CN111154006B

CN111154006B - Natural low-molecular-weight radix angelicae pubescentis polysaccharide and preparation method and application thereof

Info

Publication number: CN111154006B
Application number: CN202010064940.2A
Authority: CN
Inventors: 袁清霞; 赵龙岩; 李宏; 韦子怡
Original assignee: Guangxi University of Chinese Medicine
Current assignee: GUANGZHOU LVZAO BIOTECHNOLOGY Co.,Ltd.
Priority date: 2020-01-20
Filing date: 2020-01-20
Publication date: 2021-08-24
Anticipated expiration: 2040-01-20
Also published as: CN111154006A

Abstract

The invention discloses a natural low molecular weight radix angelicae pubescentis polysaccharide, and belongs to the technical field of medicines. The monosaccharide composition of the present invention is mannose (D-Manp), galacturonic acid (D-GalAp), glucose (D-Glcp), galactose (D-Galp) and arabinose (L-Ara), the main chain is composed of (1 → 3) -alpha-D-Glcp, (1 → 4) -beta-D-Galp, (1 → 6) -alpha-D-Manp, (1 → 3) -alpha-L-Araf, and the side chain is alpha-D-Glcp- (1 → 3) -beta-D-GalA, and is connected to the O-6 position of the main chain (1 → 4) -alpha-D-Glcp saccharide residue. The low molecular weight radix angelicae pubescentis polysaccharide has clear structure, low molecular weight, high purity, low viscosity, strong water solubility and obvious antioxidant activity, can be used as a natural non-toxic antioxidant, and is applied to the fields of medicines, foods, cosmetics and the like.

Description

Natural low-molecular-weight radix angelicae pubescentis polysaccharide and preparation method and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a natural low-molecular-weight radix angelicae pubescentis polysaccharide and a preparation method and application thereof.

Background

Free radicals are continuously generated in the metabolism process of organisms, and the excessive free radicals can damage important molecules of the organisms such as nucleic acid, protein and the like to different degrees to cause health problems. The existing research shows that the occurrence and development of various chronic diseases such as diabetes, hyperlipidemia, cancer, aging and the like are closely related to free radicals. Therefore, the organism can take natural antioxidants from foods or medicines, and is one of the important ways for delaying or reducing the oxidative damage of the human body and resisting aging.

Researches show that the active polysaccharide from natural sources has the activities of resisting oxidation, reducing blood fat, regulating immunity, resisting tumors and the like, has small toxic and side effects, and is safe and reliable. The applicant finds that some active polysaccharides derived from Chinese herbal medicines, such as mulberry leaf polysaccharide, sea cucumber polysaccharide and the like, have good antioxidant activity (Carbohydr Polym,2015,128, 52-62; polymers, 2018,23, 590). However, the structures of polysaccharides from different sources have great differences, such as the molecular weight, the monosaccharide composition, the physicochemical properties, the types of glycosidic bonds, the branching conditions and the like, so that the antioxidant activity of various polysaccharides also has great differences. The oxidation resistance of the traditional Chinese medicine polysaccharide from different sources is still to be discussed and is still a research hotspot of academia.

Radix Angelicae Pubescentis is the dried root of Angelica gigas Maxim.f. biserrata shann et Yuan of Umbelliferae plant, and is mainly used for dispelling pathogenic wind and removing dampness, and relieving pain, arthralgia and pain. Currently, the reported pubescent angelica root extract, such as the extract prepared by Chinese patent publications CN 1718578A and CN 104274505B, is generally a fat-soluble component such as volatile oil, has the effects of relieving pain, diminishing swelling, resisting inflammation and the like, is used for relieving pain and treating rheumatoid arthritis and the like, but does not contain a polysaccharide component and does not have antioxidant activity. So far, the related research reports of polysaccharide composition and physicochemical properties of one of the main active ingredients of the radix angelicae pubescentis are rarely seen at home and abroad, the research report of the exact structure of the radix angelicae pubescentis polysaccharide is not seen, and particularly the related research report that the radix angelicae pubescentis polysaccharide with the exact structure has antioxidant activity is not seen.

Researches show that the oxidation resistance of the Chinese herbal medicine active polysaccharide is closely related to the structures of the Chinese herbal medicine active polysaccharide such as viscosity, monosaccharide composition, molecular weight, uronic acid content, glycosidic bond type and the like. Especially, the molecular weight is an important structural characteristic factor influencing the strength of the antioxidant activity. Generally, the smaller the molecular weight, the more oxidation resistant the polymer. Most natural Chinese herbal medicine polysaccharide has physical characteristics such as large molecular weight, high viscosity, poor solubility and the like which are unacceptable in pharmacy. As shown in recent studies by the applicant, phellinus linteus polysaccharide has a large molecular weight (812kDa), a large viscosity (225mL/g), a poor solubility and a low antioxidant activity (Int J Biol Macromol,2018,120, 1855-.

