CN109303922B - Rosa roxburghii polysaccharide functionalized nano-selenium compound, preparation method thereof and application thereof in hypoglycemic drugs - Google Patents
Rosa roxburghii polysaccharide functionalized nano-selenium compound, preparation method thereof and application thereof in hypoglycemic drugs Download PDFInfo
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- CN109303922B CN109303922B CN201811377322.2A CN201811377322A CN109303922B CN 109303922 B CN109303922 B CN 109303922B CN 201811377322 A CN201811377322 A CN 201811377322A CN 109303922 B CN109303922 B CN 109303922B
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- 125000000969 xylosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)CO1)* 0.000 description 1
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Abstract
The invention discloses a rosa roxburghii polysaccharide functionalized nano-selenium compound, a preparation method thereof and application thereof in hypoglycemic drugs, belonging to the technical field of nano-selenium preparation. The preparation method comprises the following steps: (1) uniformly mixing a sodium selenite solution and a roxburgh rose polysaccharide solution, then adding a vitamin C solution, uniformly stirring, and carrying out oscillation reaction; (2) dialyzing the reaction solution obtained in the step (1), and drying to obtain the roxburgh rose polysaccharide functionalized nano selenium compound. According to the invention, sodium selenite is used as a selenium source, vitamin C is used as a reducing agent, rosa roxburghii polysaccharide (RTFP-3) is used as a surface modifier, surface functionalized nano selenium compound (RP 3-SeNPs) is prepared in an environmentally-friendly manner, nano selenium polysaccharide compound with good stability, narrow particle size distribution and good dispersibility is prepared, and the application of the rosa roxburghii polysaccharide nano selenium compound in preparation of drugs for inhibiting pancreatic beta cell damage is provided.
Description
Technical Field
The invention belongs to the technical field of nano-selenium preparation, and particularly relates to a roxburgh rose polysaccharide functionalized nano-selenium compound, a preparation method thereof and application thereof in hypoglycemic drugs.
Background
With the development of socioeconomic and improvement of living standard of people, the incidence rate of diabetes mellitus is gradually increased, the incidence rate and the mortality rate of the diabetes mellitus are positioned at the 3 rd position of non-infectious diseases, and particularly, the number of people with type 2 diabetes mellitus is increased more rapidly. One of the causes of type 2 diabetes is the decrease in the number of islet cells or loss of function of the human body, which results in the decrease in the amount of insulin secretion, and thus the disturbance of the blood sugar balance of the human body. At present, free radicals are found to be one of the important causes of islet beta-cell injury and apoptosis, and in normal human bodies, the free radicals are in a dynamic equilibrium state, but under the stimulation of external adverse environment, autoinflammation and the like, excessive free radicals (ROS, RNS and the like) in the bodies attack normal cells. Under oxidative stress, the composition of normal cells changes, for example, the DNA, protein and lipid components of intracellular substances change, inducing cell damage and apoptosis. Based on the relationship between oxidative stress, beta-cell apoptosis and diabetes, it was found that inhibition of the oxidative stress state of islet beta-cells is one of the effective methods for treating diabetes. Meanwhile, multiple studies show that the antioxidant supplement can eliminate free radicals, control the oxidative stress state of beta-cells and achieve the effect of protecting pancreatic islet beta-cells.
Selenium is one of essential trace elements for human body, has the functions of improving human immunity, resisting oxidation, delaying senility, preventing and resisting cancer, and is closely related to the growth, development and disease occurrence of human and animals. Research shows that selenium deficiency can cause various diseases such as cancer, heart disease, arthritis, immune system dysfunction and the like, and proper amount of selenium supplement can prevent metabolic diseases such as diabetes and the like and reduce the incidence and complications of the diabetes. Research shows that selenium as an antioxidant can participate in the synthesis of an antioxidant enzyme system (such as glutathione catalase) in a human body when entering the human body, can quickly eliminate free radicals in the human body, can effectively prevent the damage of the free radicals to cells, and has an anti-aging function. At present, selenium supplements are often added to many food supplements in the form of sodium selenite, sodium selenate, selenomethionine. Selenium nanoparticles (SeNPs) are a new research hotspot due to the characteristics of high bioavailability, low toxicity and remarkable biological activity. Chinese patent CN104310319B discloses a method for preparing nano-selenium, which comprises reacting selenite with a reducing agent under the action of a stabilizer, wherein the molar concentration of selenite solution is 15-25 mM by changing the proportion of selenite to the reducing agent, and the particle size of nano-selenium can be accurately controlled within the concentration range. Chinese patent CN104825484B discloses a method for preparing nano-selenium, which uses sodium selenite as a selenium source, potassium iodide as a stabilizer, ascorbic acid as a reducing agent and chitosan or carboxymethyl chitosan as a surface modifier to successfully prepare a nano-selenium compound with functionalized surface, wherein the particle size range of the nano-selenium compound is about 50 nm. Research shows that the bioavailability and bioactivity of selenium greatly vary with the change of the nanometer particle size, and the SeNPs which are synthesized and have no functionalized surfaces are easy to aggregate and precipitate, which becomes a bottleneck limiting the application of the SeNPs. Therefore, it is a hot spot of current research to prepare small-particle-size narrow-distribution SeNPs and functionally modify the surface of the SeNPs with polysaccharides in order to obtain selenium nanoparticles with good stability.
