CN114262390B - Achyranthes bidentata polysaccharide, preparation method and application thereof - Google Patents
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Abstract
The invention discloses achyranthes bidentata homogeneous polysaccharide AB-1-1, which consists of fructose, glucose, mannose, galactose and arabinose, consists of T-alpha-D-Glcp, → 1) -beta-D-Fruf- (2 →, → 2) -beta-D-Fruf- (6 →, → 1, 6) -beta-D-Fruf- (2 → and beta-D-Fruf- (2 →, five glycosidic linkages are connected, and the weight-average molecular weight is 3.998X 10 3 Da (+ -3.936%) and intrinsic viscosity value of 4.34mL/g (+ -2.03%); the preparation method of the homogeneous polysaccharide comprises the steps of polysaccharide extraction, deproteinization and freeze-drying, column separation, elution and drying; the invention also provides the application of the homogeneous polysaccharide AB-1-1 in the preparation of drugs for inhibiting pancreatic lipase activity and drugs for losing weight; the homogeneous polysaccharide has clear chemical composition, small toxic and side effects and obvious pancreatic lipase activity inhibition effect.
Description
Technical Field
The invention relates to the field of natural medicine, in particular to achyranthes bidentata polysaccharide, and further relates to a preparation method and application thereof.
Background
Obesity has now become a global health problem characterized by increased fat mass, adipose tissue dysfunction resulting from adipocyte proliferation and hypertrophy. Fat fed by mammals is generally digested and absorbed after being hydrolyzed by pancreatic lipase, which is a key enzyme in the fat hydrolysis process, can catalyze natural substrate grease to be hydrolyzed into glycerol and fatty acid, and can effectively inhibit the hydrolysis and absorption of the fat by inhibiting the activity of the glycerol and fatty acid, thereby achieving the effect of controlling and treating obesity; for example, semi-synthetic pancreatic lipase inhibitor Orlistat is used for clinically treating obesity, but the treatment process is accompanied by side effects such as abdominal distension, diarrhea, fatty stool and the like.
Achyranthes bidentata, a dried root of Achyranthes bidentata (Achyranthus bidentata) belonging to Amaranthaceae, is a perennial herb, is recorded in Shen nong Ben Cao Jing, is one of the well-known Chinese medicinal materials recorded in the Chinese pharmacopoeia (2020), and is mainly distributed in Henan, shanxi, hebei, shandong, sichuan and the like. Pharmacological research finds that part of polysaccharide of achyranthes bidentata has pharmacological activities of resisting tumor, regulating immunity, resisting virus, resisting oxidation and resisting osteoporosis; however, the research on the drug effect of the total polysaccharide of the medicinal materials is mainly used, the total polysaccharide is not separated and purified, the composition of the total polysaccharide is complex, the content of the polysaccharide is low, and the pharmacological action of the achyranthes bidentata fructose is difficult to embody.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the problems of complex composition and low polysaccharide content of total polysaccharides in the prior art, the invention provides achyranthes bidentata polysaccharides with clear chemical composition and high purity, and also relates to a preparation method and application thereof.
The technical scheme is as follows: the achyranthes bidentata polysaccharides of the invention are homogeneous polysaccharides AB-1-1, and consist of fructose, glucose, mannose, galactose and arabinose, wherein the mass molar ratio of the fructose to the glucose is as follows: 86:12.
Further, the weight average molecular weight of the polysaccharide is 3.998 × 10 3 Da (+ -3.936%) and number-average molecular weight of 3.902X 10 3 Da (+ -3.753%) and intrinsic viscosity value of 4.34mL/g (+ -2.03%); the polysaccharide is linked by glycosidic linkages including T- α -D-Glcp, → 1) - β -D-Fruf- (2 → 2) - β -D-Fruf- (6 → 1, 6) - β -D-Fruf- (2 → and β -D-Fruf- (2 →; wherein the ratio of the four fructose glycosides is 2.57.