Disclosure of Invention

The first purpose of the invention is to provide a natural low molecular weight radix angelicae pubescentis polysaccharide. The invention extracts and prepares a low molecular weight radix angelicae pubescentis polysaccharide from radix angelicae pubescentis for the first time, and further researches show that the natural low molecular weight radix angelicae pubescentis polysaccharide has high antioxidant activity, can obviously remove DPPH free radicals and hydroxyl free radicals within the range of experimental dosage and has strong metal ion chelating capacity. Compared with high molecular weight phellinus igniarius polysaccharide extracted earlier by the applicant, the low molecular weight radix angelicae pubescentis polysaccharide has the advantages of low viscosity, better solubility and stronger antioxidant activity. Therefore, the low molecular weight radix angelicae pubescentis polysaccharide has important application value as a natural antioxidant.

The natural Low molecular weight radix angelicae pubescentis polysaccharide (LAPP) is a natural Low molecular weight Low viscosity radix angelicae pubescentis polysaccharide (Low-molecular-weight Angelica pubescens polysaccharide) with high antioxidant activity, and the radix angelicae pubescentis polysaccharide LAPP is a mixture of homologous polysaccharides with the following structural formula:

in the above-mentioned structural formula, the compound,

A. b, D, F, G is alpha-D-glucon-1-yl;

c is alpha-D-mannosyl-1-yl;

e is alpha-L-arabinosyl-1-yl;

h is a beta-D-galactosyl-1-yl group;

i is beta-D-galacturonic acid-1-yl;

further, in the structural formula, the polysaccharide of heracleum hemsleyanum michaux contains mannose (D-Manp), galacturonic acid (D-GalAp), glucose (D-Glcp), galactose (D-Galp) and arabinose (L-Araf) in a molar ratio range of 2.0: (1.2 ± 0.3): (68.2 ± 0.3): (7.6 ± 0.3): (1.0 +/-0.3);

wherein the sum of m and n is an integer of 5-12, for example, m is 2, n is 5, and the sum is 7.

Further, in the above-mentioned case,

the weight average molecular weight (Mw) of the radix angelicae pubescentis polysaccharide LAPP is 3000-5000 Da; the molecular weight can be measured by high performance gel chromatography (HPGPC). In view of the strength of antioxidant activity, the molecular weight of LAPP selected by the present invention is about 3000-5000 Da (i.e. m + n of the homologue represented by the structural formula is an integer of about 5-12), preferably about 3000-4000 Da (m + n of the homologue represented by the structural formula is an integer of about 5-9) in terms of weight average molecular weight.

The polydispersity index of the radix angelicae pubescentis polysaccharide is between 1.0 and 2.0; the polydispersity index (PDI, ratio of weight average/number average molecular weight, Mw/Mn) is preferably between 1.0 and 1.5.

The purity of the radix angelicae pubescentis polysaccharide is more than 90% (HPGPC, area normalization method).

The viscosity of the radix angelicae pubescentis polysaccharide is 5-30 mL/g, and the radix angelicae pubescentis polysaccharide is easily dissolved in water or normal saline.

The pubescent Angelica root polysaccharide LAPP is low molecular weight polysaccharide extracted and separated from dried roots of Angelica gigas Maxim.f. biserrata Shann et Yuan which is an Umbelliferae plant.

The study of the invention finds that the radix angelicae pubescentis polysaccharide LAPP has strong antioxidant activity, can remove DPPH free radicals and hydroxyl free radicals, and has strong metal ion chelating capacity.

The second purpose of the invention is to provide a preparation method of the natural low molecular weight radix angelicae pubescentis polysaccharide LAPP. The LAPP is prepared by extracting and separating dried root (radix Angelicae Pubescentis) of Angelica gigas nakai of Umbelliferae, Angelica pubescens Maxim. f. biserrata shann et Yuan; extracting with hot water to obtain radix Angelicae Pubescentis total polysaccharide, performing fractional alcohol precipitation or ultrafiltration to obtain low molecular weight radix Angelicae Pubescentis crude polysaccharide, and further performing gel exclusion to obtain uniform polysaccharide with the molecular weight range. The method specifically comprises the following steps:

(1) extracting and preparing coarse polysaccharide of radix angelicae pubescentis: cleaning radix angelicae pubescentis, slicing, drying, crushing and sieving to obtain radix angelicae pubescentis powder, soaking or refluxing the radix angelicae pubescentis powder by using an organic solvent to remove pigments and fat-soluble micromolecule impurities to obtain impurity-removed residues, volatilizing the solvent of the impurity-removed residues, placing the impurity-removed residues in a reaction kettle, and adding water for extraction, wherein the material-liquid ratio is 1: 10-1: extracting for 1-2 times at the temperature of 30, 55-95 ℃ for 1-4 h each time, centrifuging, combining the obtained extracting solutions, and decoloring by using macroporous resin to obtain a coarse radix angelicae pubescentis polysaccharide extracting solution;

the organic solvent can be selected from, but is not limited to, absolute ethyl alcohol, acetone, ethyl acetate and the like, and preferably absolute ethyl alcohol with low toxicity and environmental friendliness.

