CN115624624A - Application of fullerene derivative in preparation of drugs for treating alcoholic liver disease - Google Patents
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Abstract
The invention discloses an application of a fullerene derivative in preparation of a medicament for treating alcoholic liver disease, belonging to the field of medicaments. In an electromagnetic therapy, the metal fullerene alcohol embedded with ferroferric oxide shows more excellent free radical scavenging capacity, improves the alcohol metabolism efficiency by influencing the activity of acetaldehyde dehydrogenase, reduces the damage of acetaldehyde to an organism, and greatly prevents and relieves the problems of liver damage, liver oxidative stress, liver steatosis and sudden weight drop caused by long-term drinking, thereby having obvious prevention and treatment effects on alcoholic liver diseases.
Description
Technical Field
The invention belongs to the field of medicines, relates to a medicine application, and particularly relates to an application of a fullerene derivative in preparation of a medicine for treating alcoholic liver disease.
Background
The alcoholic liver disease, also called as Alcoholic Liver Disease (ALD), refers to a series of pathological changes such as liver damage caused by excessive alcohol intake, according to the alcoholic liver disease diagnosis and treatment guideline established by the hepatic society of Chinese medicine in 2006 in the group of fatty liver and alcoholic liver disease, the ALD can be classified into light ALD, alcoholic fatty liver, alcoholic hepatitis, alcoholic hepatic fibrosis and alcoholic cirrhosis, and a few even liver cancers. Excessive and prolonged alcohol consumption can lead to a variety of physiological system problems, including the immune system, nervous system, cardiovascular system, and digestive system. Liver expression caused by excessive drinking is called alcoholic liver disease, and in recent years, alcoholic liver disease caused by excessive drinking is the most common chronic liver disease in the world. The pathogenesis of alcoholic liver disease is complex, but the oxidative metabolites of alcohol, including acetaldehyde and Reactive Oxygen Species (ROS), are considered the leading cause of alcoholic liver disease. At the present stage, no good medicine is available for treating alcoholic liver diseases, and the medicine treatment mainly aims at liver disease complications, but the survival rate of alcoholic liver disease patients is not obviously improved. Clinical treatment for this condition is dominated by alcohol withdrawal, nutritional support and steroid drug administration. Although there are many kinds of drugs for preventing and treating alcoholic liver disease, the use effect is not satisfactory for various reasons. Therefore, safe and effective therapeutic drugs are urgently needed for alcoholic liver diseases.
Electromagnetic therapy is a completely non-invasive treatment of redox diseases, which can be achieved by interaction with endogenous paramagnetic molecules, such as superoxide anions. The electromagnetic therapy non-invasively adjusts the redox couple of the whole body reduced glutathione and the oxidized glutathione and improves the in vivo oxidative stress response. The regulation of the redox homeostasis is a new physiological effect of static magnetic field and electrostatic field, and can be used for the noninvasive treatment of type 2 diabetes and other potential redox-related diseases. However, the daily treatment time of electromagnetic therapy is 7 hours, and the feasibility is poor, so that the application of the treatment mode is greatly limited.
Disclosure of Invention
The invention provides application of a fullerene derivative in preparation of a medicament for treating alcoholic liver disease, which aims to overcome the defects of the prior art.
In order to achieve the above object, the present invention provides an application of a fullerene derivative in the preparation of a drug for treating alcoholic liver disease, the fullerene derivative having the following characteristics: the fullerene derivative is metal fullerene alcohol embedded with ferroferric oxide.
Further, the invention provides an application of the fullerene derivative in preparing a medicament for treating alcoholic liver disease, and the fullerene derivative can also have the following characteristics: wherein the number of hydroxyl groups of the metal fullerene alcohol embedded with ferroferric oxide is 20-35 per carbon cage.
Further, the invention provides an application of the fullerene derivative in preparing a medicament for treating alcoholic liver disease, and the fullerene derivative can also have the following characteristics: wherein the diameter of the metal fullerene alcohol embedded with ferroferric oxide is 15-250 nm.
Further, the invention provides an application of the fullerene derivative in preparing a medicament for treating alcoholic liver diseases, and the fullerene derivative also has the following characteristics: the fullerene derivative is applied to preparation of a medicine for treating alcoholic liver disease by using an electromagnetic therapy.
Further, the invention provides an application of the fullerene derivative in preparing a medicament for treating alcoholic liver disease, and the fullerene derivative can also have the following characteristics: the dosage of the metal fullerene alcohol embedded with ferroferric oxide is 3.3-5.7 mg/kg.
