CN110862463A - Preparation of alfalfa root polysaccharide with bioactivity and selenizing modified polysaccharide thereof - Google Patents

Abstract

The preparation of alfalfa root polysaccharide and selenizing modified polysaccharide with biological activity relates to the preparation of natural medicine modified polysaccharide, firstly obtaining water extract from dry alfalfa root, deproteinizing, decolorizing, separating by anion exchange column chromatography, eluting with deionized water and a series of gradient NaCl solution, collecting 2 new alfalfa root polysaccharide components RAPS-1 and RAPS-2, wherein the molecular weight range of the components RAPS-1 and RAPS-2 is 10-16 KDa, and the components are mainly composed of rhamnose, arabinose, xylose, galacturonic acid, mannose, galactose and glucose. Carrying out selenylation modification on RAPS-2 with large amount to obtain new selenylation polysaccharide Se-RAPS-2, wherein the molecular weight range of the selenylation polysaccharide Se-RAPS-2 is 10-12 KDa. The DAPS-1 and Se-DAPS-2 have certain anti-HepG-2 tumor activity and are concentration-dependent, and the anti-HepG-2 tumor activity of the Se-DAPS-2 is higher than that of the DAPS-1 and the DAPS-2. The research contents lay a theoretical foundation for the activity analysis of the polysaccharide and the selenizing products thereof and the development of anti-tumor drugs and other diseases.

Description

Preparation of bioactive alfalfa root polysaccharides and their selenide-modified polysaccharides

technical field

The invention relates to the preparation of a natural medicine modified polysaccharide, in particular to the preparation of a biologically active alfalfa root polysaccharide and its selenization modified polysaccharide.

Background technique

Alfalfa ( Medicago sativa L.) is a perennial legume forage crop widely cultivated in temperate regions around the world. Alfalfa is an important industrial crop for herders. In addition, alfalfa is also eaten as a vegetable, and people around the world make it into salads, tortillas, croquettes, puddings, soups and casseroles. As a functional food, alfalfa has been recorded in many medicinal books in ancient China, such as the "Compendium of Medicine", and was regarded as the father of all foods by the ancients. When alfalfa is used as a pasture and vegetable, it exhibits potential health-promoting effects due to its rich nutritional value, including antioxidant, anti-inflammatory, anti-cancer, antibacterial, immune-enhancing, and memory-enhancing effects. Numerous studies of active ingredients in alfalfa have isolated many chemical constituents. Because alfalfa contains high-quality nutrients and a variety of different biological activities.

Alfalfa polysaccharide is a plant polysaccharide extracted from alfalfa plants. So far, in the in vitro experiments of alfalfa, it has immune-enhancing activity, anti-tumor, hypoglycemic and antioxidant activity. In addition, alfalfa polysaccharides have also been found to promote the growth of livestock and poultry. Because alfalfa polysaccharides have special biological functions and rich sources, alfalfa polysaccharides have great application potential.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a biologically active alfalfa root polysaccharide and the preparation of its selenization modified polysaccharide. The polysaccharide and selenized polysaccharide prepared by the present invention have certain anti-HepG-2 tumor activity. It lays a theoretical foundation for the structural modification of polysaccharides, the activity analysis of polysaccharides, and the development of functional health care products and drugs related to anti-tumor diseases.

The purpose of this invention is to realize through the following technical solutions:

The preparation of biologically active alfalfa root polysaccharide and its selenization modified polysaccharide includes:

(1) Take the dried alfalfa root, extract with petroleum ether and ethanol in turn to remove lipids and small molecules, add distilled water and heat under reflux for 2 to 6 times, filter, and combine the filtrates; after deproteinization and depigmentation, alfalfa crude polysaccharide is obtained The obtained crude polysaccharide was separated by anion exchange column chromatography, eluted with distilled water and 0.1-0.5 mol/L NaCl solution in turn, and 2 new alfalfa root polysaccharides RAPS-1 and RAPS were obtained after gel purification. -2;

(2) The high yield polysaccharide RAPS-2 obtained in the above step (1) is selenized modified by the nitric acid-sodium selenite (NA-SS) method to obtain a new selenopolysaccharide Se-RAPS-2.

