CN114558105A - Glucosamine calcium salt-polypeptide complex and liposome thereof - Google Patents

Glucosamine calcium salt-polypeptide complex and liposome thereof Download PDF

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CN114558105A
CN114558105A CN202210217329.8A CN202210217329A CN114558105A CN 114558105 A CN114558105 A CN 114558105A CN 202210217329 A CN202210217329 A CN 202210217329A CN 114558105 A CN114558105 A CN 114558105A
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calcium salt
glucosamine
polypeptide
liposome
compound
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毛相朝
梁梦琦
黄文灿
孙建安
王永臻
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Ocean University of China
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7008—Compounds having an amino group directly attached to a carbon atom of the saccharide radical, e.g. D-galactosamine, ranimustine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1277—Processes for preparing; Proliposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Abstract

The invention discloses a glucosamine calcium salt-polypeptide compound, a liposome embedded with the compound, and application of the glucosamine calcium salt-polypeptide compound in prevention and treatment of osteoarthritis, belonging to the technical field of pharmaceutical preparations. The glucosamine calcium salt-polypeptide complex is prepared by compounding glucosamine calcium salt and Leu-His-Trp-Lys-Ser polypeptide, wherein the mass ratio of the glucosamine calcium salt to the Leu-His-Trp-Lys-Ser polypeptide is 1: 1-3. The glucosamine calcium salt-polypeptide complex embedded liposome is prepared from a glucosamine calcium salt-polypeptide complex and a carrier. The glucosamine calcium salt-polypeptide complex and the liposome embedded with the glucosamine calcium salt-polypeptide complex are applied to or prepared into medicaments for preventing or treating osteoarthritis. The research of the invention is expected to be used for preventing and treating osteoarthritis by the liposome embedded with the glucosamine calcium salt, and the invention is a new treatment strategy for treating osteoarthritis in the future.

Description

Glucosamine calcium salt-polypeptide complex and liposome thereof
Technical Field
The invention relates to a glucosamine calcium salt-polypeptide compound, a liposome embedded with the compound, and application of the glucosamine calcium salt-polypeptide compound in prevention and treatment of osteoarthritis, belonging to the technical field of pharmaceutical preparations.
Background
Osteoarthritis (OA) is a degenerative disease, which is caused by degenerative damage of articular cartilage, reactive hyperplasia of articular margin and subchondral bone, due to various factors such as aging, obesity, strain, trauma, congenital abnormality of joints, and joint deformity. The clinical manifestations are slowly developing arthralgia, tenderness, stiffness, arthrocele, deformity, etc. This disease is commonly seen in middle-aged and elderly people, and is better at the load-bearing joints and joints with more physical activity. Excessive weight bearing or use of these joints can promote degenerative changes. Finally, the joint deformity is caused, and the life quality of the patient is seriously influenced.
The prior art medicines for treating arthritis comprise glucosamine hydrochloride, glucosamine sulfate and the like. The chelating salt formed by glucosamine sulfate and metal ions has more stable property, and has the effects of resisting inflammation, relieving pain and swelling, improving joint function and the like.
In current methods of OA treatment, local intra-articular administration is more effective than oral administration because it reduces systemic toxicity. However, the residence time of the drug administered by intra-articular injection is short, so that intra-articular concentration is high. The distearoyl phosphatidylcholine liposome (DSPC) has the advantages of strong oxidation resistance, reduced drug toxicity, slow release and targeting property. By embedding the liposome, the effect of slowly releasing the medicine can be achieved, and the defects of short action time and poor treatment effect of some medicines during treatment can be avoided.
The polypeptide is a natural protein hydrolysate, different polypeptides have the activities of resisting bacteria, resisting inflammation, resisting oxidation, reducing blood pressure, reducing blood fat, reducing uric acid and the like, have small toxic and side effects, and are widely used in the fields of functional foods, health-care products, medicines and the like.
The compound preparation is a common functional food or pharmaceutical preparation form, and is widely researched and applied because the functional activities of various components can be fully exerted and the synergistic effect can be generated after the compound preparation is compounded. Therefore, by compounding the glucosamine metal salt with high biological safety and the active polypeptide, a compound with higher functional activity for treating arthritis can be prepared.
