CN110818808B - Acidic angelica polysaccharide ASP3, acidic angelica polysaccharide-adriamycin copolymer nanoparticles, and preparation methods and applications of acidic angelica polysaccharide ASP3 and acidic angelica polysaccharide-adriamycin copolymer nanoparticles - Google Patents
Acidic angelica polysaccharide ASP3, acidic angelica polysaccharide-adriamycin copolymer nanoparticles, and preparation methods and applications of acidic angelica polysaccharide ASP3 and acidic angelica polysaccharide-adriamycin copolymer nanoparticles Download PDFInfo
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- CN110818808B CN110818808B CN201810900357.3A CN201810900357A CN110818808B CN 110818808 B CN110818808 B CN 110818808B CN 201810900357 A CN201810900357 A CN 201810900357A CN 110818808 B CN110818808 B CN 110818808B
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
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Abstract
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.
Description
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 radix Angelicae sinensis polysaccharide ASP3 is extracted from the above acidic radix Angelicae sinensis polysaccharide ASP3 by extraction method, and has 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.
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.
Example 1
This example provides a method for preparing acidic angelicae polysaccharide ASP3 and the acidic angelicae polysaccharide ASP3 obtained by the method. The preparation method specifically comprises the following steps:
s101, extracting total polysaccharides: pulverizing dried Angelica regale county, sieving with a 20-mesh sieve, adding 10 times of anhydrous ethanol, and decocting at 90-100 deg.C for three times (2 hr each time); then, after adding 10 times of distilled water by mass, the mixture was extracted by boiling with distilled water for 2 hours each time 3 times. Mixing the water extractive solutions, concentrating under reduced pressure, centrifuging at 3500r/min for 10min, and removing precipitate; adding 4 times volume of anhydrous ethanol into the supernatant, precipitating for 24h, centrifuging at 3500r/min for 10min, collecting ethanol precipitate, and directly using the obtained crude radix Angelicae sinensis polysaccharide in the next step without further treatment;
s102, deproteinizing by a Sevage method: preparing the crude angelica polysaccharide obtained by drying in the step S101 into a crude angelica polysaccharide aqueous solution with the concentration of 8mg/mL, adding 1/4 n-butyl alcohol-chloroform mixed solution (the volume ratio of n-butyl alcohol to chloroform is 1:4) of the volume of the crude angelica polysaccharide aqueous solution into the solution, violently oscillating for 20min, centrifuging at 3500r/min for 10min, collecting an upper layer solution, repeatedly operating for 8 times according to the deproteinization step, combining the upper layer solution, concentrating under reduced pressure, dialyzing, and freeze-drying to obtain the high-purity angelica polysaccharide;
s103, separation: preparing an aqueous solution with the mass concentration of 150mg/mL from the angelica polysaccharide subjected to deproteinization treatment, centrifuging for 10min at 3500r/min, taking the supernatant, carrying out primary separation by using DEAE-cellulose 52 ion column chromatography, carrying out gradient elution by using distilled water, sodium chloride solutions with the concentrations of 0.1mol/L and 0.3mol/L in sequence, collecting eluent, detecting the absorbance value of the eluent at the detection wavelength of 490nm by using a phenol-sulfuric acid method, merging the same fractions according to the elution curve (the elution curve of the angelica polysaccharide on the DEAE-cellulose 52 column is shown in figure 1), concentrating, dialyzing and freeze-drying to obtain neutral polysaccharide ASP0, acidic polysaccharide ASP1 and acidic polysaccharide ASP3 respectively;
s104, purification: weighing 100mg of neutral polysaccharide ASP0, acidic polysaccharide ASP1 and acidic polysaccharide ASP3 purified by DEAE-cellulose 52, dissolving in 10mL of distilled water, filtering with a filter membrane, purifying and separating the obtained filtrate with a Sephadex 50 chromatographic column, eluting with distilled water, collecting the eluate, detecting the absorbance value of the eluate at a detection wavelength of 490nm by a phenol-sulfuric acid method, combining the same fractions [ ASP0(a), ASP1(b) and ASP3(c) according to elution curves shown in a graph of fig. 2a, fig. 2b and fig. 2 c) on the Sephadex G-50 column, concentrating, dialyzing and freeze-drying to obtain neutral angelica polysaccharide ASP0, acidic angelica polysaccharide ASP1 and acidic angelica polysaccharide ASP 3. The three components were further dialyzed, freeze-dried, and weighed, and ASP3 was found to be the most predominant component (ratio: about 73.4. + -. 5.0%) while ASP0 and ASP1 accounted for 10.8. + -. 0.7% and 15.7. + -. 4.6%, respectively.
