CN113773406A - Preparation method and application of neutral polysaccharide composition of Korean epimedium herb - Google Patents
Preparation method and application of neutral polysaccharide composition of Korean epimedium herb Download PDFInfo
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- CN113773406A CN113773406A CN202010515436.XA CN202010515436A CN113773406A CN 113773406 A CN113773406 A CN 113773406A CN 202010515436 A CN202010515436 A CN 202010515436A CN 113773406 A CN113773406 A CN 113773406A
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Materials Engineering (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Sustainable Development (AREA)
- Toxicology (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a preparation method and application of a neutral polysaccharide composition of Korean epimedium. The epimedium koreanum neutral polysaccharide composition consists of 9.7 mol percent of rhamnose, 66.0 mol percent of glucose, 17.5 mol percent of galactose and 6.8 mol percent of arabinose, and the molecular weight is 21900 Da. The epimedium koreanum neutral polysaccharide composition has antioxidant activity, can eliminate DPPH and OH free radicals in vitro, has good metal ion chelation and reduction capacity, and can repair an antioxidant system of a D-galactose-induced aging mouse.
Description
Technical Field
The invention relates to a preparation method and application of a neutral polysaccharide composition of Korean epimedium, wherein the neutral polysaccharide composition of Korean epimedium has an antioxidant effect.
Background
Korean epimedium (E. koreanumNakai) is one of the unique epimedium herb resources in northeast China, has long medicinal history, and has the functions of tonifying kidney yang, strengthening bones and muscles and dispelling wind-damp. The literature reports its chemistryThe ingredients comprise flavone, polysaccharide, alkaloid, lignan, terpenoid and the like, but the research mainly focuses on flavonoid compounds, and the reports on the polysaccharide of the epimedium koreanum herb are very few so far.
Polysaccharides are a class of biomacromolecules ubiquitous in biological organisms, and are formed by polymerization of more than 10 monosaccharide molecules (usually, several hundred or even several thousand monosaccharide molecules), and not only participate in the formation of cytoskeletons, but also are important components of various endogenous bioactive molecules. With the research of polysaccharide, the polysaccharide is found to have complex structure and diverse activity, such as cell division and differentiation regulation, and has pharmacological actions of immunoregulation, anti-tumor, antivirus, antiulcer, antioxidation and the like. The structure of polysaccharides is surprisingly diverse, complex and microscopically heterogeneous, and the primary structure includes not only the order of arrangement of the individual glycosyl groups, but also the cyclic form of the individual glycosyl groups, the configuration of the individual glycosyl groups themselves as anomeric bodies, the manner of linkage between the individual glycosyl groups, and the sites of branching structures and the structure of branched sugar chains. Polysaccharides are good pharmaceutical resources and have become a scientific hotspot behind proteins and nucleic acids.
Disclosure of Invention
As described above, there are relatively few studies on the polysaccharide of Epimedium koreanum nakai at home and abroad, and the present invention has been made a more intensive study for searching the polysaccharide component having an application value in Epimedium koreanum nakai.
The invention aims to provide a preparation and structure characterization method of a Korean epimedium neutral polysaccharide composition with antioxidant activity, and the method is simple, simple and convenient to operate and easy to implement.
The purpose of the invention is realized by the following steps:
1. the epimedium koreanum neutral polysaccharide composition is characterized in that monosaccharide composition of the polysaccharide comprises glucose, rhamnose, galactose and arabinose, and the average molecular weight distribution of the polysaccharide is 21900 Da.
2. The neutral polysaccharide composition of epimedium koreanum according to claim 1, comprising repeating units of sugar residues of the formula.
3. The method for preparing the neutral polysaccharide composition of epimedium koreanum nakai as claimed in claims 1-2, which comprises the following steps:
a, pulverizing Korean epimedium to obtain Korean epimedium medicinal material powder;
b, extracting the epimedium koreanum medicinal material powder by 90% ethanol, and then filtering to obtain residues;
c, extracting the residue with distilled water, concentrating, and freeze-drying to obtain dry powder;
d, dissolving the dried powder obtained in the step C in distilled water, adding Sevag reagent to remove protein, and freeze-drying to obtain dried powder;
e, dissolving the dried powder obtained in the step D in distilled water, and dialyzing to obtain the crude polysaccharide of the Korean epimedium;
and F, dissolving the crude polysaccharide obtained in the step E in distilled water, and loading the solution onto an ion exchange chromatography column and a molecular sieve chromatography column to obtain the neutral polysaccharide composition of the Korean epimedium herb.