Further, the invention provides an application of the fullerene derivative in preparing a medicament for treating alcoholic liver diseases, and the fullerene derivative also has the following characteristics: the preparation method of the metal fullerene alcohol embedded with ferroferric oxide comprises the following steps: step one, preparing a hydroxylated fullerene solution; step two, adding ferric chloride into the hydroxylated fullerene solution obtained in the step one, and fully dissolving; step three, slowly dropwise adding a NaOH solution into the mixed solution obtained in the step two until the pH value of the solution is 12; step four, dripping the mixed solution obtained in the step three into ethanol to separate out a precipitate; and step five, freezing and fixing the precipitate obtained in the step four, and freeze-drying to obtain the metal fullerene alcohol powder embedded with ferroferric oxide.
Further, the invention provides an application of the fullerene derivative in preparing a medicament for treating alcoholic liver disease, and the fullerene derivative can also have the following characteristics: the preparation method of the step one comprises the following steps: adding the hydroxylated fullerene into deionized water, and stirring the solution to obtain a hydroxylated fullerene solution; the concentration of the hydroxylated fullerene solution is 0.5g/L.
Further, the invention provides an application of the fullerene derivative in preparing a medicament for treating alcoholic liver disease, and the fullerene derivative can also have the following characteristics: wherein, in the second step, the ferric chloride comprises FeCl 2 •4H 2 O and FeCl 3 •6H 2 O; hydroxylated fullerene, feCl 2 •4H 2 O and FeCl 3 •6H 2 The mass ratio of O is 25: 80: 54.
Further, the invention provides an application of the fullerene derivative in preparing a medicament for treating alcoholic liver disease, and the fullerene derivative can also have the following characteristics: wherein, in the third step, the concentration of the NaOH solution is 0.4 mol/L.
Further, the invention provides an application of the fullerene derivative in preparing a medicament for treating alcoholic liver diseases, and the fullerene derivative also has the following characteristics: wherein, in the fourth step, after the precipitate is separated out, the precipitate is washed by water and ethanol to be neutral; the specific washing method comprises the following steps: after precipitation, centrifuging at 2000 rpm for 5 min, discarding supernatant, and adding dd H 2 And O, dissolving the precipitate, performing vortex, adding ethanol, performing vortex, centrifuging at the rotating speed of 2000 rpm for 5 min, and repeating the operation until the washing solution is neutral to obtain the precipitate.
Further, the invention provides an application of the fullerene derivative in preparing a medicament for treating alcoholic liver disease, and the fullerene derivative can also have the following characteristics: wherein in the fifth step, the freezing and fixing temperature is-80 ℃; the freeze-drying time was 12 h.
The invention has the beneficial effects that:
1. the ferroferric oxide has good magnetization performance, so that the metal fullerene alcohol embedded with the ferroferric oxide can better respond to an external magnetic field, and the response can obtain better treatment effect on the alcoholic liver disease.
2. The metal fullerene alcohol has stronger free radical scavenging ability than fullerene alcohol, the metal fullerene alcohol embedded with ferroferric oxide has better hydroxyl free radical scavenging ability, and the paramagnetic molecules are introduced into the body to improve the oxidation-reduction steady state in the body, so the metal fullerene alcohol has good prospect in the aspect of liver steatosis resistance.
3. Under the synergistic effect of electromagnetic therapy, the metal fullerene alcohol has obviously enhanced regulating capacity on in-vivo redox balance and shows stronger free radical adsorption capacity; by influencing the activity of acetaldehyde dehydrogenase, the alcohol metabolism efficiency is improved, and the damage of acetaldehyde to the organism is reduced. Therefore, the transfer of hepatic triglyceride can be effectively improved, the steady state of liver metabolism is maintained, and no obvious toxicity exists, the problems of liver injury, liver oxidative stress, liver steatosis and sudden weight drop caused by long-term drinking are greatly prevented and relieved, and the obvious prevention and treatment effects on the alcoholic liver diseases are achieved.