(2) Preparing low molecular weight pubescent angelica root crude polysaccharide: and (3) carrying out grading alcohol precipitation or ultrafiltration on the crude polysaccharide extracting solution of the radix angelicae pubescentis obtained in the step (1) to obtain a radix angelicae pubescentis polysaccharide component with a low molecular weight range.

Optionally carrying out grading treatment on the decolored crude radix angelicae pubescentis polysaccharide extract by one of the following two methods:

method one (ultrafiltration method): ultrafiltering the crude radix Angelicae Pubescentis polysaccharide extractive solution with ultrafiltration membrane, collecting ultrafiltration permeate, concentrating the permeate, and lyophilizing to obtain low molecular weight radix Angelicae Pubescentis crude polysaccharide;

method two (fractional alcohol precipitation method): slowly adding 95% edible ethanol or absolute ethanol into the crude radix angelicae pubescentis polysaccharide extracting solution while stirring until the final concentration of the ethanol is 50-60%, standing for 4-12 h at 4 ℃, centrifuging to obtain a supernatant, adding 95% edible ethanol or absolute ethanol into the supernatant while stirring until the final concentration of the ethanol is 80-90%, standing for 4-12 h at 4 ℃, centrifuging to obtain polysaccharide precipitate, and performing vacuum freeze drying to obtain the low-molecular-weight crude radix angelicae pubescentis polysaccharide;

(3) preparing low molecular weight radix angelicae pubescentis polysaccharide: and (3) further separating and purifying the low molecular weight radix angelicae pubescentis crude polysaccharide obtained in the step (2) to obtain low molecular weight radix angelicae pubescentis polysaccharide which is uniform polysaccharide with target molecular weight.

Preparing the low-molecular-weight radix angelicae pubescentis crude polysaccharide obtained in the step (2) into an aqueous solution with the mass fraction of 5% -10%, passing through a treated gel column (the diameter is 2-5 cm, the length is 100-200 cm), the mobile phase is 0.01-0.2M NaCl, the sample loading volume is 1% -6% of the total volume of the column, the flow rate is 20-40 mL/h and 3-8 mL/tube, collecting elution fractions, detecting, combining low-molecular-weight radix angelicae pubescentis polysaccharide components with target molecular weight (3000-5000 Da), desalting, and carrying out vacuum freeze drying to obtain the natural low-molecular-weight radix angelicae pubescentis polysaccharide.

Further, in the step (1), the preparation of the impurity-removed residue specifically comprises the following steps: repeatedly soaking radix angelicae pubescentis powder in absolute ethyl alcohol or other organic solvents at room temperature for 3 times, or performing reflux extraction in a water bath at the temperature of 60-80 ℃ for 0.5-1 h, removing supernate, and repeating the previous operation on the lower-layer residues for 2-4 times.

Furthermore, in the step (1), protease such as papain and the like can be added for auxiliary extraction during hot water extraction at 55-95 ℃, or auxiliary extraction can be carried out by physical means of ultrasound or microwave.

Further, in the step (2), ultrafiltration is carried out by an ultrafiltration membrane of the first method, wherein the ultrafiltration membrane has a cut-off molecular weight of 8-20 kDa.

Further, in the step (2), in the second method, ethanol is firstly added into the crude polysaccharide extracting solution of the radix angelicae pubescentis until the final concentration of the ethanol is 50-60% for alcohol precipitation, most (more than 80%) of substances with the molecular weight of more than 20kDa are removed by centrifugation, and then the ethanol is added into the supernatant until the final concentration of the ethanol is 80-90%.

Further, in step (3), the Gel filler used in the Gel column chromatography may be selected from, but not limited to, a Sephadex Gel such as Sephadex G-100 Gel or Sephadex G-75 Gel, an agarose Gel such as Sepharose CL-4B or Sepharose CL-6B, a polyacrylamide Gel such as Bio-Gel P30, and the like. The elution fraction can be detected by, but not limited to, phenol-sulfuric acid method, carbazole sulfate method, anthrone sulfate method, etc., and can also be detected by the end absorption of polysaccharide at 210 nm. Desalting methods include, but are not limited to, dialysis, ultrafiltration, or gel column chromatography desalting.

The third purpose of the invention is to provide the application of the natural low molecular weight radix angelicae pubescentis polysaccharide in preparing natural antioxidants, and the natural antioxidants can be used in the fields of medicines, foods, cosmetics and the like.

