CN109248177B - Preparation of crocodile amour effective component and application of crocodile amour effective component in oxidation resistance and hepatic fibrosis resistance - Google Patents
Preparation of crocodile amour effective component and application of crocodile amour effective component in oxidation resistance and hepatic fibrosis resistance Download PDFInfo
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- CN109248177B CN109248177B CN201810968813.8A CN201810968813A CN109248177B CN 109248177 B CN109248177 B CN 109248177B CN 201810968813 A CN201810968813 A CN 201810968813A CN 109248177 B CN109248177 B CN 109248177B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/56—Materials from animals other than mammals
- A61K35/58—Reptiles
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L17/00—Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
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- Veterinary Medicine (AREA)
- Polymers & Plastics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Food Science & Technology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Nutrition Science (AREA)
- General Chemical & Material Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
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- Mycology (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- Marine Sciences & Fisheries (AREA)
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Abstract
The invention provides a preparation method of an effective component of crocodile amour, which takes a crocodile amour extract as a starting material, and obtains a component with molecular weight of more than 100kD,50-100kD and less than 50kD after ultrafiltration, crude polysaccharide precipitated by ethanol, and a fine polysaccharide component of which protein is removed after the crude polysaccharide is subjected to enzymolysis and solvent extraction. The effective components can improve the hepatic fibrosis of mice induced by carbon tetrachloride to a certain extent, and especially have the most obvious effects of crocodile nail polish polysaccharide and molecular weight of more than 100 kD; and the crocodile amour polysaccharide can effectively slow down the aging condition of the mice induced by the D-galactose. Therefore, the invention is expected to be applied to preparing the medicine for resisting oxidation, slowing down aging and preventing and treating hepatic fibrosis.
Description
Technical Field
The invention belongs to the technical field of medicines, and relates to a traditional Chinese medicine extract for treating hepatic fibrosis and delaying senescence and application thereof.
Background
Liver fibrosis (HF) is a critical stage in the development of various chronic liver diseases into cirrhosis and even liver cancer, and is a reversible stage in the development process of liver diseases, thus becoming the first-choice targeting segment for liver disease treatment. Hepatic fibrosis is a compensatory product of tissues after liver injury in the self-repair process, and most typically excessive deposition of Extracellular Matrix (ECM) in liver tissues; in addition, free radicals induce differentiation and proliferation of hepatic fibrosis cells by inducing lipid peroxidation, and are another way to stimulate hepatic fibrosis. At present, the research on the pathogenesis of hepatic fibrosis is a breakthrough in the world, but the prevention and treatment of hepatic fibrosis still remain a great challenge to the scientific community.
Modern medical research proves that the oxidative damage induced by free radicals is closely related to the occurrence and development of various diseases, so that the proper supplement of the antioxidant has important significance for improving the antioxidant capacity of organisms and improving the body health. The polysaccharide has excellent effects of resisting oxidation, eliminating free radicals and the like, has the characteristics of high efficiency, low toxicity and high bioavailability, and shows unique superiority in preventing and treating diseases caused by free radicals, such as tumors, inflammations and the like.
Ancient medical research and modern medical research show that the crocodile amour can harmonize the spleen and stomach, sooth the liver and relieve depression, contains powerful anti-cancer substances and growth factors for preventing cancer tumor new-born cells, and the cancer inhibition effect of the factors is tens of thousands times that of other reptiles. The extract is compatible with traditional Chinese medicines, has strong functions of 'walking and dispersing, activating blood circulation to dissipate blood stasis, dredging channels and collaterals and dredging orifices', can directly destroy peripheral protective factors of cancer cells and plays a role in killing the cancer cells. However, research results of the effects of resisting hepatic fibrosis and protecting liver by using crocodile amour as an active ingredient of the raw material are relatively few, and no relevant report exists so far.
Disclosure of Invention
The invention aims to provide an crocodile amour effective component for treating hepatic fibrosis and application thereof, and an anti-aging effect of the crocodile amour effective component.
The invention provides an effective component of crocodile amour, which is a fine polysaccharide component obtained by taking a crocodile amour extract as a starting material, ultrafiltering to obtain components with different molecular weights of more than 100kD,50-100kD and less than 50kD, precipitating crude polysaccharide by ethanol, and carrying out enzymolysis and solvent extraction on the crude polysaccharide to remove protein.
