CN111530425A - Lactic acid scavenger - Google Patents
Lactic acid scavenger Download PDFInfo
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- CN111530425A CN111530425A CN202010350240.XA CN202010350240A CN111530425A CN 111530425 A CN111530425 A CN 111530425A CN 202010350240 A CN202010350240 A CN 202010350240A CN 111530425 A CN111530425 A CN 111530425A
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- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 title claims abstract description 166
- 239000004310 lactic acid Substances 0.000 title claims abstract description 83
- 235000014655 lactic acid Nutrition 0.000 title claims abstract description 83
- 239000002516 radical scavenger Substances 0.000 title claims abstract description 19
- SZVJSHCCFOBDDC-UHFFFAOYSA-N iron(II,III) oxide Inorganic materials O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 claims abstract description 210
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 62
- 238000001179 sorption measurement Methods 0.000 claims abstract description 55
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 48
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 22
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 22
- GXDMUOPCQNLBCZ-UHFFFAOYSA-N 3-(3-triethoxysilylpropyl)oxolane-2,5-dione Chemical compound CCO[Si](OCC)(OCC)CCCC1CC(=O)OC1=O GXDMUOPCQNLBCZ-UHFFFAOYSA-N 0.000 claims abstract description 19
- 210000004369 blood Anatomy 0.000 claims abstract description 13
- 239000008280 blood Substances 0.000 claims abstract description 13
- 229910021577 Iron(II) chloride Inorganic materials 0.000 claims abstract description 12
- 229910021578 Iron(III) chloride Inorganic materials 0.000 claims abstract description 12
- NMCUIPGRVMDVDB-UHFFFAOYSA-L iron dichloride Chemical compound Cl[Fe]Cl NMCUIPGRVMDVDB-UHFFFAOYSA-L 0.000 claims abstract description 12
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims abstract description 12
- 238000011002 quantification Methods 0.000 claims abstract description 11
- 230000003013 cytotoxicity Effects 0.000 claims abstract description 9
- 231100000135 cytotoxicity Toxicity 0.000 claims abstract description 9
- GXGAKHNRMVGRPK-UHFFFAOYSA-N dimagnesium;dioxido-bis[[oxido(oxo)silyl]oxy]silane Chemical compound [Mg+2].[Mg+2].[O-][Si](=O)O[Si]([O-])([O-])O[Si]([O-])=O GXGAKHNRMVGRPK-UHFFFAOYSA-N 0.000 claims abstract description 6
- 239000000391 magnesium silicate Substances 0.000 claims abstract description 6
- 229940099273 magnesium trisilicate Drugs 0.000 claims abstract description 6
- 229910000386 magnesium trisilicate Inorganic materials 0.000 claims abstract description 6
- 235000019793 magnesium trisilicate Nutrition 0.000 claims abstract description 6
- 239000002994 raw material Substances 0.000 claims abstract description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 40
- 210000002381 plasma Anatomy 0.000 claims description 38
- 239000012530 fluid Substances 0.000 claims description 30
- 210000001519 tissue Anatomy 0.000 claims description 30
- 238000000034 method Methods 0.000 claims description 26
- 239000008367 deionised water Substances 0.000 claims description 25
- 229910021641 deionized water Inorganic materials 0.000 claims description 25
- 239000006228 supernatant Substances 0.000 claims description 25
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 20
- 239000002105 nanoparticle Substances 0.000 claims description 17
- 238000005406 washing Methods 0.000 claims description 17
- 238000003756 stirring Methods 0.000 claims description 16
- 239000003146 anticoagulant agent Substances 0.000 claims description 15
- 229940127219 anticoagulant drug Drugs 0.000 claims description 15
- 210000004027 cell Anatomy 0.000 claims description 15
- 238000001514 detection method Methods 0.000 claims description 15
- 239000000203 mixture Substances 0.000 claims description 15
- 238000006243 chemical reaction Methods 0.000 claims description 11
- 102000001554 Hemoglobins Human genes 0.000 claims description 10
- 108010054147 Hemoglobins Proteins 0.000 claims description 10
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 claims description 10
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 claims description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 239000012091 fetal bovine serum Substances 0.000 claims description 10
- 239000001963 growth medium Substances 0.000 claims description 10
- 238000005342 ion exchange Methods 0.000 claims description 10
- 229910001425 magnesium ion Inorganic materials 0.000 claims description 10
- 238000007885 magnetic separation Methods 0.000 claims description 10
- 229910052757 nitrogen Inorganic materials 0.000 claims description 10
- 239000013641 positive control Substances 0.000 claims description 10
- 239000002244 precipitate Substances 0.000 claims description 10
- 239000000725 suspension Substances 0.000 claims description 10
- 238000000870 ultraviolet spectroscopy Methods 0.000 claims description 10
- 210000003722 extracellular fluid Anatomy 0.000 claims description 8
- 239000000463 material Substances 0.000 claims description 8
- 238000009825 accumulation Methods 0.000 claims description 6
- 230000007246 mechanism Effects 0.000 claims description 6
- 210000003205 muscle Anatomy 0.000 claims description 6
- OAVCWZUKQIEFGG-UHFFFAOYSA-O 2-(5-methyl-2H-tetrazol-1-ium-1-yl)-1,3-thiazole Chemical compound CC1=NN=N[NH+]1C1=NC=CS1 OAVCWZUKQIEFGG-UHFFFAOYSA-O 0.000 claims description 5
- 125000000972 4,5-dimethylthiazol-2-yl group Chemical group [H]C([H])([H])C1=C(N=C(*)S1)C([H])([H])[H] 0.000 claims description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 5
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 5
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 5
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 claims description 5
- 239000012980 RPMI-1640 medium Substances 0.000 claims description 5
- 229910008051 Si-OH Inorganic materials 0.000 claims description 5
- 229910006358 Si—OH Inorganic materials 0.000 claims description 5
- 102000004142 Trypsin Human genes 0.000 claims description 5
- 108090000631 Trypsin Proteins 0.000 claims description 5
- 230000007059 acute toxicity Effects 0.000 claims description 5
- 231100000403 acute toxicity Toxicity 0.000 claims description 5
- 238000004458 analytical method Methods 0.000 claims description 5
- 238000003149 assay kit Methods 0.000 claims description 5
- 230000015572 biosynthetic process Effects 0.000 claims description 5
- 239000001110 calcium chloride Substances 0.000 claims description 5
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 5
- 235000011148 calcium chloride Nutrition 0.000 claims description 5
- 238000004113 cell culture Methods 0.000 claims description 5
- 230000003833 cell viability Effects 0.000 claims description 5
- 230000008859 change Effects 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 5
- 239000003085 diluting agent Substances 0.000 claims description 5
- 239000001257 hydrogen Substances 0.000 claims description 5
- 229910052739 hydrogen Inorganic materials 0.000 claims description 5