Has the advantages that:

the natural low molecular weight radix angelicae pubescentis polysaccharide has low viscosity, is easy to dissolve in water, has weight average molecular weight of 3000-5000 Da, consists of mannose (D-Manp), galacturonic acid (D-GalAp), glucose (D-Glcp), galactose (D-Galp) and arabinose (L-Ara), has molar ratio of D-Man: D-GalA: D-Glc: D-Gal: L-Araf of 2.0 (1.2 +/-0.3) to (68.2 +/-0.3) to (7.6 +/-0.3) to (1.0 +/-0.3), has main chain consisting of (1 → 3) -alpha-D-Glcp, (1 → 4) -beta-D-Galp, (1 → 6) -alpha-D-Manp and (1 → 3) -alpha-L-Araf, and has side chain consisting of alpha-D-Glcp- (1 → 3) -beta-Gal-A → 3), attached to the backbone (1 → 4) - α -D-Glcp sugar residue at position O-6. Proved by verification, the natural low molecular weight radix angelicae pubescentis polysaccharide can obviously remove DPPH free radicals and hydroxyl free radicals and has strong metal ion chelating capacity; compared with high molecular weight phellinus igniarius polysaccharide extracted earlier by the applicant, the low molecular weight radix angelicae pubescentis polysaccharide has the advantages of low viscosity, better solubility and stronger antioxidant activity. The radix angelicae pubescentis polysaccharide has clear structure, low molecular weight, high purity, low viscosity and strong water solubility, has obvious antioxidant activity, can be used as a natural non-toxic antioxidant, and is applied to the fields of medicines, foods, cosmetics and the like.

Drawings

FIG. 1 shows the elution profile and HPGPC chromatogram of LAPP-1;

FIG. 2 shows LAPP-1¹H NMR detection spectrum;

FIG. 3 shows LAPP-1¹³C NMR detection spectrum;

FIG. 4 shows LAPP-1¹H-¹H COSY, TOCSY and ROESY anomeric hydrogen signal area superposition map;

FIG. 5 shows LAPP-1¹H-¹H HSQC detection spectrum;

FIG. 6 shows LAPP-1¹H-¹³C HMBC detection map;

FIG. 7 is a UV spectrum of LAPP-2;

FIG. 8 is an infrared spectrum of LAPP-2;

FIG. 9 shows the result of detection of DPPH free radical scavenging action by LAPP-1;

FIG. 10 shows the result of detecting the scavenging effect of LAPP-1 on hydroxyl radicals;

FIG. 11 shows the results of the detection of the chelating ability of LAPP-1 to metal ions.

Detailed Description

The invention is further described with reference to the following figures and specific examples.

[ example 1 ]

Preparation of natural low molecular weight radix Angelicae Pubescentis polysaccharide (LAPP-1)

1.1 materials:

the root of Angelica gigas nakai of Umbelliferae, Angelica pubescens Maxim.f. biserrata Shann et Yuan, is collected from Wufeng Tujia autonomous county of Hubei province; reagents used in Sephadex G-100 and Sephadex G-10 (GE healthcare Co., U.S.A.), NaCl, ethanol, etc., are commercially available analytical reagents.

1.2 method:

(1) extracting and preparing coarse polysaccharide of radix angelicae pubescentis: washing root of Angelica gigas nakai of Umbelliferae with tap water, slicing, oven drying in 60 deg.C forced air drying oven, pulverizing, sieving with 60 mesh sieve, weighing radix Angelicae Pubescentis powder 1kg, soaking in anhydrous ethanol for 3 times to remove pigment and liposoluble small molecular impurities, volatilizing ethanol from pigment-removed residue, placing in reaction kettle, adding 20L of water (material-liquid ratio of 1:20), extracting at 92 deg.C for 2 times, 3 hr each time, centrifuging to obtain supernatant, mixing all extractive solutions, and decolorizing with ADS-7 type macroporous resin.

(2) Preparing low molecular weight pubescent angelica root crude polysaccharide: slowly adding 95% edible alcohol into the decolored radix angelicae pubescentis crude polysaccharide extracting solution obtained in the step (1) while stirring until the final concentration of the alcohol is 60%, standing for 4h at 4 ℃, centrifuging to obtain a supernatant, adding 95% edible alcohol into the supernatant while stirring until the final concentration of the alcohol is 80%, standing for 4h at 4 ℃, centrifuging to obtain polysaccharide precipitate, and performing vacuum freeze drying. The final preparation of low molecular weight radix Angelicae Pubescentis crude polysaccharide lyophilized powder is 10.1g, and the yield is about 1.0%.