Drawings
FIG. 1 is a schematic diagram of the synthesis of metallic fullerene alcohol with built-in ferroferric oxide;
FIG. 2 is an infrared spectrum of metallofullerene alcohol with ferroferric oxide embedded therein;
FIG. 3 is a hydrodynamics size distribution diagram of ferroferric oxide-embedded metal fullerene alcohol;
FIG. 4 is a hysteresis loop diagram of a metal fullerene alcohol and a fullerene alcohol embedded with ferroferric oxide;
FIG. 5 is a diagram showing the results of the detection of the scavenging ability of metal fullerene alcohol and fullerene alcohol embedded with ferroferric oxide on hydroxyl radicals under the action of a magnetic field and an electric field;
FIG. 6 is a flow chart of an in vivo experiment;
FIG. 7 is a graph of the levels of glutamate pyruvate transaminase in serum of groups of mice at days 0, 14 and 28 in a first in vivo experiment;
FIG. 8 is a graph showing serum levels of glutamic-oxaloacetic transaminase in mice of each group at days 0, 14, and 28 in a first in vivo experiment;
FIG. 9 is a graph of the levels of reduced glutathione in the liver of groups of mice at days 0, 14 and 28 in a first in vivo experiment;
FIG. 10 is a graph showing the levels of alanine aminotransferase and aspartate aminotransferase in the serum of mice in each group on days 0, 14 and 28 in experiment two in vivo;
FIG. 11 is the serum reduced glutathione levels in the second in vivo experiment in groups of mice at days 0, 14 and 28;
FIG. 12 is a graph showing the levels of alcohol dehydrogenase and acetaldehyde dehydrogenase in the serum liver of mice in each group at days 0, 14 and 28 in experiment two in vivo;
wherein representspThe value is less than or equal to 0.05pA value of 0.01 or less representspThe value is 0.001 or less.
Detailed Description
The present invention is further illustrated by the following examples.
The invention provides an application of a fullerene derivative in preparation of a medicament for treating alcoholic liver disease, wherein the fullerene derivative is metal fullerene alcohol embedded with ferroferric oxide.
The hydroxyl number of the metal fullerene alcohol embedded with ferroferric oxide is 20-35 per carbon cage. The diameter of the metal fullerene alcohol embedded with ferroferric oxide is 15-250 nm.
The synthetic schematic diagram of the ferroferric oxide-embedded metal fullerene alcohol is shown in fig. 1, and specifically, the ferroferric oxide-embedded metal fullerene alcohol can be prepared by the following method:
step one, preparing a hydroxylated fullerene solution: 25 mg of hydroxylated fullerene (Jiangsu Xiancheng nano material science and technology Co., ltd.) is weighed, 50 mL of deionized water is added, and the mixture is stirred and dissolved to obtain a hydroxylated fullerene solution with the concentration of 0.5g/L.
Step two, weighing 80 mg FeCl 2 •4H 2 O (Shanghai-derived leaf Biotech Co., ltd.) and 54 mg FeCl 3 •6H 2 O (chemical reagent of national drug group, ltd.) was added to the hydroxylated fullerene solution obtained in the first step, and stirred for 15 minutes to dissolve it sufficiently.
And step three, slowly dropwise adding 0.4 mol/L NaOH solution into the mixed solution obtained in the step two until the pH value of the solution is 12.
Step four, mixing the mixed solution obtained in the step threeDropwise adding into 300 mL ethanol, precipitating, centrifuging at 2000 rpm for 5 min, discarding supernatant, adding 2 mL dd H 2 Dissolving the precipitate, vortexing, adding 45 mL of ethanol, vortexing, centrifuging at 2000 rpm for 5 min, repeating the operation for about 8 times until the washing liquid is neutral, and discarding the supernatant to obtain the precipitate.
And step five, placing the precipitate obtained in the step four in a refrigerator at the temperature of-80 ℃, freezing and fixing the precipitate, and then carrying out freeze drying for 12 hours to obtain the metal fullerene alcohol powder embedded with ferroferric oxide.
The infrared spectrum detection is carried out on the prepared metal fullerene alcohol embedded with ferroferric oxide, the result is shown in figure 2, and the infrared spectrum shows that the infrared spectrum is 500-600 cm -1 In the region, there is a distinct vibration peak, which is Fe 3 O 4 Characteristic peak of medium Fe-O; at 3400 cm -1 The wide vibration peak of the region is the vibration peak of hydroxyl, which shows that a large number of hydroxyl groups are still connected to the surface of the nano-particles, the hydroxyl groups on the surface of the hydroxylated fullerene are not influenced in the synthesis process, and the product still has good hydrophilicity; at 1104, 1406 and 1632 cm -1 The vibration peaks at (A) are assigned to C-O, C-O-H and C-C, respectively, C = C structures, which are all assigned to hydroxylated fullerenes, which also indicates that Fe 3 O 4 Successfully embedded in a fullerene cage.
The size distribution of the metal fullerene alcohol composite nano-material embedded with ferroferric oxide is evaluated by Dynamic Light Scattering (DLS), and the result is shown in figure 3, and the result shows that the hydrodynamic diameter is 115.1 +/-6.1 nm, the particle size of the particles is mostly concentrated at 115 nm, the particle size distribution is uniform, and the metal fullerene alcohol composite nano-material has excellent dispersion effect in an aqueous solution.
