CN111530425B - Lactic acid scavenger - Google Patents
Lactic acid scavenger Download PDFInfo
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- CN111530425B CN111530425B CN202010350240.XA CN202010350240A CN111530425B CN 111530425 B CN111530425 B CN 111530425B CN 202010350240 A CN202010350240 A CN 202010350240A CN 111530425 B CN111530425 B CN 111530425B
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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 140
- 239000004310 lactic acid Substances 0.000 title claims abstract description 70
- 235000014655 lactic acid Nutrition 0.000 title claims abstract description 70
- 239000002516 radical scavenger Substances 0.000 title claims abstract description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 56
- 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 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 35
- 238000000034 method Methods 0.000 claims description 26
- 239000008367 deionised water Substances 0.000 claims description 23
- 229910021641 deionized water Inorganic materials 0.000 claims description 23
- 239000002105 nanoparticle Substances 0.000 claims description 22
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 20
- 238000003756 stirring Methods 0.000 claims description 13
- 238000006243 chemical reaction Methods 0.000 claims description 10
- 229910052757 nitrogen Inorganic materials 0.000 claims description 10
- 239000002244 precipitate Substances 0.000 claims description 10
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 4
- 238000000975 co-precipitation Methods 0.000 claims description 4
- 230000015572 biosynthetic process Effects 0.000 claims 1
- 238000003786 synthesis reaction Methods 0.000 claims 1
- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical compound O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 abstract description 164
- 238000001179 sorption measurement Methods 0.000 abstract description 49
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 abstract description 21
- 102000004169 proteins and genes Human genes 0.000 abstract description 15
- 108090000623 proteins and genes Proteins 0.000 abstract description 15
- 210000004369 blood Anatomy 0.000 abstract description 8
- 239000008280 blood Substances 0.000 abstract description 8
- 231100000135 cytotoxicity Toxicity 0.000 abstract description 6
- 230000003013 cytotoxicity Effects 0.000 abstract description 6
- 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 abstract description 5
- 239000000391 magnesium silicate Substances 0.000 abstract description 5
- 229940099273 magnesium trisilicate Drugs 0.000 abstract description 5
- 229910000386 magnesium trisilicate Inorganic materials 0.000 abstract description 5
- 235000019793 magnesium trisilicate Nutrition 0.000 abstract description 5
- 239000002994 raw material Substances 0.000 abstract description 5
- 239000000126 substance Substances 0.000 abstract description 5
- 230000009471 action Effects 0.000 abstract description 2
- 235000015872 dietary supplement Nutrition 0.000 abstract description 2
- 230000005389 magnetism Effects 0.000 abstract description 2
- 210000002381 plasma Anatomy 0.000 description 31
- 239000000243 solution Substances 0.000 description 25
- 239000012530 fluid Substances 0.000 description 23
- 210000001519 tissue Anatomy 0.000 description 23
- 239000000203 mixture Substances 0.000 description 20
- 239000006228 supernatant Substances 0.000 description 20
- 239000003146 anticoagulant agent Substances 0.000 description 16
- 229940127219 anticoagulant drug Drugs 0.000 description 16
- 230000000694 effects Effects 0.000 description 16
- 239000003463 adsorbent Substances 0.000 description 13
- 210000004027 cell Anatomy 0.000 description 12
- 238000005406 washing Methods 0.000 description 12
- 238000005342 ion exchange Methods 0.000 description 10
- 102000001554 Hemoglobins Human genes 0.000 description 8
- 108010054147 Hemoglobins Proteins 0.000 description 8
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 8
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 238000005119 centrifugation Methods 0.000 description 8
- 238000001514 detection method Methods 0.000 description 8
- 210000003722 extracellular fluid Anatomy 0.000 description 8
- 229910001425 magnesium ion Inorganic materials 0.000 description 8
- 238000007885 magnetic separation Methods 0.000 description 8
- 239000013641 positive control Substances 0.000 description 8
- 239000000725 suspension Substances 0.000 description 8
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 8
- 230000005291 magnetic effect Effects 0.000 description 7
- 239000000463 material Substances 0.000 description 7
- 238000009825 accumulation Methods 0.000 description 5
- 230000007246 mechanism Effects 0.000 description 5