The preparation of the biologically active alfalfa root polysaccharide and its selenization modified polysaccharide, the structural fragments of the two new alfalfa root polysaccharides prepared are:

RAPS-1:→2)-α-L-Rhap-(1→,α-D-GalAp-(1→,→4)-β-D-Manp-(1→,→5)-α-L- Arap-(1→,→3)-β-D-Xylp-(1→ and →6)-β-D-Glcp-(1→;

RAPS-2: →3)-α-L-Rhap-(1→,→2)-α-L-Rhap-(1→,→6)-α-D-Galp-(1→,α-D- GalAp-(1→,α-D-Glcp-(1→,→3)-β-D-Manp-(1→ and →3)-β-D-Xylp(1→.

The preparation of the biologically active alfalfa root polysaccharide and its selenization modified polysaccharide, the obtained alfalfa root polysaccharide and selenium polysaccharide both have anti-HepG-2 tumor activity, and when the concentration is 100 μM, DAPS-1 , DAPS-2 and Se-DAPS- inhibited HepG-2 tumors by 30.35%, 17.81% and 38.70%, respectively.

The invention provides alfalfa root polysaccharide and its selenization modified polysaccharide. The above polysaccharides have certain anti-HepG-2 tumor activity.

The present invention is obtained through the following steps:

Preparation of alfalfa root polysaccharide:

The alfalfa root is refluxed with petroleum ether and ethanol to remove lipids and small molecular compounds, then refluxed and extracted with distilled water for 2 to 6 times, filtered, concentrated under reduced pressure, and combined with the extract. Mix neutral protease with extract, incubate at 40-60 ^o C for 1-3 h, inactivate at 80-100 ^o C for 10-20 min, and centrifuge to obtain supernatant. Sevage reagent (1:2～1:4, v/v) prepared from n-butanol and chloroform was added to the supernatant, stirred for a certain period of time to remove the protein, centrifuged to collect the supernatant, and the protein 2 was removed by the Sevage reagent method ~ 5 times. The supernatant obtained above was concentrated, the AB-8 resin column was decolorized, eluted with distilled water, and concentrated under reduced pressure to obtain crude polysaccharide. Crude polysaccharides were separated by DEAE-52 cellulose column and eluted with distilled water and NaCl solutions of different concentrations. The sugar content was determined by the phenol-sulfuric acid method. The obtained fractions were purified by Sephadex G-200 column, and the two fractions with more sugar content were collected, concentrated and dried under reduced pressure to obtain two new polysaccharides (DAPS-1, DAPS-2).

The large amount of DAPS-2 obtained above was subjected to selenization reaction. Under the condition of nitric acid, sodium selenite and polysaccharide were reacted at 60-80 ^o C for 4-8 h, and the reaction solution was dialyzed to obtain a new selenium Polysaccharide (Se-DAPS-2).

The two new alfalfa root polysaccharides obtained above have molecular weights ranging from 10.0 KDa to 16.0 KDa, and are mainly composed of rhamnose, arabinose, xylose, glucuronic acid, mannose, glucose and galactose. The range of the ratio is (1.00～1.30):(1.50～3.20):(0～2.70):(3.50～5.50):(0～3.20):(2.70～5.10):(0～1.00). The molecular weight of a selenized polysaccharide ranged from 10.0 KDa to 12.0 KDa, and the selenium content ranged from 300 to 350 μg/g.

Two new alfalfa root polysaccharides and one selenium polysaccharide provided by the present invention were used to identify the anti-HepG-2 tumor activity of DAPS-1, DAPS-2 and Se-DAPS-2 by cytotoxicity experiments.

