CN111423484B - Beta sitosterol derivative and preparation method and application thereof - Google Patents
Beta sitosterol derivative and preparation method and application thereof Download PDFInfo
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- CN111423484B CN111423484B CN202010136016.0A CN202010136016A CN111423484B CN 111423484 B CN111423484 B CN 111423484B CN 202010136016 A CN202010136016 A CN 202010136016A CN 111423484 B CN111423484 B CN 111423484B
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- sitosterol
- derivative
- sleep
- sitosterol derivative
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Abstract
The invention belongs to the technical field of biological medicines, and discloses a beta sitosterol derivative and a preparation method and application thereof. The beta sitosterol derivative can be assembled into nanoparticles with avidin. Animal experiments prove that the beta-sitosterol derivative and the nano-drug thereof can obviously reduce the sleep latency of mice, obviously improve the sleep time and the subliminal hypnosis and sleep rate of the mice, effectively improve the sleep quality of the mice, and can be used for further preparing the drugs or health care products for improving the sleep quality.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a beta sitosterol derivative and 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.
Beta-sitosterol (BS) is one of the phytosterol components, widely existing in plant seeds such as various vegetable oils and nuts in nature, and also existing in some plant medicines. Its structure is very similar to that of cholesterol, and it has estrogenic activity. It can inhibit proliferation of human leukemia cells (G2/M block), endoreduplication, and polymerization of alpha-tubulin and microtubules. Beta-sitosterol is widely used in the pharmaceutical industry due to its characteristic biological and physicochemical properties. The beta-sitosterol is white scale-shaped, needle-shaped crystal or crystalline powder, and is odorless and tasteless. It is very soluble in chloroform and carbon disulfide, slightly soluble in ethanol or acetone, and insoluble in water. The beta-sitosterol has the functions of reducing cholesterol, relieving cough, eliminating phlegm, inhibiting tumor and repairing tissues. Can be used for treating type II hyperlipidemia, atherosclerosis and chronic tracheitis, and early stage cervical cancer and skin ulcer. Due to the extremely low bioavailability, strong hydrophobicity (insoluble in water, slightly soluble in acetone and ethanol at normal temperature, soluble in benzene, chloroform, ethyl acetate, carbon disulfide, petroleum ether, acetic acid and the like), insufficient intracellular accumulation amount and poor solubility in water, the compound has the defects of low bioavailability, in-vivo transmission, metabolism and the like, and is limited to become a potential therapeutic drug. Therefore, there is a need to improve the pharmacokinetic properties of such compounds. Therefore, the natural product analogue is synthesized by an organic synthesis method, the structure is optimized, the novel beta-sitosterol derivative with higher yield and better biological activity is constructed, and the method has important significance for the development and research of medicine and health care.
Biotin (Biotin, B) is widely applied to targeted delivery and transportation of various anti-cancer drugs and the like as a targeting ligand due to simple structure, low molecular weight, high tumor specificity and wide and high expression of a Biotin receptor on the surface of a tumor tissue membrane. Biotin, also known as vitamin H, coenzyme R, is a water-soluble beneficial vitamin, also belonging to the vitamin B group, has a molecular weight of 244.31, and has a basic structure of a bicyclic structure: the I ring is an imidazolone ring and is a binding site with avidin; II is a thiophene ring, contains a valeric acid side chain, and the terminal carboxyl group of the thiophene ring can be connected with biological macromolecules to form biotin labeled antigens, antibodies, enzymes and the like. The chemical structure of the compound has an imidazolone ring, and the imidazolone ring can be specifically combined with Avidin (AV) and streptavidin (streptavidin, SA). The binding affinity between biotin and avidin is high (binding constant about 1X 10)-15mol/L, which can be regarded as irreversible combination), strong specificity, and can be respectively combined with various types of molecules, one molecular chain mildew affinity protein can be combined with four molecular biotin, and the two streptavidin-biotin combination reactions have the superiority of multi-stage amplification and the like.
