CN109303922A - A kind of Polysaccharides from Rosa roxburghii functionalized nano selenium compound and preparation method thereof and the application in hypoglycemic medicine - Google Patents
A kind of Polysaccharides from Rosa roxburghii functionalized nano selenium compound and preparation method thereof and the application in hypoglycemic medicine Download PDFInfo
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Abstract
The invention discloses a kind of Polysaccharides from Rosa roxburghii functionalized nano selenium compounds and preparation method thereof and the application in hypoglycemic medicine, belong to nanometer selenium preparation technical field.Then vitamin c solution is added, after mixing evenly oscillating reactions the following steps are included: sodium selenite solution is uniformly mixed by (1) with Polysaccharides from Rosa roxburghii solution in the preparation method；(2) reaction solution obtained by step (1) is dialysed, is dried to get Polysaccharides from Rosa roxburghii functionalized nano selenium compound is arrived.The present invention is using sodium selenite as selenium source, vitamin C is reducing agent, Polysaccharides from Rosa roxburghii (RTFP-3) is coating material, green prepares surface-functionalized nano selenium compound (RP3-SeNPs), prepare that stability is good, particle diameter distribution is narrow, the purposes of the nanometer selenium polysaccharide compound of good dispersion and Polysaccharides from Rosa roxburghii nano selenium compound in the drug that preparation inhibits beta Cell of islet impaired.
Description
Technical field
The invention belongs to nanometer selenium preparation technical field, in particular to a kind of Polysaccharides from Rosa roxburghii functionalized nano selenium compound and
Preparation method and the application in hypoglycemic medicine.
Background technique
With socio-economic development and the improvement of people's living standards, diabetes morbidity increases increasingly, disease incidence and
The death rate has been located in the 3rd of uninfection, and especially diabetes B crowd quantity increase is rapider.Diabetes B
One of inducement is exactly that the reduction of human islet cell quantity or afunction cause amount of insulin secretion to reduce, so as to cause people
Blood glucose balance state be disturbed.It having now been found that, free radical is one of the major incentive for leading to pancreaticβ-cell damage and apoptosis,
In normal human, free radical is in dynamic balance state, but due under the stimulation such as extraneous poor environment and auto-inflammatory,
Excessive free radical (ROS, RNS etc.) can attack normal cell in vivo.Under oxidative stress status, the group of normal cell
At can change, such as the ingredients such as DNA, protein and lipid of intracellular organic matter change, and inducing cell damages
And apoptosis.Based on the relationship between oxidative stress, beta cell apoptosis and diabetes, research finds to inhibit the oxidation of pancreaticβ-cell
Stress situation is one of the effective ways for treating diabetes.Multinomial research simultaneously also indicates that supplement antioxidant can be removed certainly
By base, the oxidative stress status of beta cell can control, have the function that protect pancreaticβ-cell.
Selenium is one of the essential trace elements of the human body, and selenium, which has, to be improved the immunity of the human body, is anti-oxidant, delaying senescence and preventing
The generation of the effect of cancer, anticancer, growth, development and disease with human and animal is closely related.Studies have shown that selenium deficiency can cause
For example the various diseases such as cancer, heart disease, arthritis and immune system dysfunction, appropriate selenium-supply can prevent the generations such as diabetes
It thanks to class disease, reduces the disease incidence and complication of diabetes.Research shows that selenium can participate in body as antioxidant, into human body
The synthesis (such as Glutathione peroxidase) of interior antioxidase system, can remove intracorporal free radical, rapidly so as to have
Damage of the effect defence free radical to cell, there is the function of anti-aging.Currently, the supplement of selenium is often with sodium selenite, sodium selenate, seleno
The form of methionine makes an addition in numerous food product replenishers.Selenium nano particles (SeNPs) because its bioavilability height, toxicity it is low and
The significant characteristic of bioactivity and become new research hotspot.Chinese invention patent CN104310319B discloses a kind of nanometer selenium
Preparation method, selenite is reacted under the action of stabilizer with reducing agent, by changing selenite and reducing agent
Proportion, wherein the molar concentration of selenite solution is that 15~25 mM accurately control nanometer selenium grain in this concentration range
Diameter.Chinese invention patent CN104825484B discloses a kind of preparation method of nanometer selenium, this method using sodium selenite as selenium source,
Potassium iodide is stabilizer, and ascorbic acid is reducing agent, and chitosan or carboxymethyl chitosan are coating material, successfully prepare surface
The nano selenium compound of functionalization, particle size range is in 50 nm or so.Research shows that the bioavilability of selenium, bioactivity with
The change of its nanometer particle size and differ greatly, the surface usually synthesized does not have the SeNPs easily aggregate and precipitate of functionalization, this has become
For the bottleneck of limitation SeNPs application.Therefore, it prepares that partial size is small, SeNPs of narrowly distributing, and function is carried out to its surface using polysaccharide
Energyization modification, to obtain the good selenium nano particles of stability, becomes the hot spot studied instantly.