The dosage form of the crocodile amour effective component can be various preparations, and is preferably an oral preparation.
The invention provides application of an effective component of crocodile amour to preparation of an anti-hepatic fibrosis medicament. For this purpose, classical intraperitoneal injection of carbon tetrachloride (CCl) into mice was chosen 4 ) A hepatic fibrosis model constructed by inducing chemical liver injury adopts crocodile amour extract to treat 1.35g/kg (physique). The liver function indexes of glutamic-pyruvic transaminase (ALT), glutamic-oxalacetic transaminase (AST), albumin (ALB) and Total Bilirubin (TBIL) in the serum of a mouse are detected, hepatic fibrosis indexes of Hyaluronic Acid (HA), triple collagen (C-III), quadruple collagen (C-IV), laminin (LN) and the like in the serum are detected, and oxidation indexes of hepatic hydroxyproline (Hyp), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and the like are detected; and the pathological changes of the liver are observed through the appearance of the liver and pathological sections of liver tissues. The results show that: compared with the model group, the crocodile amour effective component can reduce the content of each detection index in the serum and the liver of the mouse to a certain extent. Therefore, the effective component of crocodile amour is to CCl 4 The liver fibrosis induction has a certain degree of protection, wherein the best anti-liver fibrosis effect is as follows: crocodile amour extract polysaccharide and a component with the molecular weight of more than 100 kD.
The invention further comprises the application of the crocodile amour polysaccharide in preparing the antioxidant drugs.
The invention adopts a model of inducing the aging of mice by injecting D-galactose subcutaneously on the back of the neck as well as exploring the in vivo antioxidant activity of crocodile amour polysaccharide. The contents of Malondialdehyde (MDA), total superoxide dismutase (SOD) and reduced Glutathione (GSH) in mouse serum and liver are used as evaluation indexes. The results show that: the crocodile amour polysaccharide can reduce the content of MDA in serum and liver of a D-galactose-induced aging model mouse, improve the content of SOD and GSH in serum and liver of the D-galactose-induced aging model mouse, reduce the peroxidation degree of lipid in vivo, reduce the damage of cells, enhance the scavenging and metabolic reaction of free radicals of an organism and achieve the antioxidation effect. Therefore, the crocodile nail polysaccharide can delay the aging of the D-galactose-induced mice.
The invention determines that each effective component of the prepared crocodile amour has an antioxidant effect through animal experiments, has obvious drug effect, is beneficial to pharmacological research and has good application prospect. The crocodile amour effective component prepared by the invention has obvious effect of resisting hepatic fibrosis, is beneficial to the development of medicaments for treating hepatic fibrosis and has wide application prospect.
Drawings
FIG. 1 crocodile amour active principle pair CCl 4 Inducing the influence of liver pathological change appearance of the liver fibrosis model mouse.
FIG. 2 Alligator turbinata active ingredient pair CCl 4 Induction of HE staining of liver tissue in liver fibrosis model mice.
FIG. 3 crocodile amour active principle pair CCl 4 Liver tissue masion staining of liver fibrosis model mice was induced.
FIG. 4 Alligator turbinata active ingredient pair CCl 4 Inducing the influence of serum liver function indexes ALB (A), TBIL (B), ALT (C) and AST (D) content of the hepatic fibrosis model mouse.
FIG. 5 Alligator turbinata active ingredient pair CCl 4 Inducing the influence of the contents of hepatic fibrosis markers HA (A), LN (B), C-III (C) and C-IV (D) in the serum of a hepatic fibrosis model mouse.
FIG. 6 crocodile amour active principle pair CCl 4 Inducing the influence of the contents of liver tissue oxidation indexes Hyp (A), SOD (B) and GSH-Px (C) of a liver fibrosis model mouse.
FIG. 7 Effect of crocodile amouse on the content of MDA in serum and liver of D-galactose-induced aging model mice.
FIG. 8 Effect of crocodile amouse on the SOD levels in serum (A) and liver (B) of D-galactose-induced aging model mice.
FIG. 9 Effect of crocodile amour polysaccharide on GSH levels in serum (A) and liver (B) of D-galactose-induced aging model mice.