- 210000003205 muscle Anatomy 0.000 description 5
- 239000002245 particle Substances 0.000 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 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 4
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 4
- 231100000002 MTT assay Toxicity 0.000 description 4
- 238000000134 MTT assay Methods 0.000 description 4
- 239000012980 RPMI-1640 medium Substances 0.000 description 4
- 229910008051 Si-OH Inorganic materials 0.000 description 4
- 229910006358 Si—OH Inorganic materials 0.000 description 4
- 102000004142 Trypsin Human genes 0.000 description 4
- 108090000631 Trypsin Proteins 0.000 description 4
- 230000001154 acute effect Effects 0.000 description 4
- 238000003149 assay kit Methods 0.000 description 4
- 239000001110 calcium chloride Substances 0.000 description 4
- 229910001628 calcium chloride Inorganic materials 0.000 description 4
- 235000011148 calcium chloride Nutrition 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 230000003833 cell viability Effects 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 239000012894 fetal calf serum Substances 0.000 description 4
- 239000001257 hydrogen Substances 0.000 description 4
- 229910052739 hydrogen Inorganic materials 0.000 description 4
- -1 hydrogen ions Chemical class 0.000 description 4
- WSSMOXHYUFMBLS-UHFFFAOYSA-L iron dichloride tetrahydrate Chemical compound O.O.O.O.[Cl-].[Cl-].[Fe+2] WSSMOXHYUFMBLS-UHFFFAOYSA-L 0.000 description 4
- NQXWGWZJXJUMQB-UHFFFAOYSA-K iron trichloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].Cl[Fe+]Cl NQXWGWZJXJUMQB-UHFFFAOYSA-K 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 239000013642 negative control Substances 0.000 description 4
- 230000002035 prolonged effect Effects 0.000 description 4
- 238000011002 quantification Methods 0.000 description 4
- 238000004088 simulation Methods 0.000 description 4
- 230000002194 synthesizing effect Effects 0.000 description 4
- 231100000419 toxicity Toxicity 0.000 description 4
- 230000001988 toxicity Effects 0.000 description 4
- 239000012588 trypsin Substances 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- 230000002378 acidificating effect Effects 0.000 description 3
- 238000004140 cleaning 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
- 230000005415 magnetization Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 230000002503 metabolic effect Effects 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000005298 paramagnetic effect Effects 0.000 description 2
- 231100000683 possible toxicity Toxicity 0.000 description 2
- 208000010444 Acidosis Diseases 0.000 description 1
- 208000025978 Athletic injury Diseases 0.000 description 1
- 206010027417 Metabolic acidosis Diseases 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000006538 anaerobic glycolysis Effects 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
- 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
- 230000000977 initiatory effect Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002207 metabolite Substances 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
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 parts), 3- (triethoxysilyl) propylsuccinic anhydride (0.1-0.3 parts), lactic acid (20-40 parts), magnesium trisilicate (5-10 parts) and BCA protein quantitative kit (10-20 parts). Compared with the common nutritional supplements, the Fe3O4@MT not only shows 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 fatigue can be well prevented.
Description
Technical Field
The invention relates to a production process of a scavenger, in particular to a lactic acid scavenger.
Background
If lactic acid is not eliminated in time after strenuous exercise, exercise fatigue is caused, and thus exercise damage is caused, and conventional lactic acid removal methods are limited in timeliness, metabolic burden and potential toxicity.
Exercise fatigue is a common physiological phenomenon during exercise training, however, if fatigue cannot be eliminated in time, exercise damage occurs. Although the mechanism of exercise-induced fatigue is controversial, there is a great deal of evidence that as lactic acid accumulation increases, fatigue deepens and LA is a metabolite produced by anaerobic glycolysis. When a large amount of LA is distributed in the muscle and blood, the balance of the internal environment of the body may be disturbed. Resulting in reduced body function and initiation of exercise fatigue. In practice, various methods have been attempted 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. Metabolism of LA takes a long time, which increases the metabolic burden of our body, and furthermore, the potential toxicity of nutritional supplements remains alarming.