4. The use according to claims 1-2, wherein the antioxidant activity is free radical scavenging ability, metal ion chelating ability, reducing ability; enhancing SOD, CAT, GSH-PX and T-AOC enzyme activities of D-galactose induced oxidative damage mice, and reducing MDA level.
The epimedium koreanum neutral polysaccharide composition can be prepared by the following specific method.
1. Removing pigments and small molecular substances: pulverizing epimedium koreanum, extracting with 30 times (w/w) of 90% ethanol at 100 deg.C for 60-120 min, filtering the extract with filter cloth (120 mesh), extracting the filtered residue twice under the same conditions, collecting the residue, and volatilizing ethanol.
2. Extracting crude polysaccharide of Korean epimedium: adding 30 times (v/v) of distilled water into the residue, and extracting at 100 deg.C for 3 times for 60, 120, and 180 min. The extract was filtered through filter cloth (120 mesh), the filtrates obtained by three extractions were combined, centrifuged, the supernatant was concentrated, and freeze-dried to obtain a powder, which was dissolved in distilled water, followed by addition of Sevag reagent [ chloroform: n-butanol =4:1 (v/v) ], centrifuging, concentrating the supernatant, and freeze-drying to obtain a powder.
3. Separation of polysaccharide of Korean epimedium: dissolving the above dry powder with 1-5 times of distilled water, dialyzing the solution, preferably for 48 hours, and using dialysis bag with recommended cut-off molecular weight below 3500 Da. Concentrating the dialyzate, and freeze-drying to obtain crude polysaccharide of Epimedium koreanum nakai.
4. Purifying crude polysaccharide of Korean epimedium: dissolving crude Epimedium koreanum polysaccharide with 2 times of distilled water, centrifuging (10000 rpm/min) for 10 min, and collecting supernatant on DEAE-cellulose (Cl)-) Eluting with 2 times of column volume of distilled water, collecting eluate, concentrating, and freeze drying to obtain neutral polysaccharide of Korean herba Epimedii; dissolving the epimedium koreanum neutral polysaccharide with 2-5 times of distilled water, centrifuging (10000 rpm/min) for 10 minutes, loading the supernatant into a Sepharose CL-6B column (2.5 multiplied by 100 cm), eluting with 2 times of column volume of distilled water, collecting the eluent, concentrating and freeze-drying to obtain the epimedium koreanum neutral polysaccharide composition.
Effects of the invention
The epimedium koreanum neutral polysaccharide composition extracted by the invention can eliminate DPPH free radicals and hydroxyl free radicals in vitro, has metal ion chelating capacity and reducing capacity, can enhance the enzyme activities of SOD, CAT, GSH-PX and T-AOC of a mouse damaged by oxidation induced by D-galactose in vivo, and reduces the MDA level, thereby having the antioxidation effect.
FIG. 1 is a high performance gel permeation chromatography and high performance liquid chromatogram of the neutral polysaccharide composition of Epimedium koreanum nakai.
FIG. 2 is a graph showing the monosaccharide composition analysis of the neutral polysaccharide composition of Epimedium koreanum.
FIG. 3 is a Fourier-Infrared (FT-IR) spectrum of a neutral polysaccharide composition of Epimedium koreanum.
Detailed Description
The specific implementation mode of the invention uses a TU-1810 ultraviolet spectrophotometer of Beijing Pujingyo company; a Diane UniMate 3000 gel permeation chromatograph, TSK-G3000 PWXL chromatography column; an agent RRLC 1200 SL liquid chromatograph, a DIKMA Inertsil ODS-3 chromatographic column and a UV-VIS DAD detector, wherein the mobile phase is PBS (0.1M), the pH value is 7.0, the flow rate is 1.0 mL/min, the sample injection amount is 20 mu L, and the detection wavelength is 245 nm; thermo Scientific TSQ 8000 triple quadrupole GC-MS from Thermo Fisher corporation; a Bruker corporation's Vertex 7.0 infrared spectrometer; bruker-600 FT-NMR spectrometer from Bruker.