The rosa roxburghii tratt is used as a medicine-food dual-purpose plant with geographical characteristics and is also eaten by people as a traditional Chinese medicine. The traditional medicine mainly uses Roxburgh rose root, leaf, fruit and the like as medicines, has the effects of promoting digestion, strengthening spleen, relieving diarrhea, relieving summer heat and the like, and has certain effects on treating diseases such as food retention, abdominal distension, hyperlipidemia, enteritis, hypertension, nyctalopia, vitamin C deficiency and the like. Polysaccharide is one of important components in the rosa roxburghii tratt fruit, and a uniform component RTFP-3 is separated by a hot water extraction method through decolorization, deproteinization and DEAE-Sepharose Fast Flow column chromatography, and has uniform molecular weight and clear structure. The obtained Rosa roxburghii polysaccharide RTFP-3 has a molecular weight of 67.2 kDa, and is composed of glucose, galactose, arabinose, xylose, fucose, etc., wherein the main glycosidic bonds comprise 33.21% of (1 → 5) -arabinose, 35.72% of (1 → 6) -galactose, 4.27% of (1 → 4) -glucose, and 17.22% of (1 → 3,4) -fucose. Also, a small portion of terminal xylose and (1 → 3,6) -mannose are included. At present, no report about the regulation and control of rosa roxburghii tratt polysaccharide for preparing nano selenium with the function of protecting islet beta-cells is found. The invention takes roxburgh rose polysaccharide as a functional modification material, takes sodium selenite and vitamin C as raw materials, and prepares the functional nano-selenium with the function of protecting islet beta-cells by an oxidation-reduction method. The product prepared by the method has high safety, and can be easily produced in large scale, has extremely high bioactivity while being used as selenium supplement, and can be developed into high-efficiency low-toxicity medicament for treating and preventing diabetes.
Disclosure of Invention
The invention aims to provide a rosa roxburghii polysaccharide functionalized nano selenium compound, a preparation method thereof and application thereof in hypoglycemic drugs.
According to the invention, sodium selenite is used as a selenium source, ascorbic acid (vitamin C) is used as a reducing agent, and rosa roxburghii tratt polysaccharide (RTFP-3) is used as a surface modifier, so that the nano selenium compound (RP 3-SeNPs) with functionalized surface is prepared in an environment-friendly manner. The nano selenium polysaccharide compound with good stability, narrow particle size distribution and good dispersibility and the application of the roxburgh rose polysaccharide nano selenium compound in preparing the medicines for inhibiting the damage of islet beta cells are prepared.
The purpose of the invention is realized by the following technical scheme.
A preparation method of a roxburgh rose polysaccharide functionalized nano selenium compound comprises the following steps:
(1) uniformly mixing a sodium selenite solution and a roxburgh rose polysaccharide solution, then adding a vitamin C solution, uniformly stirring, and carrying out oscillation reaction;
(2) dialyzing the reaction solution obtained in the step (1), and drying to obtain the roxburgh rose polysaccharide functionalized nano selenium compound.
Preferably, the concentration of the sodium selenite solution in the step (1) is 1-3 mM, and the concentration of the roxburgh rose polysaccharide solution is 0.25-4 mg/L; the concentration of the vitamin C solution is 6-10 mM.
Further preferably, the concentration of the roxburgh rose polysaccharide solution is 0.25, 0.5, 1, 2, 3 or 4 mg/L.
Preferably, the volume of the vitamin C solution in the step (1) is 6-10 times of the volume of the sodium selenite solution.
Preferably, the temperature of the reaction of step (1) is 37 ℃.
Preferably, the reaction in the step (1) is carried out for 12-36 h under the condition of 100-300 revolutions per minute.
Preferably, the dialysis in the step (2) is to transfer the reaction solution obtained in the step (1) into a 3000 Da dialysis bag, dialyze the reaction solution in water at 4 ℃, and stop the dialysis after the dialysis solution is detected to be free of selenium ions by ICP-AES; the dialysis time is 36-96 h.
The rosa roxburghii polysaccharide functionalized nano selenium compound prepared by the preparation method is provided.
The application of the rosa roxburghii polysaccharide functionalized nano selenium compound in preparing hypoglycemic drugs.
The functional nano-selenium prepared by the invention can be applied to inhibiting the oxidative damage of islet beta cells, wherein the functional nano-selenium is an active ingredient and has the activities of resisting oxidation and protecting the islet cells.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) the functionalized nano-selenium modified by the rosa roxburghii polysaccharide prepared by the invention directly takes sodium selenite and vitamin C as reaction raw materials, the rosa roxburghii polysaccharide is taken as a surface modifier, the preparation process is simple and easy to implement, the product system is simple, the particle size range of the prepared nano-selenium particles is narrow, the storage stability is good, and the particle size of the nano-selenium particles is not obviously changed within one month after storage. The energy spectrum analysis shows that the selenium atom content of the prepared roxburgh rose polysaccharide nano selenium compound is about 27.01 percent, and the selenium atom content is higher.