Rosa roxburghii Tratt is also eaten by people as Chinese medicine as the medical and edible dual purpose plant with region feature.Traditional medicine is main
It is used as medicine with roxburgh rose root, leaf and fruit etc., there is digestion-promoting spleen-invigorating, antidiarrheal, relieving summer-heat and other effects, for treats having indigestion abdominal distension, high blood
The diseases such as rouge, enteritis, hypertension, yctalopia, vitamin C deficiency have certain effect.Polysaccharide is as in fruit of Grossularia burejensis Berger
One of important component by decolourizing, takes off albumen and DEAE-Sepharose Fast Flow column by using Hot water extraction
Chromatography goes out a kind of homogeneous components RTFP-3, and molecular weight is uniform, and structure is clear.Obtained Polysaccharides from Rosa roxburghii RTFP-3 its molecular weight
For 67.2 kDa, which is made of glucose, galactolipin, arabinose, xylose, fucose etc., and main sugar glycosidic bond has respectively
33.21% (1 → 5)-arabinose, 35.72% (1 → 6)-galactolipin, 4.27% (1 → 4)-glucose, (the 1 of 17.22%
→ 3,4)-fucose.It meanwhile further including small part end group xylose and (1 → 3,6)-mannose.Regulate and control at present about Polysaccharides from Rosa roxburghii
Preparing the report with the nanometer selenium for protecting pancreaticβ-cell effect, there is not been reported.The present inventor is using Polysaccharides from Rosa roxburghii as function
Decorative material has the functionalization of protection pancreaticβ-cell with oxidation-reduction method preparation using sodium selenite and vitamin C as raw material
Nanometer selenium.Product Safety made from the method is high, method simplicity can mass production, also have while as Selenium supplement agent high
Bioactivity, can develop into treat and prevent diabetes high-efficiency low-toxicity medicament.
Summary of the invention
The purpose of the present invention is to provide a kind of Polysaccharides from Rosa roxburghii functionalized nano selenium compound and preparation method thereof with dropping
Application in sugared drug.
For the present invention using sodium selenite as selenium source, ascorbic acid (vitamin C) is reducing agent, and Polysaccharides from Rosa roxburghii (RTFP-3) is table
Face dressing agent, green prepare surface-functionalized nano selenium compound (RP3-SeNPs).Prepare that stability is good, particle diameter distribution
Narrow, good dispersion nanometer selenium polysaccharide compound and Polysaccharides from Rosa roxburghii nano selenium compound are impaired in preparation inhibition beta Cell of islet
Drug in purposes.
The purpose of the present invention is achieved through the following technical solutions.
A kind of preparation method of Polysaccharides from Rosa roxburghii functionalized nano selenium compound, comprising the following steps:
(1) sodium selenite solution is uniformly mixed with Polysaccharides from Rosa roxburghii solution, vitamin c solution is then added, shakes after mixing evenly
Swing reaction；
(2) reaction solution obtained by step (1) is dialysed, is dried to get Polysaccharides from Rosa roxburghii functionalized nano selenium compound is arrived.
Preferably, the concentration of step (1) described sodium selenite solution is 1~3 mM, the concentration of the Polysaccharides from Rosa roxburghii solution
For 0.25-4 mg/L；The concentration of the vitamin c solution is 6~10 mM.
It is further preferred that the concentration of the Polysaccharides from Rosa roxburghii solution is 0.25,0.5,1,2,3 or 4 mg/L.
Preferably, the volume of step (1) described vitamin c solution is 6~10 times of sodium selenite solution volume.
Preferably, the temperature of step (1) described reaction is 37 °C.
Preferably, step (1) reaction is 12~36 h of reaction under the conditions of 100~300 rpms.
Preferably, step (2) dialysis is that reaction solution obtained by step (1) is transferred in the bag filter of 3000 Da,
It dialyses in 4 °C of water, dialyzate stops dialysis after ICP-AES is detected without plasma selenium；The time of dialysis is 36~96 h.
A kind of Polysaccharides from Rosa roxburghii functionalized nano selenium compound as made from above-described preparation method.
A kind of above-described Polysaccharides from Rosa roxburghii functionalized nano selenium compound is preparing the application in hypoglycemic medicine.
Functionalized nano selenium prepared by the present invention can be applied to inhibit the oxidative damage of beta Cell of islet, and wherein functionalization is received
Rice selenium is active constituent, with anti-oxidant and protection islet cells activity.
The present invention compared with the existing technology, have the following advantages and the utility model has the advantages that
(1) the functionalized nano selenium of Polysaccharides from Rosa roxburghii modification prepared by the present invention is directly that reaction is former with sodium selenite and vitamin C
Material, Polysaccharides from Rosa roxburghii is coating material, and preparation process is simple and easy, and product system is simple, the nano granules of selenium partial size of preparation
Narrow range, storage stability is good, its partial size is without apparent variation in storage one month.The Polysaccharides from Rosa roxburghii of energy spectrum analysis display preparation
The content of selenium atom is about 27.01% in nano selenium compound, and selenium atom content is higher.