Example 1
Weighing 500g of crushed dry Korean epimedium medicinal material, adding 90% ethanol, heating and refluxing to remove pigment and other small molecules. Adding distilled water (1: 30) into the residue, extracting for three times (1 h, 2h and 3 h) in boiling water bath, filtering the hot extract (120 meshes), and collecting the filtrate obtained by three times. The filtrate was concentrated (60-80 ℃ C.), cooled and centrifuged (4000 rpm/min) for 10 minutes, the supernatant was collected, ethanol was added thereto until the final alcohol concentration of the solution reached 80%, and after standing overnight, centrifuged (4000 rpm/min) for 10 minutes, the precipitate was collected, and freeze-dried to obtain a powder (82.65 g).
Dissolving the above dried powder in distilled water, adding Sevag reagent (chloroform: n-butanol =4:1, v/v), vigorously shaking the mixed solution for 120 min, standing for layering, centrifuging, retaining supernatant, adding Sevag reagent again, repeating the above steps for 5 times, concentrating the supernatant under reduced pressure, adding anhydrous ethanol (1: 4, v/v), precipitating with ethanol overnight, and centrifuging.
The reaction solution was dialyzed for 48 hours using a dialysis bag having a molecular weight cutoff of 3500 Da. The dialyzed external solution was collected, concentrated under reduced pressure at 60 ℃ and freeze-dried to obtain a dry powder (29.45 g).
Weighing 10 g of the above dry powder, dissolving in 10 ml of distilled water, centrifuging, collecting supernatant, and loading on DEAE-cellulose (Cl)-) Column (7.0 × 30 cm), eluting with distilled water, collecting eluate at flow rate of 10 mL/min, collecting 12 mL per tube, and purifying with phenol-sulfuric acidThe elution was collected, concentrated and lyophilized to give EFPN (5.54 g).
Weighing 1 g of the above dry powder, dissolving in 10 mL of distilled water, centrifuging, loading the supernatant onto a Sepharose CL-6B column (2.5 multiplied by 100 cm), collecting the eluent at a flow rate of 10 mL/20 min, collecting 10 mL of the eluent in each tube, collecting the eluent, tracking and monitoring by a phenol-sulfuric acid method, analyzing to obtain a uniform narrow symmetrical elution peak with a polydispersity index of 1.1, concentrating, and freeze-drying to obtain EFPN-1 (0.60 g).
10mg of the EFPN-1 dry powder was dissolved in 10 mL of distilled water, and 20. mu.L of this solution was sampled and applied to an HPGPC system with distilled water as a mobile phase at a flow rate of 1 mL/min. The standard substance is a series of standard polysaccharides with molecular weight of 1000, 5000, 12600, 60600, 80000, 100000, 150000 Da. From the elution time, the molecular weight of EFPN-1 was found to be 21900 Da, and the molecular weight was found to be uniform.
The HPLC measurement conditions were as follows:
a chromatographic column: TSK-G3000 PWXL (7.8 mm ID X30.0 cm L)
A detector: RID-10A
Mobile phase: deionized water
Flow rate: 1.0 mL/min
Sample introduction amount: 10 μ L
Preparing 0.1 mg/ml solution from the EFPN-1 dry powder, carrying out ultraviolet spectrum scanning in the range of 190-290 nm, and finding no absorption peak at the wavelength of 260 nm and 280 nm, thereby knowing that no protein and nucleic acid exist in the EFPN-1.
Taking EFPN-1 dry powder, measuring the total sugar content by a phenol-sulfuric acid method to be 91.43%, measuring the protein content by a Coomassie brilliant blue method to be less than 1%, and measuring the phenol substances in the EFPN-1 by a Fulinol kit method.
