阅读说明：本技术 酸性当归多糖asp3、酸性当归多糖-阿霉素共聚物纳米粒以及两者的制备方法和应用 (Acidic angelica polysaccharide ASP3, acidic angelica polysaccharide-adriamycin copolymer nanoparticles, and preparation methods and applications of acidic angelica polysaccharide ASP3 and acidic angel) 是由 陈美婉 胡杰 于 2018-08-08 设计创作，主要内容包括：本发明涉及纳米药物领域,提供了一种酸性当归多糖ASP3、酸性当归多糖-阿霉素共聚物纳米粒以及两者的制备方法和应用。该酸性当归多糖ASP3的提取方法,经DEAE-cellulose离子柱、Sephadex层析柱柱层析分离、纯化得到中性多糖ASP0、酸性多糖ASP1和酸性多糖ASP3,利用酸性当归多糖作为载体,赋予纳米载药系统优良的稳定性,提高了肝癌靶向性。本发明将天然当归多糖作为载体,阿霉素通过pH敏感的腙键连接到当归多糖上,实现对阿霉素的药物递送,以期阿霉素在弱酸性的肿瘤环境中快速释放,增强药效。不仅保证了纳米载药系统在体内稳定、安全地将药物靶向递送到病灶部位,实现了对肝癌的有效治疗。(The invention relates to the field of nano-drugs, and provides acidic angelica polysaccharide ASP3, acidic angelica polysaccharide-adriamycin copolymer nanoparticles, and preparation methods and applications of the acidic angelica polysaccharide ASP3 and the acidic angelica polysaccharide-adriamycin copolymer nanoparticles. The extraction method of the acidic angelica polysaccharide ASP3 comprises the steps of carrying out column chromatography separation and purification by a DEAE-cellulose ion column and a Sephadex chromatographic column to obtain neutral polysaccharide ASP0, acidic polysaccharide ASP1 and acidic polysaccharide ASP3, and the acidic angelica polysaccharide is used as a carrier to endow a nano drug-loaded system with excellent stability and improve liver cancer targeting property. According to the invention, the natural angelica polysaccharide is used as a carrier, and the adriamycin is connected to the angelica polysaccharide through a pH-sensitive hydrazone bond, so that the adriamycin drug delivery is realized, the adriamycin is rapidly released in a weakly acidic tumor environment, and the drug effect is enhanced. Not only ensures that the nano drug-carrying system stably and safely delivers the drug to the focus part in vivo, but also realizes the effective treatment of the liver cancer.)

1. An extraction method of acidic angelica polysaccharide ASP3 is characterized by comprising the following steps:

separation: separating angelica polysaccharide by DEAE-cellulose column chromatography, performing gradient elution by using distilled water, sodium chloride solution with the concentration of 0.05-0.15mol/L and 0.25-0.35mol/L in sequence, collecting first eluent, detecting the absorbance value of the first eluent at the detection wavelength of 490nm by using a phenol-sulfuric acid method, merging the same fractions according to an elution curve, concentrating, dialyzing and freeze-drying to obtain neutral polysaccharide ASP0, acidic polysaccharide ASP1 and acidic polysaccharide ASP 3; and

and (3) purification: dissolving the separated acidic polysaccharide ASP3 in distilled water, filtering with a filter membrane, purifying and separating the obtained filtrate by Sephadex column chromatography, eluting with distilled water, and collecting a second eluent to obtain the acidic angelica polysaccharide ASP 3;

preferably, after collecting the second eluate, further comprising a second purification: detecting the absorbance value of the second eluent at detection wavelength of 490nm by phenol-sulfuric acid method, mixing the same fractions according to elution curve, concentrating, dialyzing, and lyophilizing to obtain the acidic radix Angelicae sinensis polysaccharide ASP 3.

2. The extraction process of acidic angelicase ASP3 according to claim 1, wherein said extraction process of acidic angelicase ASP3 further comprises performing, prior to said separation step:

extracting total polysaccharide: decocting radix Angelicae sinensis coarse powder with first anhydrous ethanol for 0.5-4 hr for 2-4 times; then decocting with distilled water for 2-4 times, each time for 0.5-4 hr, mixing water extractive solutions, concentrating, adding second anhydrous ethanol, and precipitating to obtain crude radix Angelicae sinensis polysaccharide; and

deproteinization: dissolving the crude radix Angelicae sinensis polysaccharide with distilled water, and deproteinizing to obtain radix Angelicae sinensis polysaccharide.