- -1 hydrogen ions Chemical class 0.000 claims description 5
- 239000013642 negative control Substances 0.000 claims description 5
- 230000002035 prolonged effect Effects 0.000 claims description 5
- 238000004088 simulation Methods 0.000 claims description 5
- 238000003786 synthesis reaction Methods 0.000 claims description 5
- 230000001988 toxicity Effects 0.000 claims description 5
- 231100000419 toxicity Toxicity 0.000 claims description 5
- 239000012588 trypsin Substances 0.000 claims description 5
- 238000005303 weighing Methods 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims 1
- 239000000126 substance Substances 0.000 abstract description 5
- 230000005389 magnetism Effects 0.000 abstract description 4
- 235000015872 dietary supplement Nutrition 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 24
- 230000000694 effects Effects 0.000 description 9
- 239000003463 adsorbent Substances 0.000 description 8
- 208000010444 Acidosis Diseases 0.000 description 3
- 206010027417 Metabolic acidosis Diseases 0.000 description 3
- 230000002378 acidificating effect Effects 0.000 description 3
- 230000006399 behavior Effects 0.000 description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 3
- 238000000975 co-precipitation Methods 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 230000005415 magnetization Effects 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 230000006378 damage Effects 0.000 description 2
- 230000002503 metabolic effect Effects 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 231100000683 possible toxicity Toxicity 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 208000025978 Athletic injury Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000006538 anaerobic glycolysis Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 238000005470 impregnation Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 238000000554 physical therapy Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000009469 supplementation Effects 0.000 description 1
- 238000012549 training Methods 0.000 description 1
Classifications
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/223—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material containing metals, e.g. organo-metallic compounds, coordination complexes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
- B01J20/28009—Magnetic properties
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
- B01J20/28016—Particle form
- B01J20/28019—Spherical, ellipsoidal or cylindrical
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- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Inorganic Chemistry (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses a lactic acid scavenger, which comprises the following raw materials in parts by weight: FeCl3 & 6H2O (8-10 parts), FeCl2 & 4H2O (3-5 parts), HCl (6-8 parts), NaOH (1-3 parts), ethanol (0.5-1 part), 3- (triethoxysilyl) propyl succinic anhydride (0.1-0.3 part), lactic acid (20-40 parts), magnesium trisilicate (5-10 parts), BCA protein quantification kit (10-20 parts). Compared with the common nutritional supplement, the Fe3O4@ MT not only shows a rapid and efficient LA adsorption behavior through physical adsorption, but also through the action of chemical bonds, and the Fe3O4@ MT has the characteristics of good blood compatibility, small cytotoxicity, good magnetism and the like, so that the occurrence of exercise-induced fatigue can be well prevented.
Description
Technical Field
The invention relates to a scavenger production process, in particular to a lactic acid scavenger.
Background
If lactic acid cannot be eliminated in time after strenuous exercise, exercise fatigue is caused, resulting in exercise damage, and the conventional lactic acid removal method is limited in timeliness, metabolic burden, and potential toxicity.
Exercise fatigue is a common physiological phenomenon in exercise training, however, if fatigue cannot be eliminated in time, exercise injuries occur. Although the mechanism of exercise-induced fatigue is controversial, there is considerable evidence that fatigue deepens with increasing accumulation of lactic acid and LA is a metabolite produced by anaerobic glycolysis. When a large amount of LA is distributed in muscles and blood, the balance of the internal environment of the body is disrupted. Resulting in reduced physical function and motor fatigue. In practice, various approaches have been tried in an effort to reduce lactic acid levels after exercise. Active and passive rest, physiotherapy and nutritional supplementation are common methods to accelerate LA elimination. These methods can prevent sports injuries and the timeliness of using these methods is limited. It takes a long time to metabolize LA, which increases our body's metabolic burden, and furthermore, the potential toxicity of nutritional supplements remains a concern.
Disclosure of Invention
The invention aims to solve the defects in the prior art, and provides a lactic acid scavenger which adopts a coprecipitation method to prepare Fe3O4 nano particles. The preparation method is characterized in that reactive carboxyl is grafted by 3- (triethoxysilyl) propyl succinic anhydride, MT is coated on nanoparticles under mild reaction conditions to form a novel LA adsorbent which is spherical and has the particle size of about 180 nanometers and good dispersibility, the magnetization intensity of Fe3O4@ MT is measured by using a superconducting quantum interference device, the saturation degree is 48.81 emu/g, and in view of the fact that the prior superparamagnetic nanoparticles have good magnetic guiding effect in biomedical application in vitro and in vivo, Fe3O4@ MT has huge potential in the aspects of exogenous magnetic guiding easy injection and extraction, and different from the traditional LA removing method, Fe3O4@ MT can directly adsorb LA in solution, LA is not metabolized into other acidic products, metabolic acidosis cannot be caused, more importantly, LA can be adsorbed by Fe3O4@ MT within a short time, fe3O4@ MT can quickly remove LA, the method is more suitable for athletes, better adsorption efficiency can be achieved at 0.5 h, more exchanged ions are used, more adsorbed LA is used, the adsorption of Fe3O4@ MT on LA is obtained, the LA can be effectively removed through Fe3O4@ MT, the adsorption amount of protein is measured through a BCA method, a small amount of protein is adsorbed by Fe3O4@ MT, the Fe3O4@ MT has good blood compatibility, MT is a good adsorbent and is applied to food and biomedicine, so that Fe3O4@ MT is a safe LA scavenging agent, the MT is coated on the surface of Fe3O4 nano particles, the MT is conveniently synthesized through Fe3O4 MT under the mild reaction condition, the Fe3O4@ MT not only represents quick and efficient LA adsorption behaviors through physical adsorption but also through the effect of chemical bonds, and the Fe3O4@ MT has good blood compatibility, Small cytotoxicity, good magnetism, and the like, and can well prevent the occurrence of exercise-induced fatigue.