EFPN-1 dry powder is taken, and subjected to complete acid hydrolysis and HPLC. Four elution peaks were analyzed and from the elution times, the composition consisted mainly of glucose (66%), and also rhamnose (9.7%), galactose (17.5%), arabinose (6.8%).
The HPLC measurement conditions were as follows:
a chromatographic column: DIKMA Inertsil ODS-3 (4.6 mm. times.150 mm)
A detector: UV-VIS DAD
Detection wavelength: 245 nm
Mobile phase: PBS (0.1M, pH 7.0): acetonitrile =82:18 (v/v), pH 7.0
Flow rate: 1.0 mL/min
Sample introduction amount: 10 μ L
Taking EFPN-1 dry powder, tabletting with KBr at 4000-400 cm-1The infrared spectrum scanning is carried out in the range of (1), and the data of the infrared spectrogram are recorded as follows: 3385cm–1And 1641cm–1The peak of the OH-treated product is in the range of 2931 cm-1Shows a CH stretching vibration absorption peak at 1422cm–1And 1042cm–1The strong absorption peaks at (B) are shown as the stretching vibration peaks of C-H and C-O, and further, 917cm–1Shows a strong absorption peak containing a large number of beta-glycosidic linkages, 815cm–1The weak absorption peak of (a) indicates the presence of an alpha-glycosidic bond.
Based on GC-MS analysis, EFPN-1 contains 8 different monosaccharide residues. Further, from the ion fragment pattern and relative retention time analysis of each monosaccharide residue, these 8 monosaccharide residues are (1 →) -Ara, respectivelyf、(1→)-Glcp、(1→)-Galp、(1→3)-Rhap、(1→4)-Glcp、(1→3)-Galp、(1→3,6)-GalpAnd (1 → 4,6) -Glcp(ii) a The molar ratio of the 8 monosaccharide residues is 7.90:8.15:12.40:1.78:44.10:6.96:7.60:11.11, (1 → 4) -Glc according to the peak area calculationpThe molar percentage of residues was 44.10%, indicating that it may be a backbone portion constituting EFPN-1, with branches consisting primarily of (1 → 4,6) -GlcpAnd (1 → 3,6) -GalpConstituted by a molar ratio of about 2:3, the terminal polysaccharide was mainly constituted by Ara, Glc and traces of Gal, with a total content of 22.10%, almost comparable to the content of branched residues (18.71%). Based on the branching content, terminal residues and linear residues, the branching Degree (DB) of EFPN-1 was calculated to be 40.40%. Thus, it was concluded that EFPN-1 is a branched polysaccharide consisting of five (1 → 4) -Glc branchespResidue repeat units.
The GC-MS measurement conditions were as follows:
a chromatographic column: DIKMA Inertsil ODS-3 (4.6 mm. times.150 mm)
A detector: UV-VIS DAD
Detection wavelength: 245 nm
Mobile phase: PBS (0.1M, pH 7.0): acetonitrile =82:18 (v/v), pH 7.0
Flow rate: 1.0 mL/min
Sample introduction amount: 10 μ L
Chromaticness online AGILENT 5975 GC/6890MS
A chromatographic column: HP-1 Quartz capillary column (23 m × 0.25 mm × 0.25 nm)
A sample inlet: 250 deg.C
An ion source: 280 deg.C
Temperature programming: holding at 120 ℃ for 5 minutes; 120 ℃ → 280 ℃ (5 ℃/min) for 2 minutes.
Detection range: 30-400 m/z
Table 1 GC-MS analysis of methylation of neutral polysaccharide composition of epimedium koreanum.
From the results of 1D and 2D NMR analyses of EFPN-1, it was found that EFPN-1 consists of 7 sugar residues, designated A-G, and is α -L-Ara, respectivelyf-(1→、→3)-α-L-Rhap-(1→、α-D-Glcp-(1→、→4)-α-D-Glcp-(1→、→3)-β-D-Galp-(1→、→4, 6)-α-D-Glcp- (1 → and → 3,6) -beta-D-Galp- (1 →. however, in the NMR spectrum, t-Gal was not foundpInformation on residues, which may be due to the content of residues in EFPN-1 being less than 5% (1.78%).