The invention discloses a preparation method of achyranthes bidentata polysaccharides, which comprises the following steps:
(1) Crushing achyranthes bidentata medicinal materials, carrying out reflux extraction by using absolute ethyl alcohol, filtering, removing small molecular impurities, taking and drying dregs, carrying out reflux extraction on the dregs, filtering and combining filtrate, carrying out reduced pressure concentration, adding absolute ethyl alcohol for precipitation, standing, removing upper clear solution, collecting precipitate, sequentially washing by using absolute ethyl alcohol, acetone and absolute ethyl ether, adding water for dissolving polysaccharide precipitate, repeatedly deproteinizing by adopting a Savage method, collecting the achyranthes bidentata polysaccharide solution from which the protein is removed, and carrying out freeze drying to obtain refined achyranthes bidentata polysaccharide;
(2) Weighing the refined achyranthes bidentata polysaccharide obtained in the step (1) to prepare a saturated solution, carrying out gradient elution through a DEAE-FF column for primary separation, developing by adopting a sulfuric acid-anthrone method, carrying out ultraviolet detection, drawing an elution curve by taking an absorbance value as a vertical coordinate and taking the number of collection tubes as a horizontal coordinate, combining and collecting sample solutions according to the elution curve, dialyzing and freeze-drying to obtain achyranthes bidentata polysaccharide powder;
(3) And (3) dissolving and centrifuging the freeze-dried achyranthes bidentata polysaccharide powder obtained in the step (2) to remove insoluble impurities, separating and purifying by a Sephadex G-25 column, eluting and collecting, developing by adopting a sulfuric acid-anthrone method, carrying out ultraviolet detection, drawing an elution curve by taking an absorbance value as a vertical coordinate and a collection pipe number as a horizontal coordinate, combining and collecting samples according to the elution curve, and carrying out freeze drying to obtain the achyranthes bidentata polysaccharide compound.
Preferably, in the step (1), 4 to 5 times volume of absolute ethyl alcohol is added to be condensed and refluxed at 50 to 90 ℃ for 2 to 5 times, each time is 0.5 to 3 hours, the filtration is carried out, after the dregs of a decoction are dried, 5 to 15 times volume of distilled water is added to be condensed and refluxed at 50 to 80 ℃ for 1 to 5 times, each time is 0.5 to 3 hours, the filtration and the combination of filtrates are carried out, and the decompression concentration is carried out at 45 to 55 ℃.
Preferably, in the step (2), the mobile phase of the gradient elution is water, 0.1M NaCl solution, 0.2M NaCl solution, 0.3M NaCl solution, 0.4M NaCl solution and 0.5M NaCl solution, the elution flow rate is 2.0-3.0mL/min, and 40-50mL/T is automatically collected.
Preferably, in the step (3), the gradient elution is carried out, the mobile phase is distilled water, the elution flow rate is 0.3-0.5mL/min, and the elution is collected at 20-35 min/T.
The invention also discloses application of the achyranthes bidentata polysaccharide in preparing a medicament for inhibiting pancreatic lipase activity and a medicament for losing weight.
The invention principle is as follows: the invention adopts water extraction and alcohol precipitation and various column chromatography separation and purification methods to obtain achyranthes bidentata homogeneous polysaccharide AB-1-1 from achyranthes bidentata medicinal materials. The invention is based on ion exchange and steric exclusion effects, firstly realizes the separation of neutral sugar and acid sugar in the achyranthes bidentata refined polysaccharide, and then carries out treatment and purification on the polysaccharide with the same property according to the molecular weight to obtain high-purity homogeneous polysaccharide AB-1-1, wherein the homogeneous polysaccharide has specific molecular weight, polymerization degree, glycosidic bond type and other physicochemical properties to endow the homogeneous polysaccharide with unique pharmacological activity. According to the traditional medicinal mode of decocting the achyranthes bidentata medicinal material by hot water, the invention furthest keeps similar characteristics, so that the activity research result is close to actual application.
