CN111423484A - β sitosterol derivative and preparation method and application thereof - Google Patents

β sitosterol derivative and preparation method and application thereof Download PDF

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CN111423484A
CN111423484A CN202010136016.0A CN202010136016A CN111423484A CN 111423484 A CN111423484 A CN 111423484A CN 202010136016 A CN202010136016 A CN 202010136016A CN 111423484 A CN111423484 A CN 111423484A
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万丹
郭新红
张水寒
曹科
曾宏亮
彭咏波
李雄
周融融
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Abstract

The invention belongs to the technical field of biological medicines, and discloses an β sitosterol derivative, a preparation method and application thereof. β sitosterol derivative can be assembled into nanoparticles with avidin. β sitosterol derivative and its nano-drug are proved by animal experiments to be capable of obviously reducing the sleep latency time of mice, obviously improving the sleep time and subliminal hypnosis and falling-asleep rate of the mice, effectively improving the sleep quality of the mice, and being used for further preparing a drug or a health care product for improving the sleep quality.

Description

β sitosterol derivative and preparation method and application thereof
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an β sitosterol derivative as well as a preparation method and application thereof.
Background
Due to the influence of factors such as working pressure, social competition, aging of population and the like, 45.4 percent of people in China have the problem of sleep disorder and are in direct proportion to anxiety mental disorder. Sleep disorders have been experienced in about 35% of the population in the united states, with insomnia being considered to severely affect their lives in 17% of the patients and ineffective treatment in 85% of the patients. The sleep disorder seriously affects the life quality and the work efficiency of people, and researches show that the sleep disorder can cause the hypoevolutism of the body and intelligence, the memory attenuation, the reduction of the immunity of the organism and the like. Persistent sleep disorders are risk factors for depressed patients and are one of the early clinical symptoms of schizophrenia and other psychiatric disorders. At present, the sleep improvement medicines mostly focus on melatonin, paroxetine, sertraline, mirtazapine, trovadone, amitriptyline and the like, and although the substances have good sleep improvement effects, adverse reactions such as dizziness, drowsiness, hypodynamia and the like can be caused during the hypnotic dose, and drug resistance and physiological and psychological dependence are generated after long-term use. Therefore, the research and development of the hypnotic medicine with high efficiency, low dependence and no drug resistance has great social and economic significance.
β -sitosterol (β -sitosterol, BS) is one of the phytosterol components, which is widely present in various plant seeds such as vegetable oils, nuts, etc. in nature, and also present in some plant drugs, its structure is very similar to cholesterol, and has estrogenic activity, it can inhibit proliferation of human leukemia cells (G2/M block), intranuclear replication, and α -polymerization of tubulin and microtubules. β -sitosterol is widely used in the pharmaceutical industry with its characteristic biological and physicochemical properties. β -sitosterol is a white scale-like, needle-like crystal or crystalline powder, odorless, tasteless, very soluble in chloroform and carbon disulfide, soluble in ethanol or acetone, insoluble in water. β -sitosterol has cholesterol-lowering, antitussive, expectorant, and tumor-suppressing and tissue-repairing effects.
Figure BDA0002397348330000021
Biotin (Biotin, B) is widely applied to targeted delivery and transportation of various anti-cancer drugs and the like as a targeted ligand due to simple structure, low molecular weight, high tumor specificity and wide high expression of a Biotin receptor on the surface of a tumor tissue membrane, the Biotin is also called vitamin H and coenzyme R, is a water-soluble beneficial vitamin, also belongs to a vitamin B group, has the molecular weight of 244.31 and has a basic structure of a bicyclic structure, wherein the ring I is an imidazolone ring which is a part combined with avidin, the ring II is a thiophene ring which contains a valeric acid side chain, and the terminal carboxyl group of the thiophene ring can be connected with a biological macromolecule to form a Biotin-labeled antigen, antibody, enzyme and the like, and the chemical structure of the Biotin has an imidazolone ring which can be specifically combined with Avidin (AV) and streptavidin (streptavidin, SA), and the combination of the Biotin and the avidin has high affinity (the binding constant of about 1 × 10)-15mol/L, and can be regarded as irreversible binding), strong specificity, each can be combined with various types of large and small molecules, one molecular chain Mycosavin can be combined with four molecular biotin, twoThe streptavidin-biotin binding reaction has the advantages of multi-stage amplification effect and the like, and related technologies of the streptavidin-biotin binding reaction are widely applied to the fields of various labeling immunoassay technologies and tumor targeted therapy medicines, and a good molecular foundation is laid for building various health products based on B.