In order to achieve the purpose, the invention provides the following technical scheme: a lactic acid scavenger comprises the following raw materials in parts by weight: FeCl3 & 6H2O (8-10 parts), FeCl2 & 4H2O (3-5 parts), HCl (6-8 parts), NaOH (1-3 parts), ethanol (0.5-1 part), 3- (triethoxysilyl) propylsuccinic anhydride (0.1-0.3 part), lactic acid (20-40 parts), magnesium trisilicate (5-10 parts), BCA protein quantification kit (10-20 parts), lactic acid assay kit (20-30 parts), 3- (4, 5-dimethylthiazol-2-yl) -2 (10-15 parts), 5-diphenyltetrazolium bromide (5-10 parts), RPMI-1640 (3-5 parts), DMEM (1-3 parts), fetal bovine serum (5-10 parts), trypsin (2-4 parts), the ionized water (20-40 parts) comprises the following steps:
s1, weighing materials: FeCl 3.6H 2O (9 parts), FeCl 2.4H 2O (4 parts), HCl (4 parts), NaOH (2 parts), ethanol (0.75 part), 3- (triethoxysilyl) propylsuccinic anhydride (0.2 part), ionized water (30 parts);
s2, Fe3O4@ MT synthesis: under nitrogen protection, 8.14 g FeCl3 & 6H2O and 3.00 g FeCl2 & 4H2O were dissolved in 30mL deionized water, followed by the addition of 0.85 mL of 12 mol/L HCl. The resulting solution was dropped into 250mL of a 1.5mol/L NaOH solution with vigorous stirring. After the reaction, the black precipitate was washed three times with deionized water and dried under vacuum, 0.1 g of prepared Fe3O4 nanoparticles was taken, ultrasonically washed with 20mL of ethanol for 2 minutes, repeated three times, 30mL of ethanol and 0.15mL of 3- (triethoxysilyl) propylsuccinic anhydride were added under nitrogen protection, and stirred at 50 ℃ for 7 hours. Washing the obtained black precipitate with deionized water for three times, storing in 50 deg.C water for 5 hr, adding dried 40mg Fe3O4 and 20.8 mg MT into ethanol, and shaking for 0.5 hr;
and (3) detecting the adsorption efficiency of S3 and Fe3O4 on lactic acid: adding Fe3O4 with different masses into plasma with high LA concentration and simulated tissue fluid respectively, putting the mixture into a four-dimensional rotary mixer for 0.5 hour, collecting supernatant by a magnetic separation method, respectively measuring the residual amounts of LA and protein in the supernatant by using a lactic acid kit and a BCA protein quantification kit, and determining the change of the adsorption efficiency of Fe3O4 with time by using an experimental group. Respectively adding Fe3O4 with the same LA quality into the blood plasma and the simulated tissue fluid, stirring the mixture again, selecting time once between 10 and 60 minutes, and measuring the residual quantity of lactic acid in the mixture;
s4, Fe3O4@ MT: incubating 10 mL of Fe3O4 suspension with different concentrations in 0.9% NaCl solution at 37 ℃ for 30 minutes, adding 0.2 mL of diluted anticoagulant, incubating for 1 hour, centrifuging at 2500 rotation speed for 5 minutes, collecting supernatant, analyzing released hemoglobin by using a 540-nanometer ultraviolet-visible spectrophotometer, taking deionized water with the same volume as a positive control, taking 0.9% NaCl solution as a negative control, adding different amounts of Fe3O4 into 0.1 mL of diluted anticoagulant, incubating for 10 minutes, adding 0.1 mL of 0.02 mol/L CaCl2 solution, recalcifying the anticoagulant, pouring 14 mL of deionized water after 0.5 hour, centrifuging at 2500 rotation speed for 5 minutes, analyzing released hemoglobin by using a 540-nanometer ultraviolet-visible spectrophotometer, and taking the diluent without Fe3O4 as a positive control;
s5, cytotoxicity detection: placing HUVEC and HSMC monolayers in a 25 cm2 cell culture bottle, placing in a corresponding culture medium containing 10% heat-inactivated FBS and 1% PS, culturing in a specific environment, inoculating the cells into a 96-well plate at a density of 5000 cells per well, adding Fe3O4 suspension with different concentrations into the culture medium after 12 hours, taking out Fe3O4 after 0.5 hours, determining the cell viability in time and 23.5 hours later by adopting an MTT (methyl thiazolyl tetrazolium) analysis method, and respectively showing acute toxicity and prolonged toxicity at 0.5 hours and 24 hours;
s6, simulation detection: preparing plasma with high lactic acid concentration and simulated tissue fluid of 3 mmol/L and 23 mmol/L respectively, simulating the accumulation of lactic acid in blood and muscle after high-intensity exercise, adding Fe3O4@ MT with different mass ratios, collecting supernatant by magnetic separation after stirring for 0.5 hour, measuring lactic acid in the supernatant by using a lactate kit, wherein the adsorption rate of the lactic acid is increased along with the increase of the mass ratio, and when the ratio reaches 1:1, different adsorption rates are obtained in the simulated tissue fluid and the plasma.