Example 2
This example provides a method for preparing acidic angelicae polysaccharide ASP3 and the acidic angelicae polysaccharide ASP3 obtained by the method. The preparation method specifically comprises the following steps:
s101, extracting total polysaccharides: pulverizing dried Angelica regale county, sieving with 20 mesh sieve, adding 11 times of anhydrous ethanol, and decocting at 90-100 deg.C for 2 times, each time for 4 hr; then, after adding 11 times of distilled water by mass, the mixture was extracted by boiling with distilled water for 4 hours each time for 2 times. Mixing the water extractive solutions, concentrating under reduced pressure, centrifuging at 4000r/min for 5min, and removing precipitate; adding 4 times volume of anhydrous ethanol into the supernatant, precipitating for 30h, centrifuging at 4000r/min for 5min, collecting ethanol precipitate, and directly using the obtained crude radix Angelicae sinensis polysaccharide in the next step without further treatment;
s102, deproteinizing by a Sevage method: preparing the crude angelica polysaccharide obtained by drying in the step S101 into a crude angelica polysaccharide water solution with the concentration of 8mg/mL, adding 1/5 n-butyl alcohol-chloroform mixed solution (the volume ratio of n-butyl alcohol to chloroform is 1:5) of the crude angelica polysaccharide water solution volume into the solution, violently oscillating for 25min, centrifuging at 4000r/min for 5min, collecting an upper layer solution, repeatedly operating for 8 times according to the deproteinization step, combining the upper layer solution, carrying out reduced pressure concentration, dialyzing, and carrying out freeze drying to obtain the high-purity angelica polysaccharide;
s103, separation: preparing aqueous solution with the mass concentration of 240mg/mL by using the angelica polysaccharide subjected to deproteinization treatment, centrifuging for 5min at 4000r/min, performing primary separation on supernatant by using DEAE-cellulose 52 ion column chromatography, performing gradient elution by using distilled water and sodium chloride solutions with the concentrations of 0.15mol/L and 0.35mol/L in sequence, collecting eluent, detecting the absorbance value of the eluent at the detection wavelength of 490nm by using a phenol-sulfuric acid method, merging same fractions according to an elution curve, concentrating, dialyzing and freeze-drying to obtain neutral ASP polysaccharide 0, acidic polysaccharide ASP1 and acidic polysaccharide ASP3 respectively;
s104, purification: weighing 100mg of neutral polysaccharide ASP0, acidic polysaccharide ASP1 and acidic polysaccharide ASP3 purified by DEAE-cellulose 52, dissolving in 10mL of distilled water, filtering with a filter membrane, purifying and separating the obtained filtrate by a Sephadex 50 chromatographic column, eluting with distilled water, collecting the eluate, detecting the absorbance value of the eluate at the detection wavelength of 490nm by a phenol-sulfuric acid method, combining the same fractions according to an elution curve, concentrating, dialyzing and freeze-drying to obtain neutral angelica polysaccharide ASP0, acidic angelica polysaccharide ASP1 and acidic angelica polysaccharide ASP 3.
Example 3
This example provides a method for preparing acidic angelicae polysaccharide ASP3 and the acidic angelicae polysaccharide ASP3 obtained by the method. The preparation method specifically comprises the following steps:
s101, separation: preparing the angelica polysaccharide extracted by a conventional method into an aqueous solution with the mass concentration of 50mg/mL, centrifuging for 15min at 3000r/min, taking supernatant, carrying out primary separation by using DEAE-cellulose 52 ion column chromatography, carrying out gradient elution by using distilled water, sodium chloride solutions with the concentrations of 0.05mol/L and 0.25mol/L in sequence, collecting eluent, detecting the absorbance value of the 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 respectively obtain neutral ASP polysaccharide 0, acidic polysaccharide ASP1 and acidic polysaccharide ASP 3;
s102, purification: weighing 100mg of neutral polysaccharide ASP0, acidic polysaccharide ASP1 and acidic polysaccharide ASP3 purified by DEAE-cellulose 52, dissolving in 10mL of distilled water, filtering with a filter membrane, purifying and separating the obtained filtrate by a Sephadex 50 chromatographic column, eluting with distilled water, collecting the eluate, detecting the absorbance value of the eluate at the detection wavelength of 490nm by a phenol-sulfuric acid method, combining the same fractions according to an elution curve, concentrating, dialyzing and freeze-drying to obtain neutral angelica polysaccharide ASP0, acidic angelica polysaccharide ASP1 and acidic angelica polysaccharide ASP 3.
