Cyclodextrin derivatives and preparation methods and applications thereof

Abstract

The invention discloses cyclodextrin derivatives and preparation methods and applications thereof, belonging to the field of pharmaceutical technology. The cyclodextrin derivatives include cyclodextrin modified with PEG or cyclodextrin modified with vitamin A. Cyclodextrin modified with PEG can improve the solubility of cyclodextrin, increase the amount of drug absorbed in the body, and significantly increase the amount of drug distribution in the liver; cyclodextrin modified with vitamin A makes the inclusion compound easily absorbed by the hepatic stellate cells of the liver, which can improve the liver targeting of the drug, thereby improving the efficacy and reducing toxic side effects.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to cyclodextrin derivatives and preparation methods and applications thereof. Background Art

[0002] Cyclodextrin (CD) is a general term for a series of cyclic oligosaccharides generated by amylose under the action of cyclodextrin glucosyltransferase produced by Bacillus. Cyclodextrin, especially amino-β-cyclodextrin, has the characteristics of being hydrophobic inside and hydrophilic outside. Drug molecules that are compatible with the size of the hydrophobic cavity partially or completely enter the cyclodextrin cavity through non-covalent bonds, thereby forming a cyclodextrin inclusion complex. Cyclodextrin, as the host molecule, can self-assemble with small molecule drugs as guest molecules to form inclusion complexes through host-guest interactions, and the hydrophobic properties of the 6-position substituent chain of cyclodextrin can further improve the ability of the hydrophobic cavity to form inclusion complexes with guest drug molecules.

[0003] Liver fibrosis is a pathological process in which the connective tissue of the liver is abnormally proliferated. Common clinical causes include hepatitis virus infection, cholestasis, alcoholism, and non-alcoholic fatty liver disease. Any liver damage will lead to the process of liver fibrosis during the process of liver repair and healing. If the damaging factors cannot be removed for a long time, the fibrosis process will continue for a long time and will develop into cirrhosis or even liver cancer. Therefore, liver fibrosis is an important link affecting the prognosis of liver disease, and early intervention treatment can reverse liver fibrosis. However, there are currently few clinically available anti-fibrosis treatment strategies, and effective therapeutic drugs are urgently needed. Existing cyclodextrins are not liver-targeted, so the inclusion complex formed by them as carriers and anti-fibrosis drugs is difficult to specifically target liver tissue, thereby exerting high-efficiency anti-fibrosis capabilities. Summary of the invention

[0004] In view of the above technical problems existing in the prior art, the present invention provides a cyclodextrin derivative with liver-targeting characteristics and a preparation method and application thereof, so as to increase the concentration of the inclusion complex formed with the cyclodextrin derivative and small molecule drugs in the liver, thereby improving the anti-liver fibrosis effect of the inclusion complex.

[0005] The present invention discloses a cyclodextrin derivative, wherein the cyclodextrin derivative is a cyclodextrin modified with PEG or a cyclodextrin modified with vitamin A, wherein the structural formula of the cyclodextrin modified with PEG is:

[0006]

[0007] The structural formula of vitamin A modified cyclodextrin is:

[0008]

[0009] Preferably, the preparation method of PEG-modified cyclodextrin comprises:

[0010] Step S1: obtaining dicarboxylated polyethylene glycol;

[0011] Step S2: dissolving dicarboxylated polyethylene glycol, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide in dimethyl sulfoxide to obtain a first mixed solution;

[0012] Step S3: dissolving amino-β-cyclodextrin in dimethyl sulfoxide to obtain a second mixed solution;

[0013] Step S4: adding the second mixed solution dropwise to the first mixed solution and stirring to react to obtain a third mixed solution;

[0014] Step S5: purify the third mixed solution by dialysis to obtain PEG-modified cyclodextrin.

[0015] Preferably, the preparation method of PEG-modified cyclodextrin comprises:

[0016] Step S2: dissolving 880 mg of dicarboxylated polyethylene glycol, 153.4 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 92 mg of N-hydroxysuccinimide in 20 mL of dimethyl sulfoxide to obtain a first mixed solution;

[0017] Step S3: dissolving 453.6 mg of amino-β-cyclodextrin in 10 mL of dimethyl sulfoxide to obtain a second mixed solution, wherein the amino-β-cyclodextrin is mono-6-O-amino-β-cyclodextrin;

[0018] Step S4: at 25° C., dropwise adding the second mixed solution into the first mixed solution, and stirring the mixture for 72 hours to obtain a third mixed solution;

[0019] Step S5: After dialyzing the third mixed solution through a 3500DA dialysis bag for 48 hours, the liquid in the dialysis bag was freeze-dried to obtain PEG-modified cyclodextrin.