(3) Preparing low molecular weight pubescent angelica root polysaccharide (LAPP-1): taking 10G of the low-molecular-weight radix angelicae pubescentis crude polysaccharide prepared in the step (2), adding 200mL of water to dissolve the crude polysaccharide, passing through a treated Sephadex G-100 gel column (phi 5 multiplied by 200cm), wherein the mobile phase is 0.1M NaCl, the sample volume per time is 50mL, the flow rate is 30mL/h and is about 5 mL/tube, collecting elution fractions, detecting by a phenol-sulfuric acid method, combining LAPP-1 components with target molecular weight (3000-5000 Da), desalting by using the Sephadex G-10 gel column, and freeze-drying in vacuum. Finally, 2.2g of purified LAPP-1 freeze-dried powder with the purity of more than 98 percent (HPGPC, area normalization method) and the weight-average molecular weight (Mw) of 3600Da is obtained.

(4) And (3) detecting physical and chemical properties and structures of LAPP-1: determining intrinsic viscosity by adopting an Ubbelohde viscometer according to a viscosity measuring method of 0633 general rules of the four pharmacopoeias (2015 edition); detecting molecular weight and distribution by high performance gel chromatography (HPGPC); monosaccharide composition was detected by pre-column derivatization HPLC.

Analysis of polysaccharide structure by NMR spectrum analysis with AVANCE AV 600 NMR spectrometer (600MHz) (Bruker, Switzerland, solvent D)₂O contains an internal Trimethylsillyl-propionic acid (TSP-d4) standard at 25 ℃ C.

1.3 results:

the elution curve and HPGPC chromatogram of LAPP-1 are shown in figure 1, and the monosaccharide composition and physicochemical parameter measurement results are shown in Table 1; the 1D and 2D NMR detection spectra are shown in the attached figures 2-6,¹H/¹³the signals attributed to the C NMR spectrum data are shown in Table 2. The results of the tests in Table 1 show that both LAPP-1 has a low molecular weight and an intrinsic viscosity.

TABLE 1 detection results of physicochemical parameters and monosaccharide composition of low molecular weight radix Angelicae Pubescentis polysaccharide LAPP-1

With LAPP-1¹H/¹³The C NMR spectrum is shown in the attached FIGS. 2-3, and the signal attribution data is shown in Table 2. The bolded chemical shifts in table 2 are sugar residue substitution sites, since the chemical shifts are significantly shifted to lower fields than those of the monosaccharide standard.

¹H-¹H COSY, TOCSY and ROESY spectrograms (figure 4) show that the prepared LAPP-1 contains ten groups of spin-coupled systems. Bonding of¹H-¹³NMR spectra such as C HSQC (FIG. 5) and the like, wherein the sugar residues and the connection modes thereof are finally assigned to (1 → 4) -alpha-D-Glcp (A), (1 → 3) -alpha-D-Glcp (B), (1 → 6) -alpha-D-Manp (C), (1 → 4,6) -alpha-D-Glcp (D), (1 → 3) -alpha-L-Araf (E) and (4 →) -alpha-D-Glcp (F)_α)、(1→)-α-D-Glcp(G)、(1→4)-β-D-Galp(H)、(4→)-β-D-Glcp(F_β) And (1 → 3) -beta-D-GalAp (I) and the like.¹H-¹³The C HMBC spectrum (FIG. 6) further confirms the linkage relationship between the constituent monosaccharides of LAPP-1.¹The molar ratio of each sugar residue can be calculated by the peak area of an anomeric hydrogen signal peak of an H NMR spectrum, and the molar ratio of each sugar residue obtained by nuclear magnetic calculation is consistent with the result obtained by analysis and calculation of monosaccharide composition. From¹H-¹³The C HMBC spectrogram can also see the anomeric hydrogen configuration of each residue, and the coupling constant is more than 170ppm and is alpha type, and the coupling constant is less than 165 and is beta type. The result of 1D and 2D nuclear magnetism attribution indicates that the reduction end of LAPP-1 is D-Glcp- (4 →, the specific gravity is large, the chemical signal of anomeric hydrogen can be clearly distinguished, the LAPP-1 is in alpha type and beta type mutual transformation in heavy water solution, therefore, two groups of spin coupling systems exist in the sugar residue F, the LAPP-1 structure obtained by the nuclear magnetism attribution can know that glucuronic acid is connected with glucose to form disaccharide which is connected with the O-6 position of the main chain → 4) -alpha-D-Glcp- (1 → as a side chain.

TABLE 2 1H/13C NMR data (δ ppm) for low molecular weight Heracleum hemsleyanum polysaccharide LAPP-1

[ example 2 ] preparation of Natural Low molecular weight Angelica pubescens polysaccharide (LAPP-2)

2.1 materials:

the root of Angelica gigas nakai of Umbelliferae, Angelica pubescens Maxim.f. biserrata Shann et Yuan, is collected from Wufeng Tujia autonomous county of Hubei province; the reagents used for papain (40 million U/g, Beijing ancient Biotechnology, Inc.), Sepharose CL-4B (Sigma-Aldrich), Bio-Gel P-2 (Burley, USA) NaCl, ethanol, etc., are commercially available analytical reagents.