Next, in this example, detailed analysis and experiments were performed on each component of angelicae sinensis polysaccharide, and specific examples of the experiments are as follows:
experimental example 1: gel Permeation Chromatography (GPC) analysis of Angelica polysaccharide fractions
Accurately weighing appropriate amount of ASP0, ASP1 and ASP3 of each component of Angelica sinensis polysaccharide to be analyzed, and adding 0.1mol/LNaNO3Solution (containing NaN with the mass fraction of 0.2 ‰ of3) Fixing the volume to 7.0mg/mL, standing in a refrigerator at 4 deg.C for 12h, taking out, returning to room temperature, and filtering with 0.22 μm filter membrane to obtain the test solution for GPC analysis of each component of radix Angelicae sinensis polysaccharide.
The chromatographic conditions of GPC analysis of each component of the angelica polysaccharide are as follows: the chromatographic column is ShodexSUGARKS-805 chromatographic column and KS-803 chromatographic column used in series (KS-805 chromatographic columnBefore, KS-803 column after), 0.1mol/LNaNO3Solution (containing NaN with the mass fraction of 0.2 ‰ of3) Elution was performed at a rate of 0.8ml/min, column and RID temperatures of 60 ℃ and 50 ℃, respectively, and sample volumes: 50 μ L. And (3) carrying out sample injection analysis on GPC chromatogram finger prints of each component of the angelica polysaccharide under the condition, and calculating the relative retention time of each peak and the relative peak area of each peak in the chromatogram.
As a result, as shown in Table 1, the weight average molecular weights of ASP0, ASP1 and ASP3 were 15169,17179 and 96258Da, respectively.
TABLE 1 determination of the molecular weight (GPC) of ASP0, ASP1 and ASP3
Experimental example 2: analysis of monosaccharide composition of each component of angelica polysaccharide
1) Complete acid hydrolysis of each component sample of angelica polysaccharide: accurately weighing 10mg of each component (ASP0, ASP1 and ASP3) of Angelica polysaccharide, placing into ampoule bottle, adding 4mL of trifluoroacetic acid solution (final concentration 2mol/L), mixing, and charging N2And putting the mixture into a 110 ℃ oven for hydrolysis for 3h, taking out the mixture and cooling the mixture to room temperature, decompressing and evaporating a hydrolysate to dryness, then adding methanol with the same volume into the hydrolysate, decompressing and evaporating to dryness again, repeating the operation for 3 times, and dissolving the mixture by using 2mL of ultrapure water.
2) PMP derivatization of angelica polysaccharide samples: 100 mu L of angelica polysaccharide hydrolysate is respectively weighed, 200 mu L of 0.3mol/L NaOH solution and 200 mu L of 0.5mol/L PMP methanol solution are sequentially added into a 2mL centrifuge tube, vortex and uniform mixing are carried out, and the mixture reacts in 70 ℃ water bath for 90 min. After cooling to room temperature, adding 200 mu L0.3mol/L HCl for neutralization, adding equal volume of chloroform for extraction, shaking, standing, discarding the organic phase, and repeating the operation for 3 times. The upper aqueous phase was filtered through a 0.45 μ M microfiltration membrane and used for HPLC analysis.
3) PMP derivatization of monosaccharide standards: weighing 100 μ L of monosaccharide standard solution (mannose Man, rhamnose Rha, Glucuronic acid (GluC acid, Gla), Galacturonic acid (Galactinic acid, GalA), glucose Glc, galactose Gal, arabinose Ara, monosaccharide concentration 5mg/ml) respectively, and performing derivatization according to 2) the PMP derivatization method of the angelica polysaccharide hydrolysate.
4) PMP derivatization of mixed monosaccharides: 100 mu L of each monosaccharide standard solution (5mg/ml) is taken, 1400 mu L of 0.3mol/L NaOH solution and 1400 mu L of 0.5mol/L PMP methanol solution are sequentially added, vortex and uniform mixing are carried out, and the mixture is reacted for 90min in 70 ℃ water bath. After cooling to room temperature, adding 1400 mu L of 0.3mol/L HCl for neutralization, adding equal volume of chloroform for extraction, oscillating, standing, discarding the organic phase, and repeating the operation for 3 times. The upper aqueous phase was filtered through a 0.45 μ M microfiltration membrane and used for HPLC analysis.