Most importantly, the improvement of solubility of β sitosterol through biotin modification and the report of sleep improvement effect of the sitosterol are not seen so far, and the specific combination of a β sitosterol derivative modified by biotin and avidin to construct nanoparticles and biomedical application are not seen.
Disclosure of Invention
To overcome the disadvantages and shortcomings of the prior art, the present invention is primarily directed to an β sitosterol derivative.
Still another object of the present invention is to provide a method for preparing the β sitosterol derivative.
Still another object of the present invention is to provide the use of the β sitosterol derivative.
Still another object of the present invention is to provide nanoparticles assembled by the β sitosterol derivatives and avidin.
It is a further object of the present invention to provide the use of the above nanoparticles.
The purpose of the invention is realized by the following technical scheme:
an β sitosterol derivative having the structure shown in formula (I):
Figure BDA0002397348330000031
the preparation method of the β sitosterol derivative comprises the following operation steps:
β sitosterol is dissolved in a solvent, the mixture is stirred and reacted for 1-6 h at 0 ℃ under the action of a dehydrating agent N, N' -dicyclohexylcarbodiimide or 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and a catalyst on dimethylaminopyridine, then biotin is added according to the molar ratio of β sitosterol being 1:1-3 times of the mixture, the temperature is raised to room temperature from an ice bath, the mixture is stirred overnight in a dark place, filtrate is concentrated, and the mixture is recrystallized by glacial ethyl ether or isopropanol, is chromatographed or is prepared into a liquid phase for purification, and is freeze-dried to obtain the β sitosterol derivative with the structure shown in the formula (I).
The solvent is CH2Cl2DMSO or DMF; the stirring reaction time is 5 h; the room temperature was 25 ℃.
The mole ratio of the β sitosterol to the dehydrating agent to the catalyst is 1:1: 1-1: 25: 25.
The β sitosterol derivative and the application of the pharmaceutically acceptable salt thereof in preparing the sleep improving medicine or the health care product are tablets, capsules, powder, granules, oral liquid, pills, powder, sustained release preparations, solutions, suspensions, injections, microneedles, ointments, creams or suppositories.
A nanoparticle is composed of β sitosterol derivative and avidin.
The application of the nano-particles in preparing the sleep-improving medicine or health-care product. The medicine is tablet, capsule, powder, granule, oral liquid, pill, powder, sustained release preparation, solution, suspension, injection, microneedle, ointment, cream or suppository.
A schematic synthesis of β sitosterol derivatives having the structure shown in formula (I) is shown in FIG. 1.
As used herein, "pharmaceutically acceptable salts" refers to salts that retain the biological potency of the free acid and free base of the specified compound, and that are biologically or otherwise not adversely affected. Salts in this application refer to acid salts formed with organic/inorganic acids, as well as basic salts formed with organic/inorganic bases.
The principle of the invention is as follows:
the invention is based on the principle of biotin and nano-drug assembly of biotin and avidin and disease cell enrichment mediated, the solubility problem of the finished drug is solved by modifying β sitosterol with biotin, and the biotin has the advantages of good water solubility, biocompatibility, no immunogenicity and the like, can improve the drug effect, increase the selectivity, reduce the toxic and side effects, and is more suitable for clinical use.
The inventor finds that β sitosterol compound chemically modified by biotin obviously improves the solubility, and particularly can completely meet the clinical administration requirement after being assembled with avidin nanometer in the design and experimental invention.