Has the advantages that: compared with the prior art, the invention has the following remarkable advantages:
(1) The achyranthes bidentata polysaccharides are homogeneous polysaccharides extracted from achyranthes bidentata medicinal materials, have clear chemical compositions and small toxic and side effects, and have remarkable pancreatic lipase activity inhibition effect;
(2) The preparation method of achyranthes bidentata polysaccharides has the advantages of being close to the traditional medicinal mode, simple and convenient in preparation method, mild in reaction condition, free of damage to sugar chain structures and convenient for process expansion production.
Drawings
FIG. 1 is a technical scheme of the preparation process of the present invention;
FIG. 2 is a high performance gel permeation chromatogram of achyranthes bidentata polysaccharides of the present invention;
FIG. 3 is a molecular weight determination profile of achyranthes bidentata polysaccharides SEC-MALS according to the present invention;
FIG. 4 is an intrinsic viscosity measurement profile of achyranthes bidentata polysaccharides of the present invention;
FIG. 5 is a monosaccharide profile of achyranthes bidentata polysaccharides according to the present invention;
FIG. 6 is a UV spectrum scan of achyranthes bidentata polysaccharides of the present invention;
FIG. 7 is an infrared spectroscopic scanning of achyranthes bidentata polysaccharides of the present invention;
FIG. 8 shows the results of in vitro inhibition of pancreatic lipase activity by achyranthes bidentata polysaccharides of the present invention;
FIG. 9 is NMR spectra of achyranthes bidentata polysaccharides of the present invention.
Detailed Description
The technical scheme of the invention is further explained by combining the attached drawings.
Example 1
Method for preparing refined achyranthes bidentata polysaccharides (AB)
Crushing achyranthes bidentata medicinal materials, weighing 500g of medicinal material powder, adding 5 times of absolute ethyl alcohol, carrying out condensation reflux extraction for 3 times at 80-90 ℃, removing small molecular impurities for 2 hours each time, filtering, collecting dregs, drying at 50 ℃, adding 10 times of distilled water, carrying out condensation reflux extraction for 3 times at 70-80 ℃, filtering and combining filtrate each time for 2 hours, carrying out reduced pressure concentration at 45-55 ℃ to about 1.0L, adding 4 times of absolute ethyl alcohol for precipitation, standing overnight at 4 ℃, removing upper clear solution, collecting precipitate, washing with absolute ethyl alcohol, acetone and absolute ethyl ether in sequence, adding a proper amount of distilled water for dissolving polysaccharide precipitate, repeatedly deproteinizing by adopting a Savage method, collecting the achyranthes bidentata polysaccharide solution after protein removal, and carrying out freeze drying to obtain refined achyranthes bidentata polysaccharide.
Example 2
Method for preparing achyranthes bidentata homogeneous polysaccharide AB-1-1
Weighing 3.0g of achyranthes bidentata polysaccharide dry powder, adding a proper amount of distilled water to prepare a saturated solution, carrying out primary separation by a DEAE-FF column (60 multiplied by 2.6 cm), carrying out gradient elution by distilled water, 0.1, 0.2, 0.3, 0.4 and 0.5M NaCl solution in sequence, carrying out automatic collection at the elution flow rate of 2.0-3.0mL/min and 40-50mL/T, developing by adopting a sulfuric acid-anthrone method, carrying out ultraviolet detection in a range of 620-630nm, drawing an elution curve by taking an absorbance value as a vertical coordinate and the number of collection tubes as a horizontal coordinate, merging and collecting samples according to the elution curve, and freeze-drying; the components eluted by the NaCl solution need to be dialyzed for 48 hours by a dialysis bag and then are freeze-dried.