(2) since Polysaccharides from Rosa roxburghii itself has antioxidant activity, by the combination of Polysaccharides from Rosa roxburghii and nano granules of selenium, with original
Nano granules of selenium is compared, and the antioxidant activity of nano granules of selenium, the antioxidant activity of compound can be greatly improved after compound
It is almost suitable with the antioxidant activity of Polysaccharides from Rosa roxburghii.
(3) Polysaccharides from Rosa roxburghii nano granules of selenium prepared by the present invention has preferable cell membrane penetration, surveys through ion chromatography
Surely the uptake for cultivating 24 h islet cells INS-1 is 12.53 μ g/108A cell, while H can be inhibited2O2The pancreas islet of induction
The apoptosis of oncocyte INS-1, improves the survival rate of cell, while can also be improved H2O2The pancreas of the insulinoma damaging cells of induction
Island element secretory volume.
Detailed description of the invention
Fig. 1 a is the grain-size graph that various concentration Polysaccharides from Rosa roxburghii modifies nanometer selenium solution.
Fig. 1 b is 1 mg/mL and the storage stability of 2 mg/mL Polysaccharides from Rosa roxburghii modification nanometer selenium solution compares figure.
Fig. 2 a is the transmission electron microscope picture of nanometer selenium solution.
Fig. 2 b is the transmission electron microscope picture of Polysaccharides from Rosa roxburghii nanometer selenium solution.
Fig. 2 c is the scanning electron microscope (SEM) photograph of nanometer selenium solution.
Fig. 2 d is the scanning electron microscope (SEM) photograph of Polysaccharides from Rosa roxburghii nanometer selenium solution.
Fig. 3 a is nanometer selenium and Polysaccharides from Rosa roxburghii nano selenium compound infrared spectroscopy (FT-IR) figure.
Fig. 3 b is the UV-visible spectrum of nanometer selenium and Polysaccharides from Rosa roxburghii nanometer selenium solution.
Fig. 3 c is the energy spectrum analysis figure (EDX) of nanometer selenium.
Fig. 3 d is the energy spectrum analysis figure (EDX) of Polysaccharides from Rosa roxburghii nano selenium compound.
Fig. 4 a is that the DPPH free radical of various concentration nanometer selenium, Polysaccharides from Rosa roxburghii and Polysaccharides from Rosa roxburghii nano selenium compound solution is clear
Removing solid capacity compares figure.
Fig. 4 b is that the ABTS free radical of various concentration nanometer selenium, Polysaccharides from Rosa roxburghii and Polysaccharides from Rosa roxburghii nano selenium compound solution is clear
Removing solid capacity compares figure.
Fig. 4 c is that the oxygen radical of various concentration nanometer selenium, Polysaccharides from Rosa roxburghii and Polysaccharides from Rosa roxburghii nano selenium compound solution absorbs
Ability compares figure.
Fig. 5 a is the cytotoxicity experiment figure of Polysaccharides from Rosa roxburghii nano selenium compound.
Fig. 5 b is the H of Polysaccharides from Rosa roxburghii nano selenium compound2O2Establish INS-1 cell oxidative damage illustraton of model.
Fig. 5 c is the protective effect figure of Polysaccharides from Rosa roxburghii and nanometer selenium to oxidative damage INS-1 cell.
Fig. 5 d is protective effect figure of the Polysaccharides from Rosa roxburghii nano selenium compound to oxidative damage INS-1 cell.
Fig. 6 is the influence diagram that Polysaccharides from Rosa roxburghii nano selenium compound acts on oxidative damage INS-1 cell insulin secretion.
Specific embodiment
Technology contents and effect of the invention, but embodiments of the present invention are further illustrated below by example and attached drawing
It is without being limited thereto.
For the present invention using sodium selenite as selenium source, vitamin C is reducing agent, and Polysaccharides from Rosa roxburghii is that coating material prepares nanometer
Selenium polysaccharide compound.