3. The extraction process of acidic angelicapolysaccharide ASP3 according to claim 2, wherein in said total polysaccharide extraction step, the mass ratio of said angelicacoarse powder to said first absolute ethanol is 1: 9-11;

preferably, before adding the second absolute ethyl alcohol into the concentrated water extracting solution, the method further comprises centrifuging the concentrated water extracting solution and discarding the precipitate, and then adding the second absolute ethyl alcohol into the supernatant;

preferably, the mass ratio of the supernatant to the second absolute ethyl alcohol is 1: 3-5.

4. The extraction method of acidic angelicae sinensis polysaccharide ASP3 according to claim 2, wherein in the deproteinization step, the crude angelicae sinensis polysaccharide is prepared into a crude angelicae sinensis polysaccharide aqueous solution with a concentration of 2-10mg/mL, n-butanol-chloroform mixed solution is added, shaking is performed for 15-25min, centrifugation is performed, an upper solution is collected, the deproteinization step is repeated for 6-10 times, the upper solutions are combined, and after concentration under reduced pressure, dialysis and freeze drying are performed to obtain angelicae angelica polysaccharide;

preferably, the volume ratio of the crude angelica polysaccharide aqueous solution to the n-butanol-chloroform mixed solution is 3-5: 1;

preferably, the volume ratio of the n-butanol to the chloroform in the n-butanol-chloroform mixed solution is 1: 3-5.

5. An acidic angelicae sinensis polysaccharide ASP3, which is obtained by the extraction method of acidic angelicae sinensis polysaccharide ASP3 according to any one of claims 1 to 4, and has a weight average molecular weight of 90-100 kDa;

preferably, the acidic angelicae polysaccharide ASP3 is a pectic polysaccharide, the backbone of the acidic angelicae polysaccharide ASP3 is → 4) - α -D-GalpA- (1 → 2) - α -L-Rhap- (1 →, and the side chains are α -T-Araf/α -1,5-Araf and β -1,4Galp attached to Rha.

6. The use of the acidic angelicae polysaccharide ASP3 of claim 5 as a carrier in the preparation of a medicament for the treatment of liver cancer.

7. An acidic angelicae sinensis polysaccharide-adriamycin copolymer nanoparticle, which is prepared by connecting the acidic angelicae sinensis polysaccharide ASP3 and adriamycin of claim 5 through an acid-sensitive hydrazone bond.

8. A preparation method of acidic angelica polysaccharide-adriamycin copolymer nanoparticles is characterized by comprising the following steps:

reacting the acidic angelicae polysaccharide ASP3 of claim 5 with hydrazine hydrate to prepare ASP 3-hydrazide;

reacting the ASP 3-hydrazide compound with hydrochloric acid adriamycin after hydrochloric acid is removed to prepare acidic angelica polysaccharide-adriamycin;

dialyzing the acidic angelica polysaccharide-adriamycin, and filtering the acidic angelica polysaccharide-adriamycin by using a microporous filter membrane to obtain the acidic angelica polysaccharide-adriamycin copolymer nanoparticles.

9. The method for preparing acidic angelicae sinensis polysaccharide-adriamycin copolymer nanoparticles according to claim 8, wherein the acidic angelicae sinensis polysaccharide-adriamycin copolymer nanoparticles are prepared from a mixture of acid-angelicae sinensis polysaccharide and adriamycin,

dissolving the acidic angelicae polysaccharide ASP3 of claim 5 in water, and stirring at room temperature; adding excessive EDC.HCl and NHS, adding hydrazine hydrate, continuing to react at room temperature, dialyzing, and freeze-drying to obtain ASP 3-hydrazide;

dissolving the ASP 3-hydrazide compound in a mixed solution of dimethyl sulfoxide and water, adding a dimethyl sulfoxide solution containing adriamycin under the protection of nitrogen, then adding triethylamine, and stirring in a dark place to obtain acidic angelica polysaccharide-adriamycin;

after the reaction is finished, dialyzing the solution, and performing a microporous membrane filtration to obtain acidic angelica polysaccharide-adriamycin copolymer nanoparticles;

preferably, the adriamycin is contained in an amount of 10-30mg per 1ml of the dimethyl sulfoxide solution containing adriamycin.