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 3- (triethoxysilyl) propyl succinic anhydride is used for grafting reactive carboxyl, MT is coated on the nano particles under the mild reaction condition to form a novel LA adsorbent, the novel adsorbent is spherical, the particle size is about 180 nanometers, the novel adsorbent has good dispersibility, the magnetization of the Fe3O4@MT is measured by utilizing a superconducting quantum interference device, the saturation of the novel adsorbent is 48.81 emu/g, the previous superparamagnetic nano particles have good magnetic guiding effect in the biomedical application in vitro and in vivo, thereby obtaining great potential of the Fe3O4@MT in the aspect of easy injection and extraction of exogenous magnetic guiding, the novel adsorbent is different from the traditional LA cleaning method, the Fe3O4@MT can directly adsorb LA in solution, the LA is not metabolized into other acidic products, more importantly, the LA can be adsorbed by the Fe3O4@MT in a short time, the Fe3O4 MT can be rapidly removed, the LA is more suitable for use, the LA can be better adsorbed by the ion exchange efficiency in the super paramagnetic nano particles in 0.5 h, the LA can be better adsorbed by the super-paramagnetic particles, the LA is more LA can be well adsorbed by the Fe3O4@MT, the novel adsorption agent has good physical and chemical adsorption effect, the specific to the Fe3O4@MT can be conveniently removed by the high, the surface of the nano particles, the specific adsorption effect of the Fe3O 4@3 MT is better than the Fe 4@3O 4 is better, the high-3O 4 is better than the specific to be adsorbed by the Fe 4, and the high-3O 4, the high-purity is better physical adsorption effect, and the high-purity is better than the high by the high-purity adsorption effect and the Fe 4 MT is better than the high 2F 3O 3 MT can be adsorbed by the better than better adsorbed by the high chemically and better than better contrast, and better than better contrast compared with the high contrast compared with the contrast compared, can well prevent the occurrence of exercise fatigue.
In order to achieve the above purpose, the present invention provides the following technical solutions: the 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 parts), 3- (triethoxysilyl) propylsuccinic anhydride (0.1-0.3 parts), 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 calf serum (5-10 parts), trypsin (2-4 parts), and ionized water (20-40 parts) comprising the steps of:
s1, weighing materials: feCl3.6H2O (9 parts), feCl2.4H2O (4 parts), HCl (4 parts), naOH (2 parts), ethanol (0.75 parts), 3- (triethoxysilyl) propyl succinic anhydride (0.2 parts), and ionized water (30 parts);
s2, synthesizing Fe3O4@ MT: 8.14g FeCl3.6H2O and 3.00g FeCl2.4H2O were dissolved in 30mL deionized water under nitrogen protection, followed by the addition of 0.85mL12mol/L HCl. The resulting solution was dropped into 250mL of 1.5mol/L NaOH solution under vigorous stirring. After the reaction, the black precipitate was washed three times with deionized water and dried under vacuum, 0.1. 0.1g of the prepared Fe3O4 nanoparticle was taken, washed with 20mL of ethanol by ultrasonic for 2 minutes, repeated three times, 30mL of ethanol and 0.15mL of 3- (triethoxysilyl) propyl succinic anhydride were added under nitrogen protection, and stirred at 50 degrees for 7 hours. Washing the obtained black precipitate with deionized water for three times, preserving in 50 ℃ water for 5 hours, adding dried 40mgFe 3O4 and 20.8mg MT into ethanol, and shaking up for 0.5 hour;
s3, detecting the adsorption efficiency of Fe3O4 to lactic acid: adding Fe3O4 with different quality into high LA concentration plasma and simulated tissue fluid respectively, placing the mixture into a four-dimensional rotary mixer for 0.5 hour, collecting supernatant by a magnetic separation method, respectively measuring residual amounts of LA and protein in the supernatant by using a lactic acid kit and a BCA protein quantitative kit, and additionally setting an experimental group to measure the change of the adsorption efficiency of Fe3O4 along with time. Adding Fe3O4 with the same mass as LA into the blood plasma and the simulated tissue fluid respectively, stirring the mixture again, selecting time for one time between 10 and 60 minutes, and measuring the residual amount of lactic acid in the mixture;
s4, haemocompatibility of fe3o4@mt: 10 mL of Fe3O4 suspension with different concentrations is incubated for 30 minutes at 37 ℃ in 0.9% NaCl solution, then 0.2 mL diluted anticoagulant is added, the mixture is incubated for 1 hour, the centrifugation is carried out at 2500 speed for 5 minutes, supernatant is collected, 540 nm ultraviolet-visible spectrophotometry is used for analyzing the released hemoglobin, the same volume of deionized water is taken as positive control, 0.9% NaCl solution is taken as negative control, different amounts of Fe3O4 are added into 0.1 mL diluted anticoagulant, the mixture is incubated for 10 minutes, 0.1 mL of 0.02 mol/L CaCl2 solution is added to recalcify the anticoagulant, after 0.5 hours, 14 mL deionized water is poured, the centrifugation is carried out at 2500 speed for 5 minutes, 540 nm ultraviolet-visible spectrophotometry is used for analyzing the released hemoglobin, and the diluted anticoagulant without adding Fe3O4 is taken as positive control;
s5, cytotoxicity detection: HUVEC and HSMC monolayers were placed in 25 cm2 cell culture flasks, placed in corresponding media containing 10% heat-inactivated FBS and 1% PS, cultured in specific environments, cells were seeded into 96-well plates at a density of 5000 cells per well, after 12 hours, fe3O4 suspensions of different concentrations were added to the media, after 0.5 hour, fe3O4 was removed, and timely and post 23.5 hours cell viability was determined by MTT assay, with acute and prolonged toxicity at 0.5 and 24 hours, respectively;
s6, simulation detection: preparing plasma with high lactic acid concentration and simulated tissue fluid with the concentration of 3 mmol/L and 23 mmol/L respectively, simulating accumulation of lactic acid in blood and muscle after high-strength exercise, then adding Fe3O4@MT with different mass ratios, collecting supernatant by a magnetic separation method after stirring for 0.5 hours, measuring lactic acid in the supernatant by using 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.