2.2 method:

(1) extracting and preparing coarse polysaccharide of radix angelicae pubescentis: washing root of Angelica gigas nakai of Umbelliferae with tap water, slicing, oven drying in a 60 deg.C forced air drying oven, pulverizing with a tissue triturator, sieving with a 60 mesh sieve, weighing 1kg of radix Angelicae Pubescentis powder, reflux-extracting with anhydrous ethanol for 3 times, 1h each time, removing pigment and liposoluble small molecular impurities, volatilizing ethanol from pigment-removed residue, adding 0.5% (w/w) papain aqueous solution 15L into a reaction kettle, stirring at 55 deg.C for reaction for 4h, boiling the enzymolysis solution for 10min to inactivate enzyme, centrifuging to obtain supernatant, decolorizing with ADS-7 type macroporous resin, adding ethanol into eluate to final concentration of 80% (v/v), standing at 4 deg.C for 12h overnight, centrifuging at 8000rpm, discarding supernatant, and collecting precipitate as radix Angelicae Pubescentis crude polysaccharide.

(2) Preparing low molecular weight pubescent angelica root crude polysaccharide: adding 2L water into the decolorized radix Angelicae Pubescentis crude polysaccharide obtained in step (1), and ultrafiltering with 10kDa ultrafiltration membrane in tangential flow ultrafiltration system (PALL, USA). Collecting the permeate, concentrating, precipitating with 80% ethanol, and finally obtaining low molecular weight radix Angelicae Pubescentis crude polysaccharide lyophilized powder 15.8g with yield of about 1.6%.

(3) Preparation of low molecular weight radix angelicae pubescentis polysaccharide (LAPP-2): taking 15g of the low molecular weight radix angelicae pubescentis crude polysaccharide prepared in the step 2, adding 300mL of water to dissolve the crude polysaccharide, passing through a pretreated Sepharose CL-4B Gel column (phi 2.5 multiplied by 150cm), wherein the mobile phase is 0.1M NaCl, the sample volume is 25mL each time, the flow rate is 25mL/h and is about 4 mL/tube, collecting elution fractions, detecting by a 210nm ultraviolet spectrophotometer, combining LAPP-2 components with target molecular weight, desalting by a Bio-Gel P-2 Gel column, and freeze-drying in vacuum. Finally, 3.5g of purified LAPP-2 freeze-dried powder with the purity of more than 98 percent (HPGPC, area normalization method) and the weight-average molecular weight (Mw) of 3600Da is obtained.

(4) And (3) detecting physical and chemical properties and structures of LAPP-2: determining intrinsic viscosity by adopting an Ubbelohde viscometer according to a viscosity measuring method of 0633 general rules of the four pharmacopoeias (2015 edition); detecting molecular weight and distribution by high performance gel chromatography (HPGPC); monosaccharide composition was detected by pre-column derivatization HPLC.

Scanning the ultraviolet and infrared spectra of LAPP-2; analysis of polysaccharide structure by NMR spectrum analysis with AVANCE AV 600 NMR spectrometer (600MHz) (Bruker, Switzerland, solvent D)₂O contains an internal Trimethylsillyl-propionic acid (TSP-d4) standard at 25 ℃ C.

2.3 results:

the monosaccharide composition and the results of the measurement of physicochemical parameters of LAPP-2 are shown in Table 3; the ultraviolet and infrared spectra are shown in figures 7-8. The molecular weight, the intrinsic viscosity and the monosaccharide composition of LAPP-2 are not obviously different from those of LAPP-1 prepared in example 1, and the LAPP-1 and the LAPP-2 are the same radix angelicae pubescentis polysaccharide compound.

TABLE 3 detection results of physicochemical parameters and monosaccharide composition of low molecular weight radix Angelicae Pubescentis polysaccharide LAPP-2

[ example 3 ] determination of antioxidant Activity of Natural Low molecular weight Angelica pubescens polysaccharide (LAPP)

3.1 materials and reagents

Detecting a sample: LAPP-1 (molecular weight 3553Da) prepared in example 1; l, 1-Diphenylpicrylphenylhydrazine (DPPH), phenanthroline, ascorbic acid (Vc), etc. are sigma company of America; disodium hydrogen phosphate, sodium dihydrogen phosphate, and ferrous chloride (FeC 1)₂) Disodium ethylenediaminetetraacetate (EDTA-2Na) and hydrogen peroxide (H)₂O₂) And the like are all domestic analytical pure reagents.