Most importantly, no method for improving the solubility of the beta-sitosterol and reporting that the beta-sitosterol has the effect of improving sleep through biotin modification is found so far, and no method for constructing nanoparticles and biomedical applications by specifically combining biotin-modified beta-sitosterol derivatives and avidin is found.
Disclosure of Invention
In order to overcome the disadvantages and shortcomings of the prior art, the present invention is primarily directed to a beta sitosterol derivative.
Still another object of the present invention is to provide a method for preparing the above beta-sitosterol derivative.
Still another object of the present invention is to provide use of the above β -sitosterol derivative.
It is still another object of the present invention to provide nanoparticles assembled from the above β -sitosterol derivative 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:
a β -sitosterol derivative having a structure represented by the following formula (I):
the preparation method of the beta sitosterol derivative comprises the following operation steps:
dissolving beta sitosterol in a solvent, and stirring and reacting 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 adding biotin according to the molar ratio of the beta-sitosterol of 1:1-3 times, raising the temperature to room temperature from an ice bath, and stirring overnight in a dark place; concentrating the filtrate, recrystallizing with glacial ethyl ether or isopropanol, purifying by chromatography or preparative liquid phase, and lyophilizing to obtain the beta-sitosterol derivative with structure shown in formula (I).
The solvent is CH2Cl2DMSO or DMF; the stirring reaction time is 5 h; the room temperature was 25 ℃.
The mol ratio of the beta sitosterol to the dehydrating agent to the catalyst is 1:1: 1-1: 25: 25.
The beta sitosterol derivative and the pharmaceutically acceptable salt thereof are applied to the preparation of the sleep improving medicine or the 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.
Nanoparticles composed of the beta 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.
The synthesis scheme of the beta-sitosterol derivative with the structure shown in the formula (I) is shown in a figure 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 the assembly of nano-drugs with the biotin and the mediation of disease cells, solves the problem of drug solubility by modifying beta-sitosterol with biotin, has the advantages of good water solubility, biocompatibility, no immunogenicity and the like, can improve drug effect, increase selectivity, reduce toxic and side effects, and is more suitable for clinical use.
The inventor finds that the beta 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 beta-sitosterol derivative or the nano preparation thereof of the invention has no erythrocyte hemolysis aggregation within 4h, and the equivalent beta-sitosterol dose is more than 60 mg. The experiment of rabbit ear vein irritation shows that the biotin-modified beta-sitosterol derivative is dissolved in normal saline and has no vascular irritation after intravenous drip.
Solubility experiments show that compared with a prototype (beta-sitosterol is insoluble in water), the water solubility of the beta-sitosterol derivative modified by biotin is obviously improved (about 5mg/ml), and particularly after the beta-sitosterol derivative is assembled into a nano preparation with avidin, the beta-sitosterol derivative has good water solubility (>20mg/ml), and is more convenient for preparation of medicament formulations, vascular administration and the like.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the biotin-modified beta-sitosterol derivative and the avidin are specifically combined to construct a nano-drug system, so that the technical problems of poor solubility, poor curative effect, lack of targeting property and the like of the beta-sitosterol are greatly solved, and the biotin-modified beta-sitosterol derivative has a 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 assembly of a β -sitosterol derivative 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 beta-sitosterol derivative (B-BS) of the invention
Dissolving beta-sitosterol (0.15mmol) in 60ml of DMSO, and stirring at 0 ℃ for 6h under the action of dehydrating agent DCC (3.0mmol) and catalyst DMAP (3.0 mmol); then, mixing the raw materials in a beta sitosterol molar ratio of 1: biotin (B) was added 3 times, the temperature was raised from the ice bath to 25 ℃ and stirred overnight in the dark. Concentrating the filtrate, recrystallizing with glacial ethyl ether or isopropanol, purifying by chromatography or preparative liquid phase, and lyophilizing to obtain beta-sitosterol derivative B-BS (yield about 48%), and identifying ion peak by mass spectrometry [ 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); characteristic peaks of β 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-1Ester carbonyl of (A)Peak, indicating that β -sitosterol and biotin are linked via an ester bond.