5) The HPLC analysis conditions were as follows: the mobile phase is A: and B is 63:17(w/w), wherein the phase A is phosphate buffer solution (0.1Mol/L, pH 6.7), and the phase B is acetonitrile. The column was reverse phase C18column (250X 4.6 mm). Column temperature: 25 ℃; detection wavelength: 245 nm; flow rate: 1.0 mL/min; sample introduction amount: 20 μ L.
As shown in FIG. 3, ASP0, ASP1 and ASP3 have similar monosaccharide compositions, wherein ASP0 is composed mainly of mannose, glucose, galactose and arabinose, and is a neutral sugar; ASP1 is composed of rhamnose, galacturonic acid, glucose, galactose and arabinose, and is acidic polysaccharide; ASP3 is composed of rhamnose, galacturonic acid, galactose and arabinose. The composition ratios of the various sugars are shown in Table 2.
TABLE 2 monosaccharide composition analysis of ASP0, ASP1 and ASP3
Experimental example 3: structural analysis of acidic angelicae polysaccharide ASP 3:
weighing acidic Angelica sinensis polysaccharide ASP3 about 40mg, and dissolving in 600 μ L deuterium oxide (D)2O), HNMR, CNMR, COSY, HSQC and HMBC thereof were characterized by a Bruker 600MHz NMR spectrometer.
Results as shown in fig. 4 a-4 e and table 3, in conjunction with the relevant literature, we concluded that the acidic angelicae ASP3 is a type of pectic polysaccharide, and that the backbone of the acidic angelicae ASP3 may be → 4) - α -D-GalpA- (1 → 2) - α -L-Rhap- (1 →, while α -T-Araf/α -1,5-Araf and β -1,4Galp are attached to Rha as side chains.
TABLE 3 chemical Shift values of 1HNMR and 13CNMR for ASP3
Experimental example 4: angelica polysaccharide components for inhibiting liver cancer activity
MTT (tetrazolium salt method) experiment: human hepatoma cells HepG2, SMMC7721 and Bel7402 cells and a human normal liver cell line L02 (purchased from cell resource center of Shanghai Life sciences research institute of Chinese academy of sciences) were cultured in a DMEM medium (Gibco) containing 10% fetal bovine serum (Gibco), 100U/mL penicillin and 100U/mL streptomycin. Cells contained 5% CO at 37 ℃2Culturing in an incubator. HepG2, SMMC7721, Bel7402 and L02 cells in logarithmic growth phase were seeded in 96-well plates at 5X 103, and after 24h incubation in an incubator, the supernatant was discarded, and 100. mu.L of a medium solution containing each component angelicae polysaccharide (prepared in example 1) was added to each of them to final concentrations of 31.25, 62.5, 125, 250, 500 and 1000. mu.g/mL, each of which was three duplicate wells, and a blank control well was additionally provided. After 24 and 48h of culture in an incubator, the cells were detected by the MTT method.
The results are shown in FIGS. 5 a-5 d, and the cytotoxicity of the components ASP0, ASP1 and ASP3 of the polysaccharide of FIGS. 5 a-5 d on HepG2(a), SMMC7721(b) and Bel7402(c) cells of liver cancer cells and L02(d) cells of normal liver cells. Wherein the vertical bar is represented by ASP0 (i.e., the first set of data in each coordinate system), the horizontal bar is represented by ASP1 (i.e., the second set of data in each coordinate system), and the diagonal bar is represented by ASP3 (i.e., the third set of data in each coordinate system).
The acidic polysaccharide ASP3 can inhibit the growth of three human liver cancer cells, namely HepG2, SMMC7721 and Bel7402, in a dose-dependent and time-dependent manner, and ASP0 and ASP1 show certain effects of promoting the growth of tumor cells. In addition, the compound has no obvious toxicity and high safety on human normal liver cell strains L02, ASP0, ASP1 and ASP 3. The ASP3 shows better anti-liver cancer activity, and the inventor finds that ASP3 can be used as a carrier for anti-liver cancer drug delivery.