The preparation of 1 Polysaccharides from Rosa roxburghii RTFP-3 of embodiment
60 meshes are crossed in dry in 60 °C of air dry ovens after Rosa roxburghii Tratt fresh fruit is cleaned, crushing, according to Rosa roxburghii Tratt dry powder and 95% second
Alcohol (v/v) is made into the mixed liquor that solid-to-liquid ratio is 1:6 g/mL, is heated to reflux 4 h under the conditions of 70 °C, residue is obtained by filtration,
It is heated to reflux and filtering is repeated 3 times.Ribes burejense powder after degreasing is put into in 45 °C of air dry oven dry 48 h.It will processing
150 g of Rosa roxburghii Tratt dry powder afterwards, addition distilled water progress hot water extraction, 95 °C of extraction temperature, 3 h of time, solid-liquid ratio 1:30(mass
Unit with volume is respectively g and mL), extraction time is 2 times；5000 g of centrifugal force, be centrifuged 10 min, collect supernatant in
Rotation is concentrated by evaporation to the 1/4 of original volume under 55 °C, obtains the concentrate of Polysaccharides from Rosa roxburghii.It is added in the concentrate of Polysaccharides from Rosa roxburghii
Sevag reagent (chloroform: n-butanol=4:1), the concentrate of Polysaccharides from Rosa roxburghii and the volume ratio of Sevag reagent are 3:1, oscillation 30
Min is centrifugated upper layer liquid glucose, 5000 g of centrifugal force, and centrifugation time is 10 min, vibrates and centrifugal process repetitive operation 12 times
More than, until visually observing no protein residue, it is concentrated under reduced pressure into the 1/3 of original volume.Macropore is added in concentrate after de- albumen
Resin A B-8 carries out decolorization, makes the volume ratio 6:1 of polysaccharide concentrate and macroreticular resin, and 37 °C of temperature, on shaking table
10 h of oscillation treatment, is obtained by filtration filtrate.Dehydrated alcohol is added in filtrate after decoloration, the volumetric concentration of ethyl alcohol is made to reach 70%,
24 h, 5000 g of centrifugal force are stood under 4 °C, 10 min of centrifugation obtain the polysaccharide precipitation of lower layer, are freeze-dried 48 h, obtain water
Dissolubility Rosa roxburghii Tratt Thick many candies.Water-soluble Rosa roxburghii Tratt Thick many candies are dissolved with distilled water, the polysaccharide for being configured to final concentration of 20 mg/mL is molten
Liquid, is splined on DEAE-Sepharose fast flow column, and applied sample amount volume and column volume ratio are 1:4, respectively with distilled water,
The NaCl solution of 0.1 mol/L, 0.2 mol/L and 0.3 mol/L carry out gradient elution, and eluent flow rate is 1.0 mL/min,
Every pipe collects 5 mL.Wherein eluted with the NaCl solution of distilled water, 0.1 mol/L, 0.2 mol/L, 0.3 mol/L, point
Not Shou Ji each component, be respectively designated as RTFP-1, RTFP-2, RTFP-3 and RTFP-4.The present invention mainly reports RTFP-3 component,
By 48 h of concentration dialysis after RTFP-3 Fraction collection, vacuum freeze drying then is carried out to RTFP-3 component, is obtained after purification
Polysaccharides from Rosa roxburghii sample.
The preparation of 2 Polysaccharides from Rosa roxburghii nanometer selenium solution (SeNPs) of embodiment
Concentration be 1 mM 10 mL of sodium selenite solution respectively with the certain density Polysaccharides from Rosa roxburghii solution of same volume (0.25,
0.5,1,2,3,4 mg/mL) or distilled water be mutually mixed, the vitamin c solution of 6 mM of 60 mL is then added, stirs evenly,
Reaction solution, is then transferred in bag filter in 4 °C of conditions by 12 h of oscillating reactions on 100 r/min shaking tables under the conditions of 37 °C
48 h of lower dialysis.Finally by the nanometer selenium solution after dialysis, freeze-drying respectively obtains Polysaccharides from Rosa roxburghii nano granules of selenium
(RP3-SeNPs), nano granules of selenium (SeNPs).Transmission electron microscope (TEM) figure is respectively adopted, high resolution scanning Electronic Speculum (SEM), swashs
Light Particle Size Analyzer, energy spectrum analysis figure (EDS) and infrared spectroscopy (FT-IR) etc. characterize the structure of nanometer selenium.
The preparation of 3 Polysaccharides from Rosa roxburghii nanometer selenium solution (SeNPs) of embodiment
Concentration be 2 mM 10 mL of sodium selenite solution and the certain density Polysaccharides from Rosa roxburghii solution of same volume (0.25,0.5,1,
2,3,4 mg/mL) or distilled water be mutually mixed, the vitamin c solution of 8 mM of 80 mL is then added, stirs evenly, at 37 °
Reaction solution, is then transferred in bag filter and dialyses under the conditions of 4 °C by 24 h of oscillating reactions on 200 r/min shaking tables under the conditions of C
72 h.Finally by the nanometer selenium solution after dialysis, freeze-drying respectively obtains Polysaccharides from Rosa roxburghii nano granules of selenium (RP3-
SeNPs), nano granules of selenium (SeNPs).Transmission electron microscope (TEM) figure, high resolution scanning Electronic Speculum (SEM), laser particle size is respectively adopted
Analyzer, energy spectrum analysis figure (EDS) and infrared spectroscopy (FT-IR) etc. characterize the structure of nanometer selenium.