Detailed Description
The invention will be further illustrated with reference to the following specific examples
EXAMPLE I preparation of active ingredient of Alligator Shell
Preparation of crocodile shell, turtle shell (as control) powder: cleaning crocodile amour and turtle shell respectively, removing non-medicinal parts, drying, pulverizing in a traditional Chinese medicine pulverizer, sieving with 80 mesh sieve, packaging, drying, sealing and storing.
1.1 preparing the effective components of the crocodile amour liquid: weighing 20g of crocodile amour powder, adding 300ml of distilled water according to a material ratio of 1.
Centrifuging the crocodile amour whole liquid through ultrafiltration tubes with different molecular weights, collecting the liquid on the upper layer of the ultrafiltration tubes, concentrating, freezing and drying to obtain components with molecular weights of more than 100kD,50-100kD and less than 50kD respectively.
1.2 preparing crocodile amour crude polysaccharide: pulverizing crocodile amour in a pulverizer, designing a single factor test from four aspects of material ratio, alkali concentration, extraction temperature and extraction time, selecting three levels on the basis of the single factor test, and performing L 9 (3 4 ) Four-factor three-level orthogonal experimental table is used for optimizing the process conditions of crocodile amour polysaccharide extraction: the optimal preparation process for finally obtaining the crocodile amour crude polysaccharide comprises the following steps: adding 20g of crocodile amour powder into 300ml of distilled water according to the material ratio of 1Mixing the filtrates, concentrating under reduced pressure to 20mL, adding anhydrous ethanol into the concentrated solution to make ethanol volume fraction reach 80%, standing for 24h, centrifuging at 4000r/min, filtering, and freeze drying the obtained precipitate in a freeze dryer at-60 deg.C under vacuum to obtain crude polysaccharide of crocodile scales. Under the condition, the yield of the crocodile amour crude polysaccharide is 5.67%.
1.3 preparing crocodile amour extract polysaccharide: accurately weighing crocodile amour crude polysaccharide powder, redissolving the crocodile amour crude polysaccharide powder into 10% aqueous solution by using distilled water, adding 1% trypsin and 1% papain to fully dissolve the crocodile amour crude polysaccharide powder, placing the solution in a water bath at 40 ℃ for heating for 2 hours for enzymolysis, performing boiling water bath for 10min after the reaction is finished to denature and inactivate enzymes, cooling the solution to room temperature, centrifuging the solution, and taking supernatant. The supernatant was concentrated, and a solvent such as Sevage reagent [ chloroform: n-butanol =4 (V/V) ] was added in an amount of 20% by volume of the crude polysaccharide aqueous solution, and the mixture was stirred well and mixed for min and transferred to a separatory funnel. The procedure was repeated to remove the protein. Collecting the upper layer liquid, concentrating, freezing and drying to prepare the crocodile amour fine polysaccharide.
Example II establishment of hepatic fibrosis animal model
Randomly dividing 120 mice into 15 groups of 8 mice, selecting one group as blank control group, performing intraperitoneal injection on the rest groups, and dividing the content of CCl by 25% of 1mL/100g volume to weight ratio 4 And (3) constructing a hepatic fibrosis model by using the solution, twice a week, modeling once every two days, continuously modeling for eight weeks, and completing modeling. Randomly extracting 2 modeling mice and 1 blank control group mouse to die after cervical vertebra dislocation, dissecting and observing the appearance and shape of liver, and judging whether the hepatic fibrosis model is successfully constructed.
EXAMPLE III grouping and administration of hepatic fibrosis animal experiments
After the modeling is successful for eight weeks, the mice in the experimental group are randomly grouped according to the body weight, each group is divided into 9 groups, and the groups are respectively a model group, a silymarin group (positive control), a turtle shell group, an alligator nail whole liquid group, an alligator nail crude polysaccharide group, an alligator nail fine polysaccharide group, an alligator nail group with the molecular weight of more than 100kD, an alligator nail group with the molecular weight of 50-100kD and a micromolecule group with the molecular weight of less than 50 kD. The administration method using gavage was determined by consulting the literature and combining the experimental dosing regimen for animals in the early stages of this subject group. The administration dose of the mice was calculated according to the mouse body surface area formula: the dosage of the mice is 9 times of that of the adults, namely the dosage is 1.35g/kg (body mass). The positive control group is administered with silymarin suspension at a body weight of 100mg/kg, carapax Trionycis is administered in the same manner as crocodile squama Manis extract to obtain 1.35g/kg, and the blank group is filled with distilled water. Each group was gavaged once a day for 1 month. In the month of intragastric treatment for hepatic fibrosis, 25% of CCl was continued to be administered to all groups of experimental mice except the blank group 4 The solution was modeled to maintain a hepatic fibrosis model, maintaining the modeled dosing interval once per week.