The magnetic hysteresis loop detection is carried out on the fullerene alcohol and the metal fullerene alcohol embedded with the ferroferric oxide, the result is shown in figure 4, the result shows that the magnetic hysteresis loop of the pure fullerene alcohol is almost a straight line, the magnetization performance is poor, the saturation magnetization is only 0.006 emu/mg, the magnetic hysteresis loop of the metal fullerene alcohol nano-particles embedded with the ferroferric oxide is a typical S-shaped curve, the saturation magnetization can reach 21.3 emu/mg, and the result shows that the nano-particles areStill maintain Fe 3 O 4 And exhibits good superparamagnetism.
Application of metal fullerene alcohol embedded with ferroferric oxide in preparation of drugs for treating alcoholic liver diseases, in particular application in preparation of drugs for treating alcoholic liver diseases by using electromagnetic therapy, wherein the electromagnetic therapy is static magnetic field and electric field (sBE) therapy.
Pharmacodynamic experiments for the above applications:
1. in vitro experiments:
OH has a very strong electron-donating, i.e. oxidizing, ability, and is considered to be the most reactive ROS induced by lipid accumulation in hepatic steatosis. Because the carbon cage has a large pi bond without electrons and has high electron affinity, the metal fullerene alcohol embedded with ferroferric oxide can be directly added with OH through an olefinic bond or provide hydrogen atoms to form water for quenching the OH. To prove the effect, the removal capability of the fullerol and the ferroferric oxide-embedded metal fulleenol on the hydroxyl radical is detected under the action of a magnetic field and an electric field, and the result is shown in figure 5. The detection result shows that more than 50% of OH is eliminated by 0.2 mg/mL of metal fullerene alcohol embedded with ferroferric oxide, and the fullerene alcohol with the same concentration can only eliminate 37% of OH; and the metal fullerene alcohol embedded with the ferroferric oxide and the fullerene alcohol are only under a magnetic field or an electric field, the change of the OH removing capacity of the metal fullerene alcohol embedded with the ferroferric oxide and the fullerene alcohol is not obviously different, but under the combined action of sBE, the OH removing capacity of the metal fullerene alcohol embedded with the ferroferric oxide and the OH removing capacity of the fullerene alcohol are both obviously enhanced, and the metal fullerene alcohol embedded with the ferroferric oxide still shows more excellent OH removing capacity than the fullerene alcohol.
2. In vivo experiments:
1. experimental methods and detection methods:
as shown in FIG. 6, the in vivo experiment included an adaptation period (4 days), a molding period (10 days) and a treatment period (28 days), and the last day of the molding period was a 42-day total period of high concentration (5 g/kg body mass) alcohol gavage.
Constructing an alcoholic liver disease mouse model: female BALB/c mice (18-20 g, 8-9 weeks, south Jiangsu Jing Weitongli, inc.) were prepared and an alcoholic liver disease mouse model was constructed. To enhance the adaptation of mice to liquid alcohol feed, the mice were fasted for one day first, then provided with liquid feed without added alcohol, after which the adaptation period for molding was started. Since mice usually eat at night and alcohol in liquid alcohol diet is volatile, liquid alcohol diet is prepared and replaced for mice at 3 pm every day. Approximately 50 mL of liquid feed was administered per cage of mice per day, and changes were made depending on the change in food intake of the mice. And (3) feeding plan of the alcohol liquid feed in the adaptation period: weighing 134 g of solid alcohol liquid feed, adding 700 mL of mouse drinking water, stirring to uniformly mix, adding 13, 26, 39 and 42 mL of 75% alcohol respectively on days 1, 2, 3 and 4, stirring to uniformly mix, adding water to a constant volume of 1L, and immediately replacing the feed for the mouse. And (3) feeding plan of alcohol liquid feed in the molding period: weighing 134 g of solid alcohol liquid feed, adding 700 mL of mouse drinking water, stirring to mix uniformly, adding 65 mL of 75% alcohol on day 5, stirring to mix uniformly, adding water to a constant volume of 1L, and immediately replacing the feed for the mouse.
After the model is successfully made, the treatment period is started. The normal mice and the model mice are divided into groups, each group of mice is fed or treated differently for 28 days continuously, and each group of mice is sampled respectively on the 0 th, 14 th and 28 th days of treatment to detect the liver injury index.