Anti-HepG-2 tumor activity showed that DAPS-1, DAPS-2 and Se-DAPS-2 inhibited HepG-2 tumor by 30.35%, 17.81% and 38.70%, respectively, at a concentration of 100 μM. The results showed that the inhibitory effect of Se-DAPS-2 on HepG-2 tumor cells was higher than that of DAPS-1 and DAPS-2.

In conclusion, the polysaccharide and selenylated polysaccharides prepared by the present invention have certain anti-HepG-2 tumor activity. It lays a theoretical foundation for the structural modification of polysaccharides, the activity analysis of polysaccharides, and the development of functional health care products and drugs related to anti-tumor diseases.

Description of drawings

Fig. 1 is the ¹ H-NMR spectrum of RAPS-1;

Fig. 2 is the ¹³ C-NMR spectrum of RAPS-1;

Fig. 3 is the HSQC pattern of RAPS-1;

Fig. 4 is the HMBC map of RAPS-1;

Fig. 5 is the ¹ H-NMR spectrum of RAPS-2;

Fig. 6 is the ¹³ C-NMR spectrum of RAPS-2;

Fig. 7 is the HSQC pattern of RAPS-2;

Fig. 8 is the HMBC map of RAPS-2;

Fig. 9 is the ¹ H-NMR spectrum of Se-RAPS-2;

Figure 10 is the ¹³ C-NMR spectrum of Se-RAPS-2;

Figure 11 is a monosaccharide composition spectrum of alfalfa root polysaccharide;

Figure 12 is the inhibitory effect of alfalfa root polysaccharides and selenized polysaccharides (RAPS-1, RAPS-2, Se-RAPS-2) on HepG-2 tumor cells.

Detailed ways

The present invention will be described below through specific embodiments, but the present invention is not limited thereto. The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and biological materials can be obtained from commercial sources unless otherwise specified.

Example 1 Extraction of polysaccharides from alfalfa roots

Alfalfa root (2.0 kg) was treated with petroleum ether and ethanol under reflux in turn, filtered, the filter residue was collected and extracted with distilled water under reflux for 2 to 6 times, filtered, concentrated under reduced pressure, and the extract was combined. Mix neutral protease and extract, incubate at 40-60 ^o C for 1-3 h, inactivate at 80-100 ^o C for 10-20 min, and centrifuge to obtain supernatant. Sevage reagent (1:2-1:4, v/v) prepared from n-butanol and chloroform was added to the supernatant, and the protein was removed by vigorous stirring, and the supernatant was collected by centrifugation. Use the Sevage method to remove the protein 2 to 5 times. The supernatant obtained above was concentrated, and the AB-8 resin column was decolorized to obtain crude polysaccharide. Crude polysaccharides were separated by DEAE-52 cellulose column and eluted with distilled water and NaCl solutions of different concentrations. The sugar content was determined by the phenol-sulfuric acid method. The obtained fractions were purified by Sephadex G-200 column to collect two fractions (RAPS-1, RAPS-2) with high sugar content, concentrated and dried under reduced pressure to obtain polysaccharides (RAPS-1, RAPS-2). The NMR spectrum (600 MHz, D ₂ O) is shown in Figure 1. It can be seen from ¹ H-NMR that RAPS-1: It can be seen from ¹ H-NMR that the signals at δ 1.23 and 1.38 ppm represent the rhamnosus in DAPS-1. The presence of sugar methyl groups. The signals at δ 2.06, 2.14, 2.22 and 2.29 are due to protons of the acetyl group. The δ 3.39−4.25ppm signal range represents H-2 to H-5 (or H-6) on the glycosidic ring. According to ¹³ C-NMR, δ 16.5 ppm represents the signal of rhamnose methyl group. The signal range of δ 60.7-75.8 ppm represents the non-anomeric carbon atoms in DAPS-2, and the signal range of δ 96.0-107.1 ppm is due to the resonance of C-2 to C-6 in sugar residues. The signal at δ 174.1 ppm indicates the presence of uronic acid in DAPS-1. Combining ¹³ C-NMR and HSQC, δ 5.23/100.8 ppm, 5.13/99.6 ppm, 4.99/100.4 ppm, 5.06/107.1 ppm, 4.60/102.6 ppm and 4.54/104.2 ppm are respectively →2)-α-L-Rhap -(1→,α-D-GalAp-(1→,→4)-β-D-Manp-(1→,→5)-α-L-Arap-(1→,→3)-β-D Correlation signals of terminal hydrogen and terminal carbon of -Xylp-(1→ and →6)-β-D-Glcp-(1→. From HMBC, δ 4.60/78.7 ppm is →3)-β-D- Correlation signals of H-1 and C-3 of Xylp-(1→, δ 5.13/81.3 ppm are H-1 and→2 of α-D-GalAp-(1→)-α-L-Arap-(1→ The correlation signal of C-2, δ 4.54/69.1 ppm is the correlation signal of →6)-β-D-Glcp-(1→H-1/C-6.