10. The use of the acidic angelicae sinensis polysaccharide-adriamycin copolymer nanoparticles of claim 7 in the preparation of a medicament for treating liver cancer.

Technical Field

The invention relates to the field of nano-drugs, in particular to acidic angelica polysaccharide ASP3, acidic angelica polysaccharide-adriamycin copolymer nanoparticles and preparation methods and application of the acidic angelica polysaccharide ASP3 and the acidic angelica polysaccharide-adriamycin copolymer nanoparticles.

Background

Angelica sinensis (oliv.) Diels is the dry root of Angelica sinensis (oliv.) Diels, which is one of the genuine herbs in Gansu province. Chinese angelica is sweet, pungent and warm in nature, is a famous common traditional Chinese medicine, has the effects of enriching and activating blood, regulating menstruation and relieving pain, and relaxing bowel, and is used for treating blood deficiency and chlorosis, dizziness and palpitation, irregular menstruation, amenorrhea and dysmenorrheal, constipation due to intestinal dryness and the like. With the increasingly intensive research and application of new technologies on angelica, a variety of chemically active ingredients have been isolated: ligustilide, ferulic acid, angelica polysaccharide, microelements, vitamins, amino acids, chalcone, sesquiterpene and alkyne compounds. In recent years, more and more researches show that angelica polysaccharide (ASP) has good medicinal efficacy, and biological activities of immunoregulation, anti-tumor, anti-aging, blood sugar reduction, anticoagulation and the like have been widely regarded and paid attention to by the medical community.

In recent years, the nano drug delivery system has made great progress in the treatment of malignant tumors. The nano drug-carrying systems such as paclitaxel-liposome, paclitaxel-albumin nano particles, adriamycin-liposome, G-CSF-polyethylene glycol nano particles, fluorouracil-polylactic acid nano particles and the like which are clinically applied at present can improve the drug curative effect to a certain extent and reduce the toxic and side effects. However, the carriers used in these nano drug delivery systems are limited to lipids and high molecular materials such as polylactic acid, polyethylene glycol or albumin, and how to develop new drug carriers for nano drug delivery is an urgent need.

Disclosure of Invention

The object of the present invention consists, for example, in providing a process for the extraction of the acidic angelicas polysaccharide ASP3, by means of which a higher purity of the acidic angelicas polysaccharide ASP3 can be obtained.

The invention also aims to provide the acidic angelica polysaccharide ASP3 which has high purity, good water solubility, biocompatibility and anti-liver cancer activity.

The invention also aims to provide the application of the acidic angelica polysaccharide ASP3 as a carrier in preparing the medicine for treating liver cancer, and the acidic angelica polysaccharide is used as the carrier to endow a nano medicine carrying system with excellent stability and improve the targeting property of the liver cancer.

The invention also aims to provide the acidic angelica polysaccharide-adriamycin copolymer nanoparticles which have good stability and strong liver cancer targeting property.

The invention also aims to provide a preparation method of the acidic angelica polysaccharide-adriamycin copolymer nanoparticles, the preparation method is simple, and the obtained acidic angelica polysaccharide-adriamycin copolymer nanoparticles have stronger stability and liver cancer targeting property.

The invention also aims to provide application of the acidic angelica polysaccharide-adriamycin copolymer nanoparticles in preparing a medicament for treating liver cancer.