The preparation of 4 Polysaccharides from Rosa roxburghii nanometer selenium solution (SeNPs) of embodiment
Concentration be 3 mM 10 mL of sodium selenite solution and the certain density Polysaccharides from Rosa roxburghii solution of same volume (0.25,0.5,1,
2,3,4 mg/mL) or distilled water be mutually mixed, the vitamin c solution of 10 mM of 100 mL is then added, stirs evenly, 37
Then reaction solution is transferred in bag filter under the conditions of 4 °C thoroughly by 36 h of oscillating reactions on 300 r/min shaking tables under the conditions of °C
Analyse 96 h.Finally by the nanometer selenium solution after dialysis, freeze-drying respectively obtains Polysaccharides from Rosa roxburghii nano granules of selenium (RP3-
SeNPs), nano granules of selenium (SeNPs).Transmission electron microscope (TEM) figure, high resolution scanning Electronic Speculum (SEM), laser particle size is respectively adopted
Analyzer, energy spectrum analysis figure (EDS) and infrared spectroscopy (FT-IR) etc. characterize the structure of nanometer selenium.
Structural Identification and activity analysis, embodiment 2 are carried out by the following method by Polysaccharides from Rosa roxburghii made from above embodiments 3
Result with 4 is similar to Example 3.
The present embodiment has investigated the Polysaccharides from Rosa roxburghii of various concentration in reaction system to nano granules of selenium partial size in reaction system
Influence.As a result as shown in Figure 1a, the present invention selects the Polysaccharides from Rosa roxburghii solution of 0.25,0.5,1,2,3,4 mg/mL to receiving respectively
Rice granules of selenium is modified.When the concentration of Polysaccharides from Rosa roxburghii RTFP-3 is 0.25 ~ 2 mg/mL, the partial size of nanometer selenium is gradually decreased；
When the concentration of Polysaccharides from Rosa roxburghii RTFP-3 is greater than 2 mg/mL, nano granules of selenium is almost unchanged.Thus may determine that Polysaccharides from Rosa roxburghii is repaired
The optium concentration for adoring nano granules of selenium is 2 mg/mL, and the average grain diameter of nano granules of selenium is 104.5 nm at this time.
As shown in Figure 1 b, the present embodiment has investigated the storage of the nanometer selenium solution of the Polysaccharides from Rosa roxburghii solution preparation of various concentration
Stability, with the increase of storage time, the partial size of the nanometer selenium solution of 1 mg/mL Polysaccharides from Rosa roxburghii solution preparation is gradually increased,
However the partial size of the nanometer selenium solution of 2 mg/mL Polysaccharides from Rosa roxburghii solution preparation is basically unchanged, and shows high concentration Polysaccharides from Rosa roxburghii solution
The stability of the nanometer selenium solution of preparation is preferable, can be conducive to exploitation into drug long-term preservation with long-time storage.
Fig. 2 a is transmission electron microscope (TEM) figure of nano selenium sol, shows that the nanometer selenium of no Rosa roxburghii Tratt polysaccharide solution modification is easy
It is agglomerated into stack structure, there are the nanometer seleniums (see Fig. 2 b) of Polysaccharides from Rosa roxburghii solution modification, and uniform particle diameter can be made, evenly dispersed
Nano granules of selenium.The nano granules of selenium of the bright no polyose modification of scanning electron microscope (SEM) chart of Fig. 2 c is gathered into honeycomb structure,
Fig. 2 d shows that the nano granules of selenium through polyose modification is dispersed in the network frame of Polysaccharides from Rosa roxburghii.
As shown in Figure 3a, compared with former Polysaccharides from Rosa roxburghii, the infrared spectrogram of Polysaccharides from Rosa roxburghii nano selenium compound is shown ,-OH
Group peak is from 3442 cm-1Red shift is to 3456 cm-1, C=O group peak is from 1610 cm-1Red shift is to 1635 cm-1, C-O-C group peak
From 1239 cm-1Red shift is to 1242 cm-1, the red shift of these characteristic peaks shows to form Se-O chemistry between Se atom and polysaccharide
Key.In addition, ultraviolet spectra (see Fig. 3 b) shows Polysaccharides from Rosa roxburghii and Polysaccharides from Rosa roxburghii nano selenium compound in 198 and 278 cm-1Place has
Identical absorption peak, the presence of these characteristic peaks show to form covalent bond between Polysaccharides from Rosa roxburghii and nanometer selenium.Energy spectrum analysis
The result shows that without selenium element in Polysaccharides from Rosa roxburghii (see Fig. 3 c) structure, selenium atom in Polysaccharides from Rosa roxburghii nano selenium compound (see Fig. 3 d)
Content is about 27.01%.