[0020] Preferably, the preparation method of vitamin A modified cyclodextrin comprises:

[0021] Step 101: obtaining the synthesis of dicarboxylated polyethylene glycol;

[0022] Step 102: dissolving dicarboxylated polyethylene glycol, 4-dimethylaminopyridine and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in dimethyl sulfoxide to obtain a fourth mixed solution;

[0023] Step 103: dissolving vitamin A in dimethyl sulfoxide to obtain a fifth mixed solution;

[0024] Step 104: adding the fifth mixed solution dropwise to the fourth mixed solution, stirring to react, and obtaining a sixth mixed solution;

[0025] Step 105: purifying the sixth mixed solution by dialysis to obtain vitamin A-modified polyethylene glycol;

[0026] Step 106: dissolving vitamin A-modified polyethylene glycol, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide in dimethyl sulfoxide to obtain a seventh mixed solution;

[0027] Step 107: dissolving amino-β-cyclodextrin in dimethyl sulfoxide to obtain a second mixed solution;

[0028] Step 108: adding the second mixed solution dropwise to the seventh mixed solution, stirring to react, and obtaining an eighth mixed solution;

[0029] Step 109: Purify the eighth mixed solution by dialysis to obtain vitamin A-modified cyclodextrin.

[0030] Preferably, the preparation method of vitamin A modified cyclodextrin comprises:

[0031] Step 102: dissolving 2.8 g of dicarboxylated polyethylene glycol, 170 mg of 4-dimethylaminopyridine and 800 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in 20 mL of dimethyl sulfoxide to obtain a fourth mixed solution;

[0032] Step 103: dissolving 364 mg of vitamin A in 20 mL of dimethyl sulfoxide to obtain a fifth mixed solution;

[0033] Step 104: under the condition of 25° C., nitrogen protection and light protection, the fifth mixed solution was added dropwise to the fourth mixed solution, and the mixture was stirred for reaction for 48 hours to obtain a sixth mixed solution;

[0034] Step 105: dialyzing the sixth mixed solution through a 2000Da dialysis bag, and freeze-drying the liquid in the dialysis bag to obtain vitamin A-modified polyethylene glycol;

[0035] Step 106: dissolving 988 mg of vitamin A-modified polyethylene glycol, 153.4 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 92 mg of N-hydroxysuccinimide in 20 mL of dimethyl sulfoxide to obtain a seventh mixed solution;

[0036] Step 107: dissolving 453.6 mg of amino-β-cyclodextrin in 10 mL of dimethyl sulfoxide to obtain a second mixed solution;

[0037] Step 108: at 25° C., dropwise add the second mixed solution to the seventh mixed solution, and stir to react for 72 hours to obtain an eighth mixed solution;

[0038] Step 109: dialyze the eighth mixed solution in a light-proof manner for 48 hours through a 3500DA dialysis bag, freeze-dry the liquid in the dialysis bag, and obtain vitamin A-modified cyclodextrin.

[0039] Preferably, the preparation method of dicarboxylated polyethylene glycol comprises:

[0040] Weigh 2 g of PEG2000 and completely dissolve it in 20 mL of dichloromethane to obtain a PEG2000-dichloromethane solution;

[0041] Weigh 0.5 g of succinic anhydride and 122 mg of 4-dimethylaminopyridine, completely dissolve in 10 mL of dichloromethane, add dropwise to the PEG2000-dichloromethane solution under magnetic stirring, and react at 60 °C for 12 h;

[0042] After the reaction is completed, deionized water is added to the reaction system for extraction to hydrate and remove excess succinic anhydride. The extracted dichloromethane layer is collected and added to color-changing silica gel for drying, and then the dichloromethane is removed by rotary evaporation; 20 mL of icy ether is added to form a precipitate, and the precipitate obtained by suction filtration is dried to obtain dicarboxylated polyethylene glycol.

[0043] Preferably, the cyclodextrin derivative is used to prepare an inclusion compound.

[0044] Preferably, the cyclodextrin derivative is used to prepare an inclusion complex of dihydrotanshinone I;

[0045] The inclusion compound of dihydrotanshinone I is used for preparing medicines or foods against liver fibrosis.