A2% rabbit erythrocyte hemolysis test shows that the biotin-modified β sitosterol derivative or the nano preparation thereof of the invention has no erythrocyte hemolysis aggregation within 4h, and the equivalent β sitosterol dosage is more than 60 mg.
Solubility experiments show that compared with a prototype (β sitosterol is insoluble in water), the β sitosterol derivative modified by biotin has obviously improved water solubility (about 5mg/ml), and particularly has good water solubility (greater than 20mg/ml) after being assembled into a nano preparation with avidin, so that the preparation of a medicament form, vascular administration and the like are more convenient.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the β sitosterol derivative modified by biotin and the β sitosterol derivative modified by biotin are specifically combined with avidin to construct a nano-drug system, so that the technical problems of poor solubility, poor curative effect, poor targeting property and the like of β sitosterol are greatly solved, and the invention has good clinical application prospect.
Drawings
FIG. 1 is a schematic diagram of B-BS synthesis.
FIG. 2 is a schematic diagram of the assembly of B-BS series and avidin into nano-drugs.
FIG. 3 is a schematic diagram of β assembly of sitosterol derivatives and avidin into nanoparticles.
Detailed description of the invention
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.
EXAMPLE 1 preparation of β sitosterol derivative (B-BS) according to the invention
β sitosterol (0.15mmol) was dissolved in 60ml DMSO in dehydrating agent DCC (3.0mmol) and catalysisStirring at 0 deg.C for 6 hr under the action of DMAP (3.0mmol), adding biotin (B) at a molar ratio of β sitosterol of 1: 3, heating to room temperature 25 deg.C in ice bath, stirring overnight in dark place, concentrating filtrate, recrystallizing with glacial ethyl ether or isopropanol, purifying by chromatography or preparative liquid phase, and lyophilizing to obtain β sitosterol derivative B-BS (yield about 48%), identifying ion peak by mass spectrum [ M + H ])]+Comprises the following steps: 642.1.
1HNMR (400MHz, DMSO): characteristic peaks of biotin: 10.76-10.82(NH, 2H, broad peak), 4.47-4.65(2C-H,2H), 2.80-3.10 (CH)2，2H)，3.24(CH，1H)，2.32(CH2，2H)，1.23-1.68(3CH26H), β characteristic peak of sitosterol 0.85-0.88(4 CH)3，12H)，0.99-1.03(CH3，3H)，1.08-1.38(7CH2，8CH,22H)，2.09-2.31(2CH2，4H)。
IR: the infrared spectrum is obviously increased by 1725cm-1The ester carbonyl peak of (a), indicating that β sitosterol and biotin are attached via an ester linkage.
The mass spectrometry data above demonstrate that the β sitosterol derivative obtained in this example has the structure shown below:
Figure BDA0002397348330000061
example 2 β preparation of sitosterol derivative avidin nanoparticles
The β sitosterol derivative (B-BS) which is equal to β sitosterol 5g and prepared in example 1 is taken, firstly dissolved in a small amount of DMSO or ethanol, then diluted by water for injection, added with sodium chloride 1.0g, added with equimolar avidin (the B-BS and avidin can be assembled according to various molar ratios of 1-4: 1 as shown in figure 2), and stirred uniformly at room temperature to obtain composite β sitosterol derivative avidin nanoparticles, and the nanoparticles are characterized by a transmission electron microscope, and have the nanometer size of about 25nm (as shown in figure 3).
Example 3 β preparation of sitosterol derivative injection
β sitosterol derivative (B-BS) which is equal to β sitosterol and is prepared in example 1 is dissolved in water for injection, 5.0g of sodium chloride is added and stirred uniformly, the pH value is adjusted to 5.0 by dilute hydrochloric acid, then 0.5% of activated carbon for injection is added, the temperature is kept constant at 60 ℃ for 30min, after the carbon is removed, water for injection is added into the filtrate to 1000ml, a sterile filter membrane with the diameter of 0.22 mu m is used for filtration, 2 ml/branch is packaged in a glass curved neck ampoule, the glass curved neck ampoule is sealed in a melting mode, steam is circulated at 100 ℃ for wet heat sterilization for 40min, and then the glass curved neck ampoule is labeled for storage.