Mechanism detection of adsorption of lactic acid by S7 and Fe3O 4: after adsorbing lactic acid, the zeta potential of Fe3O4 in plasma and simulated tissue fluid becomes negative, magnesium ions are sucked out through ion exchange, a large amount of lactic acid is adsorbed on the surface of Fe3O4, and the ion exchange of hydrogen ions and magnesium ions causes the increase of Si-OH, and the adsorption peak in FTIR is about 950 cm-1.
Preferably, an ultrasonic washing device is used in washing the ethanol.
Preferably, the high lactate plasma and the simulated interstitial fluid are mixed using a four-dimensional rotary mixer.
Preferably, the plasma and simulated tissue fluid are added to the same mass of lactic acid as Fe3O4 for 10 minutes, 20 minutes, 30 minutes, 40 minutes or 60 minutes, respectively.
Preferably, the cells are in a humidified environment at 37 ℃ with 5% CO 2.
The invention has the technical effects and advantages that:
the invention adopts a coprecipitation method to prepare Fe3O4 nano particles, reactive carboxyl is grafted by 3- (triethoxysilyl) propyl succinic anhydride, MT is coated on the nano particles under mild reaction conditions to form a novel LA adsorbent, the novel adsorbent is spherical, the particle size is about 180 nanometers, the novel LA adsorbent has good dispersibility, the magnetization intensity of Fe3O4@ MT is measured by utilizing a superconducting quantum interference device, the saturation degree is 48.81 emu/g, in view of the fact that the prior superparamagnetic nano particles have good magnetic guiding effect in biomedical application in vitro and in vivo, the Fe3O4@ MT has huge potential in the aspects of exogenous magnetic guiding easy injection and extraction, different from the traditional LA removal method, the Fe3O4 MT can directly adsorb LA in solution, the LA is not metabolized into other acidic products, and metabolic acidosis cannot be caused, more importantly, LA can be adsorbed by Fe3O4@ MT in a short time, Fe3O4@ MT can quickly remove LA, the LA is more suitable for athletes to use, better adsorption efficiency can be achieved at 0.5 h, more exchanged ions and more adsorbed LA are provided, so that the adsorption of Fe3O4@ MT on LA is obtained, the LA can be effectively removed by Fe3O4@ MT and is dependent on chemical bonds, the adsorption amount of protein is measured by adopting a BCA method, a small amount of protein is adsorbed by Fe3O4@ MT, Fe3O4@ MT has good blood compatibility, MT is a good adsorbent and is applied to food and biomedicine, so that Fe3O4@ MT is a safe LA scavenging agent, Fe3O4@ MT is conveniently synthesized by coating MT on the surface of Fe3O4 nano particles under mild reaction conditions, and Fe3O4@ MT not only can be adsorbed by physics, but also can be adsorbed by chemical bonds, and can quickly and efficiently express LA adsorption behavior, because Fe3O4@ MT has the characteristics of good blood compatibility, small cytotoxicity, good magnetism and the like, the occurrence of exercise-induced fatigue can be well prevented.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A lactic acid scavenger comprises the following raw materials in parts by weight: FeCl3 & 6H2O (8-10 parts), FeCl2 & 4H2O (3-5 parts), HCl (6-8 parts), NaOH (1-3 parts), ethanol (0.5-1 part), 3- (triethoxysilyl) propylsuccinic anhydride (0.1-0.3 part), lactic acid (20-40 parts), magnesium trisilicate (5-10 parts), BCA protein quantification kit (10-20 parts), lactic acid assay kit (20-30 parts), 3- (4, 5-dimethylthiazol-2-yl) -2 (10-15 parts), 5-diphenyltetrazolium bromide (5-10 parts), RPMI-1640 (3-5 parts), DMEM (1-3 parts), fetal bovine serum (5-10 parts), trypsin (2-4 parts), the ionized water (20-40 parts) comprises the following steps:
s1, weighing materials: FeCl 3.6H 2O (9 parts), FeCl 2.4H 2O (4 parts), HCl (7 parts), NaOH (2 parts), ethanol (0.75 part), 3- (triethoxysilyl) propylsuccinic anhydride (0.2 part), ionized water (30 parts);
s2, Fe3O4@ MT synthesis: under nitrogen protection, 8.14 g FeCl3 & 6H2O and 3.00 g FeCl2 & 4H2O were dissolved in 30mL deionized water, followed by the addition of 0.85 mL of 12 mol/L HCl. The resulting solution was dropped into 250mL of a 1.5mol/L NaOH solution with vigorous stirring. After the reaction, the black precipitate was washed three times with deionized water and dried under vacuum, 0.1 g of prepared Fe3O4 nanoparticles was taken, ultrasonically washed with 20mL of ethanol for 2 minutes, repeated three times, 30mL of ethanol and 0.15mL of 3- (triethoxysilyl) propylsuccinic anhydride were added under nitrogen protection, and stirred at 50 ℃ for 7 hours. Washing the obtained black precipitate with deionized water for three times, storing in 50 deg.C water for 5 hr, adding dried 40mg Fe3O4 and 20.8 mg MT into ethanol, and shaking for 0.5 hr;
and (3) detecting the adsorption efficiency of S3 and Fe3O4 on lactic acid: adding Fe3O4 with different masses into plasma with high LA concentration and simulated tissue fluid respectively, putting the mixture into a four-dimensional rotary mixer for 0.5 hour, collecting supernatant by a magnetic separation method, respectively measuring the residual amounts of LA and protein in the supernatant by using a lactic acid kit and a BCA protein quantification kit, and determining the change of the adsorption efficiency of Fe3O4 with time by using an experimental group. Respectively adding Fe3O4 with the same LA quality into the blood plasma and the simulated tissue fluid, stirring the mixture again, selecting time once between 10 and 60 minutes, and measuring the residual quantity of lactic acid in the mixture;