RAPS-2: The signals at δ 1.23, 1.35 and 1.46 ppm represent the presence of rhamnose methyl groups in DAPS-2 from ¹ H-NMR. The signals at δ 1.90 and 2.07 are due to protons of the acetyl group. The signal range of δ 3.33−4.26 ppm represents H-2 to H-5 (or H-6) on the glycosidic ring. Signals at δ 4.41, 4.54, 4.55, 4.57 indicate the presence of β-terminal protons; signals at δ 5.08, 5.27, 5.28, 5.77 indicate the presence of α-terminal protons. From ¹³ C-NMR, δ 16.5 and 16.7 represent the signal of rhamnose methyl group. The signal range of δ 65.8-83.4 ppm represents the non-anomeric carbon atoms in DAPS-2, and the signal range of δ 92.1-106.8 ppm is due to the resonance of C-2 to C-6 in sugar residues. Signals at delta 175.0 and 175.3 indicate the presence of uronic acid in DAPS-2. Combining ¹³ C-NMR and HSQC, δ 5.08/103.6 ppm, 5.28/101.6 ppm, 4.95/97.6 ppm, 5.18/101.0 ppm, 4.98/98.7 ppm, 4.64/101.6 ppm and 4.61/103.3 ppm are →3)- α-L-Rhap-(1→, →2)-α-L-Rhap-(1→, →6)-α-D-Galp-(1→, α-D-GalAp-(1→, α- Correlation signals of terminal hydrogen and terminal carbon of D-Glcp-(1→, →3)-β-D-Manp-(1→ and →3)-β-D-Xylp(1→. From HMBC, we can see that, δ 5.08/78.7 ppm is →3)-α-L-Rhap-(1→ Correlation signals of H-1 and C-3, δ 4.95/65.9 ppm is →6)-α-D-Galp-(1→ The correlation signal of H-1/C-6, δ 5.18/70.1 is the correlation of α-D-GalAp-(1→H-1 with →6)-α-D-Galp-(1→C-2 Signal.δ 4.98/81.1 is the correlation signal of α-D-Glcp-(1→H-1 and →3)-β-D-Manp-(1→C-4 of C-4 δ 4.64/69.5ppm and δ 4.61/ 81.1 ppm represent the H-1/C-3 correlation signals of →3)-β-D-Manp-(1→ and →3)-β-D-Xylp(1→, respectively.

Example 2 Synthesis of Selenized Polysaccharide

Take sodium selenite and RAPS-2 in a ratio of 1:0.8～1:1.5 into a round-bottomed flask, add dilute _HNO3 solution, react at 70～90 ^o C for 8～12 h after the reaction is completed, cool to room temperature, The pH of the solution was adjusted to 6-8 with NaOH solution, and small molecular compounds such as sodium selenite were dialyzed with a dialysis membrane, then the dialysis was stopped, and lyophilized to obtain selenized polysaccharide (Se-RAPS-2).