Disclosure of Invention
Aiming at the prior art, the invention provides a glucosamine calcium salt-polypeptide compound and a liposome embedding the compound. The invention researches and prepares several glucosamine metal salts, screens out the glucosamine calcium salt with optimal anti-inflammatory effect, further compounds the glucosamine calcium salt with Leu-His-Trp-Lys-Ser polypeptide, and uses the glucosamine calcium salt and the Leu-His-Trp-Lys-Ser polypeptide for preventing and treating osteoarthritis by liposome embedding, thereby improving the anti-inflammatory effect on joints and the cartilage protection effect.
The invention is realized by the following technical scheme:
a glucosamine calcium salt-polypeptide compound is prepared by compounding glucosamine calcium salt and Leu-His-Trp-Lys-Ser polypeptide, wherein the mass ratio of the glucosamine calcium salt to the Leu-His-Trp-Lys-Ser polypeptide is 1: 1-3, and 1:1 is preferred.
The preparation method of the glucosamine calcium salt-polypeptide compound comprises the following steps: adding glucosamine calcium salt and polypeptide into water, mixing uniformly to obtain a glucosamine calcium salt-polypeptide compound solution, concentrating and drying to obtain the glucosamine calcium salt-polypeptide compound, wherein the concentration of the compound in the glucosamine calcium salt-polypeptide compound solution is 0.05-0.5 percent, and the unit g/ml, preferably 0.1 percent.
A liposome embedded with glucosamine calcium salt-polypeptide complex is a liposome prepared from the glucosamine calcium salt-polypeptide complex and a carrier, wherein the proportioning relationship of the glucosamine calcium salt-polypeptide complex and the carrier is as follows: 200ml of carrier is added into every 0.02-0.20 g of glucosamine calcium salt-polypeptide compound, and 200ml of carrier is preferably added into every 0.04 of glucosamine calcium salt-polypeptide compound.
Further, the carrier is selected from distearoyl phosphatidylcholine and cholesterol, and the mass ratio of the distearoyl phosphatidylcholine to the cholesterol is 3-5: 1, and preferably 4: 1.
Further, the preparation method of the glucosamine-embedded calcium salt-polypeptide complex liposome can be as follows: adding a carrier into a container containing an organic solvent, dissolving, performing rotary evaporation to obtain a carrier film, adding an aqueous solution of the glucosamine calcium salt-polypeptide complex into the container, performing ultrasonic crushing to crush the carrier film, mixing the crushed carrier film with the aqueous solution of the glucosamine calcium salt-polypeptide complex uniformly, treating at 70-80 ℃ for 1.5-3 hours, and performing film coating to obtain the glucosamine calcium salt-polypeptide 1 complex-embedded liposome (GA-Ca-Leu-His-Trp-Lys-Ser-lip).
Further, the organic solvent is selected from methanol, chloroform or a mixture thereof.
Specifically, the preparation method is as follows: weighing 200mg of distearoyl phosphatidylcholine and 50mg of cholesterol, dissolving in a methanol-chloroform solution (consisting of methanol and chloroform in a volume ratio of 8.5:1,5), completely dissolving, and performing vacuum rotary evaporation to obtain a film; pouring 40ml of 0.05-0.5% glucosamine calcium salt-polypeptide 1 complex aqueous solution (GA-Ca-Leu-His-Trp-Lys-Ser), vibrating the membrane by ultrasound, carrying out ultrasonic crushing for 10min, carrying out water bath for 2h in a water bath kettle at 78 ℃ after the crushing is finished, and passing through the membrane to obtain the glucosamine calcium salt-polypeptide 1 complex embedded liposome (GA-Ca-Leu-His-Trp-Lys-Ser-lip).
The glucosamine calcium salt-polypeptide compound is applied to or prepared into a medicament for preventing or treating osteoarthritis.
The glucosamine calcium salt-polypeptide complex-embedded liposome is applied to or prepared into a medicament for preventing or treating osteoarthritis.
The glucosamine calcium salt-polypeptide complex and the liposome embedded with the glucosamine calcium salt-polypeptide complex have good anti-inflammatory effect, have the advantages of strong oxidation resistance, good stability and slow release, can be used for preventing and treating osteoarthritis, and improve the anti-inflammatory effect on joints and the cartilage protection effect.