[0046] Preferably, the preparation method of the inclusion compound comprises:

[0047] Add dihydrotanshinone I and a three-fold molar ratio of a cyclodextrin derivative into a mortar, add a small amount of water and an organic solvent to moisten, grind for 30 minutes, wash with anhydrous ethanol 1-3 times, collect the eluate, and vacuum dry for 12 hours to obtain an inclusion complex of dihydrotanshinone I; or

[0048] A 4 mg / mL aqueous solution of a cyclodextrin derivative was prepared; 10 mg of dihydrotanshinone I was weighed and dissolved in 1 mL of anhydrous ethanol, and then added to the aqueous solution of the cyclodextrin derivative and stirred for reaction for 3 days, wherein the molar ratio of dihydrotanshinone I to the cyclodextrin derivative was 1:3; after the reaction was completed, the mixture was filtered with a 0.22 μm microporous filter membrane, and the filtrate was freeze-dried to obtain an inclusion complex of dihydrotanshinone I.

[0049] Compared with the prior art, the beneficial effects of the present invention are as follows: PEG-modified cyclodextrin can improve the solubility of cyclodextrin, increase the amount of drug absorbed in the body, and significantly increase the amount of drug distribution in the liver; vitamin A-modified cyclodextrin makes the inclusion compound easily absorbed by hepatic stellate cells, which can improve the liver targeting of the inclusion compound, thereby improving the therapeutic effect and reducing toxic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 is a flow chart of the preparation method of the cyclodextrin derivatives of the present invention;

[0051] Figure 2 is the mass spectrum of cyclodextrin derivatives;

[0052] Figure 3 is the hydrogen spectrum of cyclodextrin derivatives;

[0053] Figure 4 is the infrared spectrum of cyclodextrin derivatives;

[0054] Figure 5 This is a scanning electron micrograph of dihydrotanshinone I loaded on cyclodextrin derivatives prepared by grinding method;

[0055] Figure 6 This is a scanning electron micrograph of dihydrotanshinone I loaded on cyclodextrin derivatives prepared by saturated solvent method;

[0056] Figure 7 This is a transmission electron micrograph of dihydrotanshinone I loaded with cyclodextrin derivative dissolved in water;

[0057] Figure 8 It is the X-ray powder diffraction pattern of dihydrotanshinone I loaded with cyclodextrin derivative;

[0058] Fig. 9 It is the differential scanning calorimetry diagram of dihydrotanshinone Ⅰ loaded on cyclodextrin derivative;

[0059] Fig.10 This is the RMSD simulation diagram of dihydrotanshinone I loaded on cyclodextrin derivatives;

[0060] Fig.11 This is the molecular docking diagram of cyclodextrin and dihydrotanshinone I;

[0061] Fig.12 It is a liver targeting test chart of cyclodextrin derivatives;

[0062] Fig.13 It is the anti-liver fibrosis efficacy diagram of cyclodextrin derivatives. DETAILED DESCRIPTION

[0063] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0064] The present invention is further described in detail below in conjunction with the accompanying drawings:

[0065] A cyclodextrin derivative, wherein the cyclodextrin derivative is PEG-modified cyclodextrin (PEG-CD), and its structural formula is:

[0066]

[0067] PEG-modified cyclodextrin can improve the solubility of cyclodextrin and increase the amount of drug absorbed in the body; in a specific test, it significantly increased the amount of drug distributed in the liver.

[0068] Vitamin A can also be connected to the end of the PEG long chain, that is, the cyclodextrin derivative is vitamin A-modified cyclodextrin (VA-PEG-CD), and its structural formula is:

[0069]

[0070] Its molecular formula can be expressed as [C 20 H 29 OOC(CH 2 ) 2 COO(CHCHO) n OC(CH 2 ) 2 COC 42 H 70 NO 34 ].

[0071] Since vitamin A tends to be easily taken up by liver cells, vitamin A modified cyclodextrin formed by grafting vitamin A on the carboxyl end of PEG modified cyclodextrin has liver targeting. Its inclusion complex formed by the main molecule and small molecule drugs is easily taken up by hepatic stellate cells (HSCs) in the liver, which can improve the liver targeting of drugs, thereby improving the efficacy and reducing toxic side effects. Among them, HSCs cells are vitamin A (VA) reserve cells, storing about 80% of VA in the human body.

[0072] Wherein, the preparation method of PEG-modified cyclodextrin includes:

[0073] Step S1: obtaining dicarboxylated polyethylene glycol.

[0074] Step S2: dissolving dicarboxylated polyethylene glycol, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCL) and N-hydroxysuccinimide (NHS) in dimethyl sulfoxide to obtain a first mixed solution.

[0075] Step S3: dissolving amino-β-cyclodextrin in dimethyl sulfoxide to obtain a second mixed solution.

[0076] Step S4: adding the second mixed solution dropwise to the first mixed solution, stirring the mixture for reaction, and obtaining a third mixed solution.