EXAMPLE four preparation of serum and liver tissue samples
After the administration of the experimental mice by gastric lavage is finished, the beards of the mice are cut off, all the experimental mice are fasted without water and fed for 12 hours, eyeballs are picked, about 1mL of blood is taken, the mice are killed after cervical vertebra is removed, the whole liver is dissected and taken out, after being rinsed by precooled physiological saline, the left lobe of the liver is taken and placed in 10% neutral formalin for fixation, and the left lobe of the liver is used for preparing pathological sections at the later stage. The same part of the right lobe of the liver tissue is taken and stored at the temperature of minus 80 ℃ for preparing liver tissue homogenate.
Example five Effect of the Experimental group on the growth and Activity of liver fibrosis model mice
The experimental period is as long as three months, and the real-time observation of the growth condition and the behavior characteristics of the mouse is particularly important in the experimental process. Observational metrics in addition to regular body weight and dietary water intake, additional concerns regarding the mice' coat, activity and mental status were required.
TABLE 1-1 Observation of mouse growth indices
TABLE 1-2 Activity status of mice in each group
Tables 1-2 show the observation results of the mental state indexes of the mice, and the blank group of mice grow well and are active and motile; the growth state of the model group mice is not good, the spirit is low, and the activity is relatively greatly reduced. The results of the experiments show that CCl 4 The hepatotoxicity of (b) causes impaired health in mice and has affected healthy growth in experimental mice. Compared with the model group, the growth indexes such as activity, food consumption and the like of each treatment group are improved to different degrees, wherein the recovery of the silymarin group, the group with the molecular weight of more than 100kD and the crocodile amour protamine polysaccharide group is the most obvious.
TABLE 1-3 weight changes in experimental mice
The results in tables 1 to 3 show that the weight gains of the mice in the respective experimental groups were substantially 20g or more before and after the experiment, and the weight gains of only the model group mice were relatively low, indicating CCl 4 The solution has inhibitory effect on normal growth of mice, and the weight increase of the mice in the crocodile nail whole liquid group, the crude polysaccharide group, the fine polysaccharide group and the mice with molecular weight of more than 100kD group and 50-100kD group is closer to that of the mice in the blank group compared with the silymarin group and the turtle shell group and the mice with molecular weight of less than 50kD group. The macromolecular substances contained in the crocodile amour can promote the growth of mice.