From EFPN-11H NMR spectrum showed that D was observed2The inhibitory effect of O, so that some of the anomeric protons of the polysaccharide have characteristic signal peaks with D2O is coincident with and is covered by D2And covering by O. In the anomeric hydrogen region, a weaker characteristic peak appears at 5.04-5.18 ppm, and the signal is classified as alpha-ArafAnd alpha-RhafAnomeric proton signal of the residue. Signals at 4.20-4.57 ppm indicate that the EFPN-1 contains beta-configuration glycosidic bonds. RhapIn residue-CH3The chemical shift of upper H was 1.27 ppm.13In the C NMR spectrum, RhapThe chemical shift of C6 in the residue is 16.8 ppm, the signal peak of delta 96.00-109.19 ppm is the characteristic peak of anomeric carbon atom of sugar residue, and the characteristic peak of C2-C5 of these residues appears in delta 60.00-84.00 ppm range.
Residue A: in that1The characteristic peak of the anomeric proton appears at delta 5.18 ppm in the H NMR spectrum, and the signal of the anomeric carbon corresponding to the anomeric proton is delta 109.19 ppm according to the HSQC spectrum, which proves that the residue is in alpha-configuration. Further analysis of the figure shows that the peaks of the HSQC map with the cross peaks of 4.14/81.9 ppm, 3.93/76.35 ppm, 4.06/83.67 ppm and 3.57/62.45 ppm are respectively assigned to the residues of H2/C2, H3/C3, H4/C4 and H5/C5, and the residue A is judged to be alpha-L-Ara according to the chemical shift values reported in the literaturef。
Residue B: δ 4.95 ppm of an anomeric proton signal peak assigned to residue B, demonstrating that residue B is an α -pyrano rhamnose residue. From the COSY spectrum, a cross signal peak of delta 4.95/delta 4.16 ppm was observed, and it was found that the chemical shift of H-2 at residue B was delta 4.16 ppm. From this we can deduce the chemical shifts of the residues H-2, H-3, H-4, H-5 and H-6 in turn. Meanwhile, according to C-H related peaks in the HSQC map, the chemical shift values of C-2-C-6 of the residue B can be obtained. Among them, the cross peak delta 3.99/76.78 ppm is ascribed to the H3/C3 cross signal peak of the residue, so that the residue B is deduced as → 3) -alpha-L-Rhap-(1→。
Residue C, D, and F: by pairs1H. COSY spectrum analysis judges that the chemical shifts of anomeric protons of C, D and F of the residues are respectively delta 4.90 ppm (C), delta 5.04 ppm (D) and delta 4.92 ppm (F), and further by HSQC spectrum, according to the cross peaks of H1-C1, H2-C2 … … H6-C6, the relative shifts of anomeric carbons of residues C, D and F are respectively delta 100.17 ppm (C), delta 99.29 ppm (D) and delta 100.24 ppm (F). The results of the analyses of anomeric protons and anomeric carbons revealed that all of these 3 residues were α -glucopyranose residues. Similarly, the carbon and hydrogen signals at other positions of these 3 residues were determined and assigned. According to the literature report, the residue C is t-alpha-D-Glcp. δ 76.35 ppm of C4 of residue D, andas a result of the shift to a low magnetic field, the residue D can be determined to be → 4) -alpha-D-Glcp- (1 →. similarly,. delta.76.35 ppm and. delta.67.87 ppm are shifts to the low field due to substitution of C4 and C6 of the residue C, and therefore the residue F is presumed to be → 4,6) - α -D-Glcp-(1→。