The mass spectrum data prove that the beta sitosterol derivative obtained in the embodiment has the following structure:
example 2 preparation of nanoparticles of beta sitosterol derivative avidin
An amount of beta-sitosterol derivative (B-BS) which is equal to 5g of beta-sitosterol and prepared in example 1 is taken, dissolved in a small amount of DMSO or ethanol, diluted with water for injection, added with 1.0g of sodium chloride, 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 beta-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 preparation of beta sitosterol derivative injection
An equivalent amount of 30g of β -sitosterol derivative (B-BS) prepared in example 1 was dissolved in water for injection, 5.0g of sodium chloride was added thereto and stirred uniformly, pH was adjusted to 5.0 with dilute hydrochloric acid, then 0.5% of activated carbon for injection was added thereto, the temperature was maintained at 60 ℃ for 30min, after decarburization, water for injection was added to the filtrate to 1000ml, the filtrate was filtered through a sterile filter of 0.22 μm, 2 ml/aliquot was packed in glass curved-neck ampoules, melt-sealed, subjected to moist heat sterilization with flowing steam at 100 ℃ for 40min, and then stored by labeling.
Example 4 preparation of beta sitosterol derivative avidin Nano injection
An amount of 30g of β sitosterol equivalent to 30g of the β sitosterol derivative (B-BS) prepared in example 1 was dissolved in water for injection, 5.0g of sodium chloride and 300.0g of avidin were added and stirred uniformly, the pH was adjusted to 5.0 with dilute hydrochloric acid, then 0.5% of activated carbon for injection was added, the temperature was maintained at 60 ℃ for 30min, after decarburization, water for injection was added to the filtrate to 1000ml, and the filtrate was filtered with a 0.22 μm sterile filter membrane, and 2 ml/branch was packed in glass curved-neck ampoules, sealed by melting, and then stored by labeling.
EXAMPLE 5 preparation of beta sitosterol derivative avidin Nano injection
An amount of 30g of β -sitosterol derivative (B-BS) prepared in example 1 equivalent to β -sitosterol was dissolved in water for injection, 5.0g of sodium chloride and 150.0g of avidin were added and stirred uniformly, pH was adjusted to 5.0 with dilute hydrochloric acid, then 0.5% of activated carbon for injection was added, the temperature was maintained at 60 ℃ for 30min, after decarburization, water for injection was added to 1000ml of the filtrate, and the filtrate was filtered with a 0.22 μm sterile filter membrane, and 2 ml/branch was packed in glass curved-neck ampoules, sealed by melting, and then labeled and stored.
EXAMPLE 6 preparation of beta sitosterol derivative lyophilized powder
An amount of 20g of β -sitosterol equivalent to 20g of β -sitosterol derivative (B-BS) powder prepared in example 1 was added to 25g of glucose powder for injection, stirred uniformly, aseptically dispensed into glass curved ampoule at 40 mg/bottle, freeze-dried, sterilized by cobalt 60 radiation, sealed and then labeled for storage.
Example 7 vascular irritation assay of nanoparticles of beta sitosterol derivatives
The beta-sitosterol derivative avidin nano injection prepared in the embodiment 4 and dissolved by trace DMSO, 40mg, of which the injection water is 50ml, is used for diluting, and 6 experimental rabbits are randomly treated in 3 groups (a beta-sitosterol derivative avidin nano injection drug group, beta-sitosterol group and normal saline group), wherein normal saline is used as a control, and biotin-beta-sitosterol injection is dripped into the left ear edge vein of the rabbits, the same volume of normal saline is dripped into the right ear edge vein of the rabbits, and the dripping is completed within 3 hours. After instillation, blood vessels and heart, liver, spleen, lung, pancreas, kidney and brain tissues 1cm below the injection point are taken for formalin fixation, paraffin embedded section, HE staining and digital pathology analysis and evaluation.