Inhibiting effect of the different samples of embodiment 3 to DPPH free radical
A certain amount of Polysaccharides from Rosa roxburghii, nano granules of selenium and Polysaccharides from Rosa roxburghii nano granules of selenium are accurately weighed, is dissolved in distilled water and being made into
The solution (0.25-2.0 mg/m L) of various concentration is spare.Take 2 mL sample solutions that (75 μ in 2 mL DPPH working solutions are added
M, 50% methanol solution are prepared), whirlpool is uniformly mixed, and is protected from light after 30 min to measure at 517 nm wavelength at room temperature and be inhaled
Light value uses distilled water to replace sample as blank control, and methanol solution replaces DPPH solution to be used as Background control, with vitamin C
It (VC) is positive control.DPPH free radical scavenging activity is calculated according to following formula:
DPPH free radical scavenging activity (%)=[1- (ASample-ABackground control)/ABlank]× 100
Fig. 4 a is the curve graph of RTFP-3, RP3-SeNPs and SeNPs to DPPH free radical inhibiting rate.As a result in 0.25- shown in
RTFP-3 and RP3-SeNPs has a preferable scavenging effect to DPPH in 2.0 mg/mL concentration ranges, and three kinds of substances is anti-oxidant
Activity enhances with the raising of concentration.When concentration is 2 mg/mL, RTFP-3, RP3-SeNPs and SeNPs are to DPPH freedom
The maximal clearance of base is respectively 66.01 %, 56.29% and 13.92%.Wherein RTFP-3 has DPPH free radical strongest clear
Except effect, RP3-SeNPs takes second place, and SeNPs shows most weak scavenging activity on DPPH.
Inhibiting effect of the different samples of embodiment 4 to ABTS free radical
ABTS working solution is by 5 mL ABTS solution (7 mM) and 5 mL K2S2O8Solution (2.45 mM) is protected from light at room temperature
It is prepared after reacting 12 h.Using it is preceding with phosphoric acid buffer sodium salt solution be diluted at 734 nm light absorption value be 0.70 ±
0.02.Then, the sample solution (0.25-2.0 mg/mL) of 0.4 mL various concentration is taken to be added to the diluted ABTS of 4 mL
In working solution, 6 min are protected from light after vibrating 30 s, absorbance value are detected under 734 nm wavelength, with same volume
Distilled water replaces sample solution to be used as blank control, using the distilled water of same volume instead of ABTS working solution as Background control,
With vitamin C (VC) it is positive control, ABTS free radical scavenging activity calculation formula is as follows:
ABTS free radical scavenging activity (%)=[1- (ASample-ABackground control)/ABlank]× 100
Test result is as shown in Figure 4 b, the half elimination ratio (IC of RTFP-3, RP3-SeNPs and SeNPs to ABTS free radical50)
Respectively 0.93 mg/mL, 1.05 mg/mL and > 10 mg/mL.It follows that nano granules of selenium SeNPs show it is extremely weak clear
Except the ability of ABTS free radical, after Polysaccharides from Rosa roxburghii RTFP-3 modification, Polysaccharides from Rosa roxburghii nano selenium compound removes ABTS free radical
Ability be obviously improved.
The evaluation of 5 Polysaccharides from Rosa roxburghii oxyradical absorbability (ORAC) of embodiment
All samples solution is prepared by phosphate buffer (75 mM, pH 7.4).20 are sequentially added in 96 hole elisa Plates
RTFP-3, SeNPs and the RP3-SeNPs solution of μ L various concentration (0.5-4.0 mg/mL) and 200 μ L fluorescein sodium salting liquids
(95.6 nM), while replacing fluorescein sodium salting liquid as a control group with phosphate buffer, use distilled water replace sample as
Blank control group.Fluorescence/chemical analyzer program is set, 37 °C are protected from light 15 min of incubation, and every hole is rapidly added 20 μ L ABAP
Solution (119.4 m M) after automatic mixing, in 535 nm of 485 nm of excitation wavelength and absorbing wavelength, records one every 2 min
It is secondary, it recycles altogether 70 times, records fluorescence intensity f respectively1、f2、f3、······f70, calculate the area (AUC) of fluorescent quenching
Value, formula are as follows: AUC=2 × [1/2 × (f1 + f35) + f2 + f3 + f4+······+ f70].While with
(6.25-100 μM) drafting standard curve of Trolox standard solution of various concentration, the ORAC value of sample indicate every gram of sample phase
When in Trolox equivalent (Trolox equivalent, TE) value, unit is μm ol TE/g.
As a result as shown in 4c, the ORAC value of RTFP-3, RP3-SeNPs and SeNPs are respectively 105.65,79.94 and 5.33
μmol TE/g.By ORAC value it is found that compared with pure nano granules of selenium, the inoxidizability of Polysaccharides from Rosa roxburghii nano selenium compound is improved
14 times.
Toxicity of the 6 Polysaccharides from Rosa roxburghii nano granules of selenium of embodiment to INS-1 cell
Experimental cell: rat Langerhans islet oncocyte INS-1 routine culture.The culture medium of culture cell includes 10% fetal calf serum,
The L-Glutamine of 2 mM, the Sodium Pyruvate of 1 mM, 50 μM of beta -mercaptoethanol, the penicillin of 100 units/mL and 100
The streptomysin of units/mL.The measurement of cell survival rate uses MTT method.