[0077] Step S5: purify the third mixed solution by dialysis to obtain PEG-modified cyclodextrin (PEG-CD).

[0078] The preparation method of vitamin A modified cyclodextrin (VA-PEG-CD) comprises:

[0079] Step 101: Obtain dicarboxylated polyethylene glycol.

[0080] Step 102: dissolving dicarboxylated polyethylene glycol, 4-dimethylaminopyridine and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in dimethyl sulfoxide to obtain a fourth mixed solution.

[0081] Step 103: Dissolve vitamin A in dimethyl sulfoxide to obtain a fifth mixed solution.

[0082] Step 104: dropwise add the fifth mixed solution into the fourth mixed solution, stir to react, and obtain a sixth mixed solution.

[0083] Step 105: Purify the sixth mixed solution by dialysis to obtain vitamin A-modified polyethylene glycol (VA-PEG-COOH).

[0084] Step 106: Dissolve vitamin A-modified polyethylene glycol, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide in dimethyl sulfoxide to obtain a seventh mixed solution.

[0085] Step 107: dissolving amino-β-cyclodextrin in dimethyl sulfoxide to obtain a second mixed solution.

[0086] Step 108: Add the second mixed solution dropwise to the seventh mixed solution, stir to react, and obtain an eighth mixed solution.

[0087] Step 109: Purify the eighth mixed solution by dialysis to obtain vitamin A-modified cyclodextrin (VA-PEG-CD).

[0088] Example 1

[0089] Preparation of PEG-CD and VA-PEG-CD.

[0090] 1. Preparation of dicarboxylated polyethylene glycol (COOH-PEG-COOH):

[0091] PEG2000 2g was weighed and completely dissolved in 20mL dichloromethane to obtain PEG2000-dichloromethane solution; succinic anhydride 0.5g and 4-dimethylaminopyridine (DMAP) 122mg were weighed and completely dissolved in 10mL dichloromethane, and added dropwise to the PEG2000-dichloromethane solution under magnetic stirring, and reacted at 60°C for 12h. After the reaction was completed, deionized water was added to the reaction system for extraction to hydrate and remove the excess succinic anhydride; deionized water was added for secondary extraction; the extracted dichloromethane layer was collected, and color-changing silica gel was added thereto for drying, and dichloromethane was removed by rotary evaporation; 20mL of icy ether was added to form a precipitate, and the precipitate obtained by suction filtration was dried to obtain dicarboxylated polyethylene glycol (COOH-PEG-COOH).

[0092] The structural formula of dicarboxylated polyethylene glycol can be expressed as:

[0093]

[0094] 2. Preparation of PEG-modified cyclodextrin (PEG-CD):

[0095] Step S2: 880 mg of dicarboxylated polyethylene glycol, 153.4 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCL) and 92 mg of N-hydroxysuccinimide (NHS) were dissolved in 20 mL of dimethyl sulfoxide (DMSO) to obtain a first mixed solution.

[0096] Step S3: dissolving 453.6 mg of amino-β-cyclodextrin in 10 mL of dimethyl sulfoxide to obtain a second mixed solution, wherein the amino-β-cyclodextrin is mono-6-O-amino-β-cyclodextrin.

[0097] Step S4: at 25° C., the second mixed solution was added dropwise to the first mixed solution, and the mixture was stirred for reaction for 72 hours to obtain a third mixed solution.

[0098] Step S5: After dialyzing the third mixed solution through a 3500Da dialysis bag for 48 hours, the liquid in the dialysis bag was freeze-dried to obtain powdered PEG-modified cyclodextrin (PEG-CD).

[0099] 3. Preparation of Vitamin A Modified Cyclodextrin (VA-PEG-CD):

[0100] Step 102: 2.8 g of dicarboxylated polyethylene glycol, 170 mg of 4-dimethylaminopyridine (DMAP) and 800 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) were dissolved in 20 mL of dimethyl sulfoxide (DMSO) to obtain a fourth mixed solution.

[0101] Step 103: Dissolve 364 mg of vitamin A in 20 mL of dimethyl sulfoxide to obtain a fifth mixed solution.

[0102] Step 104: At 25° C. under nitrogen protection and light-proof conditions, the fifth mixed solution was added dropwise to the fourth mixed solution, and the mixture was stirred for reaction for 48 hours to obtain a sixth mixed solution.

[0103] Step 105: dialyze the sixth mixed solution through a 2000Da dialysis bag, freeze-dry the liquid in the dialysis bag, and obtain vitamin A-modified polyethylene glycol (VA-PEG-COOH).