The ¹ H-NMR of Se-RAPS-2 is similar to RAPS-2, the signals at δ 1.24 and 1.30 ppm represent the presence of rhamnose methyl groups in Se-DAPS-2. The signals at δ 2.11 and 2.20 ppm are due to protons of the acetyl group. The signal range of δ3.25−4.13 ppm represents H-2 to H-5 (or H-6) on the glycosidic ring. , in the ¹³ C-NMR of Se-DAPS-2, a new peak at δ 62.5 ppm is assigned to the O-6 substituted carbon atom resonance, representing the reaction of O-6, indicating that the C-6 of RAPS-2 is replaced by selenium Atomic substitution.

Example 3 Determination of polysaccharide and monosaccharide composition of alfalfa roots

Take appropriate amount of RAPS-1 and RAPS-2 samples, dissolve them with a certain concentration of trifluoroacetic acid, and react in an oil bath of 80-120 ^o C for 4-10 h. After the completion of the reaction, use anhydrous methanol with a rotary evaporator to remove the residual trifluoroacetic acid several times. Finally, dissolve the sample with a small amount of acetonitrile: water = 70~90:10~30, and pass through a 0.22 μm organic filter.

Chromatographic conditions: LC-20AR high performance liquid chromatograph;

Chromatographic column: HP-Amino (4.6×250 mm, 5 μm);

Detector: RID-20A refractive index detector;

Mobile phase: A is acetonitrile, B is water, A:B=70～80:20～30;

Column temperature: 20～40 ^o C;

Flow rate: 0.2～0.8 mL/min;

Injection volume: 30～60 μL;

Dissolve the sample with 600 μL acetonitrile: water = 70~90:10~30, and pass through a 0.22 μm organic filter.

The chromatogram is shown in Figure 11. It can be seen from the figure that RAPS-1 is composed of rhamnose (Rha), arabinose (Ara), xylose (xyl), glucuronic acid (GlcA) and glucose (Glc), and the molar ratio is : 1.00:3.19:2.67:5.44:5.10. , DAPS-2 is composed of rhamnose (Rha), arabinose (Ara), glucuronic acid (GlcA), mannose (Man), glucose (Glc) and galactose (Gla), the molar ratio is: 1.21:1.49 :3.49:3.11:2.73:1.00.

Example 4 Determination of molecular weight of alfalfa root polysaccharides and selenized polysaccharides

Weigh out a certain amount of dextran standards and RAPS- 1. Dissolve RAPS-2 and Se-RAPS-2 in 0.5-2 mL of Wahaha pure water, mix thoroughly to obtain a standard solution, which is passed through a 0.22 μm filter membrane for use.

Chromatographic conditions: LC-20AR high performance liquid chromatograph;

Detector: RID-20A refractive index detector;

Chromatographic column: A TSK gel G 5000 PW column (I.D.=7.5mm, L=300mm);

Mobile phase: 0.02～0.05 mol/L KH ₂ PO ₄ ;

Column temperature: 20～40 ^o C;

Flow rate: 0.2～0.8 mL/min;

Injection volume: 30～60 μL;

According to the molecular weight logarithm and peak time of standard dextran, the standard curve was obtained. According to the peak time of polysaccharide and selenized polysaccharide, the molecular weights of RAPS-1, RAPS-2 and Se-RAPS-2 were 10.0 KDa, respectively. 15.8 KDa and 11.0 KDa.

Example 5 Determination of selenium content of selenized polysaccharides

Weigh a certain amount of sample into a conical flask, add 6-12 mL of nitric acid and 2-6 mL of perchloric acid, and place it in the dark for more than 12-20 h at room temperature, then heat and digest at 160-220 ^o C on a heating mantle, wait for When the solution is colorless or slightly yellow, add 6-12 mL of hydrochloric acid (0.5-1.0 mol/mL), continue to heat until the solution is about 1 mL, cool, and do a blank test at the same time. Dilute concentrated hydrochloric acid with distilled water to 6 mol/L, add the digested solution to the colorimetric tube, then add 2-8 mol/L hydrochloric acid, and place it in a constant temperature bath at 70-100 ^o C for 1-3 After h, it was taken out and cooled, diluted with distilled water, and then detected by atomic fluorescence spectrometer. The results showed that the selenium content of Se-RAPS-2 was 320 μg/g.