S7, detecting a mechanism of adsorbing lactic acid by Fe3O 4: after adsorption of lactic acid, the zeta potential of Fe3O4 in plasma and simulated tissue fluid became negative, magnesium ions were extracted by ion exchange, a large amount of lactic acid was adsorbed on the Fe3O4 surface, the ion exchange of hydrogen ions with magnesium ions resulted in an increase in Si-OH, and the adsorption peak in FTIR was about 950 cm-1.
Preferably, an ultrasonic washing apparatus is used in washing the ethanol.
Preferably, the high lactate concentration plasma and the simulated interstitial fluid are mixed using a four-dimensional rotating mixer.
Preferably, the mixing time of adding the plasma and the simulated interstitial fluid to the Fe3O4 having the same lactic acid mass is 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, 3- (triethoxysilyl) propyl succinic anhydride is used for grafting reactive carboxyl, MT is coated on the nano particles under mild reaction conditions to form a novel LA adsorbent, the novel adsorbent is spherical and has a particle size of about 180 nanometers, the nano particles have 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, the previous superparamagnetic nano particles have good magnetic guiding effect in biomedical application in vitro and in vivo in view of the good magnetic guiding effect, thus obtaining that Fe3O4@MT has great potential in the aspects of easy injection and extraction of exogenous magnetic guiding, and unlike the traditional LA cleaning method, the Fe3O4@MT can directly adsorb LA in solution, the LA is not metabolized into other acidic products and cannot cause metabolic acidosis, LA can be adsorbed by Fe3O4@MT in a short time, fe3O4@MT can quickly remove LA, is more suitable for athletes, can achieve better adsorption efficiency when in 0.5 h, and the more the exchanged ions are, so that the adsorption of Fe3O4@MT to LA is obtained, the LA is not only physically adsorbed, but also depends on chemical bonds, the LA can be effectively removed by Fe3O4@MT, the protein adsorption amount 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, thereby indicating that Fe3O4@MT is a safe LA scavenger, and Fe3O4@MT, fe3O4@MT is conveniently synthesized by coating MT on the surface of Fe3O4 nanoparticles under mild reaction conditions, and shows quick and efficient LA adsorption behavior through the action of chemical bonds, and the Fe3O4@MT has good blood compatibility, has the characteristics of small cytotoxicity, good magnetism and the like, and can well prevent the occurrence of exercise fatigue.