Cytotoxicity assay: the INS-1 cell of logarithmic growth phase digests piping and druming cell into single cell suspension with pancreatin,
750 r/min, 5 min of centrifugation remove supernatant, and cell is resuspended in culture medium, after being counted with blood cell counting plate, adjust cell concentration
According to 6 × 104A density is inoculated in 96 porocyte culture plates, is placed in 37 °C, 5% CO2Incubator in cultivate 12 h,
Then culture solution is discarded.The cell culture medium of the RP3-SeNPs containing various concentration of 100 μ L is added in every hole, is placed in incubator
In continue cultivate 24 h after, the MTT solution (5 mg/mL) of 20 μ L is added into every hole, continue incubation 4 h, then suck training
The DMSO of 100 μ L is finally added in nutrient solution, and being washed 1 time with 100 μ L PBS into every hole, vibrates after 10 min in 570 nm
The light absorption value in each hole is measured under wavelength, calculates the survival rate of cell according to the following formula.Experimental setup blank group, experimental group and right
According to group.Blank group is that cell is not added, and 100 μ L culture mediums, experimental group and control group is added, and the cell that 100 μ L are added in every hole is outstanding
Supernatant liquid, wherein control group is not loaded product.Wherein ASFor the absorbance value of experimental group, ACIt is the absorbance value of control group, A0It is blank
The absorbance value of group.
Cell survival rate (%)=(AS-A0)/(AC-A0)
It is thin to INS-1 according to gradually incremental concentration gradient (0,0.25,0.5,1,2,4 μ g/mL) detection RP3-SeNPs
The toxicity of born of the same parents, when as a result (Fig. 5 a) is shown in concentration and reaches 4 μ g/mL, the toxicity of RP3-SeNPs plays main function, starts
Promote Apoptosis, the survival rate of cell is 79.1%, significantly less than blank control group；And RP3-SeNPs is in 0 ~ 2 μ g/mL
Concentration range in, RP3-SeNPs almost no cytotoxicity.The RP3-SeNPs of low concentration has significant promotion cell Proliferation
The RP3-SeNPs of activity, high concentration is able to suppress cell Proliferation, toxic to cell, therefore RP3-SeNPs concentration is selected to be lower than
2 μ g/mL carry out subsequent experimental.
Embodiment 7 determines H2O2Induce the optium concentration of INS-1 cell oxidative damage model
According to 6 × 104The density of a cell is inoculated in 96 porocyte culture plates, is placed in 37 °C, 5% CO2Incubator in train
12 h are supported, culture solution is then discarded.The H containing various concentration of 100 μ L is added in every hole2O2Cell culture medium, be placed in culture
Continue after cultivating 12 h in case, the survival rate of cell is calculated according to MTT method.
As a result such as Fig. 5 b shown in, with H2O2The raising of concentration, H2O2Cytotoxicity it is more and more stronger, can significantly inhibit
The proliferation of INS-1 cell.Work as H2O2Concentration when being 250 μM, the survival rate of cell is 67.54%, can be used as suitable modeling
Concentration.
Embodiment 8 measures protective effect of the RP3-SeNPs to oxidative damage INS-1 cell
Measure protective effect of the RP3-SeNPs to INS-1 cell.According to 6 × 104The density of a cell is inoculated in the training of 96 hole cells
It supports in plate, is placed in 37 °C, 5% CO2Incubator in cultivate 12 h, then discard culture solution.Containing for 100 μ L is added in every hole
The RP3-SeNPs cell culture medium of various concentration is placed in incubator after continuing to cultivate 24 h, then discards culture medium.Every hole
100 μ L are added contains H2O2Cell culture medium, be placed in incubator after continuing to cultivate 12 h, calculated according to MTT method thin
The survival rate of born of the same parents.
As a result it can inhibit H after INS-1 cell is first pre-processed 24 h with RP3-SeNPs such as Fig. 5 c shown in2O2Induction
Cellular damage.When the concentration of RP3-SeNPs is 2 μ g/mL, it is H that the survival rate of cell, which is 89.34%,2O2Model group cell is deposited
1.36 times of motility rate show that RP3-SeNPs can protect islet cells from oxidative damage.
Embodiment 9 measures INS-1 cell to the intake situation of Polysaccharides from Rosa roxburghii nanometer selenium
Cell concentration is adjusted according to 5 × 105A density is inoculated in 6 porocyte culture plates, is placed in 37 °C, 5% CO2Training
It supports in case and cultivates 12 h, then discard culture solution.The RP3-SeNPs (0.5,1 containing various concentration of 2 mL is added in every hole
24 h are incubated for the cell culture medium of 2 mg/mL).Cell is collected at 8,24 h time points respectively, is washed carefully with PBS buffer solution
Born of the same parents 3 times, the content of intracellular selenium is measured with ICP-AES.