The method for measuring the liver injury index comprises the following steps:
detection of alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) in plasma: and sequentially adding corresponding reagents into the obtained plasma, rapidly mixing the reagents in each well uniformly, standing for 15 min at room temperature, setting the wavelength of an enzyme-labeling instrument to be 510 nm, measuring the OD value of each well, and obtaining the corresponding ALT and AST activities according to a standard curve by using the absolute OD value (the measured empty OD value minus the control well OD value).
Detection of activity of serum reduced Glutathione (GSH) levels: and sequentially adding the samples or the standard substances into a 96-well plate, and uniformly mixing. Adding 150 μ L of total glutathione detection working solution, mixing, and incubating at 25 deg.C or room temperature for 5 min. Add 50. Mu.l of 0.5 mg/mL NADPH solution and mix well. A412 was measured with a microplate reader 25 minutes after the addition and mixing of NADPH solution.
Detection of alcohol dehydrogenase activity in liver homogenate: placing 0.05 g of tissue block into a 1.5 mL EP tube, adding an alcohol dehydrogenase activity detection kit (AK 337) according to the weight (g) of the tissue block and the volume (mL) of AK337-A (mL) =1:10, grinding by a tissue homogenizer at 60 Hz and 90 s under the condition of ice water bath to prepare tissue homogenate, centrifuging at the temperature of 4 ℃ and 16000 Xg for 20 min, and taking supernatant and placing on ice for detection. AK337-B is placed in a water bath at 25 ℃ for heat preservation for 30 min. Taking a 96-well plate, sequentially adding reagents, obtaining a numerical value, and calculating according to the following formula: alcohol dehydrogenase (ADH, μmol/min/g) =3.215 (tangle-a assay tube-a blank tube)/W, wherein enzyme activity is defined as: the enzyme amount for oxidizing 1. Mu. Mol N alcohol dehydrogenase per gram of sample per minute was 1 enzyme activity unit.
Detection of acetaldehyde dehydrogenase activity in liver homogenate: 0.05 g of the tissue mass was put into a 1.5 mL EP tube, and the weight (g): volume (mL) =1, then ES13 was added, and the mixture was ground in a tissue homogenizer at 60 hz and 90 s in an ice water bath to form a homogenate, centrifuged at 16000 × g for 20 min at 4 ℃, and the supernatant was placed on ice for testing. AK226-A is placed in a water bath at 37 ℃ for heat preservation for 15 min. The reagent is added into a 96-well plate in turn, the well mixing is carried out, the timing is started, the absorbance value A1 control and A1 measurement are measured at 340 nm when the measurement is carried out for 15 s, then the 96-well plate is quickly placed in a water bath with the temperature of 37 ℃ for 1 min, the absorbance value A2 control and A2 measurement are measured at 340 nm, a control is tangle-solidup than A = A1 control-A2 control, a tangle-solidup than A measurement tube = A1 measurement-A2 measurement, a tangle-solidup than A measurement-A control. After obtaining the values, the following formula is calculated: acetaldehyde dehydrogenase (ALDH, mu mol/min/g) = 0.804A/W, wherein the enzyme activity is defined as the catalytic reduction of 1 mu mol NAD per gram of sample per minute + The enzyme amount of (a) is 1 enzyme activity unit.
Wherein the statistical analysis employs GraphPad Prism, includingtTest and Two-way ANOVA, trial replicates were obtained from at least 3 different individuals. * RepresentpA value of 0.05 or less representspThe value is less than or equal to 0.01pThe value is 0.001 or less.