(2) Because the roxburgh rose polysaccharide has the antioxidant activity, the antioxidant activity of the nano selenium particles can be greatly improved after the roxburgh rose polysaccharide is combined with the nano selenium particles compared with the original nano selenium particles, and the antioxidant activity of the compound is almost equivalent to that of the roxburgh rose polysaccharide.
(3) The rosa roxburghii polysaccharide nano selenium particles prepared by the invention have better cell membrane penetrability, and the absorption amount of INS-1 of islet cells cultured for 24 h determined by ion chromatography is 12.53 mug/108Can inhibit H simultaneously2O2The induced apoptosis of the insulinoma cell INS-1 improves the survival rate of the cell and can also improve H2O2Induced insulin secretion by insulinoma damaged cells.
Drawings
FIG. 1a is a particle size diagram of a solution of Rosa roxburghii polysaccharide modified with nano-selenium of different concentrations.
FIG. 1b is a graph comparing the storage stability of the Rosa roxburghii polysaccharide modified nano-selenium solution at 1 mg/mL and 2 mg/mL.
FIG. 2a is a transmission electron microscope image of the nano-selenium solution.
FIG. 2b is a transmission electron microscope image of the Rosa roxburghii polysaccharide nano selenium solution.
FIG. 2c is the scanning electron microscope image of the nano-selenium solution.
FIG. 2d is the scanning electron microscope image of the Rosa roxburghii polysaccharide nano selenium solution.
FIG. 3a is a graph of the infrared spectrum (FT-IR) of the nano selenium and Rosa roxburghii polysaccharide nano selenium compound.
FIG. 3b is the UV-VIS spectrum of the nano-selenium and Rosa roxburghii polysaccharide nano-selenium solution.
FIG. 3c is an energy spectrum analysis (EDX) of nano-selenium.
FIG. 3d is the energy spectrum analysis (EDX) of the polysaccharide-nano-selenium complex of Rosa roxburghii.
FIG. 4a is a graph comparing the DPPH radical scavenging ability of different concentrations of nano-selenium, polysaccharides from Rosa roxburghii and nano-selenium complex solutions of polysaccharides from Rosa roxburghii.
FIG. 4b is a graph comparing ABTS free radical scavenging ability of different concentrations of nano-selenium, polysaccharide from Rosa roxburghii and polysaccharide from Rosa roxburghii nano-selenium complex solution.
FIG. 4c is a graph comparing the oxygen radical absorbance capacity of different concentrations of nano-selenium, polysaccharides from Rosa roxburghii and polysaccharides from Rosa roxburghii.
FIG. 5a is a cytotoxicity test chart of Rosa roxburghii polysaccharide nano selenium compound.
FIG. 5b shows H of Rosa roxburghii polysaccharide nano-selenium complex2O2And establishing a map of INS-1 cell oxidative damage model.
FIG. 5c is a graph showing the protective effect of Rosa roxburghii polysaccharide and nano-selenium on oxidative damage of INS-1 cells.
FIG. 5d is a graph showing the protective effect of the polysaccharide-nano-selenium complex of Rosa roxburghii on INS-1 cells damaged by oxidation.
FIG. 6 is a graph showing the effect of Rosa roxburghii polysaccharide nano-selenium complex on the insulin secretion of INS-1 cells by oxidative damage.
Detailed Description
Technical contents and effects of the present invention will be further described below by way of examples and drawings, but embodiments of the present invention are not limited thereto.
The invention takes sodium selenite as a selenium source, vitamin C as a reducing agent and rosa roxburghii tratt polysaccharide as a surface modifier to prepare the nano selenium polysaccharide compound.
EXAMPLE 1 preparation of Ribes burejense polysaccharide RTFP-3
Cleaning fresh fructus Rosae Normalis, drying in a 60 deg.C air-blast drying oven, pulverizing, sieving with 60 mesh sieve, mixing with 95% ethanol (v/v) to obtain a mixture with solid-to-liquid ratio of 1:6 g/mL, heating and refluxing at 70 deg.C for 4 hr, filtering to obtain residue, and repeating heating, refluxing and filtering for 3 times. And (3) putting the degreased roxburgh rose powder into a blowing drying oven at 45 ℃ for drying for 48 h. Adding distilled water into 150 g of the treated roxburgh rose dry powder for hot water extraction, wherein the extraction temperature is 95 ℃, the extraction time is 3 h, the material-liquid ratio is 1:30 (the unit of mass and volume is g and mL respectively), and the extraction frequency is 2 times; centrifuging at centrifugal force of 5000 g for 10 min, collecting supernatant, rotary evaporating at 55 deg.C to concentrate to 1/4 of original volume to obtain fructus Rosae Normalis polysaccharide concentrated solution. Adding Sevag reagent (chloroform: n-butanol =4: 1) into the concentrated solution of the roxburgh rose polysaccharide, wherein the volume ratio of the concentrated solution of the roxburgh rose polysaccharide to the Sevag reagent is 3:1, oscillating for 30min, centrifuging to separate supernatant sugar solution, centrifuging for 10 min at a centrifugal force