Detailed Description
The present invention will be described in further detail with reference to specific embodiments in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The 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 parts), 3- (triethoxysilyl) propylsuccinic anhydride (0.1-0.3 parts), 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 calf serum (5-10 parts), trypsin (2-4 parts), and ionized water (20-40 parts) comprising the steps of:
s1, weighing materials: feCl3.6H2O (9 parts), feCl2.4H2O (4 parts), HCl (7 parts), naOH (2 parts), ethanol (0.75 parts), 3- (triethoxysilyl) propyl succinic anhydride (0.2 parts), and ionized water (30 parts);
s2, synthesizing Fe3O4@ MT: 8.14g FeCl3.6H2O and 3.00g FeCl2.4H2O were dissolved in 30mL deionized water under nitrogen protection, followed by the addition of 0.85mL12mol/L HCl. The resulting solution was dropped into 250mL of 1.5mol/L NaOH solution under vigorous stirring. After the reaction, the black precipitate was washed three times with deionized water and dried under vacuum, 0.1. 0.1g of the prepared Fe3O4 nanoparticle was taken, washed with 20mL of ethanol by ultrasonic for 2 minutes, repeated three times, 30mL of ethanol and 0.15mL of 3- (triethoxysilyl) propyl succinic anhydride were added under nitrogen protection, and stirred at 50 degrees for 7 hours. Washing the obtained black precipitate with deionized water for three times, preserving in 50 ℃ water for 5 hours, adding dried 40mgFe 3O4 and 20.8mg MT into ethanol, and shaking up for 0.5 hour;
s3, detecting the adsorption efficiency of Fe3O4 to lactic acid: adding Fe3O4 with different quality into high LA concentration plasma and simulated tissue fluid respectively, placing the mixture into a four-dimensional rotary mixer for 0.5 hour, collecting supernatant by a magnetic separation method, respectively measuring residual amounts of LA and protein in the supernatant by using a lactic acid kit and a BCA protein quantitative kit, and additionally setting an experimental group to measure the change of the adsorption efficiency of Fe3O4 along with time. Adding Fe3O4 with the same mass as LA into the blood plasma and the simulated tissue fluid respectively, stirring the mixture again, selecting time for one time between 10 and 60 minutes, and measuring the residual amount of lactic acid in the mixture;
s4, haemocompatibility of fe3o4@mt: 10 mL of Fe3O4 suspension with different concentrations is incubated for 30 minutes at 37 ℃ in 0.9% NaCl solution, then 0.2 mL diluted anticoagulant is added, the mixture is incubated for 1 hour, the centrifugation is carried out at 2500 speed for 5 minutes, supernatant is collected, 540 nm ultraviolet-visible spectrophotometry is used for analyzing the released hemoglobin, the same volume of deionized water is taken as positive control, 0.9% NaCl solution is taken as negative control, different amounts of Fe3O4 are added into 0.1 mL diluted anticoagulant, the mixture is incubated for 10 minutes, 0.1 mL of 0.02 mol/L CaCl2 solution is added to recalcify the anticoagulant, after 0.5 hours, 14 mL deionized water is poured, the centrifugation is carried out at 2500 speed for 5 minutes, 540 nm ultraviolet-visible spectrophotometry is used for analyzing the released hemoglobin, and the diluted anticoagulant without adding Fe3O4 is taken as positive control;
s5, cytotoxicity detection: HUVEC and HSMC monolayers were placed in 25 cm2 cell culture flasks, placed in corresponding media containing 10% heat-inactivated FBS and 1% PS, cultured in specific environments, cells were seeded into 96-well plates at a density of 5000 cells per well, after 12 hours, fe3O4 suspensions of different concentrations were added to the media, after 0.5 hour, fe3O4 was removed, and timely and post 23.5 hours cell viability was determined by MTT assay, with acute and prolonged toxicity at 0.5 and 24 hours, respectively;
s6, simulation detection: preparing plasma with high lactic acid concentration and simulated tissue fluid with the concentration of 3 mmol/L and 23 mmol/L respectively, simulating accumulation of lactic acid in blood and muscle after high-strength exercise, then adding Fe3O4@MT with different mass ratios, collecting supernatant by a magnetic separation method after stirring for 0.5 hours, measuring lactic acid in the supernatant by using 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.
S7, detecting a mechanism of adsorbing lactic acid by Fe3O 4: after adsorption of lactic acid, the zeta potential of Fe3O4 in plasma and simulated tissue fluid became negative, magnesium ions were extracted by ion exchange, a large amount of lactic acid was adsorbed on the Fe3O4 surface, the ion exchange of hydrogen ions with magnesium ions resulted in an increase in Si-OH, and the adsorption peak in FTIR was about 950 cm-1.
Preferably, an ultrasonic washing apparatus is used in washing the ethanol.
Preferably, the high lactate concentration plasma and the simulated interstitial fluid are mixed using a four-dimensional rotating mixer.
Preferably, the mixing time of adding the plasma and the simulated interstitial fluid to the Fe3O4 having the same lactic acid mass 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% and 39.33%, and reaches good adsorption effect within the standard adsorption rates of 50.36+/-1.98% and 38.72+/-1.69%.