Dissolving the freeze-dried achyranthes bidentata polysaccharide powder 400mg in 2.0mL of distilled water, centrifuging for 5-10min at 9000-12000rpm/min, removing insoluble impurities, further separating and purifying by a Sephadex G-25 column (120 multiplied by 2.0 cm), taking distilled water as a mobile phase, carrying out elution and collection at the flow rate of 0.3-0.5mL/min and 20-35min/T, developing by adopting a sulfuric acid-anthrone method, carrying out ultraviolet detection in the range of 620-630nm, drawing an elution curve by taking an absorbance value as a vertical coordinate and the number of collection tubes as a horizontal coordinate, combining and collecting samples according to the elution curve, and carrying out freeze drying to obtain a homogeneous achyranthes bidentata polysaccharide sample AB-1-1.
Example 3
Physicochemical Properties of achyranthes bidentata Hovenia Spreng AB-1-1
1. Purity and molecular weight determination
High performance gel permeation chromatography: an Agilent 1290HPLC system is provided with a G4260BELSD detector, agilent PL aquagel-OH MIXED-H, agilent PL aquagel-OH MIXED-M (300 multiplied by 7.5mm,8 um) series columns, the flowing phase detection of 0.6mL/min ultra-pure water is carried out at 25 ℃, the sample concentration is 1.0mg/mL, the sample introduction is 10uL, the detection time is 40min, and as shown in figure 2, the result shows that AB-1-1 is high-purity homogeneous polysaccharide;
an SEC-MALS system is adopted to determine AB-1-1 weight average molecular weight, number average molecular weight and intrinsic viscosity value, 1200HPLC is combined with MALS, viscostar and dRI detectors, shodex Ohpak-806M HQ chromatographic column, normal saline is used as a mobile phase, the sample concentration is 4.5mg/mL, and 100uL of sample injection is carried out. As shown in FIGS. 3 and 4, the results showed that the uniform polysaccharide had a weight average molecular weight of 3.998X 10 3 Da (+ -3.936%) and a number average molecular weight of 3.902X 10 3 Da (+ -3.753%) and intrinsic viscosity value of 4.34mL/g (+ -2.03%).
2. Optical rotation analysis
Drying a sample to be tested to constant weight, preparing a solution of 1.0mg/mL, measuring the optical rotation by using a polarimeter, and calculating the specific rotation by a formula [ alpha ] Dt =100 alpha/L c.
Wherein c: solution concentration (g/100 mL); α: optical rotation; l: glass tube length (dm). The specific optical rotation of radix Achyranthis homogeneous polysaccharide AB-1-1 is-33.33 °.
3. Monosaccharide composition analysis
Weighing 10.0mg of sample polysaccharide, adding 2.0mL of distilled water for dissolving, adding 1.0mL of sample solution into 1.0mL of 0.1M THA solution in an ampoule bottle, sealing with an alcohol torch, and hydrolyzing at 110 deg.C for 0.5h. The hydrolyzate was blown dry with nitrogen and 3 additional 2.0mL portions of methanol to remove TFA. The sample was dissolved by adding 2.0mL of ultrapure water, centrifuged at 12000rpm/min for 5min, and fructose was detected. In addition, 1.0mLAB-1-1 sample solution is added with 4.0mL 4M TFA solution in an ampoule bottle, an alcohol blast lamp is sealed, the mixture is hydrolyzed at 110 ℃ for 3 hours, hydrolysate is dried by nitrogen, 2.0mL methanol is blown for 3 times, residual TFA is removed, 2.0mL ultrapure water is added for dissolving, the mixture is centrifuged at 12000rpm/min for 5min, 100uL of supernatant is taken to be mixed with 100uL0.5mol/L PMP methanol solution, 100uL0.3mol/L NaOH solution is added for vortex mixing, 70 ℃ water bath reaction is carried out for 30min, the mixture is cooled to room temperature, 100uL0.3mol/L HCl solution is added for neutralization, reaction solution 300uL and 900uL chloroform are taken for extraction for 3 times, residual PMP reagent is removed, upper layer water phase is taken for HPLC detection, and the wavelength is detected to be 250nm. Calculating the molar mass ratio of monosaccharide components in AB-1-1 according to the following formula:
formula (la)
Ax: sample peak area; ai: standard peak area; ci: the molar concentration of the standard substance; vx: volume of sample solution.