[0104] The structural formula of VA-PEG-COOH can be expressed as:

[0105]

[0106] Step 106: 988 mg of vitamin A-modified polyethylene glycol (VA-PEG-COOH), 153.4 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCL) and 92 mg of N-hydroxysuccinimide (NHS) are dissolved in 20 mL of dimethyl sulfoxide (DMSO) to obtain a seventh mixed solution.

[0107] Step 107: Dissolve 453.6 mg of amino-β-cyclodextrin in 10 mL of dimethyl sulfoxide to obtain a second mixed solution.

[0108] Step 108: At 25° C., add the second mixed solution dropwise to the seventh mixed solution, stir and react for 72 hours to obtain an eighth mixed solution.

[0109] Step 109: The eighth mixed solution is dialyzed in a 3500DA dialysis bag for 48 hours in a dark environment, and the liquid in the dialysis bag is freeze-dried to obtain vitamin A-modified cyclodextrin (VA-PEG-CD).

[0110] The prepared COOH-PEG-COOH, VA-PEG-COOH and VA-PEG-CD were subjected to mass spectrometry (MS), nuclear magnetic resonance spectroscopy ( 1 H-NMR) and infrared spectroscopy (IR) characterization detection. The mass spectrometry characterization results are as follows Figure 2The charge of COOH-PEG-COOH, VA-PEG-COOH and VA-PEG-CD is 1, the average mass-to-charge ratio / average molecular weight of dicarboxylated polyethylene glycol (COOH-PEG-COOH) is 2200, the average mass-to-charge ratio / average molecular weight of VA-PEG-COOH is 2400, and the average mass-to-charge ratio / average molecular weight of VA-PEG-CD is 3500. The characterization results of H NMR are shown in Figure 3 , 3.52ppm characteristic peak belongs to the methylene in the PEG repeating unit, 2.50ppm characteristic peak belongs to the methylene in the succinic anhydride end group reacting with PEG; 1.07ppm characteristic peak belongs to the grafted VA methyl peak, 2.13ppm is the methyl peak on the double bond structure connecting VA and PEG; 4.83ppm is the cyclodextrin H-1 peak, 3.34 and 3.64ppm are the H-2 and H-6 peaks in the cyclodextrin derivatives, and 5.70ppm is the OH peak of the cyclodextrin derivative. According to the above nuclear magnetic resonance hydrogen spectrum results, it can be confirmed that the double carboxylated PEG molecules are successfully connected to VA and cyclodextrin respectively. The infrared spectroscopy characterization results are shown in Figure 4 , 2800~3000cm -1 The peaks of CH stretching vibration of methyl and methylene groups are shown in Table 1. Compared with the COOH-PEG-COOH spectrum, the peaks at 1730 cm-1 in the VA-PEG-COOH spectrum are -1 The C=O absorption of 1631cm -1 The C=C stretching vibration peak of VA was newly added at 3418cm, which proved that VA was successfully grafted onto COOH-PEG-COOH. Compared with the infrared spectrum of VA-PEG-COOH, the peak of VA-PEG-CD at 3418cm -1 The absorption of the stretching vibration peak of the hydroxyl group was enhanced, indicating that cyclodextrin was successfully grafted with VA-PEG-COOH.

[0111] Example 2

[0112] Preparation of dihydrotanshinone (DHI) inclusion complexes (CD-DHI, PEG-CD-DHI and VA-PEG-CD-DHI). DHI inclusion complexes were prepared by grinding method and saturated solvent method respectively:

[0113] Preparation of inclusion complex by grinding method:

[0114] DHI and a three-fold molar ratio of a cyclodextrin derivative were added to a mortar respectively, a small amount of water and an organic solvent were added to moisten them, and after grinding for 30 min, they were washed with anhydrous ethanol for 1-3 times, the eluate was collected, and vacuum dried for 12 h to obtain the cyclodextrin derivative DHI inclusion complex.

[0115] The above method was used to prepare cyclodextrin-loaded dihydrotanshinone inclusion complex (CD-DH I), PEG-modified cyclodextrin-loaded dihydrotanshinone inclusion complex (PEG-CD-DH I) and VA-modified cyclodextrin-loaded dihydrotanshinone inclusion complex (VA-PEG-CD-DHI).