Mouse liver appearance observation
FIG. 1 is an appearance of mouse liver, which is injected intraperitoneally with CCl 4 The appearance and the shape of the liver are changed by inducing chemical liver injury to construct a liver fibrosis model, and the appearance graph of the liver shows that the liver of a blank group of mice has ruddy and glossy color and smooth surface, a layer of film is covered on the surface of the mice, the liver is soft in texture and sharp in edge. The liver of the model group mouse is dull and lusterless, the surface is uneven and has rough particles, the texture of the liver is hard, the edge is blunt and round, and even the liver is adhered with the surrounding organs. By using positive medicines and crocodile amourAfter the stomach perfusion treatment, the color and the appearance of the liver are improved to a certain degree, after the silymarin treatment, the shape of the liver is closest to a blank group, the shapes of the crocodile nail fine polysaccharide group and the group with the molecular weight of more than 100kD are closer to the silymarin group, and the rest groups are slightly improved compared with the model group. (see FIG. 1)
Sixth example, liver tissue HE staining results of mouse liver fibrosis model by experimental group
FIG. 2 is a pathological result of HE staining of mice, and shows that the sections of the blank mice are red stained, the cytoplasm of the cells is abundant, and the cells are arranged in a radial manner in veins surrounding the center. The liver of the model group has disordered lobular structure and obvious fibroplasia, the hyperplastic tissue forms fibrosepta to divide the lobular liver into a plurality of false lobular structures, and a great deal of steatosis and vesicular necrosis appear after lesion. Blank and model groups are significantly different. The positive medicine and crocodile nail components of the experimental group have certain recovery of liver tissues compared with the model group. The silymarin group has the best recovery effect, and the crocodile amour polysaccharide group and the group with the molecular weight of more than 100kD have obvious improvement effect on hepatic fibrosis. (see FIG. 2)
Example seven, masson staining results of mouse liver fibrosis model liver tissue by experimental group
FIG. 3 shows the result of Masson staining, which indicates that the lobular structure of the liver of the normal group of mice is complete and has no fibrous interval; the hepatic lobule structure of the mouse in the model group is obviously damaged, fibrous tissues in the hepatic tissues are obviously proliferated, and thick and compact fibrous intervals are generated to wrap the hepatic structures into a plurality of fake hepatic lobules. The liver tissues of mice in each experimental group are scattered in fibrous tissue hyperplasia and have hepatic fibrosis areas, but the fibrous intervals are reduced to different degrees compared with those in the model group. The fibrosis reversal effects of the silymarin group, the crocodile amour protamine group and the group with the molecular weight of more than 100kD are the most remarkable, and the crocodile amour protamine and the component with the molecular weight of more than 100kD have more remarkable effects on reversing liver fibrosis. (see FIG. 3)
EXAMPLE VIII, effect of the groups on Biochemical indices of hepatic fibrosis model liver function (AST, ALT, ALB, TBIL) in mice
Glutamate-oxaloacetate transaminase (AST) and glutamate-pyruvate transaminase (ALT) are known as human body substance metabolism catalysts, are rich in the cytoplasm of liver cells and are also an important index for measuring whether the liver is normal or not, and when the liver is damaged by various stimulations, the AST and ALT contents in serum can be increased, so that clinical diagnosis is performed. Albumin (ALB) synthesis can only be accomplished in the liver, and thus the liver becomes the only site for ALB production, and when the liver is damaged, ALB synthesis is hindered and its content in serum is reduced. High Total Bilirubin (TBIL) is commonly found in hepatitis, extrahepatic biliary obstruction, hemolytic diseases and the like, and when the liver is damaged by the outside, the TBIL content is increased due to the induction of hepatitis.
TABLE 1-4 liver function (AST, ALT, ALB, TBIL) index test results
Fig. 4 shows the changes of ALB, TBIL, ALT and AST content in the mouse serum, and the results show that the ALB content in the model group is significantly reduced and the TBIL, ALT and AST content in the normal group is significantly increased. Compared with the model group, the positive drug silymarin group has the most obvious improvement effect on the indexes, which shows that the positive drug has very good reversion effect on hepatic fibrosis. All components of the crocodile amour selected by the subject group have certain improvement on hepatic fibrosis, wherein the crocodile amour essential polysaccharide group and the group with the molecular weight of more than 100kD have relatively good improvement effect on hepatic fibrosis, and the trend displayed by the experimental result is consistent with the trend presented by in vitro pharmacodynamic research. The experimental result proves that the effective component of the crocodile amour plays a role in resisting hepatic fibrosis through the approach of reducing enzyme and protecting the liver. (see FIG. 4)
EXAMPLE nine Effect of the Experimental group on mouse liver fibrosis model serum markers (HA, LN, C-III, C-IV)
HA. LN, C-III and C-IV are four golden indexes of clinical detection of hepatic fibrosis, and the results are authoritative and representative. HA is a component of ECM, is synthesized by mesenchymal cells, is mainly eliminated in liver, can accurately and sensitively reflect the amount of hepatic fibrosis and the damage degree of the liver, and is the most sensitive one of four detection indexes; LN is a non-collagenous structural protein which is unique in basement membrane, and has great correlation with the activity degree of hepatic fibrosis and the pressure of portal vein; the serum volume of C-III is consistent with the degree of hepatic fibrosis, and if the content of C-III rises rapidly, it indicates that the liver disease is worsening and progressing to the formation of liver cirrhosis. C-IV is the main component of liver basement membrane and can reflect the renewal rate of liver basement membrane collagen. The four indexes can be used for judging the process of hepatic fibrosis.