Residues E and G:1residues E and G are in the beta-configuration as evidenced by delta 4.57 ppm and delta 4.45 ppm in H and COSY maps. According to HSQC map analysis, the H1/C1 cross peaks at residues E and G are delta 4.57/103.86 ppm and delta 4.45/102.93 ppm, and the cross signal peak delta 3.68/80.02 ppm is assigned to the H3/C3 at residue E. According to the literature, beta-D-GalpThe chemical shift of C3 in (b) is δ 71 to 73 ppm, and it is understood that the residue E is → 3) - β -D-Gal when C3 of the residue E is substituted and moves to the earth's magnetic fieldp- (1 →, similarly, it was judged that the residue G was 3,6) - β -D-Gal because C3 (δ 80.96 ppm) and C6 (δ 67.0 ppm) were substituted in the residue Gp-(1→。
TABLE 2 neutral polysaccharide composition of Epimedium koreanum1HNMR and13chemical shift of CNMR spectroscopic signals
The order of attachment of the 7 residues (A-G) of EFPN-1 can be determined from HMBC mapping results. Based on HMBC mapping, we found peaks of crossing signals for AH1-EC3, CH1-BC3 and CH1-GC3, from which it was concluded that EFPN-1 contains the following sugar residue building blocks: → 3) -beta-D-Galp-(1→α-L-Araf、→3)-α-L-Rhap-(1→α-D-GlcpAnd 4) -alpha-D-Glcp-(1→α-D-Glcp. Similarly, in HMBC map, we find out the cross signal peaks of DH1-DC4, DH1-FC4, EH1-GC6, BH1-GC3 and FH1-DC4, and prove that EFPN-1 contains the following sugar residue structural unit → 4) -alpha-D-Glcp-(1→4,6)-α-D-Glcp、→3)- β-D-Galp-(1→3, 6)-β-D-Galp、→3)-α-L-Rhap-(1→3)-α-L-Rhap。
Test of drug efficacy
An in vitro antioxidant activity test was performed using the neutral polysaccharide composition of epimedium koreanum prepared in example 1.
1. In vitro antioxidant experiment of neutral polysaccharide composition of Korean epimedium
1.1 hydroxyl radical scavenging experiments
0.1 ml of the EFPN-1 solution was mixed with 0.6 ml of a reaction buffer [ 0.2M phosphate buffer (pH = 7.4) 2.67 mM deoxyribose and 0.13 mM EDTA ], 0.2 ml of 0.4 mM ferrous sulfate, 0.05 ml of 12 mM vitamin C and 0.05 ml of 20 mM hydrogen peroxide to prepare a reaction solution. The mixed solution was water-bathed at 37 ℃ for 15 minutes, and then 1 ml of 1% thiobarbituric acid and 1 ml of 2.0% trichloroacetic acid were added. The mixture was boiled in a water bath for 15 minutes, cooled in ice and the absorbance A sample was measured at 532 nm. Distilled water was used as a blank solution a blank and vitamin C was used as a positive control. Lower absorbance values measured indicate higher scavenging capacity. And half maximal inhibitory concentration IC50 values were calculated.
The ability to scavenge hydroxyl radicals is calculated using the following formula:
the clearance S (%) = (1-sample absorbance/control absorbance) × 100%.
As shown in Table 1, EFPN and EFPN-1 have good clearing effect within the range of 1-8 mg/mL, and are dose-dependent, and the IC50 values are respectively 6.55 mg/mL and 1.54 mg/mL. EFPN and EFPN-1 reach maximum values at 8 mg/mL, wherein the EFPN-1 has the best effect, the inhibition rate reaches 90.05 percent, and the clearance rate is almost close to that of Vc (92.46 percent). EFPN-1 has a stronger hydroxyl radical scavenging ability than EFPN over the range of the tested dose.
Table 3. korean epimedium neutral polysaccharide composition hydroxyl radical scavenging ability.
DPPH free radical scavenging experiments:
1.5 ml of 0.1 mmol/L DPPH solution was added to this solution 4.5 ml of EFPN-1 solution, and mixed well with shaking. After 30 minutes, the absorbance value A of the sample was determined at 517 nm. Lower absorbance values of the reactants represent higher radical scavenging capability. Distilled water was used as a blank for the blank solution a. Vitamin C was assayed as a positive control. And half maximal inhibitory concentration IC50 values were calculated. The ability to clear DPPH is calculated using the following equation:
the ability to scavenge DPPH radicals is calculated using the following formula:
clearance S (%) =1- (a sample/a blank) × 100%.