[0116] Preparation of inclusion compounds by saturated solvent method:

[0117] A 4 mg / mL aqueous solution of a cyclodextrin derivative was prepared; 10 mg of dihydrotanshinone I was weighed and dissolved in 1 mL of anhydrous ethanol, and then added to the aqueous solution of the cyclodextrin derivative and stirred for reaction for 3 days, wherein the molar ratio of dihydrotanshinone I to the cyclodextrin derivative was 1:3; after the reaction was completed, the mixture was filtered with a 0.22 μm microporous filter membrane, and the filtrate was freeze-dried to obtain an inclusion complex of dihydrotanshinone I.

[0118] The cyclodextrin derivative-loaded dihydrotanshinone I inclusion complex prepared by the above method was measured by scanning electron microscopy (SEM), transmission electron microscopy (TEM), differential scanning calorimetry (DSC) and X-ray powder diffraction (XRD). Figure 5 and Figure 6 As shown in Figure 2, the inclusion complex prepared by the grinding method is in the form of irregular blocks, while the inclusion complex prepared by the saturated solvent method is in the form of flakes after freeze-drying; the transmission electron microscopy results are shown in Figure 2. Figure 7 As shown, compared with the unmodified cyclodextrin inclusion complex, the VA-modified cyclodextrin-loaded dihydrotanshinone inclusion complex (dissolved in water) exhibits a more regular spherical shape.

[0119] The XRD and DSC results are shown in Figure 8 and Fig. 9 DSC is composed of Fig. 9 It can be seen that DHI has a strong endothermic peak at 230℃; Figure 8 It can be seen that DHI has a strong characteristic peak; while the endothermic peak and characteristic peak of the inclusion complex formed by cyclodextrin derivatives loaded with DHI disappear, indicating that DHI no longer exists in a crystalline form after grinding.

[0120] AutoDock4.2 was used to perform docking calculations and analyze the interaction mode between the compound and the receptor, and the interaction between the compound and the receptor was obtained, such as the generated hydrogen bonding, π stacking, hydrophobic interaction, etc., and then the stability of the formed complex was inferred by referring to the docking score of the compound. The molecular docking results are shown in Table 1.

[0121] Table 1

[0122]

[0123] To further investigate the interaction between small molecules and cyclodextrin, molecular dynamics simulations of the cyclodextrin-small molecule complex were performed for 20 ns. Monitoring the root mean square deviation (RMSD) of cyclodextrin and the small molecule complex can provide insight into their structural conformational changes throughout the simulation. RMSD analysis can indicate whether the simulation has reached equilibrium; the RMSD value is usually stable near a fixed value. If the RMSD of cyclodextrin is still increasing or decreasing on average at the end of the simulation, the system has not yet reached equilibrium, and the simulation time may not be sufficient for rigorous analysis. Similarly, RMSD also reflects the stability of cyclodextrin and small molecules. The larger the RMSD, the more unstable the cyclodextrin system.

[0124] The molecular docking results showed that the compound DHI had a good binding effect with the target receptor and a high matching degree, with a binding energy of -5.78 kcal / mol. The complex formed by the docking compound and the receptor was visualized using Pymol2.1 software to obtain the binding mode of the compound and the receptor. According to the binding mode, it can be clearly seen that there are three hydrogen bond interactions between DHI and CD, and the hydrogen bond distances are Much smaller than traditional hydrogen bonds The strong binding ability makes an important contribution to promoting the formation of a stable complex between the two molecules. In addition, since DHI contains multiple rings and has a certain hydrophobicity, it can form a good hydrophobic interaction with the hydrophobic cavity of cyclodextrin, which also contributes to the stabilization of small molecules.

[0125] like Fig.10 As shown, the average RMSD of cyclodextrin is about Moreover, the complex reached dynamic equilibrium in a relatively short period of time, indicating that the small molecule DHI has a good match with the cyclodextrin target and can form a stable complex with cyclodextrin. In addition, it can be seen from the results in the figure that there is no obvious fault problem in the conformational change of the complex, which also indicates that the small molecule DHI can bind well to cyclodextrin and will not leave the active pocket of cyclodextrin. Fig.11 It can be seen that during the entire simulation process, the small molecule did not leave the cyclodextrin cavity, and within 5ns ( Fig.11 A), 10ns( Fig.11 B), 15ns( Fig.11 C) and 20ns( Fig.11 D) were further analyzed and found that these compounds can form hydrogen bonds, conjugation, hydrophobic and other interactions with the cyclodextrin cavity, and these interactions effectively promote the formation of a stable complex between the small molecule DHI and cyclodextrin. In summary, DHI and cyclodextrin derivatives can form multiple interactions, high matching and stable inclusion complexes.

[0126] Liver targeting test of cyclodextrin derivatives

[0127] Preparation of cyclodextrin derivative-loaded Nile red inclusion complex: Nile red was used instead of DHI to prepare cyclodextrin derivative-loaded Nile red inclusion complex.