s4, Fe3O4@ MT: incubating 10 mL of Fe3O4 suspension with different concentrations in 0.9% NaCl solution at 37 ℃ for 30 minutes, adding 0.2 mL of diluted anticoagulant, incubating for 1 hour, centrifuging at 2500 rotation speed for 5 minutes, collecting supernatant, analyzing released hemoglobin by using a 540-nanometer ultraviolet-visible spectrophotometer, taking deionized water with the same volume as a positive control, taking 0.9% NaCl solution as a negative control, adding different amounts of Fe3O4 into 0.1 mL of diluted anticoagulant, incubating for 10 minutes, adding 0.1 mL of 0.02 mol/L CaCl2 solution, recalcifying the anticoagulant, pouring 14 mL of deionized water after 0.5 hour, centrifuging at 2500 rotation speed for 5 minutes, analyzing released hemoglobin by using a 540-nanometer ultraviolet-visible spectrophotometer, and taking the diluent without Fe3O4 as a positive control;
s5, cytotoxicity detection: placing HUVEC and HSMC monolayers in a 25 cm2 cell culture bottle, placing in a corresponding culture medium containing 10% heat-inactivated FBS and 1% PS, culturing in a specific environment, inoculating the cells into a 96-well plate at a density of 5000 cells per well, adding Fe3O4 suspension with different concentrations into the culture medium after 12 hours, taking out Fe3O4 after 0.5 hours, determining the cell viability in time and 23.5 hours later by adopting an MTT (methyl thiazolyl tetrazolium) analysis method, and respectively showing acute toxicity and prolonged toxicity at 0.5 hours and 24 hours;
s6, simulation detection: preparing plasma with high lactic acid concentration and simulated tissue fluid of 3 mmol/L and 23 mmol/L respectively, simulating the accumulation of lactic acid in blood and muscle after high-intensity exercise, adding Fe3O4@ MT with different mass ratios, collecting supernatant by magnetic separation after stirring for 0.5 hour, measuring lactic acid in the supernatant by using a lactate kit, wherein the adsorption rate of the lactic acid is increased along with the increase of the mass ratio, and when the ratio reaches 1:1, different adsorption rates are obtained in the simulated tissue fluid and the plasma.
Mechanism detection of adsorption of lactic acid by S7 and Fe3O 4: after adsorbing lactic acid, the zeta potential of Fe3O4 in plasma and simulated tissue fluid becomes negative, magnesium ions are sucked out through ion exchange, a large amount of lactic acid is adsorbed on the surface of Fe3O4, and the ion exchange of hydrogen ions and magnesium ions causes the increase of Si-OH, and the adsorption peak in FTIR is about 950 cm-1.
Preferably, an ultrasonic washing device is used in washing the ethanol.
Preferably, the high lactate plasma and the simulated interstitial fluid are mixed using a four-dimensional rotary mixer.
Preferably, the mixing time for adding the same amount of Fe3O4 to the plasma and simulated interstitial fluid is 10 minutes.
Preferably, the cells are in a humidified environment at 37 ℃ with 5% CO 2.
The adsorption rates of Fe3O4@ MT obtained by the method in simulated tissue fluid and plasma are respectively as follows: 51.22 percent and 39.33 percent, and achieves good adsorption effect within the ranges of standard adsorption rates of 50.36 +/-1.98 percent and 38.72 +/-1.69 percent.
Example 2
A lactic acid scavenger comprises the following raw materials in parts by weight: FeCl3 & 6H2O (8-10 parts), FeCl2 & 4H2O (3-5 parts), HCl (6-8 parts), NaOH (1-3 parts), ethanol (0.5-1 part), 3- (triethoxysilyl) propylsuccinic anhydride (0.1-0.3 part), lactic acid (20-40 parts), magnesium trisilicate (5-10 parts), BCA protein quantification kit (10-20 parts), lactic acid assay kit (20-30 parts), 3- (4, 5-dimethylthiazol-2-yl) -2 (10-15 parts), 5-diphenyltetrazolium bromide (5-10 parts), RPMI-1640 (3-5 parts), DMEM (1-3 parts), fetal bovine serum (5-10 parts), trypsin (2-4 parts), the ionized water (20-40 parts) comprises the following steps:
s1, weighing materials: FeCl3 & 6H2O (8 parts), FeCl2 & 4H2O (3 parts), HCl (6 parts), NaOH (1 part), ethanol (0.5 part), 3- (triethoxysilyl) propylsuccinic anhydride (0.1 part), ionized water (20 parts);
s2, Fe3O4@ MT synthesis: under nitrogen protection, 8.14 g FeCl3 & 6H2O and 3.00 g FeCl2 & 4H2O were dissolved in 30mL deionized water, followed by the addition of 0.85 mL of 12 mol/L HCl. The resulting solution was dropped into 250mL of a 1.5mol/L NaOH solution with vigorous stirring. After the reaction, the black precipitate was washed three times with deionized water and dried under vacuum, 0.1 g of prepared Fe3O4 nanoparticles was taken, ultrasonically washed with 20mL of ethanol for 2 minutes, repeated three times, 30mL of ethanol and 0.15mL of 3- (triethoxysilyl) propylsuccinic anhydride were added under nitrogen protection, and stirred at 50 ℃ for 7 hours. Washing the obtained black precipitate with deionized water for three times, storing in 50 deg.C water for 5 hr, adding dried 40mg Fe3O4 and 20.8 mg MT into ethanol, and shaking for 0.5 hr;
and (3) detecting the adsorption efficiency of S3 and Fe3O4 on lactic acid: adding Fe3O4 with different masses into plasma with high LA concentration and simulated tissue fluid respectively, putting the mixture into a four-dimensional rotary mixer for 0.5 hour, collecting supernatant by a magnetic separation method, respectively measuring the residual amounts of LA and protein in the supernatant by using a lactic acid kit and a BCA protein quantification kit, and determining the change of the adsorption efficiency of Fe3O4 with time by using an experimental group. Respectively adding Fe3O4 with the same LA quality into the blood plasma and the simulated tissue fluid, stirring the mixture again, selecting time once between 10 and 60 minutes, and measuring the residual quantity of lactic acid in the mixture;