As a result such as Fig. 5 d shown in, INS-1 cell is to the intake of Polysaccharides from Rosa roxburghii nanometer selenium, as incubation time and polysaccharide are received
The raising of rice selenium concentration, intake gradually increase.When RP3-SeNPs concentration is 0.5 μ g/mL, and incubation time is 24 h,
INS-1 intracellular selenium concentration is 5.68 mg/ 108A cell.When it is 2 μ g/mL that RP3-SeNPs concentration, which increases, INS-1
Cellular uptake selenium amount improves 2.21 times.
The insulin secretion situation of 10 glucose of embodiment stimulation insulinoma cell INS-1
Cell concentration is adjusted according to 6 × 105A density is inoculated in 6 porocyte culture plates, is placed in 37 °C, 5% CO2Training
It supports in case and cultivates 12 h, then discard culture solution.RP3-SeNPs (0.5, the 1 and 2 μ g/ of 2 mL various concentrations are added in every hole
ML after cell culture medium) is incubated for 24 h, culture medium is discarded.Except for the normal group, every hole, which is added, contains 250 μM of H2O2Cell
After culture medium is incubated for 12 h, then with the glucose solution that concentration is 5.6 mmol/L and 16.7 mmol/L, (Hanks is flat respectively
The salting liquid that weighs is prepared) it is incubated for INS-1 cell 30 min, contained with insulin in insulinase linked immunoassay reagent kit measurement culture medium
Amount.
As a result such as Fig. 6, H shown in2O2The function of INS-1 cells secrete insulin, H can be damaged2O2Model group with normal group
It compares, at 5.6 mmol/L or 16.7 mmol/L glucose stimulations, the ability of INS-1 cells secrete insulin is aobvious
Decline is write, shows H2O2INS-1 cell oxidative damage can be induced, it is impaired so as to cause its insulin secretion function.Work as INS-1
After the RP3-SeNPs pre-treatment of cell various concentration, as a result, it has been found that RP3-SeNPs can protect the insulin of INS-1 cell
Secreting function.It is wherein 2 μM of 24 h of RP3-SeNP pre-treatment INS-1 cell with concentration, in the glucose (16.7 of higher concentration
Mmol/L) under stimulation, the amount of insulin of INS-1 cell secretion is 2.23 times of model group.However in the Portugal of low concentration
Under grape sugar (5.6 mmol/L) stimulation, after handling INS-1 cell with RP3-SeNP, the amount and model group of excreting insulin
It compares, without apparent difference.As can be known from the above results, RP3-SeNPs can protect the insulin secretion function of insulinoma cell
Energy.
Claims (10)
1. a kind of preparation method of Polysaccharides from Rosa roxburghii functionalized nano selenium compound, which comprises the following steps:
(1) sodium selenite solution is uniformly mixed with Polysaccharides from Rosa roxburghii solution, vitamin c solution is then added, shakes after mixing evenly
Swing reaction；
(2) reaction solution obtained by step (1) is dialysed, is dried to get Polysaccharides from Rosa roxburghii functionalized nano selenium compound is arrived.
2. preparation method according to claim 1, which is characterized in that the concentration of step (1) described sodium selenite solution is 1
~3 mM, the concentration of the Polysaccharides from Rosa roxburghii solution are 0.25-4 mg/L；The concentration of the vitamin c solution is 6~10 mM.
3. preparation method according to claim 2, which is characterized in that the concentration of the Polysaccharides from Rosa roxburghii solution be 0.25,
0.5,1,2,3 or 4 mg/L.
4. preparation method according to claim 1, which is characterized in that step (1) sodium selenite solution and Rosa roxburghii Tratt are more
The volume ratio of sugar juice is 1:1 ~ 1:3.
5. preparation method according to claim 1, which is characterized in that the volume of step (1) described vitamin c solution is Asia
5~10 times of sodium selenate liquor capacity.
6. preparation method according to claim 1, which is characterized in that the temperature of step (1) described reaction is 37 °C.
7. preparation method according to claim 1, which is characterized in that step (1) reaction is every at 100~300 turns
12~36 h are reacted under the conditions of minute.
8. preparation method according to claim 1, which is characterized in that step (2) dialysis is will be anti-obtained by step (1)
It answers liquid to be transferred in bag filter, dialyses in water, dialyzate stops dialysis after ICP-MS is detected without plasma selenium；The dialysis
Temperature be 2~8 °C；The drying is freeze-drying, and dry temperature is -20~-50 °C.
9. a kind of Polysaccharides from Rosa roxburghii functionalized nano selenium compound as made from the described in any item preparation methods of claim 1-8.
10. a kind of Polysaccharides from Rosa roxburghii functionalized nano selenium compound as claimed in claim 9 is preparing the application in hypoglycemic medicine.
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