2. In vivo experiment one:
grouping experiments:
(1) normal group: 15 normal test mice without any treatment;
(2) non-treatment group: 15 alcoholic liver disease mice constructed by the method are not treated;
(3) electromagnetic field group: the static magnetic field and the electric field are independently applied to an alcoholic liver disease mouse model, and the specific operation is as follows: placing 15 alcoholic liver disease mice constructed according to the above operation in a device simultaneously applying a magnetic field (3 mT) and an electric field (7 kv/m) for 7 hours, and continuously treating for 28 days;
(4) a fullerene alcohol group: independently applying fullerene alcohol without embedded ferroferric oxide to an alcoholic liver disease mouse model, and specifically operating as follows: injecting an injection reagent (prepared by fullerene alcohol powder and normal saline or PBS, and the concentration is 0.2 mg/mL) containing fullerene alcohol without embedded ferroferric oxide into 15 alcoholic liver disease mice constructed according to the operation every day, wherein the injection dosage of the fullerene alcohol without embedded ferroferric oxide is 4.5 mg/kg;
(5) a metallofullerene alcohol group: the metal fullerene alcohol embedded with ferroferric oxide is independently applied to an alcoholic liver disease mouse model, and the specific operation is as follows: injecting an injection reagent containing metallic fullerene alcohol embedded with ferroferric oxide (prepared by metallic fullerene alcohol powder embedded with ferroferric oxide and normal saline or PBS (phosphate buffer solution) into 15 alcoholic liver disease mice constructed according to the above operation every day, wherein the concentration of the injection reagent is 0.2 mg/mL), and the injection dosage of the fullerene alcohol embedded with ferroferric oxide is 4.5 mg/kg;
(6) electromagnetic field treatment group of fullerene alcohols: fullerenol without embedded ferroferric oxide and an electromagnetic field are jointly applied to an alcoholic liver disease mouse model, and the specific operation is as follows: injecting 15 alcoholic liver disease mice constructed according to the above operation with Fullerenol without embedded ferroferric oxide into abdominal cavity every day, wherein the dose is 4.5 mg/kg, and simultaneously placing the mice in a device for applying a magnetic field (3 mT) and an electric field (7 kv/m) for 3 hours for continuous treatment for 28 days;
(7) electromagnetic field treatment group of metallofullerene alcohols: the method is characterized in that ferroferric oxide embedded metal fullerene alcohol and an electromagnetic field are jointly applied to an alcoholic liver disease mouse model, and the specific operation is as follows: 15 alcoholic liver disease mice constructed according to the above operation are intraperitoneally injected with metallic fullerene alcohol embedded with ferroferric oxide every day, the dose is 4.5 mg/kg, and the mice are placed in a device for applying a magnetic field (3 mT) and an electric field (7 kv/m) for 3 hours and are continuously treated for 28 days.
To evaluate the therapeutic effect of the above treatment strategy on alcoholic liver disease, the activities of ALT and AST in serum, and GSH in liver were measured in each group of mice on days 0, 14, and 28 of the experiment, respectively.
The results are shown in figures 7, 8 and 9 and demonstrate that both ALT and AST levels were significantly higher in the day 0 non-treated mice than in normal mice, indicating that a model of chronic alcoholic liver disease was successfully constructed by feeding liquid alcohol diet for 14 days. After 14 days of treatment, the pure fullerene alcohol and the metallic fullerene alcohol embedded with the ferroferric oxide both have good treatment effects in the electromagnetic therapy, and the metallic fullerene alcohol embedded with the ferroferric oxide can keep the normal ALT and AST levels of mice in the electromagnetic therapy. After 28 days of treatment, the metallofullerene electromagnetic field treatment group was still the most effective and excellent group with 20.583 + -4.35U/L and 23.789 + -5.745U/L ALT and AST, while normal mice had 11.298 + -3.773U/L and 15.519 + -3.939U/L ALT and AST.
The magnetization of pure fullerene alcohol is poorer, and the magnetization intensity of the metal fullerene alcohol nano-particle embedded with ferroferric oxide is greatly enhanced, which shows that the nano-particle still keeps the magnetic property of the ferroferric oxide and shows good superparamagnetism. Meanwhile, under the action of a magnetic field or an electric field, the change of the removal capacity of the hydroxyl radicals of pure fullerene alcohol and the metal fullerene alcohol embedded with ferroferric oxide has no significant difference, but under the combined action of the electromagnetic field, the removal capacities of the hydroxyl radicals of the pure fullerene alcohol and the metal fullerene alcohol embedded with the ferroferric oxide are both significantly enhanced, and the metal fullerene alcohol embedded with the ferroferric oxide still shows more excellent removal capacity of the hydroxyl radicals than the fullerene alcohol.
Under the action of an external magnetic field, the pure fullerene alcohol, the metal fullerene alcohol embedded with the ferroferric oxide and the hydroxyl radical all generate weak magnetism, and the magnetism promotes the mutual attraction of the metal fullerene alcohol embedded with the ferroferric oxide and the pure fullerene alcohol and the hydroxyl radical, so that the scavenging capacity of the metal fullerene alcohol embedded with the ferroferric oxide and the pure fullerene alcohol to the hydroxyl radical is enhanced.
In conclusion, the metal fullerene embedded with ferroferric oxide has good treatment effect in the treatment of alcoholic liver diseases by electromagnetic therapy.