s4, Fe3O4@ MT: incubating 10 mL of Fe3O4 suspension with different concentrations in 0.9% NaCl solution at 37 ℃ for 30 minutes, adding 0.2 mL of diluted anticoagulant, incubating for 1 hour, centrifuging at 2500 rotation speed for 5 minutes, collecting supernatant, analyzing released hemoglobin by using a 540-nanometer ultraviolet-visible spectrophotometer, taking deionized water with the same volume as a positive control, taking 0.9% NaCl solution as a negative control, adding different amounts of Fe3O4 into 0.1 mL of diluted anticoagulant, incubating for 10 minutes, adding 0.1 mL of 0.02 mol/L CaCl2 solution, recalcifying the anticoagulant, pouring 14 mL of deionized water after 0.5 hour, centrifuging at 2500 rotation speed for 5 minutes, analyzing released hemoglobin by using a 540-nanometer ultraviolet-visible spectrophotometer, and taking the diluent without Fe3O4 as a positive control;
s5, cytotoxicity detection: placing HUVEC and HSMC monolayers in a 25 cm2 cell culture bottle, placing in a corresponding culture medium containing 10% heat-inactivated FBS and 1% PS, culturing in a specific environment, inoculating the cells into a 96-well plate at a density of 5000 cells per well, adding Fe3O4 suspension with different concentrations into the culture medium after 12 hours, taking out Fe3O4 after 0.5 hours, determining the cell viability in time and 23.5 hours later by adopting an MTT (methyl thiazolyl tetrazolium) analysis method, and respectively showing acute toxicity and prolonged toxicity at 0.5 hours and 24 hours;
s6, simulation detection: preparing plasma with high lactic acid concentration and simulated tissue fluid of 3 mmol/L and 23 mmol/L respectively, simulating the accumulation of lactic acid in blood and muscle after high-intensity exercise, adding Fe3O4@ MT with different mass ratios, collecting supernatant by magnetic separation after stirring for 0.5 hour, measuring lactic acid in the supernatant by using a lactate kit, wherein the adsorption rate of the lactic acid is increased along with the increase of the mass ratio, and when the ratio reaches 1:1, different adsorption rates are obtained in the simulated tissue fluid and the plasma.
Mechanism detection of adsorption of lactic acid by S7 and Fe3O 4: after adsorbing lactic acid, the zeta potential of Fe3O4 in plasma and simulated tissue fluid becomes negative, magnesium ions are sucked out through ion exchange, a large amount of lactic acid is adsorbed on the surface of Fe3O4, and the ion exchange of hydrogen ions and magnesium ions causes the increase of Si-OH, and the adsorption peak in FTIR is about 950 cm-1.
Preferably, the material grinding and crushing time is 30 minutes, the material activation time is 45 minutes, and the material impregnation time is 30 minutes.
Preferably, an ultrasonic washing device is used in washing the ethanol.
Preferably, the high lactate plasma and the simulated interstitial fluid are mixed using a four-dimensional rotary mixer.
Preferably, the mixing time for adding the same amount of Fe3O4 to the plasma and simulated interstitial fluid is 30 minutes.
Preferably, the cells are in a humidified environment at 37 ℃ with 5% CO 2.
The adsorption rates of Fe3O4@ MT obtained by the method in simulated tissue fluid and plasma are respectively as follows: 51.12 percent and 39.41 percent, and achieves good adsorption effect within the ranges of standard adsorption rates of 50.36 +/-1.98 percent and 38.72 +/-1.69 percent.
Example 3
A lactic acid scavenger comprises the following raw materials in parts by weight: FeCl3 & 6H2O (8-10 parts), FeCl2 & 4H2O (3-5 parts), HCl (6-8 parts), NaOH (1-3 parts), ethanol (0.5-1 part), 3- (triethoxysilyl) propylsuccinic anhydride (0.1-0.3 part), lactic acid (20-40 parts), magnesium trisilicate (5-10 parts), BCA protein quantification kit (10-20 parts), lactic acid assay kit (20-30 parts), 3- (4, 5-dimethylthiazol-2-yl) -2 (10-15 parts), 5-diphenyltetrazolium bromide (5-10 parts), RPMI-1640 (3-5 parts), DMEM (1-3 parts), fetal bovine serum (5-10 parts), trypsin (2-4 parts), the ionized water (20-40 parts) comprises the following steps:
s1, weighing materials: FeCl 3.6H 2O (10 parts), FeCl 2.4H 2O (5 parts), HCl (8 parts), NaOH (3 parts), ethanol (1 part), 3- (triethoxysilyl) propyl succinic anhydride (0.3 part), and ionized water (40 parts);
s2, Fe3O4@ MT synthesis: under nitrogen protection, 8.14 g FeCl3 & 6H2O and 3.00 g FeCl2 & 4H2O were dissolved in 30mL deionized water, followed by the addition of 0.85 mL of 12 mol/L HCl. The resulting solution was dropped into 250mL of a 1.5mol/L NaOH solution with vigorous stirring. After the reaction, the black precipitate was washed three times with deionized water and dried under vacuum, 0.1 g of prepared Fe3O4 nanoparticles was taken, ultrasonically washed with 20mL of ethanol for 2 minutes, repeated three times, 30mL of ethanol and 0.15mL of 3- (triethoxysilyl) propylsuccinic anhydride were added under nitrogen protection, and stirred at 50 ℃ for 7 hours. Washing the obtained black precipitate with deionized water for three times, storing in 50 deg.C water for 5 hr, adding dried 40mg Fe3O4 and 20.8 mg MT into ethanol, and shaking for 0.5 hr;
and (3) detecting the adsorption efficiency of S3 and Fe3O4 on lactic acid: adding Fe3O4 with different masses into plasma with high LA concentration and simulated tissue fluid respectively, putting the mixture into a four-dimensional rotary mixer for 0.5 hour, collecting supernatant by a magnetic separation method, respectively measuring the residual amounts of LA and protein in the supernatant by using a lactic acid kit and a BCA protein quantification kit, and determining the change of the adsorption efficiency of Fe3O4 with time by using an experimental group. Respectively adding Fe3O4 with the same LA quality into the blood plasma and the simulated tissue fluid, stirring the mixture again, selecting time once between 10 and 60 minutes, and measuring the residual quantity of lactic acid in the mixture;