In order to achieve at least one of the above purposes, the invention adopts the following technical scheme:

an extraction method of acidic angelica polysaccharide ASP3 comprises the following steps:

separation: separating angelica polysaccharide by DEAE-cellulose column chromatography, performing gradient elution by using distilled water, sodium chloride solution with the concentration of 0.05-0.15mol/L and 0.25-0.35mol/L in sequence, collecting first eluent, detecting the absorbance value of the first eluent at the detection wavelength of 490nm by using a phenol-sulfuric acid method, merging the same fractions according to an elution curve, concentrating, dialyzing and freeze-drying to obtain neutral polysaccharide ASP0, acidic polysaccharide ASP1 and acidic polysaccharide ASP 3; and

and (3) purification: dissolving the separated acidic polysaccharide ASP3 in distilled water, filtering with a filter membrane, purifying and separating the obtained filtrate by Sephadex column chromatography, eluting with distilled water, and collecting second eluate to obtain acidic polysaccharide ASP 3;

preferably, after collecting the second eluate, further comprising a second purification: detecting the absorbance value of the second eluate at detection wavelength of 490nm by phenol-sulfuric acid method, mixing the same fractions according to elution curve, concentrating, dialyzing, and lyophilizing to obtain acidic radix Angelicae sinensis polysaccharide ASP 3.

An acidic angelicae sinensis polysaccharide ASP3 is extracted from the acidic angelicae sinensis polysaccharide ASP3, and has a weight average molecular weight of 90-100kDa, preferably, the acidic angelicae sinensis polysaccharide ASP3 is pectin polysaccharide, and the main chain of acidic angelicae sinensis polysaccharide ASP3 is → 4) - α -D-GalpA- (1 → 2) - α -L-Rhap- (1 →, and the side chain is α -T-Araf/α -1,5-Araf and β -1,4Galp connected to Rha.

The acidic Angelica sinensis polysaccharide ASP3 can be used as carrier for preparing medicine for treating hepatocarcinoma.

An acidic radix Angelicae sinensis polysaccharide-adriamycin copolymer nanoparticle is prepared by connecting the acidic radix Angelicae sinensis polysaccharide ASP3 and adriamycin via acid-sensitive hydrazone bond.

A method for preparing acidic angelica polysaccharide-adriamycin copolymer nanoparticles comprises the following steps: reacting the acidic angelicapolysaccharides ASP3 with hydrazine hydrate to prepare ASP 3-hydrazide compound; reacting ASP 3-hydrazide compound with hydrochloric acid adriamycin after hydrochloric acid is removed to prepare acidic angelica polysaccharide-adriamycin; dialyzing the acidic angelica polysaccharide-adriamycin, and filtering the acidic angelica polysaccharide-adriamycin by a microporous filter membrane to obtain the acidic angelica polysaccharide-adriamycin copolymer nanoparticles.

The acidic angelica polysaccharide-adriamycin copolymer nanoparticles are applied to preparing a medicament for treating liver cancer.

The beneficial effects provided by the embodiment of the invention comprise:

according to the extraction method of the acidic angelica polysaccharide ASP3, the neutral polysaccharide ASP0, the acidic polysaccharide ASP1 and the acidic polysaccharide ASP3 are obtained through column chromatography separation and purification of a DEAE-cellulose 52 ion column and a Sephadex G-50 chromatographic column, systematic analysis and identification are carried out on the physicochemical properties, the molecular weight, the monosaccharide composition and the like of the three components of the polysaccharide, and a solid foundation is laid for application in the fields of food, health products, medicine and the like in the future; the acidic angelica polysaccharide is used as a carrier, so that the nano drug delivery system has excellent stability, and the targeting property of liver cancer is improved. According to the invention, the natural angelica polysaccharide is used as a carrier, and the adriamycin is connected to the angelica polysaccharide through a pH-sensitive hydrazone bond, so that the adriamycin drug delivery is realized, the adriamycin is rapidly released in a weakly acidic tumor environment, and the drug effect is enhanced. Not only ensures that the nano drug-carrying system stably and safely delivers the drug to the focus part in vivo, but also realizes the effective treatment of the liver cancer.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1: elution profile of the crude polysaccharide in example 1 on a DEAE-cellulose 52 column. Respectively by dd H₂Taking O, 0.1M NaCl and 0.3M NaCl as mobile phases, and detecting the polysaccharide content in the mobile phases by a sulfuric acid phenol method;

fig. 2a, 2b and 2 c: elution profiles of ASP0(a), ASP1(b) and ASP3(c) on Sephadex G-50 column in example 1. dd H₂Taking O as a mobile phase, and detecting the polysaccharide content in the O by a sulfuric acid phenol method;