Example 6 In vitro anti-tumor experiment

Human hepatoma cells (HepG2) in the logarithmic growth phase were taken and placed in RPMI-1640 (containing 10% fetal bovine serum) medium, 5000 cells per well, 100 μL of cell suspension per well were inoculated in 96-well plates in 5% CO ₂ , incubator at 37 °C overnight for 12 h. The supernatant (medium) was then aspirated and discarded. Add 100 μL of culture medium containing polysaccharide samples, the polysaccharide concentrations are 0, 6.25, 12.5, 25, 50, and 100 μM, and each concentration is set to 3 parallels. After culturing for 48 hours, 10 μL of CCK-8 reagent is added. Continue to cultivate for 2 h, and measure the absorbance value of each well at 450 nm with a microplate reader. At the same time, the blank control group was set as no hepatoma cells (HepG2) and polysaccharide samples, only medium and CCK-8 solution. Calculate the cancer cell inhibition rate according to the following formula:

Cancer cell inhibition rate (%)=1-[(A _s -A ₀ )/(A ₁ -A ₀ )]×100%;

In the formula: A _s is the OD value of the experimental group; A ₁ is the OD value of the experimental group whose polysaccharide solubility is 0 μM; A ₀ is the OD value of the blank control group.

The results showed that DAPS-1 and Se-DAPS-2 had a certain inhibitory effect on HepG-2 tumor cells in a concentration-dependent manner. The inhibitory effect of Se-DAPS-2 on HepG-2 tumor cells was higher than that of DAPS-1 and DAPS-2.

Claims (3)

1. the preparation of alfalfa root polysaccharide with biological activity and selenization modified polysaccharide thereof, is characterized in that, its preparation process comprises: (1) Take the dried alfalfa root, extract with petroleum ether and ethanol in turn to remove lipids and small molecules, add distilled water and heat under reflux for 2 to 6 times, filter, and combine the filtrates; after deproteinization and depigmentation, alfalfa crude polysaccharide is obtained The obtained crude polysaccharide was separated by anion exchange column chromatography, eluted with distilled water and 0.1-0.5 mol/L NaCl solution in turn, and 2 new alfalfa root polysaccharides RAPS-1 and RAPS were obtained after gel purification. -2; (2) The high yield polysaccharide RAPS-2 obtained in the above step (1) is selenized modified by the nitric acid-sodium selenite (NA-SS) method to obtain a new selenopolysaccharide Se-RAPS-2. 2. the preparation of biologically active alfalfa root polysaccharide and selenization modified polysaccharide thereof according to claim 1, is characterized in that, the 2 new alfalfa root polysaccharides that make, its structural fragment are respectively: RAPS-1: →2)-α-L-Rhap-(1→,α-D-GalAp-(1→,→4)-β-D-Manp-(1→,→5)-α-L- Arap-(1→,→3)-β-D-Xylp-(1→ and →6)-β-D-Glcp-(1→; RAPS-2: →3)-α-L-Rhap-(1→,→2)-α-L-Rhap-(1→,→6)-α-D-Galp-(1→,α-D- GalAp-(1→,α-D-Glcp-(1→,→3)-β-D-Manp-(1→ and →3)-β-D-Xylp(1→. 3. the preparation of biologically active alfalfa root polysaccharide and selenization-modified polysaccharide thereof according to claim 1, wherein the obtained alfalfa root polysaccharide and selenium polysaccharide both have anti-HepG-2 tumor activity, At a concentration of 100 μM, the inhibitory effects of DAPS-1, DAPS-2 and Se-DAPS- on HepG-2 tumors were 30.35%, 17.81% and 38.70%, respectively.

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