram of the inhibitory effect of polysaccharide R1 on human liver cancer cell HepG2 cells. R1 can inhibit the proliferation of liver cancer cell HepG2 in a dose-dependent manner. When the concentration of polysaccharide R1 reaches 1 mg/mL, the survival rate of HepG2 cells after treatment is 69.37%.

Figure 5 is a diagram showing the inhibitory effect of polysaccharide R1 on colon cancer cell HCT116 cells. When the R1 concentration reached 1mg/mL, the HCT116 cell survival rate was 73.36%.

Example 5

The anti-inflammatory activity of the raspberry polysaccharide R1 obtained in Example 1 was determined. The specific experimental method is as follows:

The ELISA double-antibody sandwich method was used to test the effect of raspberry polysaccharide R1 on the expression of IL-6, IL-1β, and TNF-α inflammatory factors in RAW264.7 cells.

1. Cell culture

(1) Pipette and resuspend the RAW264.7 cells in the logarithmic growth phase into a single cell suspension, discard the culture supernatant after centrifugation, and add an appropriate amount of fresh complete culture medium (10% FBS+DMEM) to resuspend the cells. Sample count;

(2) Seed cells (1.25×10 ⁵ cells/0.5mL/well) in a 24-well plate and culture in a cell culture incubator for 24 hours.

2.Polysaccharide treatment

(1) Prepare culture medium: 5% FBS + 94% DMEM + 1% P/S (dual antibodies, penicillin and streptomycin)

(2) Plate and culture for about 24 hours, and observe and record the confluence and morphology of the cells;

(3) Based on the culture medium in step (1), prepare in advance loading culture medium with different R1 concentrations (100, 200, 400 μg/mL). The blank control is the culture medium containing 10% sterile water, and the positive control is 0.1 μg/mL LPS in 10% sterile water.

(4) Discard the medium, add 0.5 mL of loading medium to each well according to groups, and continue culturing in the cell culture incubator for 24 hours.

(5) After adding the drug and culturing for 24 hours, observe and record the degree of fusion and morphology of the cells;

(6) Cross-shake the well plate, collect the supernatant into a 1.5 mL centrifuge tube, centrifuge at 12000 rpm and 4°C for 10 min, aliquot, and immediately store in a -80°C refrigerator for later use.

3. Detection of inflammatory factors

Preparation before testing:

(1) The kit and pre-coated enzyme plate should be taken out at least 20 minutes in advance and equilibrated to room temperature.

(2) Use sterile water to dilute 20× concentrated washing solution into 1× washing working solution.

(3) Standard and sample dilution.

According to the following table, in the detection of different inflammatory factors, use the sample diluent in the ELISA detection kit to dilute the samples of each group:

Table 1 Dilution concentration information of inflammatory factors

Experimental steps

(1) Add samples and common specimen diluents to blank wells (1 well), standards of different concentrations (1 well each), sample wells (3 duplicate wells per group), add 100 μL liquid to each well, and seal with sealing tape Reaction wells were placed in a 37°C incubator and incubated for 90 minutes;

(2) 20 minutes in advance, prepare biotinylated antibody working solution: dilute 30× concentrated biotinylated antibody into 1× working solution with biotinylated antibody diluent;

(3) Wash the plate 5 times;

(4) Add biotinylated antibody diluent to the blank wells, add biotinylated antibody working solution to the remaining wells, 100 μL/well, seal the reaction wells with new sealing tape, and incubate in a 37°C incubator for 60 minutes;

(5) Prepare the enzyme conjugate working solution 20 minutes in advance: dilute the 30× concentrated enzyme conjugate into 1× working solution with the enzyme conjugate diluent, and place it at room temperature (22-25°C) away from light;

(6) Wash the plate 5 times;

(7) Add enzyme conjugate diluent to the blank wells, add enzyme conjugate working solution to the remaining holes, 100 μL/well, seal the reaction wells with new sealing tape, and incubate in a 37°C incubator in the dark for 30 minutes;

(8) Turn on the microplate reader, set the program, and preheat the machine;

(9) Wash the plate 5 times;

(10) Add 100 μL of chromogenic substrate TMB to each well, and incubate in a 37°C incubator in the dark for 15 minutes;

(11) Add 100 μL of reaction stop solution to each well, mix well (use a microplate reader to shake for 15 seconds), and measure OD450 immediately (complete within 3 minutes).

R1 has a good anti-inflammatory factor effect. Figure 6 is a diagram showing the effect of polysaccharides on IL-6 expression. Figure 7 is a diagram showing the effect of polysaccharides on IL-1β expression. Figure 8 is a diagram showing the effect of polysaccharides on TNF-α expression. It can be seen from the figure that compared with the blank control group, the positive control group LPS prompted RAW264.7 cells to be activated to release a large number of inflammatory factors, while the polysaccharide component could significantly inhibit the production of inflammatory factors, and the inhibitory effect was negatively correlated with the polysaccharide concentration. R1 has the strongest inhibitory effect at 100 μg/mL, the expression level of IL-6 is 4.01pg/mL, the release amount of IL-1β is 2.32pg/mL, and the expression level of TNF-α is 385.80pg/mL.