Example 2
The 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 parts), 3- (triethoxysilyl) propylsuccinic anhydride (0.1-0.3 parts), 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 calf serum (5-10 parts), trypsin (2-4 parts), and ionized water (20-40 parts) comprising the steps of:
s1, weighing materials: feCl3.6H2O (8 parts), feCl2.4H2O (3 parts), HCl (6 parts), naOH (1 part), ethanol (0.5 part), 3- (triethoxysilyl) propyl succinic anhydride (0.1 part), and ionized water (20 parts);
s2, synthesizing Fe3O4@ MT: 8.14g FeCl3.6H2O and 3.00g FeCl2.4H2O were dissolved in 30mL deionized water under nitrogen protection, followed by the addition of 0.85mL12mol/L HCl. The resulting solution was dropped into 250mL of 1.5mol/L NaOH solution under vigorous stirring. After the reaction, the black precipitate was washed three times with deionized water and dried under vacuum, 0.1. 0.1g of the prepared Fe3O4 nanoparticle was taken, washed with 20mL of ethanol by ultrasonic for 2 minutes, repeated three times, 30mL of ethanol and 0.15mL of 3- (triethoxysilyl) propyl succinic anhydride were added under nitrogen protection, and stirred at 50 degrees for 7 hours. Washing the obtained black precipitate with deionized water for three times, preserving in 50 ℃ water for 5 hours, adding dried 40mgFe 3O4 and 20.8mg MT into ethanol, and shaking up for 0.5 hour;
s3, detecting the adsorption efficiency of Fe3O4 to lactic acid: adding Fe3O4 with different quality into high LA concentration plasma and simulated tissue fluid respectively, placing the mixture into a four-dimensional rotary mixer for 0.5 hour, collecting supernatant by a magnetic separation method, respectively measuring residual amounts of LA and protein in the supernatant by using a lactic acid kit and a BCA protein quantitative kit, and additionally setting an experimental group to measure the change of the adsorption efficiency of Fe3O4 along with time. Adding Fe3O4 with the same mass as LA into the blood plasma and the simulated tissue fluid respectively, stirring the mixture again, selecting time for one time between 10 and 60 minutes, and measuring the residual amount of lactic acid in the mixture;
s4, haemocompatibility of fe3o4@mt: 10 mL of Fe3O4 suspension with different concentrations is incubated for 30 minutes at 37 ℃ in 0.9% NaCl solution, then 0.2 mL diluted anticoagulant is added, the mixture is incubated for 1 hour, the centrifugation is carried out at 2500 speed for 5 minutes, supernatant is collected, 540 nm ultraviolet-visible spectrophotometry is used for analyzing the released hemoglobin, the same volume of deionized water is taken as positive control, 0.9% NaCl solution is taken as negative control, different amounts of Fe3O4 are added into 0.1 mL diluted anticoagulant, the mixture is incubated for 10 minutes, 0.1 mL of 0.02 mol/L CaCl2 solution is added to recalcify the anticoagulant, after 0.5 hours, 14 mL deionized water is poured, the centrifugation is carried out at 2500 speed for 5 minutes, 540 nm ultraviolet-visible spectrophotometry is used for analyzing the released hemoglobin, and the diluted anticoagulant without adding Fe3O4 is taken as positive control;
s5, cytotoxicity detection: HUVEC and HSMC monolayers were placed in 25 cm2 cell culture flasks, placed in corresponding media containing 10% heat-inactivated FBS and 1% PS, cultured in specific environments, cells were seeded into 96-well plates at a density of 5000 cells per well, after 12 hours, fe3O4 suspensions of different concentrations were added to the media, after 0.5 hour, fe3O4 was removed, and timely and post 23.5 hours cell viability was determined by MTT assay, with acute and prolonged toxicity at 0.5 and 24 hours, respectively;
s6, simulation detection: preparing plasma with high lactic acid concentration and simulated tissue fluid with the concentration of 3 mmol/L and 23 mmol/L respectively, simulating accumulation of lactic acid in blood and muscle after high-strength exercise, then adding Fe3O4@MT with different mass ratios, collecting supernatant by a magnetic separation method after stirring for 0.5 hours, measuring lactic acid in the supernatant by using 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.
S7, detecting a mechanism of adsorbing lactic acid by Fe3O 4: after adsorption of lactic acid, the zeta potential of Fe3O4 in plasma and simulated tissue fluid became negative, magnesium ions were extracted by ion exchange, a large amount of lactic acid was adsorbed on the Fe3O4 surface, the ion exchange of hydrogen ions with magnesium ions resulted in an increase in Si-OH, and the adsorption peak in FTIR was 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 apparatus is used in washing the ethanol.