of 5000 g, repeating the oscillation and centrifugation processes for more than 12 times until no protein residue is observed by naked eyes, and concentrating under reduced pressure to 1/3 of the original volume. Adding macroporous resin AB-8 into the deproteinized concentrated solution, decolorizing at 37 deg.C with polysaccharide concentrated solution and macroporous resin volume ratio of 6:1, shaking on a shaking table for 10 hr, and filtering to obtain filtrate. Adding anhydrous ethanol into the decolorized filtrate to make the volume concentration of ethanol reach 70%, standing at 4 deg.C for 24 hr, centrifuging at 5000 g for 10 min to obtain lower layer polysaccharide precipitate, and freeze drying for 48 hr to obtain water soluble fructus Rosae Normalis crude polysaccharide. Dissolving the water-soluble roxburgh rose crude polysaccharide with distilled water to prepare a polysaccharide solution with the final concentration of 20 mg/mL, loading the polysaccharide solution on a DEAE-Sepharose fast flow column, wherein the ratio of the loading volume to the column volume is 1:4, performing gradient elution by using distilled water, 0.1 mol/L NaCl solution, 0.2 mol/L NaCl solution and 0.3 mol/L NaCl solution respectively, wherein the flow rate of an eluent is 1.0 mL/min, and collecting 5 mL in each tube. Wherein, distilled water, 0.1 mol/L, 0.2 mol/L and 0.3 mol/L NaCl solution are used for elution, and all components are respectively collected and named as RTFP-1, RTFP-2, RTFP-3 and RTFP-4. The invention mainly reports an RTFP-3 component, the RTFP-3 component is collected, concentrated and dialyzed for 48 hours, and then the RTFP-3 component is subjected to vacuum freeze drying to obtain a purified roxburgh rose polysaccharide sample.
EXAMPLE 2 preparation of Rosa roxburghii polysaccharide Nano selenium solution (SeNPs)
10 mL of sodium selenite solution with the concentration of 1 mM is respectively mixed with the same volume of roxburgh rose polysaccharide solution (0.25, 0.5, 1, 2, 3,4 mg/mL) with a certain concentration or distilled water, then 60 mL of vitamin C solution with the concentration of 6 mM is added, the mixture is stirred uniformly, the shaking table is shaken on a 100 r/min shaking table under the condition of 37 ℃ for 12 hours, and then the reaction solution is transferred into a dialysis bag to be dialyzed for 48 hours under the condition of 4 ℃. Finally, freeze drying the dialyzed nano selenium solution to obtain roxburgh rose polysaccharide nano selenium particles (RP 3-SeNPs) and nano selenium particles (SeNPs) respectively. And respectively adopting a Transmission Electron Microscope (TEM) picture, a high-resolution Scanning Electron Microscope (SEM), a laser particle size analyzer, an energy spectrum analysis (EDS) picture, an infrared spectrum (FT-IR) picture and the like to characterize the structure of the nano selenium.
EXAMPLE 3 preparation of Rosa roxburghii polysaccharide Nano selenium solution (SeNPs)
Mixing 10 mL of sodium selenite solution with the same volume and a certain concentration of roxburgh rose polysaccharide solution (0.25, 0.5, 1, 2, 3 and 4 mg/mL) or distilled water, adding 80 mL of 8 mM vitamin C solution, stirring uniformly, carrying out shaking reaction on a shaking table at 200 r/min at 37 ℃ for 24 hours, and then transferring the reaction solution into a dialysis bag for dialysis for 72 hours at 4 ℃. Finally, freeze drying the dialyzed nano selenium solution to obtain roxburgh rose polysaccharide nano selenium particles (RP 3-SeNPs) and nano selenium particles (SeNPs) respectively. And respectively adopting a Transmission Electron Microscope (TEM) picture, a high-resolution Scanning Electron Microscope (SEM), a laser particle size analyzer, an energy spectrum analysis (EDS) picture, an infrared spectrum (FT-IR) picture and the like to characterize the structure of the nano selenium.
EXAMPLE 4 preparation of Rosa roxburghii polysaccharide Nano selenium solution (SeNPs)
Mixing 10 mL of sodium selenite solution with the same volume and certain concentration of fructus Rosae Normalis polysaccharide solution (0.25, 0.5, 1, 2, 3,4 mg/mL) or distilled water, adding 100 mL of 10 mM vitamin C solution, stirring, shaking on a shaker at 300 r/min at 37 deg.C for 36 h, and dialyzing the reaction solution at 4 deg.C for 96 h. Finally, freeze drying the dialyzed nano selenium solution to obtain roxburgh rose polysaccharide nano selenium particles (RP 3-SeNPs) and nano selenium particles (SeNPs) respectively. And respectively adopting a Transmission Electron Microscope (TEM) picture, a high-resolution Scanning Electron Microscope (SEM), a laser particle size analyzer, an energy spectrum analysis (EDS) picture, an infrared spectrum (FT-IR) picture and the like to characterize the structure of the nano selenium.
The polysaccharide of Rosa roxburghii prepared as in example 3 above was subjected to structural identification and activity analysis by the following methods, and the results of examples 2 and 4 were similar to those of example 3.