Example 4
The embodiment provides acidic angelica polysaccharide-adriamycin copolymer nanoparticles and a preparation method thereof, and the preparation method specifically comprises the following steps:
100mg of the acidic angelicae sinensis polysaccharide ASP3 was dissolved in 10mL of distilled water, 256mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) was added thereto with magnetic stirring, and after reaction at room temperature for 30min, 146mg of N-hydroxysuccinimide (NHS) was added thereto, and the reaction was continued for 2 h. Meanwhile, 3mL of hydrazine hydrate is directly added into the angelica polysaccharide activation system, and the mixture is magnetically stirred to react for 24 hours. After the reaction was completed, the reaction mixture was dialyzed against distilled water (MWCO, 3500) for 2 days, and freeze-dried to obtain an acidic angelicae polysaccharide-hydrazide compound.
100mg of the ASP 3-hydrazide compound obtained above was dissolved in 5mL of a mixed solution (1:1) of dimethyl sulfoxide and water, and 1mL of a dimethyl sulfoxide solution containing 10 to 30mg of doxorubicin was added under nitrogen protection, followed by 50. mu.L of triethylamine, and the mixture was stirred for 24 hours in the dark. After the reaction is finished, dialyzing the reaction mixture with distilled water (MWCO, 3500) for 2 days, and filtering the dialyzed against filtering membrane of 0.45 micron to obtain acid angelica polysaccharide-adriamycin copolymer nanometer particle. The HNMR characterization of the synthesized product is shown in fig. 6.
The preparation process of the acidic angelicae sinensis polysaccharide-adriamycin copolymer nanoparticles is shown in table 4. 10mg ASP-DOX (different mass ratios, 10:0.5, 10:1 and 10:2 react to obtain acid angelica polysaccharide-adriamycin copolymer nanoparticles) is dissolved in 5mL dd H2And O, performing ultrasonic treatment for 5min (power 100W, ultrasonic treatment for 3s, stopping for 4s), and performing dialysis for several hours. Finally, the obtained product is filtered by a 0.45 mu m microporous filter membrane to obtain ASP-DOX nanoparticles. Measuring the particle diameter by Dynamic Light Scattering (DLS) when ASP-NH2When the mass ratio of the/DOX is 10:1, the nanoparticles have better particle size and potential (199.4 +/-8.5 nm and-21.3 +/-2.32 respectively), and the Pdi is smaller, so that the ASP-DOX nanoparticles are prepared by selecting the ratio. Transmission electron microscope TEM shows that the ASP-DOX polymer nanoparticles with the proportion have better appearance morphology (a and b in figure 7).
TABLE 4 influence of ASP 3-hydrazide compound/doxorubicin mass ratio on the particle size, Pdi, potential, etc. of ASP-DOX copolymer nanoparticles formed
In view of the weak acidity of the tumor microenvironment, in the research, doxorubicin is connected to the angelica polysaccharide through a pH-sensitive hydrazone bond, so that doxorubicin can be rapidly released in the weak acidity tumor environment, and the drug effect is enhanced. We simulated different pH environments by in vitro experiments. As can be seen from c in fig. 7, under acidic conditions, the nanoparticle size increases, which is likely to be caused by nanoparticle aggregation; in addition, nanoparticle fragmentation was found to be multimodal, which may be caused by breakage of the nanoparticles under acidic conditions.
Next, detailed analysis and experiments are performed on the acidic angelicae sinensis polysaccharide-adriamycin copolymer nanoparticles in this example, and specific examples of the experiments are as follows:
experimental example 5: in vitro release behavior of acidic angelicae sinensis polysaccharide-adriamycin copolymer nanoparticles
The in vitro drug release behavior of the acidic angelicae sinensis polysaccharide-adriamycin copolymer nanoparticles prepared in example 4 was examined by dialysis. Precisely measuring 2mL of acidic angelicae sinensis polysaccharide-adriamycin copolymer nanoparticles (the concentration is 200 mug/mL), placing the acidic angelicae sinensis polysaccharide-adriamycin copolymer nanoparticles into a dialysis bag (the cut-off molecular weight is 3500), immersing the acidic angelicae sinensis polysaccharide-adriamycin copolymer nanoparticles into 30mL of PBS (PBS) containing 1% Tween 80(w/v) and having pH values of 7.4 and 5.5 respectively, shaking the acidic angelicae sinensis polysaccharide-adriamycin copolymer nanoparticles at 37 ℃ and 100r/min, sampling 1mL of acidic angelicae sinensis polysaccharide-adriamycin copolymer nanoparticles for 0.5, 1, 2, 4, 6, 8, 12, 18, 24, 36h and 48h respectively, and simultaneously supplementing fresh media with the same pH value, volume and temperature. The sample was filtered through a 0.45 μm filter membrane, the initial filtrate was discarded, the doxorubicin content was measured by UV method, and the cumulative drug release percentage was calculated, and the result is shown in d in fig. 7. In the release medium with pH of 7.4 and 5.5, the cumulative drug release amount for 12h of the acidic angelicae sinensis polysaccharide-adriamycin copolymer nanoparticles is 45.5 percent and 70.2 percent respectively, which shows that the acidic angelicae sinensis polysaccharide-adriamycin copolymer nanoparticles are stable in a physiological environment (pH of 7.4), and can release drugs more quickly when entering an acidic environment (pH of 5.0-6.0) of tumor cell endosomes.