Example 6

The moisturizing properties of the raspberry polysaccharide R1 obtained in Example 1 were measured. The specific experimental methods are as follows:

Moisture absorption experiment: Place 100 mL of saturated ammonium sulfate solution in a dry and sealed container. At 20°C, the relative humidity in the container can be maintained at 81%. Accurately weigh a certain amount of polysaccharide R1 and place it in a 3cm diameter glass flat weighing dish, and then place the weighing dish in a high-humidity container. Place the sealed container into an incubator with a temperature of (20±0.1)℃. The weighing dish was taken out and weighed every 3 hours, and the moisture absorption rate was calculated by measuring the weight difference of the polysaccharide before and after the experiment.

Moisture absorption rate (%) = (M _n -M ₀ )/M ₀ ×100 (1-1)

In the formula, M ₀ is the mass of polysaccharide before moisture absorption; M _n is the mass of polysaccharide after being left for n hours.

Moisturizing test: Keep the ambient temperature at 20°C, put 100% dry color-changing silica gel in a dry and sealable container, place the polysaccharide sample and weighing dish for the moisture absorption test on the silica gel, and then seal the container. Take out the weighing dish and weigh it every 12 hours, and calculate the moisture retention rate through formula 1-2.

Moisture retention rate (%) = [1-(M _n -M ₀ )/M ₀ ]×100 (1-2)

In the formula, M ₀ is the mass of the polysaccharide before being placed; M _n is the mass of the polysaccharide after being placed for n hours.

Figure 9 shows the hygroscopicity of polysaccharides. In a high humidity environment of 81%, the moisture absorption rate of R1 reached the maximum value in 9 hours, with the highest moisture absorption rate of 21.76%. Figure 10 shows the moisturizing properties of polysaccharides. Within 48 hours in a dry environment, R1 had the lowest moisturizing capacity of 88.18%.

The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, etc. may be made without departing from the spirit and principles of the present invention. All simplifications should be equivalent substitutions, and are all included in the protection scope of the present invention.

Claims (7)

1. The application of raspberry polysaccharide R1 in preparing anti-tumor drugs and anti-inflammatory preparations is characterized in that:

the raspberry polysaccharide R1 is prepared by the following steps:

(1) Pulverizing dry fructus Rubi, sieving, adding water, heating for leaching, concentrating the leaching solution, and filtering to obtain crude polysaccharide;

(2) Deproteinizing crude raspberry polysaccharide, loading on DEAE agarose gel column, eluting at least three column volumes with deionized water, eluting with 0.2mol/L NaCl solution until the eluent does not contain polysaccharide, mixing 0.2mol/L NaCl solution eluents, and dialyzing to obtain raspberry polysaccharide R1;

the tumors are cervical cancer, liver cancer and colon cancer; the monosaccharide composition of the raspberry polysaccharide R1 is as follows: arabinose (Ara): galactose (Gal): xylose (Xyl): glucose (Glc): mannose (Man): glucuronic acid (Glc-UA): fucose (Fuc): guluronic acid (Gul-UA): galacturonic acid (Gal-UA): ribose (Rib) =31.15: 27.64:13.61:13.48:10.60:1.34:0.76:0.59:0.54:0.29, wherein the proportion of the monosaccharide is calculated by mass percent;

the deproteinization of step (2) comprises the following steps:

adding ultrapure water into raspberry crude polysaccharide for dissolution, slowly adding trichloroacetic acid solution into ice bath, fully mixing and standing; then adjusting the pH value to 7; centrifuging for 5-10 min at a speed of 5000-10000 r/min, removing gelatinous protein precipitate, and filtering to obtain polysaccharide filtrate; concentrating, adding 95% ethanol solution, continuously stirring to uniformly precipitate polysaccharide, standing at 4 ℃ for 8-24 hours, taking out, and vacuum filtering to obtain precipitate, thereby obtaining deproteinized raspberry polysaccharide;

the raspberry is Rubus palmatusRubus idaeus L.）。

2. The use according to claim 1, characterized in that: and (2) the filtering step (1) is to add absolute ethyl alcohol or an ethanol solution into the concentrated solution, so that polysaccharide is subjected to alcohol precipitation to form brown flocculent precipitate, and the precipitate is obtained by suction filtration after standing.

3. The use according to claim 1, characterized in that: the heating leaching in the step (1) is leaching at 80 ℃.

4. The use according to claim 1, characterized in that: the dialysis of step (2) comprises the steps of:

concentrating the NaCl solution eluent, transferring into a dialysis bag, dialyzing at 4 ℃ for 24-96 h, removing small molecular impurities, and keeping the liquid in the dialysis bag containing the purified raspberry polysaccharide R1.

5. The use according to claim 4, characterized in that: the molecular weight cut-off of the dialysis bag is 3000Da.

6. The use according to claim 1, characterized in that: the antitumor and anti-inflammatory preparation also contains other active ingredients and auxiliary materials.

7. The use according to claim 6, characterized in that: the auxiliary materials are sustained release agent, filler, adhesive, wetting agent, disintegrating agent, absorption promoter, adsorption carrier, surfactant or lubricant.