3. In vivo experiment two:
grouping experiments:
(1) normal group: 15 normal test mice without any treatment;
(2) non-treatment group: 15 alcoholic liver disease mice constructed by the method are not treated;
(3) metadoxine group: 15 alcoholic liver disease mice constructed according to the method are treated with metadoxine alone, and are intragastrically administrated with 0.29 mg of metadoxine every day;
(4) electromagnetic field treatment group of metallofullerene alcohols: the injection reagent containing the metal fullerene alcohol embedded with the ferroferric oxide (prepared by the metal fullerene alcohol powder embedded with the ferroferric oxide and physiological saline or PBS (phosphate buffer solution) with the concentration of 0.2 mg/mL) is injected into 15 alcoholic liver disease mice constructed according to the operation every day, wherein the injection dose of the fullerene alcohol embedded with the ferroferric oxide is 4.5 mg/kg, and meanwhile, the mice are placed in a device capable of applying a magnetic field (3 mT) and an electric field (7 kv/m) for 3 hours for continuous treatment for 28 days.
In particular, metadoxine is mainly used for treating acute and chronic alcoholism and alcoholic fatty liver in clinic. In animal experiments, metadoxine can improve the alcohol metabolism rate, shorten the time of alcohol exerting toxic effect in organisms and contribute to improving alcoholism. Therefore, metadoxine medication group was set up.
In order to evaluate whether the metallic fullerene alcohol embedded with ferroferric oxide can effectively relieve the liver injury caused by alcohol in the electromagnetic therapy, the serum levels of ALT and aspartate aminotransferase AST of each group of mice are measured, and the result is shown in figure 10. The results show that after 14 and 28 days of treatment, the electromagnetic field treatment group of the metal fullerene alcohol achieves good treatment effect, and the diseased mice return to normal ALT and AST levels after treatment, and the treatment effect is not obviously different from that of the metadoxine group.
In order to evaluate the effect of ferroferric oxide-embedded metallofullerene alcohol on the redox system in the electromagnetic therapy, the activities of ALT and AST in the serum of each group of mice, and the levels of GSH in the serum were measured, and the results are shown in fig. 11. The results showed that GSH of mice in the electromagnetic field treatment group of metallofullerene alcohol and the metadoxine group was restored to normal level at the 14 th and 28 th days of treatment.
The results show that the metallofullerene alcohol embedded with ferroferric oxide in the electromagnetic therapy well prevents and relieves liver injury and liver oxidative stress caused by long-term drinking.
In order to evaluate the influence of the metal fullerene alcohol embedded with ferroferric oxide on alcohol metabolism in the electromagnetic therapy, the influence of treatment on alcohol metabolism is evaluated by detecting and counting data of liver ADH, ALDH activity and the like of each group of mice, and the result is shown in FIG. 12. The results show that on day 0, ADH activity in the non-treated mice is significantly improved compared to normal mice, while ALDH activity is reduced compared to normal, and since ADH activity is increased, in addition, alcohol up-regulates the expression and activity of CYP2E1, further promoting the conversion of ethanol to acetaldehyde, while the reduction of ALDH activity inhibits the further metabolism of acetaldehyde, so long-term alcohol consumption "stagnates" alcohol metabolism during the acetaldehyde step, and aggravates direct damage caused by acetaldehyde. However, as liver damage increased, there was no significant difference between groups at days 14 and 28, with each group having a tendency to decrease ADH activity compared to normal mice. However, the ALDH activity of each group of mice was reduced to a different extent than that of the normal group of mice, and the difference was significant. More importantly, the ALDH activities of mice in the metadoxine group and the metal fullerene electromagnetic field treatment group are higher than those in the non-treatment group, on the 28 th day, the ALDH activities of the two groups are respectively 6.06 +/-0.229 mu mol/min/g and 6.371 +/-0.408 mu mol/min/g, no significant difference exists between the two groups, and the metal fullerene electromagnetic field treatment group has a dominant trend compared with the metadoxine group.
In conclusion, after treatment for 14 and 28 days, the metal fullerene alcohol embedded with ferroferric oxide has good treatment effect in the electromagnetic therapy, so that the diseased mice return to normal ALT and AST levels after treatment, and the curative effect of the diseased mice is not obviously different from that of metadoxine.
In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Also, the reagents, materials and procedures used herein are those that are widely used in the corresponding fields.
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 changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The application of the fullerene derivative in preparing the medicine for treating the alcoholic liver disease is characterized in that:
the fullerene derivative is metal fullerene alcohol embedded with ferroferric oxide.
2. The use of a fullerene derivative according to claim 1 in the preparation of a medicament for the treatment of alcoholic liver disease, wherein:
wherein the number of hydroxyl groups of the metal fullerene alcohol embedded with ferroferric oxide is 20-35 per carbon cage.