Preferably, the high lactate concentration plasma and the simulated interstitial fluid are mixed using a four-dimensional rotating mixer.
Preferably, the mixing time of adding the plasma and the simulated interstitial fluid to the Fe3O4 having the same lactic acid quality 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% and 39.41%, and reaches good adsorption effect within the standard adsorption rates of 50.36+/-1.98% and 38.72+/-1.69%.
Example 3
The 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 parts), 3- (triethoxysilyl) propylsuccinic anhydride (0.1-0.3 parts), 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 calf serum (5-10 parts), trypsin (2-4 parts), and ionized water (20-40 parts) comprising the steps of:
s1, weighing materials: feCl3.6H2O (10 parts), feCl2.4H2O (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, synthesizing Fe3O4@ MT: 8.14g FeCl3.6H2O and 3.00g FeCl2.4H2O were dissolved in 30mL deionized water under nitrogen protection, followed by the addition of 0.85mL12mol/L HCl. The resulting solution was dropped into 250mL of 1.5mol/L NaOH solution under vigorous stirring. After the reaction, the black precipitate was washed three times with deionized water and dried under vacuum, 0.1. 0.1g of the prepared Fe3O4 nanoparticle was taken, washed with 20mL of ethanol by ultrasonic for 2 minutes, repeated three times, 30mL of ethanol and 0.15mL of 3- (triethoxysilyl) propyl succinic anhydride were added under nitrogen protection, and stirred at 50 degrees for 7 hours. Washing the obtained black precipitate with deionized water for three times, preserving in 50 ℃ water for 5 hours, adding dried 40mgFe 3O4 and 20.8mg MT into ethanol, and shaking up for 0.5 hour;
s3, detecting the adsorption efficiency of Fe3O4 to lactic acid: adding Fe3O4 with different quality into high LA concentration plasma and simulated tissue fluid respectively, placing the mixture into a four-dimensional rotary mixer for 0.5 hour, collecting supernatant by a magnetic separation method, respectively measuring residual amounts of LA and protein in the supernatant by using a lactic acid kit and a BCA protein quantitative kit, and additionally setting an experimental group to measure the change of the adsorption efficiency of Fe3O4 along with time. Adding Fe3O4 with the same mass as LA into the blood plasma and the simulated tissue fluid respectively, stirring the mixture again, selecting time for one time between 10 and 60 minutes, and measuring the residual amount of lactic acid in the mixture;
s4, haemocompatibility of fe3o4@mt: 10 mL of Fe3O4 suspension with different concentrations is incubated for 30 minutes at 37 ℃ in 0.9% NaCl solution, then 0.2 mL diluted anticoagulant is added, the mixture is incubated for 1 hour, the centrifugation is carried out at 2500 speed for 5 minutes, supernatant is collected, 540 nm ultraviolet-visible spectrophotometry is used for analyzing the released hemoglobin, the same volume of deionized water is taken as positive control, 0.9% NaCl solution is taken as negative control, different amounts of Fe3O4 are added into 0.1 mL diluted anticoagulant, the mixture is incubated for 10 minutes, 0.1 mL of 0.02 mol/L CaCl2 solution is added to recalcify the anticoagulant, after 0.5 hours, 14 mL deionized water is poured, the centrifugation is carried out at 2500 speed for 5 minutes, 540 nm ultraviolet-visible spectrophotometry is used for analyzing the released hemoglobin, and the diluted anticoagulant without adding Fe3O4 is taken as positive control;
s5, cytotoxicity detection: HUVEC and HSMC monolayers were placed in 25 cm2 cell culture flasks, placed in corresponding media containing 10% heat-inactivated FBS and 1% PS, cultured in specific environments, cells were seeded into 96-well plates at a density of 5000 cells per well, after 12 hours, fe3O4 suspensions of different concentrations were added to the media, after 0.5 hour, fe3O4 was removed, and timely and post 23.5 hours cell viability was determined by MTT assay, with acute and prolonged toxicity at 0.5 and 24 hours, respectively;
s6, simulation detection: preparing plasma with high lactic acid concentration and simulated tissue fluid with the concentration of 3 mmol/L and 23 mmol/L respectively, simulating accumulation of lactic acid in blood and muscle after high-strength exercise, then adding Fe3O4@MT with different mass ratios, collecting supernatant by a magnetic separation method after stirring for 0.5 hours, measuring lactic acid in the supernatant by using 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.