As can be seen from Table 2, the scavenging ability of EFPN and EFPN-1 against DPPH radicals is concentration-dependent within the range of 0.05-0.4 mg/mL. The maximum values of the clearance rates of Vc, EFPN and EFPN-1 are 76.68%, 53.80% and 71.83% respectively, the clearance rate of EFPN-1 is almost close to that of Vc, and the IC50 is 0.98, 0.18 and 0.12 mg/mL respectively. EFPN-1 has greater antioxidant activity than EFPN over the range of the tested dose.
Table 4 DPPH radical scavenging ability of neutral polysaccharide composition of epimedium koreanum.
Experiment of chelating ability of Metal ion
1 ml of EFPN-1 solution, 3.7 ml of methanol and 0.1 ml of 2 mM FeCl were added2·4H2O, 0.2 ml of 5 mM felodizine was added, and the absorbance value was measured at 562 nm immediately after 10 minutes of reaction at room temperature. Lower absorbance indicates greater ability to chelate ferrous ions. The assay was performed with distilled water as a blank solution and vitamin C as a positive control solution. And half maximal inhibitory concentration IC50 values were calculated.
The metal ion chelating ability was calculated by the following equation:
clearance S (%) =1- (a sample/a blank) × 100%.
As shown in Table 3, the concentration of EFPN was 0-5 mg/mL in Fe2+The chelating rate is 40.62% -64.40%, the EFPN-1 is 37.54% -85.25%, and the IC50 is 1.32 mg/mL and 1.15 mg/mL respectively. The maximum chelating efficiency of EDTA was 97.40% at a concentration of 1.5 mg/mL. Thus EFPN-1 has a stronger chelating activity than EFPN.
Table 5 chelating ability of the epimedium koreanum neutral polysaccharide composition to metal ions.
Reduction Capacity (FRAP) experiment
FeSO4Drawing a standard curve: taking FeSO4·7H25ml of O solution is fixedly contained in a 50 ml volumetric flask, namely 8 mmo1/L FeSO4And (4) standard solution. Preparing 7.2, 6.4, 5.6, 4.8, 4.0, 3.2 and 0.8 mmol/L standard solutions in sequence, and drawing a standard curve.
0.3 ml of EFPN-1 solution is taken, 2.7 ml of FRAP working solution preheated to 37 ℃ is added, after shaking up, the mixture is placed for 10 minutes, and the absorbance value A of the mixture is measured at the wavelength of 593 nm. Distilled water was used as the blank solution a. According to the absorbance value after reaction, the corresponding FeSO is obtained on the standard curve4 Is defined as the FRAP value. The greater the FRAP value, the greater the antioxidant activity.
The standard curve of ferrous sulfate is y =0.0865x +0.0971, R2= 0.9994. Calculating the antioxidant capacity of the sample according to a linear standard curve, and calculating the FeSO per gram of the sample4The equivalent weight is expressed. FRAP values for EFPN and EFPN-1 (5 mg/mL) were 4.40 mm FeSO respectively4(g) and 7.04 mm FeSO4The EFPN reducing power is lower than that of EDTA (0.04 mg/mL, 5.58 mmFeSO)4/g), the EFPN-1 reducing capability is higher than that of EDTA, which shows that the EFPN-1 has stronger reducing capability.
2. Research on in-vivo antioxidant activity of neutral polysaccharide composition of Korean epimedium herb
The model making method of the D-galactose animal model with aging is simple, convenient and feasible, has low price and relatively stable result, and becomes a recognized animal model with aging. D-galactose can cause oxidative stress injury in animals, and therefore an index of antioxidation is further determined on the basis of this model.
2.1 model building
Male Kunming mice (20 + -2 g, 8 weeks old), 21 + -1 deg.C and 50-60% relative humidity, freely ingest food, water and maintain a light/dark 12 hour circulating environment. The mice are randomly divided into six groups, including a normal group (gavage 15mL/kg of physiological saline), a D-galactose model control group (intraperitoneal injection of D-galactose 1.35g/kg and gavage 15mL/kg of physiological saline), a positive control group (intraperitoneal injection of D-galactose 1.35g/kg and gavage Vc 100mg/kg), experimental groups 10, 50 and 100mg/kg (intraperitoneal injection of D-galactose 1.35g/kg, and respective gavage of Korean epimedium neutral polysaccharide compositions 10, 100, 200 and 400 mg/kg), and are continuously gavage for 42 days.