FIG. 3: PMP pre-column derivatization HPLC method in Experimental example 2 analyzed monosaccharide composition of Angelica sinensis polysaccharide. a. Mixing curves of monosaccharide standards; b.asp0, c.asp1, d.asp3. Man mannose, Rha rhamnose, GlcA glucuronic acid, GalA galacturonic acid, Glc glucose, Gal galactose, Ara galactose;

fig. 4a, 4b, 4c, 4d and 4 e: HNMR (a), CNMR (b), COSY (c), HSQC (d) and HMBC (e) structural analysis of ASP3 in Experimental example 3;

fig. 5a, 5b, 5c and 5 d: the cytotoxicity of each component of angelicae sinensis polysaccharide ASP0, ASP1 and ASP3 on hepatoma cells HepG2(a), SMMC7721(b) and Bel7402(c) and normal hepatoma cells L02(d) in Experimental example 4;

FIG. 6: synthetic characterization of acidic angelicae sinensis polysaccharide-doxorubicin copolymer (ASP-DOX) in example 4;

FIG. 7: characterization of acidic angelicae polysaccharide-doxorubicin copolymer nanoparticles in example 4 and experimental example 5, (a) particle size (b) potential (c) particle size as a function of pH (d) drug release profile of ASP-DOX at pH7.4 and 5.5, respectively;

fig. 8a, 8b and 8 c: the acidic angelicae sinensis polysaccharide-adriamycin copolymer nanoparticles in the experimental example 6 have anti-liver cancer activity. Liver cancer cells HepG2(a), SMMC7721(b) and normal liver cells L02 (c);

fig. 9A, 9B, and 9C: in experimental example 7, flow cytometry detection of the acid angelicae sinensis polysaccharide-adriamycin copolymer nanoparticles by uptake investigation of liver cancer cells HepG2(a), SMMC7721(b) and normal liver cells L02(c) shows that DOX: doxorubicin; ASP-DOX acidic angelicae sinensis polysaccharide-adriamycin copolymer nanoparticles; gal galactose, asialoglycoprotein receptor inhibitor;

FIG. 10: IN experimental example 7, the acidic angelicae sinensis polysaccharide-adriamycin copolymer nanoparticles were detected by the uptake of hepatoma cells HepG2 and SMMC7721 by a high content analysis Cell imaging system (IN Cell Analyzer 2000). DOX: doxorubicin; ASP-DOX acidic angelicae sinensis polysaccharide-adriamycin copolymer nanoparticles.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The acidic angelicae sinensis polysaccharide ASP3, the preparation method and the application thereof, and the acidic angelicae sinensis polysaccharide-adriamycin copolymer nanoparticles, the preparation method and the application thereof according to the embodiment of the invention are specifically described below.

This example provides an acidic angelicae polysaccharide ASP3 and a method for preparing the same.

The preparation method specifically comprises the following steps:

s101, total polysaccharide extraction: decocting radix Angelicae sinensis coarse powder with first anhydrous ethanol for 0.5-4 hr for 2-4 times; then decocting with distilled water for 2-4 times, each time for 0.5-4 hr, mixing water extractive solutions, concentrating, adding second anhydrous ethanol, and precipitating to obtain crude radix Angelicae sinensis polysaccharide.

Wherein the mass ratio of the angelica coarse powder to the first absolute ethyl alcohol is 1: 9-11; the boiling temperature is 90-100 ℃. When the Chinese angelica is boiled and extracted by distilled water, the adding amount of the distilled water is 9 to 11 times of the mass of the Chinese angelica coarse powder. The inventor researches and discovers that the extraction is complete by adopting the solvent with the mass of 9-11 times of the angelica coarse powder for extraction. After the water extracting solutions are combined, the operation of decompression and concentration is carried out, the centrifugation is carried out for 5 to 15min at the rotating speed of 3000-4000r/min, the sediment is discarded, and then second absolute ethyl alcohol with the volume ratio of 3 to 5 times of that of the supernatant is added into the supernatant; precipitating for 12-36h, centrifuging at 3000-4000r/min for 5-15min, and collecting alcohol precipitate to obtain crude radix Angelicae sinensis polysaccharide.

In this embodiment, the first absolute ethyl alcohol is first used for boiling extraction, then the distilled water is used for boiling extraction, and different solvents are used for boiling extraction respectively, so that the total polysaccharide extraction rate is higher in this embodiment compared with that of single ethyl alcohol boiling extraction or distilled water boiling extraction.