3.2 methods

(1) LAPP-1 DPPH radical scavenging Activity assay (Carbohydr Polym,2015,128, 52-62): absorbing 50 mu L of LAPP polysaccharide sample solution with different concentrations into a 96-well plate, adding 25 mu L of 0.4mmol/L DPPH absolute ethyl alcohol solution and 100 mu L of ultrapure water, oscillating the 96-well plate, mixing uniformly, reacting for 30min in a dark place at 30 ℃, and measuring the light absorption value at 517nm by using an enzyme-labeling instrument. Vc is a positive control. The scavenging activity is according to the formula: clearance (%) ([ 1- (Abs) ]₁-Abs₂)]/Abs₀X l 00%, where: abs₀Absorbance values for control experiments (water instead of sample); abs₁The light absorption value of the sample experiment is obtained; abs₂The absorbance value of the sample interference experiment (absolute ethyl alcohol replaces DPPH solution).

(2) LAPP-1 hydroxyl radical scavenging activity assay (Carbohydr Polym,2015,128, 52-62): the reaction system contains 50 μ L of sample solution with different concentrations, 50 μ L of phenanthroline (0.75mM), 75 μ L of phosphate buffer (0.15M, pH 7.4), and Fe_SO₄(0.75mM) 50. mu.L and H₂O₂(0.01%, w/v) 50. mu.L, after mixing well, reacted in a water bath at 37 ℃ for 30min, and absorbance was measured at 536 nm. According to the following formula: hydroxyl radical clearance (%) - (Abs)₂-Abs₀)/(Abs₁-Abs₀)]X l00 calculating hydroxyl radical clearance, wherein: abs₀Is the light absorption value of the control group experiment (water replaces the sample solution); abs₁Is water instead of H₂O₂And the light absorption value of the sample; abs₂Is the absorbance of the sample set.

(3) And (3) determining the chelating capacity of LAPP-1 metal ions: 50 mu L of sample solutions with different concentrations are put into a 96-well plate, and then 2.5 mu L of ferrous chloride solution with 2.0mmol/L, 10 mu L of phenazine solution with 5.0mmol/L and 137 mu L of deionized water are added. Mixing, reacting for 10min, and measuring absorbance at 562 nm. EDTA-2Na is a positive control. Chelating ability (%) as a formula chelating rate [ < 1- (Abs) ]₁-Abs₂)]/Abs₀X l 00%, where: abs0 is the absorbance of the control experiment (sample solution replaced with water); abs₁Is the absorbance value of the sample experiment; abs₂The absorbance of the sample interference experiment (ferrous chloride solution replaced with water).

3.3 results

(1) LAPP-1 scavenging DPPH free radical activity: in the accompanying figure 9, Vc is a positive control, and the result shows that LAPP-1 can effectively remove DPPH free radicals in the experimental dose range, the DPPH free radical removal activity shows a stable increasing trend along with the increase of the polysaccharide concentration, and the maximum removal rate can reach 58.6%.

(2) LAPP-1 hydroxyl radical scavenging activity: in the attached figure 10, Vc is a positive control, and the result shows that LAPP-1 has a good effect of eliminating hydroxyl radicals in the experimental dose range, the hydroxyl radical eliminating capacity is increased along with the increase of the polysaccharide concentration, and the maximum eliminating rate can reach 65.1%.

(3) And (3) determining the chelating capacity of LAPP-1 metal ions: the EDTA-2Na in the attached figure 11 is a positive control, and the result shows that the LAPP-1 has stronger metal ion chelating capacity in the experimental dose range, and the maximum chelating rate can reach 48.7 percent, which indicates that the antioxidant activity of the LAPP-1 is possibly related to chelating metal ions.

The research results show that the low molecular weight radix angelicae pubescentis polysaccharide has stronger antioxidant activity and is probably related to the fact that the polysaccharide with higher molecular weight contains more reducing terminals. The antioxidant activity of the low molecular weight radix angelicae pubescentis polysaccharide prepared by the invention is stronger than that of other Chinese herbal medicine source polysaccharides such as phellinus igniarius polysaccharide reported by the applicant in recent years (Int J Biol Macromol,2018,120, 1855-. Under the same detection conditions, the applicant reports that the maximum clearance rates of DPPH free radicals and hydroxyl free radicals of phellinus igniarius polysaccharides are 42.8 percent and 58.0 percent respectively, the maximum chelating rate of metal ions is only 23.59 percent, and each index of antioxidant activity is lower than that of LAPP-1 prepared by the invention. The antioxidant activity of LAPP-1 prepared by the invention is also obviously higher than that of the reported angelica polysaccharide (the DPPH and the maximum clearance rate of hydroxyl free radicals of the angelica polysaccharide are 43.8 percent and 33.4 percent respectively) (Int J Biol Macromol,2017,97, 46-54). Therefore, the low molecular weight radix angelicae pubescentis polysaccharide LAPP has important application value in the fields of medicine, food, cosmetics and the like.