The invention induces mouse macrophage by lipopolysaccharide, constructs a cell inflammation model, and screens glucosamine calcium salt with optimal anti-inflammatory effect by detecting the anti-inflammatory activity of several glucosamine metal salts through medicine adding intervention. Then, the cytotoxicity and the anti-inflammatory activity of the glucosamine calcium salt, the three polypeptides and the compound of the glucosamine calcium salt and the polypeptides in different proportions are respectively researched. The glucosamine calcium salt and polypeptide Leu-His-Trp-Lys-Ser (the ratio is 1:1) complex (GA-Ca-Leu-His-Trp-Lys-Ser) are used for embedding distearoyl phosphatidylcholine liposome to obtain the GA-Ca-Leu-His-Trp-Lys-Ser embedded liposome (GA-Ca-Leu-His-Trp-Lys-Ser-lip), and the liposome is subjected to related characterization, in-vitro release research and cytotoxicity research, and the protection effect on inflammation-induced chondrocytes is detected through in-vitro experiments. According to the invention, researches show that the GA-Ca-Leu-His-Trp-Lys-Ser has the best anti-inflammatory effect, and the glucosamine calcium salt and the polypeptide Leu-His-Trp-Lys-Ser have a possible synergistic effect when the ratio is 1: 1. The drug is embedded by distearoyl phosphatidylcholine liposome, and the results of in vitro experiments show that GA-Ca-Leu-His-Trp-Lys-Ser-lip can realize better anti-inflammatory and slow drug release effects, and has a protective effect on inflammation-induced chondrocyte degeneration. The research of the invention is expected to be used for preventing and treating osteoarthritis by the liposome embedded with the glucosamine calcium salt, and is probably a new treatment strategy for treating osteoarthritis in the future.
The various terms and phrases used herein have the ordinary meaning as is well known to those skilled in the art.
Drawings
FIG. 1: and (4) detecting the infrared spectrum of the glucosamine calcium salt.
FIG. 2: and (4) infrared spectrum detection of the glucosamine magnesium salt.
FIG. 3: and (4) infrared spectrum detection of the glucosamine zinc salt.
FIG. 4: and (4) detecting the infrared spectrum of the glucosamine iron salt.
FIG. 5: and (4) detecting glucosamine potassium salt by infrared spectroscopy.
FIG. 6: and (4) detecting the infrared spectrum of the glucosamine sodium salt.
FIG. 7: each group was cytotoxic.
FIG. 8: TNF-alpha content for each group.
FIG. 9: IL-6 content in each group.
FIG. 10: NO content in each group.
FIG. 11: the cytotoxicity of glucosamine calcium salt, polypeptide and each compound (the ratio is 1: 1).
FIG. 12: the cytotoxicity of the glucosamine calcium salt polypeptide compound (the mixture ratio is 1:2 and 1: 3).
FIG. 13: the TNF- α content of glucosamine calcium salts, polypeptides and different compounds, wherein, group 1: blank; and (2) group: LPS; and 3, group: a polypeptide 2; 4 groups are as follows: a polypeptide 3; and 5, group: 1, a polypeptide; 6 groups are as follows: a calcium salt; 7 groups of: calcium salt polypeptide 2(1: 1); and 8 groups: calcium salt polypeptide 3(1: 1); 9 groups of: calcium salt polypeptide 1(1: 1); 10 groups: calcium salt polypeptide 2(1: 2); 11 groups: calcium salt polypeptide 3(1: 2); 12 groups of: calcium salt polypeptide 1(1: 2); 13 groups: calcium salt polypeptide 2(1: 3); 14 groups of: calcium salt polypeptide 3(1: 3); 15 groups of: calcium salt polypeptide 1(1: 3).
FIG. 14: IL-6 content of glucosamine calcium salt, polypeptides and different compounds, wherein, group 1: blank; and 2, group: LPS; and 3, group: a polypeptide 2; 4 groups are as follows: a polypeptide 3; and 5 groups of: 1, a polypeptide; 6 groups are as follows: a calcium salt; 7 groups of: calcium salt polypeptide 2(1: 1); and 8 groups: calcium salt polypeptide 3(1: 1); 9 groups of: calcium salt polypeptide 1(1: 1); 10 groups: calcium salt polypeptide 2(1: 2); 11 groups: calcium salt polypeptide 3(1: 2); 12 groups of: calcium salt polypeptide 1(1: 2); 13 groups: calcium salt polypeptide 2(1: 3); 14 groups of: calcium salt polypeptide 3(1: 3); 15 groups of: calcium salt polypeptide 1(1: 3).