S7, detecting a mechanism of adsorbing lactic acid by Fe3O 4: after adsorption of lactic acid, the zeta potential of Fe3O4 in plasma and simulated tissue fluid became negative, magnesium ions were extracted by ion exchange, a large amount of lactic acid was adsorbed on the Fe3O4 surface, the ion exchange of hydrogen ions with magnesium ions resulted in an increase in Si-OH, and the adsorption peak in FTIR was about 950 cm-1.
Preferably, an ultrasonic washing apparatus is used in washing the ethanol.
Preferably, the high lactate concentration plasma and the simulated interstitial fluid are mixed using a four-dimensional rotating mixer.
Preferably, the mixing time of adding the plasma and the simulated interstitial fluid to the Fe3O4 having the same lactic acid mass is 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% and 37.86%, and reaches good adsorption effect within the standard adsorption rates of 50.36+/-1.98% and 38.72+/-1.69%.
To sum up: 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 3- (triethoxysilyl) propyl succinic anhydride is used for grafting reactive carboxyl, MT is coated on the nano particles under the mild reaction condition to form a novel LA adsorbent, the novel adsorbent is spherical, the particle size is about 180 nanometers, the novel adsorbent has good dispersibility, the magnetization of the Fe3O4@MT is measured by utilizing a superconducting quantum interference device, the saturation of the novel adsorbent is 48.81 emu/g, the previous superparamagnetic nano particles have good magnetic guiding effect in the biomedical application in vitro and in vivo, thereby obtaining great potential of the Fe3O4@MT in the aspect of easy injection and extraction of exogenous magnetic guiding, the novel adsorbent is different from the traditional LA cleaning method, the Fe3O4@MT can directly adsorb LA in solution, the LA is not metabolized into other acidic products, more importantly, the LA can be adsorbed by the Fe3O4@MT in a short time, the Fe3O4 MT can be rapidly removed, the LA is more suitable for use, the LA can be better adsorbed by the ion exchange efficiency in the super paramagnetic nano particles in 0.5 h, the LA can be better adsorbed by the super-paramagnetic particles, the LA is more LA can be well adsorbed by the Fe3O4@MT, the novel adsorption agent has good physical and chemical adsorption effect, the specific to the Fe3O4@MT can be conveniently removed by the high, the surface of the nano particles, the specific adsorption effect of the Fe3O 4@3 MT is better than the Fe 4@3O 4 is better, the high-3O 4 is better than the specific to be adsorbed by the Fe 4, and the high-3O 4, the high-purity is better physical adsorption effect, and the high-purity is better than the high by the high-purity adsorption effect and the Fe 4 MT is better than the high 2F 3O 3 MT can be adsorbed by the better than better adsorbed by the high chemically and better than better contrast, and better than better contrast compared with the high contrast compared with the contrast compared, can well prevent the occurrence of exercise fatigue.
Finally, it should be noted that: the foregoing description is only illustrative of the preferred embodiments of the present invention, and 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 described, or equivalents may be substituted for elements thereof, and any modifications, equivalents, improvements or changes may be made without departing from the spirit and principles of the present invention.
Claims (1)
1. Use of a lactic acid scavenger for adsorbing lactic acid, characterized in that: the lactic acid scavenger adopts a coprecipitation method to prepare Fe 3 O 4 The nanoparticle is grafted with 3- (triethoxysilyl) propyl succinic anhydride to form a reactive carboxyl group, and MT is coated on the nanoparticle under mild reaction conditions, and the method specifically comprises the following steps:
Fe 3 O 4 synthesis of @ MT: under nitrogen, 8.14g FeCl 3 ·6H 2 O and 3.00g FeCl 2 ·4H 2 O was dissolved in 30mL of deionized water, followed by the addition of 0.85mL12mol/LHCl;
the resulting solution was dropped into 250mL of 1.5mol/LNaOH solution with vigorous stirring;
after the reaction, the black precipitate was washed three times with deionized water and dried under vacuum to obtain 0.1g of prepared Fe 3 O 4 The nanoparticles were washed with 20ml ethanol for 2 minutes, repeated three times, 30ml ethanol and 0.15ml3- (triethoxysilyl) propylsuccinic anhydride were added under nitrogen protection, and stirred at 50 ℃ for 7 hours;
the black precipitate obtained was washed three times with deionized water, stored in 50℃water for 5 hours, and dried 40mgFe 3 O 4 And 20.8mgMT were added to ethanol and shaken well for 0.5 hours.
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