TABLE 1-5 Change in serum markers (HA, LN, C-III, C-IV) for hepatic fibrosis
FIG. 5 shows the measurement results of HA, LN, C-III, and C-IV contents in mouse serum. The graph data shows that the levels of the four indexes of HA, LN, C-III and C-IV in the model group are obviously increased compared with the blank group; compared with the model group, the content of the four indexes in each experimental group is reduced to different degrees, wherein each index of the silymarin group and the crocodile amour sperm polysaccharide group and the index of the group with the molecular weight of more than 100Kd have significant difference compared with the model group, and the other groups have certain improvement effect compared with the model group. The experimental result shows that the effective component of the crocodile amour can inhibit the deposition of collagen in liver tissues and the proliferation of collagen cells so as to play the role of resisting hepatic fibrosis. (see FIG. 5)
EXAMPLE ten Effect of Experimental groups on liver tissue Oxidation index (Hyp, SOD, GSH-PX) of mouse hepatic fibrosis model
Liver cell damage is the first link in the process of hepatic fibrosis, and oxygen free radical increase in vivo is an important factor causing liver cell damage, so that the removal of oxygen free radicals in vivo also becomes one of effective measures capable of reversing and preventing hepatic fibrosis. SOD is an important enzyme for removing Reactive Oxygen Species (ROS) in an organism, has unique physiological activity, and the activity level can reflect the ROS removing capability of the organism; hydroxyproline (Hyp) is a unique amino acid in collagen, so that the content of hydroxyproline can indirectly reflect the degree of hepatic fibrosis and can be used for evaluating the degree of hepatic fibrosis and the content of collagen; GSH-Px can reduce the peroxidation capacity of toxic components through oxidation-reduction reaction, prevent sulfydryl-containing enzyme from being activated by oxidant and heavy metal, reduce oxidized sulfydryl-containing enzyme through oxidation-reduction reaction, remove excessive lipid peroxide in vivo, and finally protect the integrity of cell membrane structure and function.
The experimental results in fig. 6 show that the content of Hyp, SOD and GSH-Px in the model group is significantly increased compared with the blank group, the differences between the positive drug silymarin group and the crocodile nail polish polysaccharide group and the three groups of drugs with molecular weight more than 100kD are significant, and the rest groups have certain improvement on hepatic fibrosis. These results indicate that the effect of improving liver fibrosis is more obvious in the seminal polysaccharide group and the group with molecular weight of more than 100 kD. The results show that the crocodile amour effective component can reduce the fibrosis degree of liver tissues, can enhance the oxidation resistance of the liver tissues to a certain degree, further protect liver cells and play a role in resisting hepatic fibrosis. (see FIG. 6)
TABLE 1-6 Change in Oxidation index (Hyp, SOD, GSH-PX) for each group of mice
EXAMPLE eleven, replication and administration of D-galactose model mouse laboratory animals
Healthy male ICR mice 35 are selected, aged for 4 weeks, are adapted to the environment for one week before the experiment, are injected with D-galactose (200 mg/kg) subcutaneously at the neck and back, establish a mouse aging model, and are injected with physiological saline with the same volume in a normal group. Then, 7 individuals in each group are divided into a model group and crocodile amour polysaccharide low, medium and high dose groups, the administration is performed by intragastric administration once every day after modeling for 4 hours, the experimental period is 42 days, the gavage of the low, medium and high groups is performed by intragastric administration of 100, 200mg/mL and 400mg/mL crocodile amour polysaccharide solutions, the intragastric administration volume is 20mL/kg body weight, and the normal group and the model group are subjected to intragastric administration of the same volume of physiological saline.
TABLE 2-1 mice grouping
EXAMPLE twelve, serum and liver tissue sample preparation
The eyes of each group of mice are bled, centrifuged at 3 000r/mim for 15min at 4 ℃, and the supernatant (namely serum) is taken for standby. The mice are dislocated and killed after blood is taken, liver tissues are quickly taken out, and the extravasated blood is washed away by physiological saline at the temperature of 0-4 ℃. Precisely weighing 0.5g of liver tissue, adding 4.5mL of 0 ℃ physiological saline, placing the liver tissue into a homogenizer for ice water bath and rapidly grinding to prepare tissue homogenate, centrifuging the liver tissue for 10min at 4 ℃ at 4000r/min, and taking supernatant for later use.