FIG. 15 is a schematic view of: the NO content of glucosamine calcium salt, polypeptides and different compounds, wherein, group 1: blank; and 2, group: LPS; and 3, group: a polypeptide 2; 4 groups are as follows: a polypeptide 3; and 5, group: 1, a polypeptide; 6 groups are as follows: a calcium salt; 7 groups of: calcium salt polypeptide 2(1: 1); and 8 groups: calcium salt polypeptide 3(1: 1); 9 groups of: calcium salt polypeptide 1(1: 1); 10 groups: calcium salt polypeptide 2(1: 2); 11 groups: calcium salt polypeptide 3(1: 2); 12 groups of: calcium salt polypeptide 1(1: 2); 13 groups: calcium salt polypeptide 2(1: 3); 14 groups of: calcium salt polypeptide 3(1: 3); 15 groups of: calcium salt polypeptide 1(1: 3).
FIG. 16: transmission electron micrograph of blank liposome.
FIG. 17: GA-Ca-Leu-His-Trp-Lys-Ser-lip transmission electron micrograph.
FIG. 18: in vitro drug release pattern.
FIG. 19 is a schematic view of: GA-Ca-Leu-His-Trp-Lys-Ser and the cytotoxicity of both liposomes.
FIG. 20: COX-2 content in each group.
FIG. 21: IL-6 content in each group.
FIG. 22: NO content in each group.
FIG. 23: PGE2 content for each group.
FIG. 24: TNF-alpha content for each group.
FIG. 25 is a schematic view of: IL-1. beta. mRNA expression.
FIG. 26: mRNA expression of IL-6.
FIG. 27 is a schematic view showing: mRNA expression of TAC 1.
FIG. 28: mRNA expression of MMP 1.
FIG. 29: mRNA expression of Agg.
FIG. 30: mRNA expression of Col2 α.
Detailed Description
The present invention will be further described with reference to the following examples. However, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention.
The instruments, reagents, materials and the like used in the following examples are conventional instruments, reagents, materials and the like in the prior art and are commercially available in a normal manner unless otherwise specified. Unless otherwise specified, the experimental methods, detection methods, and the like described in the following examples are conventional experimental methods, detection methods, and the like in the prior art.
Example 1 preparation and characterization of several glucosamine Metal salts
Dissolving 5g of glucosamine hydrochloride and 7.5g of anhydrous calcium sulfate in 100mL of water, stirring for 2h at room temperature, adjusting the pH value to 6.8 by using a sodium hydroxide solution, and then heating to 50 ℃ for reaction for 4 h; pouring into a beaker, adding acetone with the volume about 3 times that of the solution after stirring, standing for 30min, pouring out supernatant, adding sufficient absolute ethyl alcohol, continuously stirring by using a glass rod, standing, performing suction filtration, recrystallizing a filter cake by absolute ethyl alcohol to obtain a solid, and performing vacuum drying.
The preparation method of other glucosamine metal salts (such as glucosamine zinc sulfate salt, glucosamine iron sulfate salt, glucosamine magnesium sulfate salt, glucosamine potassium sulfate salt and glucosamine sodium sulfate salt) is the same as above.
Infrared spectrum characterization is respectively carried out on the prepared glucosamine metal salts, and the following results are obtained: as shown in FIGS. 1 to 6, the glucosamine calcium salt is 3071cm-1、2939cm-1、2805cm-1、2734cm-1The peak of the telescopic vibration absorption of hydroxyl and amino appears at 1668cm-1The absorption peaks appear at the left and right, and the existence of crystal water is further proved. D, D-glucosamine zinc salt is 2597.1m-1、2561.9cm-1、2446.6cm-1、2142.3cm-1The peak of the stretching vibration absorption of hydroxyl and amino appears at 1661.9cm-1The absorption peaks appear at the left and right, and the existence of crystal water is further proved. Glucosamine magnesium salt is 2555.5cm-1、2446.6cm-1、2155.1cm-1、2135.9cm-1: the peak of absorption of stretching vibration of hydroxyl and amino appears at 1533.8cm-1An absorption peak appeared, further demonstrating the production of crystalline water. The content of glucosamine iron salt is 2593.9cm-1、2552.3cm-1、2443.4cm-1、2180.8cm-1The peak of absorption of stretching vibration of hydroxyl and amino appears at 1530.6cm-1The absorption peaks at the left and right sides prove that the crystal water is generated.