The results show that as shown in fig. 5, the homopolysaccharide is mainly composed of fructose and glucose, and the mass molar ratio is about: 86, contains trace mannose, galactose and arabinose at the same time,
4. ultraviolet spectral analysis
Taking a proper amount of AB-1-1 to prepare 1.0mg/mL solution to be detected, and scanning the ultraviolet spectrum within the range of 200-700 nm. As shown in FIG. 6, AB-1-1 showed no absorption at 280nm, indicating that the sample contained no impurities such as protein nucleic acid.
5. Infrared spectroscopic analysis
Weighing 1.0mg polysaccharide powder, mixing with 200mg KBr, grinding into powder, tabletting, and scanning with infrared spectrum of 4000-400cm-1. As shown in figure 7, at 3394.101, 2935.126, 1639.197cm- 1 The absorption peak of (2) is a characteristic absorption peak of the polysaccharide compound, and is 3394.101cm -1 The strong broad peak of (2) is OH absorption peak in polysaccharide, 2935.126cm -1 The absorption peak is CH stretching vibration peak, 1639.197cm -1 The absorption peak is mainly caused by moisture, 929.5029cm -1 And 813.2524cm -1 The absorption peak at (A) indicates the presence of a furan ring, 817.1669cm -1 The absorption peaks are attributed to the beta configuration in the polysaccharide, and the results show that the polysaccharide has beta configurationPyranose and furanose are present.
6. Methylation analysis
Weighing 20mg of dried polysaccharide sample, rapidly adding and flushing N 2 The test tube with the plug is sealed, 50mg of NaOH powder is added for ultrasonic treatment for 0.5h, the mixture is kept stand for 2h at room temperature, and 1.5mL of CH is slowly dripped into the test tube after the reaction is finished 3 And I, reacting for 2 hours in a dark environment, adding 1mL of pure water after the reaction is finished, stopping the reaction, extracting for 5 times by using 3.0mL of trichloromethane, collecting a trichloromethane phase, drying by blowing nitrogen, and repeatedly methylating for 3 times. Dissolving the obtained reaction product with chloroform, dripping a small amount of the solution on dried KBr tablets, scanning with a Fourier infrared spectrometer, and observing at 400-4000cm -1 In the wavelength range, 3400cm -1 Whether the broad peak at (b) disappears or not indicates that the methylation of the sample is complete.
Adding 2M TFA into completely methylated sample to dissolve the sample, hydrolyzing at 110 deg.C for 3 hr, distilling methanol to remove TFA, adding 2mL pure water solution hydrolyzed sample, transferring to clean tube, adding 50mg NaBH 4 The reaction was carried out at room temperature for 12 hours, and after completion of the reaction, a 10% acetic acid solution was slowly added dropwise to the test tube until no significant air bubbles were generated. Repeatedly co-steaming the reaction solution and methanol until white powder appears after complete evaporation, transferring the powder into a clean test tube, drying for 1h at 50 ℃, adding 3mL of acetic anhydride-pyridine mixed solution (1). The results showed that glucose exists mainly in the form of 1,5-Di-O-acetyl-2,3,4,6-tetra-O-methyl-d-glucitol glycosidic bond.