Experimental example 6: acidic angelica polysaccharide-adriamycin copolymer nanoparticle for inhibiting liver cancer activity in vitro
Human hepatoma cells HepG2(a), SMMC7721(b) and normal hepatocytes L02(c) were seeded in a 96-well plate DMEM medium (Gibco) containing 10% fetal bovine serum (Gibco), 100U/mL penicillin and 100U/mL streptomycin. Cells were incubated at 37 ℃ in the presence of 5% CO2Is incubated in a humid atmosphere. The medium was replaced with fresh medium 24 hours after inoculation. ASP3, doxorubicin, ASP-DOX were dissolved in PBS and diluted with cell culture medium (ASP-DOX groups of pH7.4 and 6.5 refer to ASP-DOX diluted with pH7.4 and pH6.5 medium, respectively). For each well, 100 μ L of cell culture medium with different indicated drug concentrations was added. A negative control was generated by adding 100. mu.L of medium. After 24 and 48h of culture in an incubator, the cells were detected by the MTT method.
As shown in FIGS. 8 a-8 c, the cytotoxicity of ASP-DOX on HepG2 cell was significantly better than that of doxorubicin, while the toxicity of ASP-DOX on SMMC7721 and L02 cell was approximately equivalent to that of doxorubicin. In addition, the antiproliferative activity of ASP-DOX at pH6.5 was superior to that of the pH7.4 group, which may be associated with a faster and more abundant release of doxorubicin from ASP-DOX under acidic conditions. Notably, the empty carrier ASP3 without drug loading did not show significant anti-tumor activity at the corresponding doxorubicin concentration (ASP3 maximum concentration of about 30 μ g/mL), suggesting better biocompatibility.
Experimental example 7: in vitro cellular uptake of acidic angelicae sinensis polysaccharide-adriamycin copolymer nanoparticles
Hepatoma cells HepG2(a), SMMC7721(b) and normal hepatocyte L02(c) were inoculated into 12-well plates and 24 hours later, the three cells were incubated with DOX, ASP-DOX and ASP-DOX + Gal1 for 3,6 hours, respectively. Then, cells were detached using trypsin and washed three times with PBS. Cells were analyzed for doxorubicin fluorescence intensity using a BD Beckman Coulter flow cytometer. As shown by the corresponding a-f in fig. 9A-B, the ability of ASP-DOX to be taken up by HepG2, SMMC7721 hepatoma cells and L02 normal hepatocytes was significantly better than the original drug DOX, especially the most significant difference at 3h and 6 h. Further studies showed that doxorubicin prodrug reached almost maximal values in HepG2 and SMMC7721 cells at 12h, whereas ASP-DOX could enter cells more, even not at longer times, e.g. 24h (g, h in fig. 9C). This suggests that ASP-DOX promotes the accumulation of the drug in tumor cells. In addition, the Incell high-content cellular imager is used for researching the uptake and distribution of DOX and ASP-DOX by HepG2 and SMMC7721 cells, and the ASP-DOX uptake capacity by two kinds of liver cancer cells is higher than that of adriamycin raw drug (figure 10).