After the administration, the rabbit has normal diet, hair, anus, respiration, central nervous system, four limbs activity state, etc. and no toxic manifestation. Until about 48h, the sacrificed animals were observed to have smooth and flat rectal mucosa with no abnormalities; the rest rabbits are kept for daily monitoring without abnormal conditions. By day seven animals were sacrificed and body weight was observed and vascular stimulation was graded according to the New drug research guidelines. The pathological histological examination result of the rabbit vascular irritation test shows that auricle and epidermis are not abnormal, endothelial cells of dermal blood vessels are not swollen, the walls of capillary vessels are not subjected to hemorrhage, necrosis or inflammatory cell infiltration, cartilage layers and cartilage cells are not proliferated or necrotic, and the cartilage cells are arranged in order; liver, myocardial tissue, brain tissue, lung, kidney and pancreatic tissue are all free of abnormalities. The normal saline control group has no abnormal auricle epidermis, no swelling of endothelial cells of dermal blood vessels, no bleeding, necrosis or inflammatory cell infiltration of capillary walls, regular arrangement of cartilage cells, no hyperplasia or necrosis, and no hyperplasia or necrosis of cartilage layers and cartilage cells. The beta sitosterol derivative avidin nanometer injection drug group has no obvious difference with the beta sitosterol and normal saline group on pathological tissues, but the solubility is obviously improved.
Example 8 experimental study of beta-sitosterol derivatives prepared in example 1 in mice with improved 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: after the animals are pre-fed for 1 week, the animals are randomly divided into 3 experimental groups, the first group is subjected to a direct sleep experiment and an experiment for prolonging the sleep time of the sodium pentobarbital, the second group is subjected to a subthreshold dose hypnosis experiment of the sodium pentobarbital, and the third group is subjected to a sleep latency experiment of the sodium pentobarbital. For each experimental group, 50 mice were randomly divided by body weight into 5 groups of 10 animals each. And (3) setting a control group, a low-dosage group, a medium-dosage group and a high-dosage group of beta-sitosterol derivatives (dosage groups are set according to the weight of 50, 100 and 200 mg/kg), a beta-sitosterol group (200 mg/kg), an ursolic acid group (50 mg/kg), and the control group is administered by intragastric administration, and the control group is administered by the same solvent for 1 time/d for 30 days continuously.
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 sleep time of sodium pentobarbital: after 15 minutes of last gastric lavage, 50mg/kg of sodium pentobarbital is injected into the abdominal cavity of each group of animals, the injection amount is 0.2mL/20g of the animals, the disappearance of the righting reflex is taken as a sleep-falling judgment standard, and whether the test sample prolongs the sleep time of the sodium pentobarbital or not is observed.
4) Pentobarbital sodium subthreshold dose hypnosis test: after 15 minutes of the last gastric lavage, 25mg/kg of sodium pentobarbital is injected into the abdominal cavity of each group of animals, the injection amount is 0.2mL/20g of the animals, 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 experiments: after 15 minutes of the last gastric lavage, 280mg/kg of sodium pentobarbital is injected into the abdominal cavity of each group of animals, the injection amount is 0.2mL/20g of the animals, the influence of the tested sample on the sleep latency of the sodium pentobarbital is observed by taking the disappearance of the righting reflex as an index.
The experimental results are as follows:
1) effect on mouse development growth body weight: as shown in table 1 below, beta-sitosterol had little effect compared to the control group; the ursolic acid group and the beta sitosterol derivative medium and high dose groups have obvious growth promotion effects, the beta sitosterol derivative high dose group has the best effect, the weight of the mouse is increased by more than 25%, and the difference is very obvious and statistically different.
Table 1: effect on mouse body weight
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) Observation of the effects of direct sleep in mice: as shown in table 2, in the direct sleep test, the number of sleeping animals and the sleep time of each dose group of the β -sitosterol group, the ursolic acid group and the β -sitosterol derivative group are all 0, which indicates that the two substances have no direct hypnotic effect on mice and can be used for detecting the following three indexes.