This example examined the effect of different concentrations of rosa roxburghii tratt polysaccharide in the reaction system on the particle size of the nano-selenium particles in the reaction system. As shown in FIG. 1a, the present invention selects Rosa roxburghii polysaccharide solutions of 0.25, 0.5, 1, 2, 3,4 mg/mL to modify the nano-selenium particles. When the concentration of the rosa roxburghii tratt polysaccharide RTFP-3 is 0.25-2 mg/mL, the particle size of the nano selenium is gradually reduced; when the concentration of the rosa roxburghii tratt polysaccharide RTFP-3 is more than 2 mg/mL, the nano selenium particles are almost unchanged. Therefore, the optimal concentration of the roxburgh rose polysaccharide modified nano selenium particles is determined to be 2 mg/mL, and the average particle size of the nano selenium particles is 104.5 nm.
As shown in fig. 1b, in this embodiment, the storage stability of the nano-selenium solution prepared from the rosa roxburghii polysaccharide solutions with different concentrations is considered, and as the storage time increases, the particle size of the nano-selenium solution prepared from 1 mg/mL rosa roxburghii polysaccharide solution gradually increases, but the particle size of the nano-selenium solution prepared from 2 mg/mL rosa roxburghii polysaccharide solution is basically unchanged, which indicates that the nano-selenium solution prepared from the high-concentration rosa roxburghii polysaccharide solution has better stability, can be stored for a long time, and is beneficial to long-term storage of developed drugs.
Fig. 2a is a Transmission Electron Microscope (TEM) image of the nano-selenium sol, which shows that the nano-selenium modified by the polysaccharide solution without rosa roxburghii tratt is easy to agglomerate into a stacked structure, and the nano-selenium modified by the polysaccharide solution with rosa roxburghii tratt (see fig. 2b) can be prepared into nano-selenium particles with uniform particle size and uniform dispersion. Fig. 2c is a Scanning Electron Microscope (SEM) image showing that the polysaccharide-free nano-selenium particles are aggregated into a honeycomb structure, and fig. 2d shows that the polysaccharide-modified nano-selenium particles are uniformly dispersed in the network framework of the rosa roxburghii tratt polysaccharide.
As shown in FIG. 3a, the IR spectrum of the fructus Rosae Normalis polysaccharide nano selenium complex shows that the peak of-OH group is from 3442 cm-1Red shift to 3456 cm-1C = O radical peak from 1610 cm-1Red shift to 1635 cm-1Peak of C-O-C group from 1239 cm-1Red shift to 1242 cm-1The red-shift of these characteristic peaks indicates the formation of a Se-O chemical bond between the Se atom and the polysaccharide. In addition, the UV spectrum (see FIG. 3b) shows that the complex of Rosa roxburghii polysaccharide and Rosa roxburghii polysaccharide nano-selenium is 198 and 278 cm-1Has the same absorption peak, and the existence of the characteristic peaks indicates that covalent bonding is formed between the rosa roxburghii polysaccharide and the nano selenium. The energy spectrum analysis result shows that the structure of the rosa roxburghii tratt polysaccharide (shown in figure 3 c) does not contain selenium element, and the selenium atom content of the rosa roxburghii tratt polysaccharide nano selenium compound (shown in figure 3 d) is about 27.01 percent.
EXAMPLE 3 inhibition of DPPH free radical by different samples
Accurately weighing a certain amount of Rosa roxburghii polysaccharide, nanometer selenium particles and Rosa roxburghii polysaccharide nanometer selenium particles, and dissolving in distilled water to prepare solutions (0.25-2.0 mg/m L) with different concentrations for use. Adding 2 mL of sample solution into 2 mL of DPPH working solution (prepared by 75 mu M of 50% methanol solution), vortex mixing uniformly, reacting for 30min in a dark place at room temperature, measuring the light absorption value at the wavelength of 517 nm, using distilled water to replace the sample as a blank control, using the methanol solution to replace the DPPH solution as a background control, and using Vitamin C (VC) as a positive control. DPPH radical clearance was calculated according to the following formula:
DPPH radical clearance (%) = [1- (a)Sample (I)-ABackground control)/ABlank space]× 100
FIG. 4a is a graph of the inhibition rate of the RTFP-3, RP3-SeNPs and SeNPs against DPPH radicals. The results show that RTFP-3 and RP3-SeNPs have better effect of eliminating DPPH in the concentration range of 0.25-2.0 mg/mL, and the antioxidant activity of the three substances is enhanced along with the increase of the concentration. The maximum DPPH free radical clearance rates of RTFP-3, RP3-SeNPs and SeNPs at a concentration of 2 mg/mL were 66.01%, 56.29% and 13.92%, respectively. Among them, RTFP-3 has the strongest scavenging effect on DPPH free radicals, and RP3-SeNPs are the second order, and SeNPs show the weakest activity of scavenging DPPH.