Notably, the crude angelicae polysaccharide was obtained and used in the next step without further treatment.

In other embodiments of the present invention, if the raw material angelica is not powder, the method may further comprise the step of crushing the angelica: pulverizing dried radix Angelicae sinensis, and sieving with 20-40 mesh sieve.

S102, deproteinization: dissolving the crude radix Angelicae sinensis polysaccharide with distilled water, and deproteinizing to obtain radix Angelicae sinensis polysaccharide.

The deproteinization method can be carried out by various methods, for example, one or more methods selected from Sevage method, repeated freeze-thaw method and isoelectric precipitation method can be used together. In this embodiment, the Sevage method is preferably used for deproteinization, and the specific method is as follows:

preparing the crude angelica polysaccharide obtained in the step S101 into a crude angelica polysaccharide aqueous solution with the concentration of 2-10mg/mL, adding 1/5-1/3 n-butyl alcohol-chloroform mixed solution (the volume ratio of n-butyl alcohol to chloroform is 1:3-4) of the crude angelica polysaccharide aqueous solution into the crude angelica polysaccharide aqueous solution, violently oscillating for 15-25min, centrifuging for 5-15min at 3000-4000r/min, collecting an upper layer solution, repeatedly operating for 8 times according to the deproteinization step, merging the upper layer solution, dialyzing after reduced pressure concentration, and freeze-drying to obtain the high-purity angelica polysaccharide.

S103, separation: separating the angelica polysaccharide by DEAE-cellulose column chromatography, carrying out gradient elution by using distilled water, sodium chloride solution with the concentration of 0.05-0.15mol/L and 0.25-0.35mol/L in sequence, collecting first eluent, detecting the absorbance value of the first eluent at the detection wavelength of 490nm by using a phenol-sulfuric acid method, merging the same fractions according to an elution curve, concentrating, dialyzing and freeze-drying to obtain neutral polysaccharide ASP0, acidic polysaccharide ASP1 and acidic polysaccharide ASP 3. In other embodiments of the invention, the concentration of the sodium chloride solution having a concentration of 0.05-0.15mol/L may be, for example, any one of or a range of values between any two of 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, or 0.15 mol/L; the concentration of the 0.25-0.35mol/L sodium chloride solution may be, for example, any one of 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, or 0.35mol/L or a range between any two.

Before the angelica polysaccharide is subjected to column separation, the angelica polysaccharide is prepared into an aqueous solution with the mass concentration of 50-200mg/mL, and the aqueous solution is centrifuged for 5-15min at 3000-4000r/min, and supernatant is taken. The supernatant was then subjected to preliminary separation by DEAE-cellulose 52 ion column chromatography. In other embodiments of the invention, the mass concentration of the aqueous angelica polysaccharide solution is, for example, any one of or a range of values between 50, 60, 80, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 and 200 mg/mL. The rotational speed of the centrifugation may be, for example, any one of 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, and 4000r/min or a range value therebetween.

In the embodiment, the neutral polysaccharide ASP0, the acidic polysaccharide ASP1 and the acidic polysaccharide ASP3 are separated better by performing gradient elution by distilled water, a sodium chloride solution with the concentration of 0.05-0.15mol/L and a sodium chloride solution with the concentration of 0.25-0.35mol/L in sequence.

The column used in this example was DEAE-cellulose 52.

S104, purification: dissolving the separated acidic polysaccharide ASP3 in distilled water, filtering with a filter membrane, purifying and separating the obtained filtrate by Sephadex column chromatography, eluting with distilled water, and collecting second eluate to obtain acidic polysaccharide ASP 3;

preferably, after collecting the second eluate, further comprising a second purification: detecting the absorbance value of the second eluate at detection wavelength of 490nm by phenol-sulfuric acid method, mixing the same fractions according to elution curve, concentrating, dialyzing, and lyophilizing to obtain acidic radix Angelicae sinensis polysaccharide ASP 3.

The chromatographic column used in this example was Sephadex 50.

Of course, it will be readily understood that if purification of the neutral polysaccharide ASP0 or the acid polysaccharide ASP1 is also desired in this example, it can be performed according to the purification steps described above.