EXAMPLE 4 β preparation of sitosterol derivative avidin Nano injection
Equal to β g of sitosterol 30g of the β sitosterol derivative (B-BS) prepared in example 1 is dissolved in water for injection, 5.0g of sodium chloride and 300.0g of avidin are added and stirred uniformly, the pH value is adjusted to 5.0 by dilute hydrochloric acid, then 0.5% of activated carbon for injection is added, the temperature is kept constant at 60 ℃ for 30min, after the carbon is removed, water for injection is added into the filtrate to 1000ml, sterile filter membrane filtration with the diameter of 0.22 mu m is carried out, 2 ml/branch is packed in a glass curved neck ampoule, and the ampoule is sealed by melting and then is labeled for storage.
EXAMPLE 5 β preparation of sitosterol derivative avidin Nano injection
Equal to β g of sitosterol 30g of the β sitosterol derivative (B-BS) prepared in example 1 is dissolved in water for injection, 5.0g of sodium chloride and 150.0g of avidin are added and stirred uniformly, the pH value is adjusted to 5.0 by dilute hydrochloric acid, then 0.5% of activated carbon for injection is added, the temperature is kept constant at 60 ℃ for 30min, after the carbon is removed, water for injection is added into the filtrate to 1000ml, sterile filter membrane filtration with the diameter of 0.22 mu m is carried out, 2 ml/branch is packed in a glass curved neck ampoule, and the ampoule is sealed by melting and then is labeled for storage.
Example 6 β preparation of Freeze-dried powder of sitosterol derivatives
β sitosterol derivative (B-BS) powder prepared in example 1, which is equal to β sitosterol 20g, is added with glucose powder for injection 25g and stirred uniformly, and the mixture is aseptically subpackaged in glass curved-neck ampoules according to 40 mg/bottle, freeze-dried, sterilized by cobalt 60 radiation, sealed and then labeled for storage.
Example 7 β vascular irritation test of sitosterol derivative nanoparticles
β sitosterol derivative avidin nano injection prepared in example 4 and dissolved by trace DMSO, 40mg of water for injection, 6 experimental rabbits, 3 random groups (β sitosterol derivative avidin nano injection drug group, β sitosterol group and normal saline group), wherein normal saline is used as a control, biotin- β sitosterol injection is instilled into the left ear edge vein of the rabbits, normal saline with the same volume is instilled into the right ear edge vein, instillation is completed within 3h, blood vessels, heart, liver, spleen, lung, pancreas, kidney and brain tissues at the position of 1cm below the injection point are taken after instillation is completed, formalin solution is fixed, paraffin is embedded into slices, HE staining is performed, and digital pathology analysis and evaluation are performed.
After the administration, the rabbit is observed that the rabbit is normal in diet, hair, anus, respiration, central nervous system, four limbs activity state every day, and has no toxic manifestation, when the rabbit is about 48 hours, the killed animal is observed that the rectal mucosa is smooth and flat, and has no abnormal appearance, the rest rabbits are kept to monitor day by day, and no abnormal appearance appears, when the animal is killed in the seventh day, the animal is observed for weight and the vascular stimulation is graded according to new drug research guidelines, the pathological histological examination result of the rabbit vascular irritation test is that auricle and epidermis have no abnormality, dermal vascular endothelial cells have no swelling, capillary walls have no bleeding, necrosis or inflammatory cell infiltration, cartilage layers and cartilage cells have no hyperplasia or necrosis, cartilage cells are arranged orderly, liver, myocardial tissue, brain tissue, lung, kidney and pancreas tissue have no abnormality, physiological saline has no abnormality compared with auricle epidermis, dermal vascular endothelial cells have no swelling, vascular capillary walls have no bleeding, necrosis or inflammatory cell infiltration, the cartilage cells are arranged orderly, the cartilage layers and cartilage cells have no hyperplasia or necrosis, β, and the physiological saline solution injection shows obvious difference with β nano saline solution.