s4, Fe3O4@ MT: incubating 10 mL of Fe3O4 suspension with different concentrations in 0.9% NaCl solution at 37 ℃ for 30 minutes, adding 0.2 mL of diluted anticoagulant, incubating for 1 hour, centrifuging at 2500 rotation speed for 5 minutes, collecting supernatant, analyzing released hemoglobin by using a 540-nanometer ultraviolet-visible spectrophotometer, taking deionized water with the same volume as a positive control, taking 0.9% NaCl solution as a negative control, adding different amounts of Fe3O4 into 0.1 mL of diluted anticoagulant, incubating for 10 minutes, adding 0.1 mL of 0.02 mol/L CaCl2 solution, recalcifying the anticoagulant, pouring 14 mL of deionized water after 0.5 hour, centrifuging at 2500 rotation speed for 5 minutes, analyzing released hemoglobin by using a 540-nanometer ultraviolet-visible spectrophotometer, and taking the diluent without Fe3O4 as a positive control;
s5, cytotoxicity detection: placing HUVEC and HSMC monolayers in a 25 cm2 cell culture bottle, placing in a corresponding culture medium containing 10% heat-inactivated FBS and 1% PS, culturing in a specific environment, inoculating the cells into a 96-well plate at a density of 5000 cells per well, adding Fe3O4 suspension with different concentrations into the culture medium after 12 hours, taking out Fe3O4 after 0.5 hours, determining the cell viability in time and 23.5 hours later by adopting an MTT (methyl thiazolyl tetrazolium) analysis method, and respectively showing acute toxicity and prolonged toxicity at 0.5 hours and 24 hours;
s6, simulation detection: preparing plasma with high lactic acid concentration and simulated tissue fluid of 3 mmol/L and 23 mmol/L respectively, simulating the accumulation of lactic acid in blood and muscle after high-intensity exercise, adding Fe3O4@ MT with different mass ratios, collecting supernatant by magnetic separation after stirring for 0.5 hour, measuring lactic acid in the supernatant by using a lactate kit, wherein the adsorption rate of the lactic acid is increased along with the increase of the mass ratio, and when the ratio reaches 1:1, different adsorption rates are obtained in the simulated tissue fluid and the plasma.
Mechanism detection of adsorption of lactic acid by S7 and Fe3O 4: after adsorbing lactic acid, the zeta potential of Fe3O4 in plasma and simulated tissue fluid becomes negative, magnesium ions are sucked out through ion exchange, a large amount of lactic acid is adsorbed on the surface of Fe3O4, and the ion exchange of hydrogen ions and magnesium ions causes the increase of Si-OH, and the adsorption peak in FTIR is about 950 cm-1.
Preferably, an ultrasonic washing device is used in washing the ethanol.
Preferably, the high lactate plasma and the simulated interstitial fluid are mixed using a four-dimensional rotary mixer.
Preferably, the plasma and simulated tissue fluid are added to the same mass of lactic acid as Fe3O4 for 10 minutes, 20 minutes, 30 minutes, 40 minutes or 60 minutes, respectively.
Preferably, the cells are in a humidified environment at 37 ℃ with 5% CO 2.
The adsorption rates of Fe3O4@ MT obtained by the method in simulated tissue fluid and plasma are respectively as follows: 49.58 percent and 37.86 percent, and achieves good adsorption effect within the range of standard adsorption rates of 50.36 +/-1.98 percent and 38.72 +/-1.69 percent.
In summary, the following steps: compared with other treatment processes, the lactic acid scavenger provided by the invention has the following advantages: fe3O4 nano particles are prepared by adopting a coprecipitation method. The preparation method is characterized in that reactive carboxyl is grafted by 3- (triethoxysilyl) propyl succinic anhydride, MT is coated on nanoparticles under mild reaction conditions to form a novel LA adsorbent which is spherical and has the particle size of about 180 nanometers and good dispersibility, the magnetization intensity of Fe3O4@ MT is measured by using a superconducting quantum interference device, the saturation degree is 48.81 emu/g, and in view of the fact that the prior superparamagnetic nanoparticles have good magnetic guiding effect in biomedical application in vitro and in vivo, Fe3O4@ MT has huge potential in the aspects of exogenous magnetic guiding easy injection and extraction, and different from the traditional LA removing method, Fe3O4@ MT can directly adsorb LA in solution, LA is not metabolized into other acidic products, metabolic acidosis cannot be caused, more importantly, LA can be adsorbed by Fe3O4@ MT within a short time, fe3O4@ MT can quickly remove LA, the method is more suitable for athletes, better adsorption efficiency can be achieved at 0.5 h, more exchanged ions are used, more adsorbed LA is used, the adsorption of Fe3O4@ MT on LA is obtained, the LA can be effectively removed through Fe3O4@ MT, the adsorption amount of protein is measured through a BCA method, a small amount of protein is adsorbed by Fe3O4@ MT, the Fe3O4@ MT has good blood compatibility, MT is a good adsorbent and is applied to food and biomedicine, so that Fe3O4@ MT is a safe LA scavenging agent, the MT is coated on the surface of Fe3O4 nano particles, the MT is conveniently synthesized through Fe3O4 MT under the mild reaction condition, the Fe3O4@ MT not only represents quick and efficient LA adsorption behaviors through physical adsorption but also through the effect of chemical bonds, and the Fe3O4@ MT has good blood compatibility, Small cytotoxicity, good magnetism, and the like, and can well prevent the occurrence of exercise-induced fatigue.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments or portions thereof without departing from the spirit and scope of the invention.