Although the present invention has been described with respect to the preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. A preparation method of natural low molecular weight radix angelicae pubescentis polysaccharide is characterized by comprising the following steps:

(1) extracting and preparing coarse polysaccharide of radix angelicae pubescentis: cleaning radix angelicae pubescentis, slicing, drying, crushing and sieving, soaking or refluxing radix angelicae pubescentis powder by using an organic solvent to remove pigments and fat-soluble small molecular impurities to obtain impurity-removed residues, volatilizing the solvent of the impurity-removed residues, placing the impurity-removed residues in a reaction kettle, and adding water for extraction, wherein the material-liquid ratio is 1: extracting for 1-2 times at 55-95 ℃ for 1-4 h each time at 10-30 ℃, centrifuging and combining the obtained extracting solutions, and decolorizing with macroporous resin to obtain a coarse radix angelicae pubescentis polysaccharide extracting solution;

(2) preparing low molecular weight pubescent angelica root crude polysaccharide: optionally treating the decolorized radix angelicae pubescentis crude polysaccharide extract by one of the following two methods:

the method comprises the following steps: ultrafiltering the crude radix Angelicae Pubescentis polysaccharide extractive solution with ultrafiltration membrane, concentrating the filtrate, and lyophilizing to obtain low molecular weight radix Angelicae Pubescentis crude polysaccharide;

the second method comprises the following steps: adding 95% edible ethanol or absolute ethanol into the crude radix angelicae pubescentis polysaccharide extracting solution while stirring until the final concentration of the ethanol is 50-60%, standing for 4-12 h at 4 ℃, centrifuging to obtain a supernatant, adding 95% edible ethanol or absolute ethanol into the supernatant while stirring until the final concentration of the ethanol is 80-90%, standing for 4-12 h at 4 ℃, centrifuging to obtain polysaccharide precipitate, and performing vacuum freeze drying to obtain the low-molecular-weight crude radix angelicae pubescentis polysaccharide;

(3) preparing low molecular weight radix angelicae pubescentis polysaccharide: preparing a water solution with the mass fraction of 5-10% from low-molecular-weight radix angelicae pubescentis crude polysaccharide, passing through a gel column, enabling a mobile phase to be 0.01-0.2M NaCl, enabling a sample loading volume to be 1-6% of the total volume of the column, enabling the flow rate to be 20-40 mL/h and enabling the flow rate to be 3-8 mL/tube, collecting elution fractions, detecting, combining low-molecular-weight radix angelicae pubescentis polysaccharide components with target molecular weight, desalting, and carrying out vacuum freeze drying to obtain natural low-molecular-weight radix angelicae pubescentis polysaccharide; the natural low-molecular-weight radix angelicae pubescentis polysaccharide has a weight average molecular weight (Mw) of 3000-5000 Da.

2. The method of claim 1, wherein the natural low molecular weight radix Angelicae Pubescentis polysaccharide is prepared by: in the step (1), the preparation steps of the impurity removal residue are as follows: repeatedly soaking the radix angelicae pubescentis powder in absolute ethyl alcohol or other organic solvents at room temperature for 3 times, or performing reflux extraction in a water bath at the temperature of 60-80 ℃ for 0.5-1 h, removing supernate, and repeating the last operation on the lower-layer residues for 2-4 times.

3. The method of claim 1, wherein the natural low molecular weight radix Angelicae Pubescentis polysaccharide is prepared by: in the step (1), protease is added for auxiliary extraction during hot water extraction at 55-95 ℃, or auxiliary extraction is carried out by using an ultrasonic/microwave physical means.

4. The method of claim 1, wherein the natural low molecular weight radix Angelicae Pubescentis polysaccharide is prepared by: in the step (2), ultrafiltration is carried out by using an ultrafiltration membrane of the first method, wherein the ultrafiltration membrane has a molecular weight cutoff of 8-20 kDa.

5. The method of claim 1, wherein the natural low molecular weight radix Angelicae Pubescentis polysaccharide is prepared by: the Gel filler used in the step (3) is selected from Sephadex G-100 Gel or Sephadex G-75 Gel, Sepharose CL-4B or Sepharose CL-6B, and polyacrylamide Gel Bio-Gel P30.

6. Natural low molecular weight pubescent angelica root polysaccharide prepared by the preparation method of any one of claims 1 to 5;

the polydispersity index of said pubescent angelica root polysaccharide is between 1.0 and 2.0;

the purity of the radix angelicae pubescentis polysaccharide is more than 90%;

the viscosity of the radix angelicae pubescentis polysaccharide is 5-30 mL/g.

7. Use of the natural low molecular weight heracleum hemsleyanum michaux polysaccharide of claim 6 in the preparation of natural antioxidants.