Example 2 cytotoxicity assays
Culturing mouse mononuclear macrophage RAW264.7 by using a 96-well plate, wherein each well is about 6000-7000 cells, 100 mu l of cell culture solution (gibco, DMEM/F12) is added into each well, and the mixture is placed inIncubator 37 ℃ CO2Culturing for 24h under the condition of 5% concentration, adding medicines (namely glucosamine metal salt, glucosamine hydrochloride, glucosamine sulfate and calcium chloride) according to the concentrations of 0.1%, 0.25% and 0.5% (g/ml, the same below) after 24h, detecting the absorbance of 450nm after 12h, and respectively calculating the cytotoxicity of different samples according to a cell survival rate formula.
The detection results are shown in fig. 7, the cell survival rates of 0.25% and 0.5% of the glucosamine zinc salt are lower than 80%, and the cell survival rates of other substances are higher than 80%, so that the concentration of 0.1% of each substance is selected for the inflammation screening test.
EXAMPLE 3 detection and screening of several inflammatory factors
To screen the anti-inflammatory effects of several glucosamine metal salts, RAW264.7 cells were plated at 3X 10 per well5The individual cells were inoculated in a 12-well plate, placed in an incubator for 12 hours, cultured for 12 hours, and then the cell culture fluid was aspirated, and a prepared lipopolysaccharide solution (LPS) of 1. mu.g/mL was added to construct an inflammation model. Wherein, after the blank group is sucked out of the culture solution, the lipopolysaccharide solution is not added, and the cell culture solution without serum and double antibody is added. Adding 0.1% of each sample into each hole in sequence, intervening by adding medicines (namely glucosamine metal salt, glucosamine hydrochloride, glucosamine sulfate and calcium chloride), incubating for 12h, and taking 300 μ l of each hole for detecting several inflammatory factors.
The detection results of several inflammation factors are shown in figures 8, 9 and 10, and the results show that the contents of TNF-alpha, NO and IL-6 of glucosamine calcium salt are the lowest compared with other substances, and the contents constitute significant differences.
Example 4 cytotoxicity Studies on glucosamine calcium salt, three polypeptides, and combinations of glucosamine calcium salt and polypeptides at different ratios
The invention discloses three polypeptides Leu-His-Trp-Lys-Ser (polypeptide 1) (shown as SEQ ID NO. 1), Tyr-Asp-Leu-Cys-Ile (polypeptide 2) (shown as SEQ ID NO. 2) and Pro-Ala-Arg-Leu (polypeptide 3) (shown as SEQ ID NO. 3) with antioxidant activity, which are discovered from protein hydrolysate of penaeus vannamei boone, and the three polypeptides are synthesized by adopting a solid phase synthesis method, and compounding with glucosamine calcium salt (adding glucosamine calcium salt and polypeptide into water, mixing uniformly to obtain the compound preparation; investigating three proportion relations, namely the mass ratio of the glucosamine calcium salt to the polypeptide is 1:1, 1:2 and 1:3, investigating three concentrations, namely 0.1%, 0.25% and 0.5%, which refer to the concentration of the compound in the aqueous solution and are in unit g/ml), and using the compound preparation for experimental research on cytotoxicity and anti-inflammatory activity.
Culturing mouse mononuclear macrophage RAW264.7 by using a 96-well plate, culturing 6000-7000 cells in each well, adding 100 mu l of cell culture solution in each well, placing in an incubator at 37 ℃ under CO2Culturing for 24h under the condition of 5% concentration, and adding medicines into several samples according to the concentration of 0.1%, 0.25% and 0.5% after 24h, namely: glucosamine calcium salt, polypeptide1, polypeptide 2, polypeptide 3, glucosamine calcium salt-polypeptide 1 complex (ratio 1:1), glucosamine calcium salt-polypeptide 1 complex (ratio 1:2), glucosamine calcium salt-polypeptide 1 complex (ratio 1:3), glucosamine calcium salt-polypeptide 2 complex (ratio 1:1), glucosamine calcium salt-polypeptide 2 complex (ratio 1:2), glucosamine calcium salt-polypeptide 2 complex (ratio 1:3), glucosamine calcium salt-polypeptide 3 complex (ratio 1:1), glucosamine calcium salt-polypeptide 3 complex (ratio 1:2), and glucosamine calcium salt-polypeptide 3 complex (ratio 1:3), 10 mu l of each well, detecting the absorbance at 450nm after 12h, and respectively calculating the cytotoxicity of different samples according to a cell survival rate formula.