Table 2: observation of the Effect of direct sleep in mice
3) Effect on prolonging sleep time of sodium pentobarbital: as shown in table 3, in the experiment for prolonging the sleep time of pentobarbital sodium, compared with the control group, the beta-sitosterol derivative dose groups and the ursolic acid group can prolong the sleep time of pentobarbital sodium, so that the beta-sitosterol derivative 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
Remarking: statistical differences were significant compared to control group (p < 0.05); statistical differences were very significant (p < 0.01);
4) effect on subliminal dose hypnotic effect of sodium pentobarbital in mice: as shown in table 4, the beta-sitosterol derivative and ursolic acid in each dose group significantly improved the sleep rate of mice, which was improved by 20%, 50%, 60% and 20% respectively, compared with the control group, and the effect was the best in the high dose group.
Table 4: effect on subliminal dose hypnosis of sodium pentobarbital in mice
Remarking: statistical differences were significant compared to control group (p < 0.05); statistical differences were very significant (p < 0.01);
5) effect on sleep latency in mice induced by sodium pentobarbital: as shown in table 5, the sleep latency of the control group mice is 26.99min, and the sleep latency of the mice is shortened by each dosage group of the beta-sitosterol derivative and the ursolic acid group, wherein the latency of the high dosage group is the shortest and has a significant statistical difference with the control group.
Table 5: influence on sleep latency of mice induced by sodium pentobarbital
Remarking: statistical differences were significant compared to control group (p < 0.05); statistical differences were very significant (p < 0.01);
combining the results, the beta-sitosterol derivative has obvious efficacy of improving the sleep of mice, and has better dose-dependent presentation effect.
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 (5)
1. An application of beta sitosterol derivative or nanoparticles assembled by the beta sitosterol derivative and avidin in preparing a medicine or health care product for improving sleep is characterized in that: the beta sitosterol derivative has a structure shown as the following formula (I):
2. use according to claim 1, characterized in that it comprises the following operative steps:
dissolving beta sitosterol in a solvent, and stirring and reacting 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 adding biotin according to the molar ratio of the beta-sitosterol of 1:1-3 times, raising the temperature to room temperature from an ice bath, and stirring overnight in a dark place; concentrating the filtrate, recrystallizing with glacial ethyl ether or isopropanol, purifying by chromatography or preparative liquid phase, and lyophilizing to obtain the beta-sitosterol derivative with structure shown in formula (I).
3. Use according to claim 2, characterized in that: the solvent is CH2Cl2DMSO or DMF; the stirring reaction time is 5 h; the room temperature was 25 ℃.
4. Use according to claim 2, characterized in that: the mol ratio of the beta sitosterol to the dehydrating agent to the catalyst is 1:1: 1-1: 25: 25.
5. Use according to claim 1, 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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| JPH0296581A (en) * | 1988-09-29 | 1990-04-09 | Shiseido Co Ltd | Biotin ester and skin ointment and hair tonic using the ester |
| CN104098594A (en) * | 2014-07-16 | 2014-10-15 | 中国科学院昆明植物研究所 | Biotin-podophyllotoxin esterified derivative and pharmaceutical composition thereof, as well as preparation methods and applications of derivative and pharmaceutical composition |
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|---|---|---|---|---|
| JPH0296581A (en) * | 1988-09-29 | 1990-04-09 | Shiseido Co Ltd | Biotin ester and skin ointment and hair tonic using the ester |
| CN104098594A (en) * | 2014-07-16 | 2014-10-15 | 中国科学院昆明植物研究所 | Biotin-podophyllotoxin esterified derivative and pharmaceutical composition thereof, as well as preparation methods and applications of derivative and pharmaceutical composition |
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| DCC法合成胆甾醇酯;陈经佳等;《浙江化工》;20051231;第36卷(第2期);17-19 * |
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