2.2 detection of enzymatic Activities and MDA levels of SOD, CAT, GSH-Px, T-AOC
Serum was obtained by centrifuging (4000 rpm, 10 minutes) after blood was collected from the eyeballs of all mice 24h after the last administration. The liver was removed rapidly, homogenized to a 10% (w/v) homogenate after washing with physiological saline, centrifuged (4000 rpm, 10 minutes) to remove cell debris, and the supernatant was collected. The enzymatic activities of SOD, CAT, T-AOC, GSH-Px and the MDA level in the serum and liver homogenates were determined according to the procedures of the kit instructions.
Tables 4 and 5 show that the enzyme activities of SOD, CAT, GSH-Px and T-AOC and the MDA levels in mice with serum and liver homogenates are obviously inhibited and the MDA levels are increased compared with those in a blank group (the statistical significance is shown in the table (A) (B))P<0.01 orP<0.05), which shows that the subacute aging model prepared by injecting large dose D-Gal into the abdominal cavity is successful; the EFPN-1 administration groups increased the enzyme activities of SOD, CAT, GSH-Px, and T-AOC in the serum and liver of mice to different degrees compared with the model group (P<0.01 orP<0.05), MDA is reducedP<0.01 orP<0.05), indicating that EFPN-1 has obvious antioxidant activity in vivo, wherein the effect of the group administered with 10mg/kg is most remarkable (P<0.01)。
TABLE 6 Effect of Epimedium koreanum neutral polysaccharide composition on serum SOD (U/mL), CAT (U/mL), T-AOC (U/mL), GSH-Px (U/mL) and MDA (nmol/mL) levels in oxidatively damaged mice
a P<0.05 vs. negative control;b P<0.01 vs. negative control.
TABLE 7 Effect of Epimedium koreanum neutral polysaccharide composition on the levels of SOD (U/mg protein), CAT (U/mg protein), T-AOC (U/mg protein), GSH-Px (U/mg protein) and MDA (nmol/mg protein) in liver homogenates of oxidatively injured mice
a P<0.05 vs. negative control;b P<0.01 vs. negative control.
Claims (5)
1. A neutral polysaccharide composition of epimedium koreanum, wherein the monosaccharide composition of the polysaccharide comprises 9.7% of rhamnose, 66.0% of glucose, 17.5% of galactose and 6.8% of arabinose, and the average molecular weight of the polysaccharide is 21900 Da.
2. The neutral polysaccharide composition of epimedium koreanum according to claim 1, comprising repeating units of sugar residues of the formula.
3. The method for preparing the neutral polysaccharide composition of epimedium koreanum nakai as claimed in claims 1-2, which comprises the following steps:
a, pulverizing Korean epimedium to obtain Korean epimedium medicinal material powder;
b, extracting the epimedium koreanum medicinal material powder by 90% ethanol, and then filtering to obtain residues;
c, extracting the residue with distilled water, concentrating, and freeze-drying to obtain dry powder;
d, dissolving the dried powder obtained in the step C in distilled water, adding Sevag reagent to remove protein, and freeze-drying to obtain dried powder;
e, dissolving the dried powder obtained in the step D in distilled water, and dialyzing to obtain the crude polysaccharide of the Korean epimedium;
and F, dissolving the crude polysaccharide obtained in the step E in distilled water, and loading the solution onto an ion exchange chromatography column and a molecular sieve chromatography column to obtain the neutral polysaccharide composition of the Korean epimedium herb.
4. The use of the neutral polysaccharide composition of Epimedium koreanum nakai of claims 1-2 in the preparation of a medicament having antioxidant activity.
5. The use according to claim 4, wherein the antioxidant activity is DPPH, OH radical scavenging ability, metal ion chelating ability, reducing ability; enhancing the activities of SOD, CAT and GSH-PX of mice with oxidative damage induced by D-galactose and reducing the MDA level.
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