EXAMPLE thirteen Effect of Alligator Phosphoeropolysaccharide on weight and Activity in D-galactose model mice
TABLE 2-2 weight changes in mice
Tables 2-2 show the weight change before and after the mouse experiment, indicating that the weight of the model group mice increases more slowly than that of the blank group during the experiment, and the weight of each group of mice with gavage crocodile amour polysaccharide increases to a certain extent than that of the model group, but does not exceed that of the blank group.
Tables 2-3 Activity status of groups of mice
The results of the mouse activity comparison tables in tables 2-3 show that the mouse activity of the model group is reduced, and the hair color is relatively dull and rough, which indicates that the mouse aging model is successfully constructed. When the gavage drug was administered, the mice in the drug group recovered to some extent compared to the model group, and showed a dose-dependent relationship.
Example fourteen effects of crocodile amour polysaccharide on D-galactose senescence model mouse model GSH Activity
Tables 2-4 changes in GSH Activity in groups of mice
Example fifteen Effect of crocodile amour polysaccharide on SOD activity in model mouse model of D-galactose-induced aging
Tables 2-5 variation in SOD Activity in groups of mice
EXAMPLE sixteen Effect of crocodile amour polysaccharide on D-galactose-induced aging model mouse model MDA Activity
Tables 2-6 changes in MDA Activity in groups of mice
The results (tables 2-6, fig. 7-9) of the three antioxidant indexes of SOD, MDA and GSH prove that the model group has significant differences compared with the blank group, which indicates that the model is successfully constructed. The results of each treatment group of crocodile amour polysaccharide on the model group are improved to a certain extent, and particularly the treatment effect of the treatment group of crocodile amour polysaccharide is more remarkable. The crocodile amour polysaccharide has a certain treatment effect on a D-galactose-induced aging model and shows dose dependence. Is expected to be applied to antioxidant and anti-aging preparations.
Claims (2)
1. An application of an active component of crocodile amour in preparing a medicament for preventing and treating liver fibrosis, resisting oxidation and delaying aging is disclosed, wherein the component refers to a component with a molecular weight of more than 100KD obtained by ultrafiltration of crocodile amour whole liquid, or a component with a molecular weight of 50-100KD obtained by ultrafiltration of crocodile whole liquid, or a component with a molecular weight of less than 50KD obtained by ultrafiltration of crocodile whole liquid, or crude polysaccharide obtained by ethanol precipitation of crocodile whole liquid, or refined polysaccharide obtained by enzymolysis of crude polysaccharide, wherein the preparation method of the active component of crocodile amour comprises the following steps:
1) Adding distilled water into crocodile amour powder according to a certain proportion, stirring and soaking, boiling and reflux-extracting for multiple times under an alkaline condition, concentrating the filtrate on a rotary evaporator, and freeze-drying to obtain crocodile amour whole solution;
2) Centrifuging the crocodile amour whole liquid through ultrafiltration tubes with different molecular weights, collecting the liquid on the upper layer of the ultrafiltration tubes, concentrating, freezing and drying to respectively obtain components with molecular weights of more than 100kD,50-100kD and less than 50 kD;
3) Concentrating the whole liquid under reduced pressure, adding anhydrous ethanol to make ethanol volume fraction reach 80%, standing for 24h, centrifuging at 4000r/min, filtering, and vacuum freeze drying the obtained precipitate at-60 deg.C to obtain crude polysaccharide of crocodile amour;
4) Dissolving crocodile amour crude polysaccharide in water solution, adding trypsin and papain to fully dissolve, performing enzymolysis, then adding a solvent, fully stirring and uniformly mixing, removing protein, collecting supernatant, concentrating, freeze-drying, and preparing to obtain crocodile amour refined polysaccharide.
2. The use of claim 1, wherein the medicament is a pharmaceutical composition comprising an effective component of alligator nail and a pharmaceutically acceptable carrier.
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