The detection results are shown in fig. 11 and 12, and the cell survival rates of the substances with the concentrations of 0.1%, 0.25% and 0.5% are all higher than 80%, and the substances are not cytotoxic.
Example 5 anti-inflammatory Activity Studies of glucosamine calcium salt, three Polypeptides, and combinations of glucosamine calcium salt and Polypeptides at different ratios
RAW264.7 cells were plated at 3X 10 per well5Inoculating each cell in a 12-hole plate, placing in an incubator for 12h, culturing for 12h, sucking out cell culture fluid, adding 1 mu g/mL lipopolysaccharide solution, and constructing an inflammation model. Wherein, after the blank group is sucked out of the culture solution, the lipopolysaccharide solution is not added, and the cell culture solution without serum and double antibody is added. Each hole is respectively arranged according to the sequence0.1% of each sample (same as example 4) was added for dosing intervention, and after incubation for 12h, 300. mu.l of each well was used for the detection of several inflammatory factors (TNF-. alpha., NO, IL-6).
The detection results are shown in fig. 13, 14 and 15, and the contents of the three proinflammatory factors of the glucosamine calcium salt-polypeptide 1 compound (the proportion is 1:1) are the lowest compared with the contents of other substances, so that the obvious difference is formed.
Compared with glucosamine calcium salt, the anti-inflammatory activity of the three polypeptides is researched, and the research finds that the three polypeptides have certain anti-inflammatory activity but are lower than the glucosamine calcium salt. The anti-inflammatory activity of the three polypeptides and the calcium glucosamine salt is improved after the three polypeptides and the calcium glucosamine salt are compounded according to different mixture ratios, wherein the anti-inflammatory effect of the Polypeptide1 and calcium glucosamine salt compound (GA-Ca-Polypeptide1) with the compound ratio of 1:1(9 groups) is optimal, is obviously superior to that of other groups, and has obvious difference. The glucosamine calcium salt is compounded with the polypeptide with a specific sequence for use, can generate the effect similar to glucosamine polypeptide, supposedly has synergistic effect and can improve the anti-inflammatory activity of the product.
Example 6 Liposome preparation and characterization
Preparing liposome by adopting a film hydration method: weighing 200mg distearoyl phosphatidylcholine and 50mg cholesterol, dissolving in 10ml methanol-chloroform solution (8.5ml:1.5ml), ultrasonic treating for 20min, completely dissolving, and vacuum rotary steaming at 55 deg.C to obtain film; pouring 40mL of 0.1% (unit g/mL) glucosamine calcium salt-polypeptide 1 compound aqueous solution (GA-Ca-Leu-His-Trp-Lys-Ser), ultrasonically vibrating the film, ultrasonically crushing for 10min, after the crushing is finished, putting the film in a water bath kettle at 78 ℃ for 2h, and passing through the film to obtain the glucosamine calcium salt-polypeptide 1 compound embedded liposome (GA-Ca-Leu-His-Trp-Lys-Ser-lip).
Blank liposomes (lipopomes) were prepared using the same experimental procedure.
The two kinds of liposomes were subjected to transmission electron microscopy scanning and particle size and potential detection, respectively, and the results are shown in fig. 16 and 17, and the liposomes prepared were round and relatively uniform. The particle sizes of the two kinds of liposome are respectively 61.5 + -0.47 nm and 87.4 + -0.85 nm. The potential is-45.4 mv and-17.2 mv respectively.
Example 7 in vitro Release study
In vitro release studies were performed on GA-Ca-Leu-His-Trp-Lys-Ser and GA-Ca-Leu-His-Trp-Lys-Ser-lip (prepared in example 6) by dialysis. The two substances are respectively weighed into 10mL of dialysis bag, and the two ends are tied tightly to remove air bubbles. The mixture was placed in 200mL of PBS solution at 37 ℃ and pH 7.0, and samples were taken at 0.5, 1, 1.5, 2, 2.5, 3, 6, 9, 12, 18, 24, 30, 36, 48, 60, and 72 hours at 37 ℃ and were supplemented with an isothermal and equal volume of dialysis medium. And (4) respectively centrifuging and filtering the taken dialysate, measuring the absorbance, substituting the absorbance into a standard curve to calculate the concentration, calculating the accumulated release amount, and drawing a release curve.