7. NMR analysis
60mg of dried polysaccharide sample was weighed and 1.0ml LD was added 2 Dissolving O, freeze drying, repeating for 3 times, and collecting 0.5mLD 2 The O-solubilized polysaccharide solution was added to a nuclear magnetic tube and analyzed using a Bruker AVANCE3HD600MHZ nuclear magnetic spectrometer. The results are shown in FIG. 9, where 1 H NMR (FIG. 9A) and 13 in C NMR (FIG. 9B), an anomeric hydrogen signal of delta 5.31ppm was correlated with an anomeric carbon signal of delta 92.49ppm in HSQC (FIG. 9C), indicating grapesThe sugar exists in the form of T-alpha-D-Glcp and consists of monosaccharide and 13 CNMR analysis showed that the presence of four different fructose residues in AB-1-1 was indicated by δ 103.77, 104.16, 103.61, 104.05ppm, designated sequentially as a, b, C, D, and as indicated by the ratio of peak heights of anomeric carbon signals in the inverted gated coupled carbon spectrum, that a: b: C: D = 2.57. Based on the above analysis, AB-1-1 is composed mainly of → 1) - δ 0-D-Fruf- (2 → 2) - δ 1-D-Fruf- (6 → 1, 6) - δ 2-D-Fruf- (2 → linked fructose residue backbone, and terminated by T- δ 3-D-Glcp and δ 4-D-Fruf- (2 → the specific spectrum mapping analysis is shown in Table 1.
TABLE 1 chemical shifts of achyranthes bidentata polysaccharides AB-1-1 glycosyl residues in 1H and 13C NMR spectra
| Sugar residue | H1/C1 | H2/C2 | H3/C3 | H4/C4 | H5/C5 | H6/C6 |
| →1)-β-D-Fruf-(2→a | 60.03 | 103.77 | 76.48 | 74.61 | 81.13 | 63.33 |
| 3.75 | 4.11 | 4.15 | 3.79 | 3.50 | ||
| →2)-β-D-Fruf-(6→b | 60.38 | 104.16 | 76.3 | 75.19 | 80.26 | 62.48 |
| 3.69 | 4.09 | 3.87 | 3.87 | 3.60 | ||
| β-D-Fruf-(2→c | 60.41 | 103.61 | 76.41 | 74.22 | 81.09 | 62.52 |
| 3.66 | 4.12 | 4.01 | 3.73 | 3.57 | ||
| →1，6)-β-D-Fruf-(2→d | 59.92 | 104.05 | 76.72 | 74.67 | 80.17 | 62.28 |
| 3.59 | 4.10 | 4.10 | 3.73 | 3.67 | ||
| α-D-Glcp-(1→e | 92.49 | 71.11 | 72.61 | 69.17 | 72.40 | 63.19 |
| 5.31 | 3.48 | 3.70 | 3.38 | 3.73 | 3.85 |
Example 4
In vitro inhibition of pancreatic lipase Activity
Weighing 100mg AB-1-1, dissolving in distilled water, sequentially diluting with distilled water to obtain series of different administration concentrations, weighing 1.0mg Orlistat distilled water to obtain 1.0mg/mL as positive drug, and using distilled water as blank control group. 5mL of olive oil and 10mL of 1% Tween 20 were mixed, and sonicated for 10min to prepare a lipid emulsion. The lipid emulsion was diluted 4-fold with 0.1M Tris-HCl buffer (pH 8.4) to obtain a lipid emulsion dilution. 50uL of sample solutions with different concentrations are taken to incubate for 10min in 20uL of pancreatic lipase (1.0 mg/mL), 50uL of lipid emulsion diluent is added, and after incubation for 10min at 37 ℃, the lipase activity is determined according to the instructions of the lipase activity kit. The results show that as shown in fig. 8, pancreatic lipase activity decreased with increasing polysaccharide administration dose, and pancreatic lipase inhibition was significant when the polysaccharide administration dose reached 25mg/mL, compared to the model group. The results show that the homogeneous polysaccharide has obvious pancreatic lipase inhibiting activity and is concentration-dependent.
The invention adopts various column chromatography separation and purification methods to obtain homogeneous polysaccharide AB-1-1 with higher purity from radix achyranthis bidentatae medicinal materials, and adopts the technologies of chromatography, spectrometry, wave spectrum method and the like to comprehensively analyze the structural characteristics of AB-1-1; on the basis, it is further studied that AB-1-1 inhibits the activity of pancreatic lipase and is useful for the treatment of diseases such as obesity.