Example 8 experimental study in mice of β sitosterol derivatives prepared in example 1 to improve sleep
Experimental animals: 180 healthy male Kunming mice are purchased by SPF experimental animal center, the weight of the mice is 18-22 g, and the mice and feed are purchased in SPF experimental animal center, 4-5 weeks; placing the mixture in a feeding room with the temperature of 22-25 ℃ and the relative humidity of 50-70 percent.
The test method comprises the following steps:
1) grouping and administration, wherein animals are randomly divided into 3 experimental groups after being pre-fed for 1 week, the first group is subjected to a direct sleep experiment and an experiment for prolonging the sleep time of the pentobarbital sodium, the second group is subjected to a subliminal dose hypnosis experiment of the pentobarbital sodium, and the third group is subjected to a sleep latency experiment of the pentobarbital sodium, 50 mice are randomly divided into 5 groups according to the body weight in each experimental group, 10 animals in each group are set as a control group, β low, medium and high dose groups of sitosterol derivatives (dose groups are set according to body weights of 50, 100 and 200 mg/kg), β sitosterol (200mg/kg body weight dose group), ursolic acid group (50mg/kg body weight dose group), and the animals are subjected to gastric lavage administration, and the control group is subjected to the same solvent drenching for 1 time/d for 30.
2) Direct sleep experiments: after the stomach is not irrigated again, whether the sleep phenomenon appears is observed, and the sleep takes the disappearance of the righting reflex as an index. When the mouse is placed in the back lying position, the mouse can turn over to the right position immediately, if the mouse cannot turn over for more than 60 seconds, the turning-over reflection is considered to disappear, and the mouse enters sleep. The recovery of the righting reflex is the awakening of the animal, the period from disappearance of the righting reflex to recovery is the sleeping time of the animal, and the number of sleeping animals and the sleeping time of the control group and each dosage group are recorded.
3) Experiment for prolonging the sleep time of the sodium pentobarbital comprises the steps of injecting 50mg/kg of sodium pentobarbital into the abdominal cavity of each group of animals 15 minutes after last intragastric administration, wherein the injection amount is 0.2m L/20 g of the animals, and observing whether the test sample prolongs the sleep time of the sodium pentobarbital by taking disappearance of positive turning reflex as a sleep-entering judgment standard.
4) Pentobarbital sodium subthreshold dose hypnosis experiment includes injecting pentobarbital sodium of 25mg/kg body weight into abdominal cavity of animals of each group 15 minutes after last gastric lavage, wherein the injection amount is 0.2m L/20 g body weight, and the number of the animals falling asleep within 30 minutes is recorded by taking the disappearance of righting reflex for more than 1 minute as a sleep judgment standard.
5) Pentobarbital sodium sleep latency period experiment, after 15 minutes of last gastric lavage, 280mg/kg of pentobarbital sodium is injected into the abdominal cavity of each group of animals, the injection amount is 0.2m L/20 g of body weight, the disappearance of righting reflex is taken as an index, and the influence of the tested sample on the sleep latency period of the pentobarbital sodium is observed.
The experimental results are as follows:
1) the influence on the development and growth weight of mice is shown in the following table 1, compared with a control group, β sitosterol has almost no effect, the ursolic acid group and the β sitosterol derivative in and at high dose have obvious growth promotion effect, the β sitosterol derivative high dose group has the best effect, the weight of the mice is increased by more than 25%, and the difference is very obvious and statistically different.
Table 1: effect on mouse body weight
Figure BDA0002397348330000091
Remarking: statistical differences were significant compared to control group (p < 0.05); statistical differences were very significant (p < 0.01); AV + -SD is the mean + -error value.