As shown in FIG. 18, it was found from FIG. 18 that GA-Ca-Leu-His-Trp-Lys-Ser was released in the first 3 hours in a percentage of (96.2. + -. 2.12)%, and was almost completely released. And the release curve of GA-Ca-Leu-His-Trp-Lys-Ser-lip shows the trend of short-time quick release at the beginning and slow release after 24 h. The release rate at 24h is (46.3 +/-3.09)%.
Example 8 biocompatibility study
Cytotoxicity: cytotoxicity detection was carried out by the CCK-8 method for GA-Ca-Leu-His-Trp-Lys-Ser, GA-Ca-Leu-His-Trp-Lys-Ser-lip and liposomes at concentrations of 0.1%, 0.25%, 0.5%, 1% and 2.5%, respectively. Mouse articular chondrocytes were cultured in an incubator at 37 ℃ in the presence of CO in an amount of about 7000 cells/well in 100. mu.l of cell culture medium2Culturing for 24h under the condition of 5% concentration, adding medicines into several samples according to different concentrations (0.1%, 0.25%, 0.5%, 1%, 2.5%) after 24h, detecting absorbance at 450nm after 12h, and calculating cytotoxicity of different samples according to a cell survival rate formula. The results are shown in fig. 19, and show that the cell survival rate of several substances is higher than 80%, and three substances are not cytotoxic.
Example 9 influence of GA-Ca-Polypeptide1 and two liposomes on the expression level of proinflammatory factors
By culturing mouse articular chondrocytes, constructing an inflammation model, and respectively detecting the concentration of GA-Ca-Leu-His-Trp-Lys-Ser and the concentration of 0.1 percent of two liposomes to 5 inflammatory factors (COX-2, IL-6, NO,PGE2TNF-. alpha.) expression level. Mouse articular chondrocytes were plated at 4X 10 per well4Perwell inoculation in 6-well plates, in incubators (temperature 37 ℃ C., CO)2 Concentration 5%) for 24h, adding 1ml lipopolysaccharide (1 μ g/ml) into the wells except the control group, and incubating in an incubator to construct an inflammation model. Adding 0.1% concentration samples into each well in sequence, incubating for 12 hr, and sampling at different stages (wherein TNF-alpha sampling time is 12 hr, NO and IL-6 sampling time is 16 hr, COX-2 and PGE are obtained2Sampling time was 18h) for the detection of several inflammatory factors.
The results are shown in FIGS. 20-24, and show that the expression levels of several inflammatory factors are the lowest in the GA-Ca-Leu-His-Trp-Lys-Ser group, and that significant differences are formed in the GA-Ca-Leu-His-Trp-Lys-Ser-lip group. This is due to the slow release properties of liposomes. The encapsulated calcium glucosamine salt formulation released more slowly over the corresponding sampling period for several inflammatory factors. This is consistent with previous in vitro release profiles.
Example 10 real-time fluorescent quantitation of mRNA expression for several factors
RNA was first extracted from various groups of cells (cells obtained after drug-added dry culture in example 9), then converted into DNA by reverse transcription of cDNA, and the expression of the transcription levels of IL-1. beta., IL-6, TAC1, MMP1, Agg, and Col 2. alpha. was analyzed by QRT-PCR.
The four factors of IL-1 beta, IL-6, TAC1 and MMP1 are low in normal articular chondrocyte, and the content of the four factors is increased due to the occurrence of inflammatory reaction. As shown in FIGS. 25 to 30, the results showed that the group GA-Ca-Leu-His-Trp-Lys-Ser had the lowest content of 4 factors, and that the group GA-Ca-Leu-His-Trp-Lys-Ser-lip was the next group, which was significantly different. The two factors of Agg and Col2 alpha are contained in normal chondrocytes in a large amount, and when an inflammatory reaction occurs, the contents of the two factors are significantly reduced. The results show that the contents of two factors of GA-Ca-Leu-His-Trp-Lys-Ser are the highest, and the group of GA-Ca-Leu-His-Trp-Lys-Ser-lip is the next, which forms obvious difference. Because of the embedding of liposome, GA-Ca-Leu-His-Trp-Lys-Ser-lip is slowly released, so the expression of several factors is different from that of the GA-Ca-Leu-His-Trp-Lys-Ser group. The result shows that the glucosamine-polypeptide 1 compound liposome can realize the effects of resisting inflammation and slowly releasing medicaments, and has a protective effect on inflammation-induced cartilage cell degeneration.