[0128] Animal BDL model establishment: SD male rats (weighing 180-200g) were used as experimental animals. They were fasted for 12h before surgery. After anesthesia with isoflurane, the abdomen was opened under aseptic conditions, the liver edge was raised, the duodenum was pulled open, and the common bile duct was separated by 2-3cm. Two ligatures were made near the duodenum and near the liver hilum, respectively, with No. 000 silk thread. The common bile duct was cut between the two ligatures, and the liver was restored to its original state and the incision was sutured. After the animals woke up from anesthesia, they ate normally and drank water freely to obtain BDL rats.

[0129] BDL rats were intraperitoneally injected with 2 mg / kg of Nile red (free), cyclodextrin-loaded Nile red inclusion complex, and PEG-modified cyclodextrin-loaded Nile red inclusion complex. After the injection, the rats were subjected to in vivo imaging analysis. The animals were then killed by injection of chloral hydrate solution, and their liver tissues were collected and imaged. Fig.12 As shown in A and 12B, the fluorescence intensity / average radiance of the PEG-modified cyclodextrin-loaded Nile red inclusion complex at 8 hours was significantly higher than that of the cyclodextrin-loaded Nile red inclusion complex and free Nile red, indicating that PEG-modified cyclodextrin can increase the drug distribution amount of the inclusion complex in liver tissue; Fig.12 As shown in A, 12B and 12C, the accumulation of Nile red in the liver was further enhanced after Nile red was loaded on VA-modified cyclodextrin.

[0130] Anti-liver fibrosis drug efficacy test

[0131] BDL rats were randomly selected as BDL control group, dihydrotanshinone administration group, cyclodextrin-loaded dihydrotanshinone administration group, PEG-modified cyclodextrin-loaded dihydrotanshinone administration group, and VA-modified cyclodextrin-loaded dihydrotanshinone administration group. Each administration group was intraperitoneally injected with dihydrotanshinone (10 mg / kg dihydrotanshinone dose) on the second day after surgery, once a day, and after 14 days of administration, blood, bile, liver and other samples were collected after fasting for 12 hours; the BDL control group was intraperitoneally injected with normal saline on the second day after surgery, once a day, and after 14 days of normal saline administration, blood, bile, liver and other samples were collected after fasting for 12 hours.

[0132] Sham operation group: experimental animals were fasted for 12 hours before surgery; surgery: after anesthesia with isoflurane, the abdomen was opened under sterile conditions, and then the incision was sutured. After the animals woke up from anesthesia, they ate normally and drank water freely; on the second day after the operation, normal saline was injected intraperitoneally once a day. After 14 days of normal saline administration, blood, bile, liver and other samples were collected after fasting for 12 hours.

[0133] Liver tissue sections were stained with hematoxylin-eosin, Sirius red and Masson's staining. Fig.13 As shown in the results, compared with the sham operation group, the bile duct hyperplasia and necrosis in the BDL control group increased significantly; after administration of dihydrotanshinone and its cyclodextrin derivative inclusion complex, the VA-modified cyclodextrin inclusion complex can significantly improve the phenomenon of bile duct hyperplasia and necrosis in the liver. Sirius red and Masson staining results can reflect the collagen deposition in the liver. The more collagen deposition, the more severe the liver fibrosis. Severe collagen deposition can be seen in the BDL control group. After administration of PEG-modified cyclodextrin inclusion complex, collagen deposition is reduced; after administration of VA-modified cyclodextrin inclusion complex, collagen deposition is significantly reduced, indicating that VA-modified cyclodextrin inclusion complex can significantly improve the degree of liver fibrosis in BDL rats. Therefore, the inclusion complex of dihydrotanshinone I prepared with VA-modified cyclodextrin as a carrier can be used to prepare drugs or foods for anti-liver fibrosis.

[0134] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A cyclodextrin derivative, characterized in that: The cyclodextrin derivative is vitamin A-modified cyclodextrin, The structural formula of vitamin A modified cyclodextrin is: Here, n is a natural number.