Claims (3)
1. The application of achyranthes bidentata polysaccharides is characterized in that the achyranthes bidentata polysaccharides are used for preparing a medicament for inhibiting pancreatic lipase activity and a medicament for losing weight; the achyranthes bidentata polysaccharides are uniform polysaccharides AB-1-1, and comprise fructose, glucose, mannose, galactose and arabinose, and the weight average molecular weight of the achyranthes bidentata polysaccharides is 3.998 × 10 3 Da±3.998×10 3 Da x 3.936% connected by glycosidic linkages, the glycosidic linkages including T- α -D-Glcp, → 1) - β -D-Fruf- (2 → 2) - β -D-Fruf- (6 → 1, 6) - β -D-Fruf- (2 → and β -D-Fruf- (2 →;
the preparation method of achyranthes bidentata polysaccharide is characterized by comprising the following steps:
(1) Crushing achyranthes bidentata medicinal materials, carrying out reflux extraction by using absolute ethyl alcohol, filtering, removing small molecular impurities, taking and drying dregs, carrying out reflux extraction on the dregs, filtering and combining filtrate, carrying out reduced pressure concentration, adding absolute ethyl alcohol for precipitation, standing, removing upper clear solution, collecting precipitate, sequentially washing by using absolute ethyl alcohol, acetone and absolute ethyl ether, adding water for dissolving polysaccharide precipitate, repeatedly deproteinizing by adopting a Savage method, collecting the achyranthes bidentata polysaccharide solution from which the protein is removed, and carrying out freeze drying to obtain refined achyranthes bidentata polysaccharide;
(2) Weighing the refined achyranthes bidentata polysaccharide obtained in the step (1) to prepare a saturated solution, carrying out gradient elution through a DEAE-FF column for primary separation, developing by adopting a sulfuric acid-anthrone method, carrying out ultraviolet detection, drawing an elution curve by taking an absorbance value as a vertical coordinate and taking the number of collection tubes as a horizontal coordinate, combining and collecting sample solutions according to the elution curve, dialyzing and freeze-drying to obtain achyranthes bidentata polysaccharide powder;
(3) Taking the achyranthes bidentata polysaccharide powder freeze-dried in the step (2), dissolving and centrifuging to remove insoluble impurities, separating and purifying by a Sephadex G-25 column, eluting and collecting, developing by adopting a sulfuric acid-anthrone method, carrying out ultraviolet detection, drawing an elution curve by taking an absorbance value as a vertical coordinate and a collection pipe number as a horizontal coordinate, combining and collecting samples according to the elution curve, and carrying out freeze drying to obtain the achyranthes bidentata polysaccharide compound;
wherein, in the step (2), the gradient elution, the mobile phase adopts water, 0.1M NaCl solution, 0.2M NaCl solution, 0.3M NaCl solution, 0.4M NaCl solution and 0.5M NaCl solution in turn, the elution flow rate is 2.0-3.0mL/min, and 40-50mL/T is automatically collected; in the step (3), the gradient elution is carried out, the mobile phase is distilled water, the elution flow rate is 0.3-0.5mL/min, and the elution is collected at 20-35 min/T.
2. Use according to claim 1, wherein the polysaccharide has a number average molecular weight of 3.902 x 10 3 Da±3.902×10 3 Da X3.753%, intrinsic viscosity 4.34 mL/g. + -. 4.34mL/g X2.03%.
3. The use as claimed in claim 1, wherein in step (1), 4-5 times volume of anhydrous ethanol is added for condensing reflux extraction at 50-90 deg.C for 2-5 times, each time for 0.5-3h, filtering is carried out, the residue is dried, then 5-15 times volume of distilled water is added for condensing reflux extraction at 50-80 deg.C for 1-5 times, each time for 0.5-3h, filtering is carried out, the filtrates are combined, and vacuum concentration is carried out at 45-55 deg.C.
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| "怀牛膝对去卵巢肥胖大鼠体重和肿瘤坏死因子-A的影响";郭姣红等;《成都医学院学报》;20080630;第3卷(第2期);第100-102,106页 * |
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