EXAMPLE 4 inhibition of ABTS free radicals by different samples
ABTS working solution is prepared from 5 mL of ABTS solution (7 mM) and 5 mL of K2S2O8The solution (2.45 mM) was prepared after 12 hours of reaction at room temperature in the dark. Before use, the solution is diluted with a phosphate buffered sodium salt solution to an absorbance of 0.70. + -. 0.02 at 734 nm. Then, 0.4 mL of sample solution with different concentrations (0.25-2.0 mg/mL) is added into 4 mL of diluted ABTS working solution, the solution is oscillated for 30 s and then is reacted for 6 min in the dark, the absorbance value is detected under the wavelength of 734 nm, the sample solution is replaced by distilled water with the same volume as a blank control, the ABTS working solution is replaced by distilled water with the same volume as a background control, and vitamin C (V) is usedC) For positive control, the formula for ABTS free radical clearance calculation is as follows:
ABTS free radical clearance (%) = [1- (A)Sample (I)-ABackground control)/ABlank space]× 100
The results of the tests are shown in FIG. 4b, where RTFP-3, RP3-SeNPs and SeNPs have half the clearance (IC) of ABTS free radicals50) 0.93 mg/mL, 1.05 mg/mL and>10 mg/mL. Therefore, the nano-selenium particles SeNPs show extremely weak ABTS free radical scavenging capability, and the ABTS free radical scavenging capability of the roxburgh rose polysaccharide nano-selenium compound is remarkably improved after the roxburgh rose polysaccharide RTFP-3 is modified.
Example 5 evaluation of the Oxidative Radical Absorption Capacity (ORAC) of Rosa polysaccharide
All sample solutions were prepared from phosphate buffer (75 mM, pH 7.4). 20 μ L of RTFP-3, SeNPs and RP3-SeNPs solutions and 200 μ L of fluorescein sodium salt solution (95.6 nM) were sequentially added to a 96-well microplate in different concentrations (0.5-4.0 mg/mL), while a phosphate buffer solution was used as a control instead of the fluorescein sodium salt solution, and distilled water was used as a blank control instead of the sample. Setting fluorescence/chemical analyzer program, incubating at 37 deg.C in dark for 15 min, rapidly adding 20 μ L ABAP solution (119.4 m M) into each well, automatically mixing, and excitingRecording the emission wavelength of 485 nm and the absorption wavelength of 535 nm every 2 min, circulating for 70 times, and respectively recording the fluorescence intensity f1、f2、f3、······f70The area of fluorescence quenching (AUC) value was calculated, and the formula is as follows: AUC = 2 × [1/2 × (f)1 + f35) + f2 + f3 + f4+······+ f70]. And simultaneously drawing a standard curve by using Trolox standard solutions (6.25-100 mu M) with different concentrations, wherein the ORAC value of the sample represents that each gram of sample is equivalent to a Trolox Equivalent (TE) value, and the unit is mu mol TE/g.
As a result, the ORAC values of RTFP-3, RP3-SeNPs and SeNPs were 105.65, 79.94 and 5.33. mu. mol TE/g, respectively, as shown in FIG. 4 c. From the ORAC value, compared with pure nano selenium particles, the oxidation resistance of the roxburgh rose polysaccharide nano selenium compound is improved by 14 times.
Example 6 toxicity of Rosa polysaccharide Nano-selenium particles to INS-1 cells
Experimental cells: rat insulinoma cell INS-1 was routinely cultured. The medium for culturing the cells comprises 10% of fetal bovine serum, 2 mM of L-glutamine, 1 mM of sodium pyruvate, 50. mu.M of beta-mercaptoethanol, 100 units/mL of penicillin and 100 units/mL of streptomycin. The cell viability was determined by the MTT method.
The cytotoxicity determination comprises collecting INS-1 cells in logarithmic growth phase, blowing and beating the cells into single cell suspension with pancreatin, centrifuging for 5 min to remove supernatant, resuspending the cells in culture medium, counting with blood cell counting plate, adjusting cell concentration to 6 × 104The density of the cells was inoculated in a 96-well cell culture plate and placed at 37 ℃ in 5% CO2The culture was performed for 12 hours, and then the culture solution was discarded. Adding 100 mu L of cell culture medium containing RP3-SeNPs with different concentrations into each well, placing the cell culture medium into an incubator for continuous culture for 24 h, adding 20 mu L of MTT solution (5 mg/mL) into each well, continuing incubation for 4 h, then sucking out the culture solution, washing for 1 time by using 100 mu L of PBS, finally adding 100 mu L of DMSO into each well, measuring the light absorption value of each well at the wavelength of 570 nm after oscillating for 10 min, and calculating the survival rate of the cells according to the following formula. Experimental setup blank, experimental group andand (4) a control group. The blank group is not added with cells, 100 muL of culture medium is added, the experimental group and the control group are added with 100 muL of cell suspension liquid in each hole, wherein the control group is not added with samples. Wherein A isSAbsorbance values for the experimental groups, ACIs the absorbance value of the control group, A0Is the absorbance value of the blank.
Cell survival rate (%) = (a)S-A0)/(AC-A0)
The toxicity of RP3-SeNPs to INS-1 cells was measured at progressively increasing concentration gradients (0, 0.25, 0.5, 1, 2, 4. mu.g/mL), and the results (FIG. 5 a) showed that at concentrations up to 4. mu.g/mL, the toxicity of RP3-SeNPs exerted a major role, beginning to promote apoptosis, with cell survival rates of 79.1%, significantly less than the blank control; and the concentration range of the RP3-SeNPs is 0-2 mug/mL, and the RP3-SeNPs have almost no cytotoxicity. The low-concentration RP3-SeNPs have remarkable cell proliferation promoting activity, and the high-concentration RP3-SeNPs can inhibit cell proliferation and are toxic to cells, so that the concentration of the RP3-SeNPs is lower than 2 mug/mL for subsequent experiments.