2. A method for preparing a cyclodextrin derivative as claimed in claim 1, characterized in that: The preparation method of vitamin A modified cyclodextrin comprises: Step 101: obtaining dicarboxylated polyethylene glycol; Step 102: dissolving dicarboxylated polyethylene glycol, 4-dimethylaminopyridine and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in dimethyl sulfoxide to obtain a fourth mixed solution; Step 103: dissolving vitamin A in dimethyl sulfoxide to obtain a fifth mixed solution; Step 104: adding the fifth mixed solution dropwise to the fourth mixed solution, stirring to react, and obtaining a sixth mixed solution; Step 105: purifying the sixth mixed solution by dialysis to obtain vitamin A-modified polyethylene glycol; Step 106: dissolving vitamin A-modified polyethylene glycol, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide in dimethyl sulfoxide to obtain a seventh mixed solution; Step 107: dissolving amino-β-cyclodextrin in dimethyl sulfoxide to obtain a second mixed solution; Step 108: adding the second mixed solution dropwise to the seventh mixed solution, stirring to react, and obtaining an eighth mixed solution; Step 109: Purify the eighth mixed solution by dialysis to obtain vitamin A-modified cyclodextrin.

3. The preparation method according to claim 2, characterized in that: The preparation method of vitamin A modified cyclodextrin comprises: Step 102: dissolving 2.8 g of dicarboxylated polyethylene glycol, 170 mg of 4-dimethylaminopyridine and 800 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in 20 mL of dimethyl sulfoxide to obtain a fourth mixed solution; Step 103: dissolving 364 mg of vitamin A in 20 mL of dimethyl sulfoxide to obtain a fifth mixed solution; Step 104: under the condition of 25° C., nitrogen protection and light protection, the fifth mixed solution was added dropwise to the fourth mixed solution, and the mixture was stirred for reaction for 48 hours to obtain a sixth mixed solution; Step 105: dialyzing the sixth mixed solution through a 2000Da dialysis bag, and freeze-drying the liquid in the dialysis bag to obtain vitamin A-modified polyethylene glycol; Step 106: dissolving 988 mg of vitamin A-modified polyethylene glycol, 153.4 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 92 mg of N-hydroxysuccinimide in 20 mL of dimethyl sulfoxide to obtain a seventh mixed solution; Step 107: dissolving 453.6 mg of amino-β-cyclodextrin in 10 mL of dimethyl sulfoxide to obtain a second mixed solution; Step 108: at 25° C., dropwise add the second mixed solution to the seventh mixed solution, and stir to react for 72 hours to obtain an eighth mixed solution; Step 109: dialyze the eighth mixed solution in a light-proof manner for 48 hours through a 3500DA dialysis bag, freeze-dry the liquid in the dialysis bag, and obtain vitamin A-modified cyclodextrin.

4. The preparation method according to claim 2, characterized in that: The preparation method of dicarboxylated polyethylene glycol comprises: Weigh 2 g of PEG2000 and dissolve it in 20 mL of dichloromethane to obtain a PEG2000-dichloromethane solution; Weigh 0.5 g of succinic anhydride and 122 mg of 4-dimethylaminopyridine, dissolve in 10 mL of dichloromethane, add dropwise to the PEG2000-dichloromethane solution under magnetic stirring, and react at 60 °C for 12 h; After the reaction is completed, deionized water is added to the reaction system for extraction to hydrate and remove excess succinic anhydride. The extracted dichloromethane layer is collected and added to color-changing silica gel for drying, and then the dichloromethane is removed by rotary evaporation; 20 mL of icy ether is added to form a precipitate, and the precipitate obtained by suction filtration is dried to obtain dicarboxylated polyethylene glycol.

5. A use of the cyclodextrin derivative as claimed in claim 1, characterized in that: The cyclodextrin derivative is used for preparing inclusion compound.

6. The use according to claim 5, characterized in that: The cyclodextrin derivative is used for preparing the inclusion compound of dihydrotanshinone I.

7. The use according to claim 6, characterized in that: The inclusion compound of dihydrotanshinone I is used for preparing medicines or foods against liver fibrosis.

8. The use according to claim 6, characterized in that: The preparation method of the inclusion compound comprises: Add dihydrotanshinone I and a three-fold molar ratio of a cyclodextrin derivative into a mortar, add a small amount of water and an organic solvent to moisten, grind for 30 minutes, wash with anhydrous ethanol 1-3 times, collect the washing liquid, and vacuum dry for 12 hours to obtain an inclusion complex of dihydrotanshinone I; or A 4 mg / mL aqueous solution of a cyclodextrin derivative was prepared; 10 mg of dihydrotanshinone I was weighed and dissolved in 1 mL of anhydrous ethanol, and then added to the aqueous solution of the cyclodextrin derivative and stirred for reaction for 3 days, wherein the molar ratio of dihydrotanshinone I to the cyclodextrin derivative was 1:3; after the reaction was completed, the solution was filtered with a 0.22 μm microporous filter membrane, and the filtrate was freeze-dried to obtain an inclusion complex of dihydrotanshinone I.

Images