3. Use of a fullerene derivative according to claim 1 in the preparation of a medicament for the treatment of alcoholic liver disease, wherein:
wherein the diameter of the metal fullerene alcohol embedded with ferroferric oxide is 15-250 nm.
4. Use of a fullerene derivative according to claim 1 in the preparation of a medicament for the treatment of alcoholic liver disease, wherein:
wherein, the fullerene derivative is applied to the preparation of drugs for treating alcoholic liver diseases by electromagnetic therapy.
5. The use of a fullerene derivative according to claim 1 in the preparation of a medicament for the treatment of alcoholic liver disease, wherein:
the preparation method of the metal fullerene alcohol embedded with ferroferric oxide comprises the following steps:
step one, preparing a hydroxylated fullerene solution;
step two, adding ferric chloride into the hydroxylated fullerene solution obtained in the step one, and fully dissolving;
step three, dropwise adding a NaOH solution into the mixed solution obtained in the step two until the pH value of the solution is 12;
step four, dripping the mixed solution obtained in the step three into ethanol to separate out a precipitate;
and step five, freezing and fixing the precipitate obtained in the step four, and freeze-drying to obtain the metal fullerene alcohol powder embedded with ferroferric oxide.
6. Use of a fullerene derivative according to claim 5 in the preparation of a medicament for the treatment of alcoholic liver disease, wherein:
the preparation method of the step one comprises the following steps: adding the hydroxylated fullerene into deionized water, and stirring the solution to obtain a hydroxylated fullerene solution;
the concentration of the hydroxylated fullerene solution is 0.5g/L.
7. Use of a fullerene derivative according to claim 5 in the preparation of a medicament for the treatment of alcoholic liver disease, wherein:
wherein, in the second step, the ferric chloride comprises FeCl 2 •4H 2 O and FeCl 3 •6H 2 O；
Hydroxylated fullerene, feCl 2 •4H 2 O and FeCl 3 •6H 2 Mass of OThe ratio is 25: 80: 54.
8. Use of a fullerene derivative according to claim 5 in the preparation of a medicament for the treatment of alcoholic liver disease, wherein:
in the third step, the concentration of the NaOH solution is 0.4 mol/L.
9. The use of a fullerene derivative according to claim 5 in the preparation of a medicament for the treatment of alcoholic liver disease, wherein:
wherein, in the fourth step, after the precipitate is separated out, the precipitate is washed by water and ethanol to be neutral;
the specific washing method comprises the following steps: after precipitation, centrifuging at 2000 rpm for 5 min, discarding supernatant, and adding dd H 2 And O, dissolving the precipitate, performing vortex, adding ethanol, performing vortex, centrifuging at the rotating speed of 2000 rpm for 5 min, and repeating the operation until the washing solution is neutral to obtain the precipitate.
10. Use of a fullerene derivative according to claim 5 in the preparation of a medicament for the treatment of alcoholic liver disease, wherein:
wherein in the fifth step, the freezing and fixing temperature is-80 ℃; the freeze-drying time was 12 h.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080206222A1 (en) * | 2004-12-07 | 2008-08-28 | Nobuhiko Miwa | Preventive/Therapeutic Composition For Free Radical Disease |
| CN108201543A (en) * | 2016-12-19 | 2018-06-26 | 北京福纳康生物技术有限公司 | Application of the water-soluble fullerene structure in the drug for preparing treatment fatty liver |
| US20190038667A1 (en) * | 2016-01-21 | 2019-02-07 | Beijing Fu Na Kang Biotechnology Co., Ltd. | Application of fullerene/metal-fullerene for preparing pharmaceutical product |
| CN111517372A (en) * | 2020-05-11 | 2020-08-11 | 山西医科大学 | Fullerene coated Fe3O4Composite nano material and preparation method thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080206222A1 (en) * | 2004-12-07 | 2008-08-28 | Nobuhiko Miwa | Preventive/Therapeutic Composition For Free Radical Disease |
| US20190038667A1 (en) * | 2016-01-21 | 2019-02-07 | Beijing Fu Na Kang Biotechnology Co., Ltd. | Application of fullerene/metal-fullerene for preparing pharmaceutical product |
| CN108201543A (en) * | 2016-12-19 | 2018-06-26 | 北京福纳康生物技术有限公司 | Application of the water-soluble fullerene structure in the drug for preparing treatment fatty liver |
| CN111517372A (en) * | 2020-05-11 | 2020-08-11 | 山西医科大学 | Fullerene coated Fe3O4Composite nano material and preparation method thereof |
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