Example 7 determination of H2O2Optimal concentration for inducing INS-1 cell oxidative damage model
According to 6X 104The density of individual cells was seeded in 96-well cell culture plates at 37 ℃ in 5% CO2The culture was performed for 12 hours, and then the culture solution was discarded. Adding 100 mu L of H containing different concentrations into each hole2O2After the cell culture medium is placed in an incubator and continuously cultured for 12 hours, the survival rate of the cells is calculated according to the MTT method.
The results are shown in FIG. 5b, with H2O2Increase in concentration, H2O2The cytotoxicity of the INS-1 cell is stronger and stronger, and the proliferation of the INS-1 cell can be obviously inhibited. When H is present2O2The cell survival rate was 67.54% at a concentration of 250. mu.M, and the molding concentration was determined to be suitable.
Example 8 determination of the protective Effect of RP3-SeNPs on oxidatively damaged INS-1 cells
The protective effect of RP3-SeNPs on INS-1 cells was determined. According to 6X 104The density of individual cells was seeded in 96-well cell culture plates at 37 ℃ in 5% CO2The culture was performed for 12 hours, and then the culture solution was discarded. Adding 100 mu L of RP3-SeNPs cell culture medium containing different concentrations into each well, placing the cells in an incubator for continuous culture for 24 h, and then discarding the culture medium. Adding 100 mu L of H-containing solution into each hole2O2After the cell culture medium is placed in an incubator and continuously cultured for 12 hours, the survival rate of the cells is calculated according to the MTT method.
The results are shown in FIG. 5c, where INS-1 cells were pre-treated with RP3-SeNPs for 24H to inhibit H2O2Induced cell damage. When the concentration of RP3-SeNPs was 2 μ g/mL, the survival rate of the cells was 89.34%, which is H2O2The survival rate of the model group cells is 1.36 times, and RP3-SeNPs can protect islet cells from oxidative damage.
Example 9 determination of the uptake of Nerium album polysaccharide Nanosenium by INS-1 cells
Adjusting cell concentration to 5 × 105The density of the cells was inoculated in 6-well cell culture plates and incubated at 37 ℃ in 5% CO2The culture was performed for 12 hours, and then the culture solution was discarded. 2 mL of cell culture medium containing different concentrations of RP3-SeNPs (0.5, 1 and 2 mg/mL) were added to each well and incubated for 24 h. The cells were harvested at 8 and 24 h time points, washed 3 times with PBS buffer, and the intracellular selenium content was measured by ICP-AES.
As shown in FIG. 5d, the uptake of polysaccharide nanoselen by INS-1 cells gradually increased with the incubation time and the increase in polysaccharide nanoselen concentration. When the concentration of RP3-SeNPs is 0.5 mug/mL and the incubation time is 24 h, the selenium concentration in the INS-1 cells is 5.68 mg/108And (4) cells. When the concentration of RP3-SeNPs is increased to 2 mug/mL, the selenium intake of INS-1 cells is increased by 2.21 times.
Example 10 glucose stimulation of insulin secretion by insulinoma cell INS-1
Adjusting cell concentration to 6 × 105The density of the cells was inoculated in 6-well cell culture plates and incubated at 37 ℃ in 5% CO2The culture was performed for 12 hours, and then the culture solution was discarded. 2 mL per wellAfter incubation for 24 h with the same concentration of RP3-SeNPs (0.5, 1 and 2 μ g/mL), the medium was discarded. Adding M H containing 250 mu per well except for normal group2O2After 12 h incubation, INS-1 cells were incubated with 5.6 mmol/L and 16.7 mmol/L glucose solutions (in Hanks balanced salt solution) for 30min and the insulin content of the medium was determined using an insulin enzyme-linked immunoassay kit.
The results are shown in FIG. 6, H2O2Can impair the insulin-secreting function of INS-1 cells, H2O2Compared with the normal group, the INS-1 cell insulin secretion capacity is remarkably reduced under the stimulation of 5.6 mmol/L or 16.7 mmol/L glucose in the model group, which indicates that H2O2Can induce INS-1 cells to undergo oxidative damage, thereby causing impaired insulin secretion. After INS-1 cells are pretreated by RP3-SeNPs with different concentrations, RP3-SeNPs are found to protect the insulin secretion function of INS-1 cells. Wherein INS-1 cells were pretreated with 2. mu.M RP3-SeNP for 24 h, and the amount of insulin secreted by INS-1 cells was 2.23 times that of the model group under the stimulation of higher concentration glucose (16.7 mmol/L). However, the insulin secretion amount of INS-1 cells treated with RP3-SeNP was not significantly different from that of the model group after stimulation with glucose at a lower concentration (5.6 mmol/L). From the above results, it is clear that RP3-SeNPs can protect the insulin secretion function of insulinoma cells.