In order to verify that ASP-DOX can target ASGPR receptors on the surfaces of liver cancer cells, the expression of the ASGPR receptors on the surfaces of three liver cancer cells, namely HepG2, SMMC7721 and Bel7402 and normal liver cells L02, is firstly studied. As shown by i in fig. 9C, the ASGPR receptor was overexpressed on the surface of HepG2 cells, whereas SMMC7721 and Bel7402 cells underexpressed the receptor. By adding a competitive inhibitor (small molecule galactose Gal, 80mM), ASP-DOX uptake by HepG2 cells was found to be greatly reduced, especially at 6h with the most significant difference. While no decrease in ASP-DOX uptake was detected in other SMMC7721 and L02 cells with low or no ASGPR expression. The competitive experiment shows that the ASP-DOX copolymer nanoparticles can target liver cancer cells through ASGPR mediation. This is probably the reason why the antitumor activity of ASP-DOX is more remarkable than that of the original drug in HepG2 cells.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Claims (14)
1. The acidic angelica polysaccharide-adriamycin copolymer nanoparticle is characterized in that the acidic angelica polysaccharide ASP3 and adriamycin are connected through an acid-sensitive hydrazone bond to prepare the acidic angelica polysaccharide-adriamycin copolymer nanoparticle, wherein the extraction method of the 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 filter membrane, purifying and separating the filtrate by Sephadex column chromatography, eluting with distilled water, and collecting second eluate to obtain acidic polysaccharide ASP3
Wherein said acidic angelicae polysaccharide ASP3 has a weight average molecular weight of 90-100 kDa.
2. The acidic angelicae sinensis polysaccharide-adriamycin copolymer nanoparticles as claimed in claim 1, wherein the extraction method of acidic angelicae sinensis polysaccharide ASP3 further comprises performing secondary purification after collecting the second eluent: 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.
3. The acidic angelicae sinensis polysaccharide-adriamycin copolymer nanoparticle according to claim 1, wherein the extraction method of acidic angelicae sinensis polysaccharide ASP3 further comprises performing the following steps before the 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.
4. The acidic angelicae sinensis polysaccharide-adriamycin copolymer nanoparticle as claimed in claim 3, wherein the weight ratio of angelicae sinensis coarse powder to the first absolute ethyl alcohol in the total polysaccharide extraction step is 1: 9-11.
5. The acidic angelicae sinensis polysaccharide-adriamycin copolymer nanoparticle according to claim 4, wherein before adding the second absolute ethyl alcohol to the concentrated water extract, the method further comprises centrifuging the concentrated water extract and discarding the precipitate, and then adding the second absolute ethyl alcohol to the supernatant.
6. The acidic angelicae sinensis polysaccharide-adriamycin copolymer nanoparticle as claimed in claim 5, wherein the mass ratio of the supernatant to the second absolute ethyl alcohol is 1: 3-5.
7. The acidic angelicae sinensis polysaccharide-adriamycin copolymer nanoparticles as claimed in claim 3, 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, an n-butanol-chloroform mixed solution is added, shaking is carried out for 15-25min, centrifugation is carried out, an upper layer solution is collected, the deproteinization step is repeatedly carried out for 6-10 times, the upper layer solution is combined, and dialysis and freeze drying are carried out after reduced pressure concentration to obtain the angelicae sinensis polysaccharide.
8. The acidic angelicae sinensis polysaccharide-adriamycin copolymer nanoparticle as claimed in claim 7, wherein the volume ratio of the crude angelicae sinensis polysaccharide aqueous solution to the n-butanol-chloroform mixed solution is 3-5: 1.
9. The acidic angelicae sinensis polysaccharide-adriamycin copolymer nanoparticle as claimed in claim 8, wherein the volume ratio of n-butanol to chloroform in the n-butanol-chloroform mixed solution is 1: 3-5.
10. The acidic angelicae sinensis polysaccharide-adriamycin copolymer nanoparticle according to claim 1, wherein the acidic angelicae sinensis polysaccharide ASP3 is a pectic polysaccharide, the main chain of the acidic angelicae sinensis 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 linked to Rha.
11. The method for preparing acidic angelicae sinensis polysaccharide-adriamycin copolymer nanoparticles according to any one of claims 1 to 10, wherein the method comprises the following steps:
reacting the acidic angelicapolysaccharides ASP3 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.
12. The production method according to claim 11,
dissolving the acidic angelica polysaccharide ASP3 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 the acidic angelica polysaccharide-adriamycin copolymer nanoparticles.
13. The method according to claim 12, wherein 10 to 30mg of doxorubicin is contained in 1ml of the doxorubicin-containing dimethylsulfoxide solution.
14. The use of the acidic angelicae sinensis polysaccharide-adriamycin copolymer nanoparticles according to any one of claims 1 to 10 in the preparation of a medicament for treating liver cancer.
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