The acidic angelicae sinensis polysaccharide ASP3 extracted by the extraction method of the acidic angelicae sinensis polysaccharide ASP3 can be used as a carrier in preparation of a medicament for treating liver cancer. According to the extraction method of the acidic angelica polysaccharide ASP3, the neutral polysaccharide ASP0, the acidic polysaccharide ASP1 and the ASP3 are obtained by column chromatography separation and purification of a DEAE-cellulose 52 ion column and a Sephadex G-50 chromatographic column, systematic analysis and identification are carried out on the physicochemical properties, the molecular weight, the monosaccharide composition and the like of the three components of the polysaccharide, and a solid foundation is laid for application in the fields of food, health products, medicine and the like in the future; the acidic angelica polysaccharide is used as a carrier, so that the nano drug delivery system has excellent stability, and the targeting property of liver cancer is improved.

In addition, the embodiment of the invention also provides acidic angelica polysaccharide-adriamycin copolymer nanoparticles and a preparation method and application thereof.

The acidic angelica polysaccharide-adriamycin copolymer nanoparticle is prepared by connecting the acidic angelica polysaccharide ASP3 and adriamycin through an acid-sensitive hydrazone bond. Specifically, the method comprises the following steps:

s201, reacting the acidic angelica polysaccharide ASP3 with hydrazine hydrate to prepare an ASP 3-hydrazide compound;

specifically, acidic angelica polysaccharide ASP3 is dissolved in water and stirred at room temperature; adding excessive EDC.HCl and NHS, adding hydrazine hydrate, reacting at room temperature, dialyzing, and freeze-drying to obtain ASP 3-hydrazide.

More specifically, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) is added firstly, and after reacting for 30-40min at room temperature, N-hydroxysuccinimide (NHS) is added, and the reaction is continued for 1-3 h. Meanwhile, hydrazine hydrate is directly added into the angelica polysaccharide activation system, and the mixture is magnetically stirred to react for 20 to 30 hours. After the reaction was completed, the reaction mixture was dialyzed against distilled water (MWCO, 3500) for 2 to 3 days, and lyophilized to obtain ASP 3-hydrazide.

S202, reacting the ASP 3-hydrazide compound with hydrochloric acid adriamycin after hydrochloric acid is removed to prepare acidic angelica polysaccharide-adriamycin;

specifically, dissolving ASP 3-hydrazide compound in a mixed solution of dimethyl sulfoxide and water, adding a dimethyl sulfoxide solution containing adriamycin under the protection of nitrogen, then adding triethylamine, and stirring in a dark place to obtain acidic angelicapolysaccharide-adriamycin; preferably, the adriamycin is contained in an amount of 10-30mg per 1ml of the dimethyl sulfoxide solution containing adriamycin.

S203, dialyzing the acidic angelica polysaccharide-adriamycin, and filtering the acidic angelica polysaccharide-adriamycin through a microporous filter membrane to obtain the acidic angelica polysaccharide-adriamycin copolymer nanoparticles.

The adriamycin is connected to the angelica polysaccharide through a pH sensitive hydrazone bond, so that the adriamycin is expected to be quickly released in a weakly acidic tumor environment, and the drug effect is enhanced.

Application of acidic angelicae sinensis polysaccharide-adriamycin copolymer nanoparticles in preparation of medicines for treating liver cancer. The invention takes the natural angelica polysaccharide as a carrier to realize the drug delivery of the adriamycin, thereby not only ensuring that a nano drug delivery system stably and safely delivers the drug to the focus part in vivo in a targeted manner, but also realizing the effective treatment of the liver cancer.

The acidic angelicae sinensis polysaccharide ASP3, the preparation method and the application thereof, and the acidic angelicae sinensis polysaccharide-adriamycin copolymer nanoparticles, the preparation method and the application thereof are further illustrated in the following examples.

In the following examples, DEAE-Cellulose 52, Sephadex G-50 was obtained from Solebao Biotech, Inc. of Shanghai; angelica sinensis was purchased from Min county, Min, Gansu province and identified as a dry root of Angelica sinensis (Angelica sinensis diel), a plant of Umbelliferae.