Claims (7)
1. A preparation method of a rosa roxburghii polysaccharide functionalized nano selenium compound is characterized by comprising the following steps:
(1) uniformly mixing a sodium selenite solution and a roxburgh rose polysaccharide solution, then adding a vitamin C solution, uniformly stirring, and carrying out oscillation reaction;
(2) dialyzing the reaction solution obtained in the step (1), and drying to obtain a rosa roxburghii polysaccharide functionalized nano selenium compound;
the concentration of the roxburgh rose polysaccharide solution is 0.25-4 mg/L;
the concentration of the sodium selenite solution in the step (1) is 1-3 mM, and the concentration of the vitamin C solution is 6-10 mM;
the volume ratio of the sodium selenite solution to the rosa roxburghii polysaccharide solution in the step (1) is 1: 1-1: 3;
the temperature of the reaction in the step (1) is 37 ℃;
the Rosa roxburghii polysaccharide in the step (1) is RTFP-3, and the preparation of the RTFP-3 comprises the following steps:
(a) cleaning fresh rosa roxburghii tratt fruits, drying and crushing the rosa roxburghii tratt fruits in a 60-DEG C forced air drying box, sieving the rosa roxburghii tratt fruits by a 60-mesh sieve to obtain rosa roxburghii tratt dry powder, preparing mixed liquid with the solid-to-liquid ratio of 1:6 g/mL according to the rosa roxburghii tratt dry powder and 95% ethanol, heating and refluxing for 4 hours under the condition of 70 ℃, filtering to obtain residues, and repeating the heating, refluxing and filtering for 3 times to obtain degreased rosa roxburghii tratt dry powder;
(b) putting the degreased roxburgh rose dry powder in the step (a) into a 45-DEG C forced air drying oven for drying for 48 h; adding distilled water into 150 g of dried roxburgh rose powder for hot water extraction, wherein the extraction temperature is 95 ℃, the extraction time is 3 hours, the volume ratio of the mass of the dried roxburgh rose powder to the distilled water is 1:30 according to the mass g of the dried roxburgh rose powder and the volume ml of the distilled water, and the extraction frequency of the hot water extraction is 2 times; centrifuging at centrifugal force of 5000 g for 10 min, collecting supernatant, rotary evaporating at 55 deg.C to concentrate to 1/4 of original volume to obtain fructus Rosae Normalis polysaccharide concentrate;
(c) adding Sevag reagent into the concentrated solution of the roxburgh rose polysaccharide in the step (b), wherein the Sevag reagent is chloroform: n-butyl alcohol =4:1, the volume ratio of the concentrated solution of the roxburgh rose polysaccharide to the Sevag reagent is 3:1, the oscillation is carried out for 30min, the upper layer sugar solution is centrifugally separated, the centrifugal force is 5000 g, the centrifugal time is 10 min, the oscillation and centrifugation processes are repeatedly carried out for more than 12 times until no protein residue is observed by naked eyes, and the concentrated solution is decompressed and concentrated to 1/3 of the original volume to obtain the concentrated solution after deproteinization;
(d) adding macroporous resin AB-8 into the deproteinized concentrated solution obtained in the step (C), carrying out decoloring treatment, wherein the volume ratio of the deproteinized concentrated solution to the macroporous resin is 6:1, the temperature is 37 ℃, carrying out oscillation treatment on a shaking table for 10 hours, and filtering to obtain filtrate; adding anhydrous ethanol into the decolorized filtrate to make the volume concentration of ethanol reach 70%, standing at 4 deg.C for 24 hr, centrifuging at 5000 g for 10 min to obtain lower layer polysaccharide precipitate, and freeze drying for 48 hr to obtain water soluble fructus Rosae Normalis crude polysaccharide;
(e) dissolving the water-soluble roxburgh rose crude polysaccharide obtained in the step (d) by using distilled water to prepare a polysaccharide solution with the final concentration of 20 mg/mL, loading the polysaccharide solution on a DEAE-Sepharose fast flow column, wherein the ratio of the loading volume to the column volume is 1:4, performing gradient elution by using 0.2 mol/L NaCl solution, the flow rate of an eluent is 1.0 mL/min, and collecting 5 mL of eluent in each tube; and concentrating and dialyzing the collected solution for 48 h, and then carrying out vacuum freeze drying to obtain the purified roxburgh rose polysaccharide RTFP-3.
2. The method of claim 1, wherein the concentration of the Rosa roxburghii polysaccharide solution is 0.25, 0.5, 1, 2, 3 or 4 mg/L.
3. The method according to claim 1, wherein the volume of the vitamin C solution in the step (1) is 5 to 10 times that of the sodium selenite solution.
4. The preparation method of claim 1, wherein the reaction in step (1) is carried out at 100-300 rpm for 12-36 h.
5. The preparation method of claim 1, wherein the dialysis in step (2) is carried out by transferring the reaction solution obtained in step (1) into a dialysis bag, dialyzing in water, and stopping dialysis after the dialysate is free of selenium ions through ICP-MS detection; the dialysis temperature is 2-8 ℃; the drying is freeze drying, and the drying temperature is-20 to-50 ℃.
6. A Rosa roxburghii polysaccharide functionalized nano selenium compound prepared by the preparation method of any one of claims 1-5.
7. The use of the rosa roxburghii polysaccharide functionalized nano-selenium compound of claim 6 in the preparation of hypoglycemic drugs.
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