The above examples are provided to those of ordinary skill in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications apparent to those skilled in the art are intended to be within the scope of the appended claims.
Sequence listing
<110> China oceanic university
<120> glucosamine calcium salt-polypeptide complex and liposome thereof
<141> 2022-03-07
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 5
<212> PRT
<213> Artificial Sequence
<400> 1
Leu His Trp Lys Ser
1 5
<210> 2
<211> 5
<212> PRT
<213> Artificial Sequence
<400> 2
Tyr Asp Leu Cys Ile
1 5
<210> 3
<211> 4
<212> PRT
<213> Artificial Sequence
<400> 3
Pro Ala Arg Leu
1

Claims (10)

1. A glucosamine calcium salt-polypeptide complex, which is characterized in that: the composition is prepared by compounding glucosamine calcium salt and Leu-His-Trp-Lys-Ser polypeptide, wherein the mass ratio of the glucosamine calcium salt to the Leu-His-Trp-Lys-Ser polypeptide is 1: 1-3.
2. The method for preparing a glucosamine calcium salt-polypeptide complex according to claim 1, wherein the method comprises the following steps: adding glucosamine calcium salt and polypeptide into water, mixing uniformly to obtain a glucosamine calcium salt-polypeptide compound solution, concentrating and drying to obtain the glucosamine calcium salt-polypeptide compound, wherein the concentration of the compound in the glucosamine calcium salt-polypeptide compound solution is 0.05-0.5%.
3. A glucosamine calcium salt-polypeptide complex-embedded liposome is characterized in that: is a liposome prepared from the glucosamine calcium salt-polypeptide compound as defined in claim 1 and a carrier.
4. The glucosamine-embedded liposome of calcium salt-polypeptide complex of claim 3, wherein: the proportion relationship between the glucosamine calcium salt-polypeptide compound and the carrier is as follows: 200ml of carrier is added into every 0.02-0.20 g of glucosamine calcium salt-polypeptide compound.
5. The glucosamine calcium salt-polypeptide complex-embedding liposome according to claim 3 or 4, wherein: the carrier is selected from distearoyl phosphatidylcholine and cholesterol, and the mass ratio of the distearoyl phosphatidylcholine to the cholesterol is 3-5: 1.
6. The process for preparing glucosamine-embedded calcium salt-polypeptide complex liposomes according to claim 3, 4 or 5, wherein the liposome comprises: adding a carrier into a container containing an organic solvent, dissolving, performing rotary evaporation to obtain a carrier film, adding an aqueous solution of the glucosamine calcium salt-polypeptide complex into the container, performing ultrasonic crushing to crush the carrier film, mixing the crushed carrier film with the aqueous solution of the glucosamine calcium salt-polypeptide complex, uniformly mixing, processing at 70-80 ℃ for 1.5-3 hours, and performing film coating to obtain the glucosamine calcium salt-polypeptide 1 complex-embedded liposome.
7. The method of claim 6, wherein: the organic solvent is selected from methanol, chloroform or their mixture.
8. The preparation method according to claim 6, wherein the specific preparation method is as follows: weighing 200mg of distearoyl phosphatidylcholine and 50mg of cholesterol, dissolving in a methanol-chloroform solution, and performing vacuum rotary evaporation after complete dissolution to obtain a film; and pouring 40ml of 0.05-0.5% glucosamine calcium salt-polypeptide 1 compound aqueous solution, vibrating the membrane by ultrasound, ultrasonically crushing, carrying out water bath in a water bath kettle at 78 ℃ for 2h after the crushing is finished, and passing through the membrane to obtain the glucosamine calcium salt-polypeptide 1 compound embedded liposome.
9. Use of the calcium glucosamine-polypeptide complex of claim 1 in the preparation of a medicament for the prevention or treatment of osteoarthritis.
10. Use of the glucosamine embedded calcium salt-polypeptide complex liposome of claim 3, 4 or 5 in the preparation of a medicament for preventing or treating osteoarthritis.
CN202210217329.8A 2022-03-07 2022-03-07 Glucosamine calcium salt-polypeptide complex and liposome thereof Pending CN114558105A (en)

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