Claims (5)
1. A lactic acid scavenger characterized by: comprises the following raw materials in parts by weight: FeCl3 & 6H2O (8-10 parts), FeCl2 & 4H2O (3-5 parts), HCl (6-8 parts), NaOH (1-3 parts), ethanol (0.5-1 part), 3- (triethoxysilyl) propylsuccinic anhydride (0.1-0.3 part), lactic acid (20-40 parts), magnesium trisilicate (5-10 parts), BCA protein quantification kit (10-20 parts), lactic acid assay kit (20-30 parts), 3- (4, 5-dimethylthiazol-2-yl) -2 (10-15 parts), 5-diphenyltetrazolium bromide (5-10 parts), RPMI-1640 (3-5 parts), DMEM (1-3 parts), fetal bovine serum (5-10 parts), trypsin (2-4 parts), ionized water (20-40 parts);
the method comprises the following steps:
s1, weighing materials: FeCl 3.6H 2O (9 parts), FeCl 2.4H 2O (4 parts), HCl (4 parts), NaOH (2 parts), ethanol (0.75 part), 3- (triethoxysilyl) propylsuccinic anhydride (0.2 part), ionized water (30 parts);
s2, Fe3O4@ MT synthesis: under the protection of nitrogen, 8.14 g of FeCl3 & 6H2O and 3.00 g of FeCl2 & 4H2O are dissolved in 30mL of deionized water, and then 0.85 mL of 12 mol/L HCl is added;
dropping the obtained solution into 250mL of 1.5mol/L NaOH solution under vigorous stirring;
after the reaction, washing the black precipitate with deionized water for three times, drying in vacuum, taking 0.1 g of prepared Fe3O4 nano particles, ultrasonically washing with 20mL of ethanol for 2 minutes, repeating for three times, adding 30mL of ethanol and 0.15mL of 3- (triethoxysilyl) propyl succinic anhydride under the protection of nitrogen, and stirring for 7 hours at 50 ℃;
washing the obtained black precipitate with deionized water for three times, storing in 50 deg.C water for 5 hr, adding dried 40mg Fe3O4 and 20.8 mg MT into ethanol, and shaking for 0.5 hr;
and (3) detecting the adsorption efficiency of S3 and Fe3O4 on lactic acid: adding Fe3O4 with different masses into plasma with high LA concentration and simulated tissue fluid respectively, putting the mixture into a four-dimensional rotary mixer for 0.5 hour, collecting supernatant by a magnetic separation method, respectively determining the residual amounts of LA and protein in the supernatant by adopting a lactic acid kit and a BCA protein quantification kit, and determining the change of the adsorption efficiency of Fe3O4 along with time by additionally arranging an experimental group;
respectively adding Fe3O4 with the same LA quality into the blood plasma and the simulated tissue fluid, stirring the mixture again, selecting time once between 10 and 60 minutes, and measuring the residual quantity of lactic acid in the mixture;
s4, Fe3O4@ MT: incubating 10 mL of Fe3O4 suspension with different concentrations in 0.9% NaCl solution at 37 ℃ for 30 minutes, adding 0.2 mL of diluted anticoagulant, incubating for 1 hour, centrifuging at 2500 rotation speed for 5 minutes, collecting supernatant, analyzing released hemoglobin by using a 540-nanometer ultraviolet-visible spectrophotometer, taking deionized water with the same volume as a positive control, taking 0.9% NaCl solution as a negative control, adding different amounts of Fe3O4 into 0.1 mL of diluted anticoagulant, incubating for 10 minutes, adding 0.1 mL of 0.02 mol/L CaCl2 solution, recalcifying the anticoagulant, pouring 14 mL of deionized water after 0.5 hour, centrifuging at 2500 rotation speed for 5 minutes, analyzing released hemoglobin by using a 540-nanometer ultraviolet-visible spectrophotometer, and taking the diluent without Fe3O4 as a positive control;
s5, cytotoxicity detection: placing HUVEC and HSMC monolayers in a 25 cm2 cell culture bottle, placing in a corresponding culture medium containing 10% heat-inactivated FBS and 1% PS, culturing in a specific environment, inoculating the cells into a 96-well plate at a density of 5000 cells per well, adding Fe3O4 suspension with different concentrations into the culture medium after 12 hours, taking out Fe3O4 after 0.5 hours, determining the cell viability in time and 23.5 hours later by adopting an MTT (methyl thiazolyl tetrazolium) analysis method, and respectively showing acute toxicity and prolonged toxicity at 0.5 hours and 24 hours;
s6, simulation detection: preparing plasma with high lactic acid concentration and simulated tissue fluid, wherein the concentration is respectively 3 mmol/L and 23 mmol/L, simulating the accumulation of lactic acid in blood and muscle after high-intensity exercise, adding Fe3O4@ MT with different mass ratios, collecting supernatant by a magnetic separation method after stirring for 0.5 hour, measuring the lactic acid in the supernatant by a lactate kit, increasing the adsorption rate of the lactic acid along with the increase of the mass ratio, and obtaining different adsorption rates in the simulated tissue fluid and the plasma when the ratio reaches 1: 1;
mechanism detection of adsorption of lactic acid by S7 and Fe3O 4: after adsorbing lactic acid, the zeta potential of Fe3O4 in plasma and simulated tissue fluid becomes negative, magnesium ions are sucked out through ion exchange, a large amount of lactic acid is adsorbed on the surface of Fe3O4, and the ion exchange of hydrogen ions and magnesium ions causes the increase of Si-OH, and the adsorption peak in FTIR is about 950 cm-1.
2. A lactic acid scavenger according to claim 1, wherein: ultrasonic washing equipment is used in washing ethanol.
3. A lactic acid scavenger according to claim 1, wherein: high lactate plasma and simulated interstitial fluid were mixed using a four-dimensional rotary mixer.
4. A lactic acid scavenger according to claim 1, wherein: the plasma and simulated interstitial fluid were added to the same mass of lactic acid as Fe3O4 for 10 minutes, 20 minutes, 30 minutes, 40 minutes, or 60 minutes, respectively.
5. A lactic acid scavenger according to claim 1, wherein: the cells were maintained in a humidified atmosphere at 37 ℃ with 5% CO 2.
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