2) The observation of the direct sleep effect of the mice shows that in the direct sleep test, the number of sleeping animals and the sleep time of each dose group of β sitosterol group, ursolic acid group and β sitosterol derivative group are 0, which indicates that the two substances have no direct hypnotic effect on the mice and can be used for detecting the following three indexes.
Table 2: observation of the Effect of direct sleep in mice
Figure BDA0002397348330000101
3) As shown in Table 3, in the experiment for prolonging the sleep time of sodium pentobarbital, β sitosterol derivatives in each dose group and ursolic acid group can prolong the sleep time of sodium pentobarbital compared with the control group, so that β sitosterol derivatives in middle and high dose groups have the best effect and have statistically significant difference.
Table 3: effect on prolonging sleep time of pentobarbital sodium in mice
Figure BDA0002397348330000102
Remarking: statistical differences were significant compared to control group (p < 0.05); statistical differences were very significant (p < 0.01);
4) the influence on the subliminal dose hypnosis effect of the sodium pentobarbital in the mice is shown in the table 4, β sitosterol derivatives and ursolic acid can obviously improve the sleep rate of the mice, compared with a control group, the sleep rate of the mice is respectively improved by 20 percent, 50 percent, 60 percent and 20 percent, and the effect is best in a high-dose group.
Table 4: effect on subliminal dose hypnosis of sodium pentobarbital in mice
Figure BDA0002397348330000111
Remarking: statistical differences were significant compared to control group (p < 0.05); statistical differences were very significant (p < 0.01);
5) the influence on the sleep latency of mice induced by sodium pentobarbital in the mice is shown in Table 5, the sleep latency of the mice in a control group is 26.99min, β sitosterol derivatives in each dose group and ursolic acid in the mice are shortened, wherein the latency of the high dose group is shortest and has a statistical difference with the control group.
Table 5: influence on sleep latency of mice induced by sodium pentobarbital
Figure BDA0002397348330000112
Remarking: statistical differences were significant compared to control group (p < 0.05); statistical differences were very significant (p < 0.01);
combining the results, β sitosterol derivative has obvious efficacy of improving the sleep of mice, and has better effect in dose dependence.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (9)

1. An β sitosterol derivative, characterized in that the derivative has the following structure:
Figure FDA0002397348320000011
2. the process for the preparation of β sitosterol derivatives according to claim 1, characterized by comprising the following operating steps:
β sitosterol is dissolved in a solvent, the mixture is stirred and reacted for 1-6 h at 0 ℃ under the action of a dehydrating agent N, N' -dicyclohexylcarbodiimide or 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and a catalyst on dimethylaminopyridine, then biotin is added according to the molar ratio of β sitosterol being 1:1-3 times of the mixture, the temperature is raised to room temperature from an ice bath, the mixture is stirred overnight in a dark place, filtrate is concentrated, and the mixture is recrystallized by glacial ethyl ether or isopropanol, is chromatographed or is prepared into a liquid phase for purification, and is freeze-dried to obtain the β sitosterol derivative with the structure shown in the formula (I).
3. The method of claim 2, wherein: the solvent is CH2Cl2DMSO or DMF; the stirring reaction time is 5 h; the room temperature was 25 ℃.
4. The method according to claim 2, wherein the molar ratio of the β sitosterol to the dehydrating agent to the catalyst is 1:1:1 to 1:25: 25.
5. The use of β sitosterol derivatives and their pharmaceutically acceptable salts according to claim 1 for the preparation of a medicament or health product for improving sleep.
6. Use according to claim 5, characterized in that: the medicine is tablet, capsule, powder, granule, oral liquid, pill, powder, sustained release preparation, solution, suspension, injection, microneedle, ointment, cream or suppository.
7. A nanoparticle assembled from the β sitosterol derivative of claim 1 and avidin.
8. Use of the nanoparticle according to claim 7 for the preparation of a medicament or health product for improving sleep.
9. Use according to claim 8, characterized in that: the medicine is tablet, capsule, powder, granule, oral liquid, pill, powder, sustained release preparation, solution, suspension, injection, microneedle, ointment, cream or suppository.
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