阅读说明：本技术 萝卜硫素或其纳米粒在制备改善孕妇杂环胺摄入导致的胚胎神经系统发育不良药物中的应用 (Application of sulforaphane or nanoparticles thereof in preparation of drugs for improving embryonic nervous system dysplasia caused by intake of heterocyclic amine in pregnant women ) 是由 王广 杨雪松 张萍 穆斯塔法·辛迪 刘畅 程欣 于 2020-07-09 设计创作，主要内容包括：本发明公开了萝卜硫素或其纳米粒在制备改善孕妇杂环胺摄入导致的胚胎神经系统发育不良药物中的应用。本发明提供了一种萝卜硫素的新用途-胚胎发育保护用途,不仅扩大了萝卜硫素的应用范围,提高了其应用价值,还有助于进一步开发新的改善孕妇杂环胺摄入诱导的胚胎神经系统发育不良的纳米药物,具有良好的应用前景。(The invention discloses application of sulforaphane or nanoparticles thereof in preparing a medicine for improving embryonic nervous system dysplasia caused by intake of heterocyclic amine in pregnant women. The invention provides a new application of sulforaphane, namely an embryonic development protection application, which not only expands the application range of the sulforaphane and improves the application value of the sulforaphane, but also is beneficial to further developing a new nano-medicament for improving embryonic nervous system dysplasia induced by heterocyclic amine intake of pregnant women, and has good application prospect.)

1. Application of sulforaphane or nanoparticles thereof in preparing a medicine for improving embryonic nervous system dysplasia caused by intake of heterocyclic amine in pregnant women.

2. The use of sulforaphane or nanoparticles thereof according to claim 1 for the preparation of a medicament for improving embryonic nervous system dysplasia caused by the ingestion of heterocyclic amines by pregnant women, characterized in that:

the effective concentration of the sulforaphane or the nanoparticles thereof is 0-10 mu mol/L.

3. The use of sulforaphane or nanoparticles thereof according to claim 1 for the preparation of a medicament for improving embryonic nervous system dysplasia caused by the ingestion of heterocyclic amines by pregnant women, characterized in that:

the medicine comprises at least one of sulforaphane and a pharmaceutically acceptable modifier synthesized by the sulforaphane as a lead compound;

the modification comprises at least one of salification modification, esterification modification, amidation modification, cyclization modification and ring-opening modification.

4. The use of sulforaphane or nanoparticles thereof according to claim 1 for the preparation of a medicament for improving embryonic nervous system dysplasia caused by the ingestion of heterocyclic amines by pregnant women, characterized in that:

the sulforaphane nanoparticles comprise a nano-carrier and sulforaphane encapsulated in the nano-carrier.

5. The use of sulforaphane or nanoparticles thereof according to claim 4 for the preparation of a medicament for ameliorating embryonic nervous system dysplasia caused by the ingestion of heterocyclic amines by pregnant women, wherein:

the nano-carrier is liposome or polymer capable of forming micelle; further methoxy polyethylene glycol-polyglutamic acid; wherein the molecular weight of the methoxy polyethylene glycol segment is 3000-7000, and the molecular weight of the polyglutamic acid segment is 8000-12000.

6. The use of sulforaphane or nanoparticles thereof according to claim 4 for the preparation of a medicament for ameliorating embryonic nervous system dysplasia caused by the ingestion of heterocyclic amines by pregnant women, wherein:

the sulforaphane nanoparticles are prepared by the following method:

1) dissolving sulforaphane in dimethyl sulfoxide to obtain a sulforaphane solution;

2) dissolving methoxypolyethylene glycol-polyglutamic acid in sodium lactate ringer's solution to obtain methoxypolyethylene glycol-polyglutamic acid solution;

3) mixing the methoxypolyethylene glycol-polyglutamic acid solution obtained in the step 2) with the sulforaphane solution obtained in the step 1), performing ultrasonic treatment, and standing; then centrifuging, washing the precipitate, and obtaining the precipitate which is the sulforaphane nano-particle encapsulated by the methoxypolyethylene glycol-polyglutamic acid.

7. The use of sulforaphane or nanoparticles thereof according to claim 6 for the preparation of a medicament for ameliorating embryonic nervous system dysplasia caused by the ingestion of heterocyclic amines by pregnant women, wherein:

the concentration of the sulforaphane solution in the step 1) is 10-100 mmol/L;

the concentration of the methoxypolyethylene glycol-polyglutamic acid solution in the step 2) is 0.01-0.1 mug/muL;

the preferred ratio of the methoxypolyethylene glycol-polyglutamic acid solution to the sulforaphane solution in the step 3) is 1: 5-20 by volume;

the specific operation of mixing in the step 3) is to drop the methoxypolyethylene glycol-polyglutamic acid solution into the sulforaphane solution;

the specific operation of the ultrasonic treatment in the step 3) is to perform ultrasonic treatment on the obtained mixed solution for 30-40 minutes, and then adding the mixed solution into the mixed solution according to the following mixed solution: adding sodium lactate ringer 'solution into the sodium lactate ringer' solution in a volume ratio of 1: 10-15, shaking for 5-8 minutes, and performing ultrasonic treatment again for 30-40 minutes;

standing in a dark room at 4 ℃ for overnight in the step 3);

the condition of the centrifugal treatment in the step 3) is preferably 15000-20000 g for 10-30 minutes;

the operations of centrifugation and washing of the precipitate in step 3) need to be repeated more than 5 times.

8. The application of the sulforaphane or the nanoparticles thereof according to any one of claims 1 to 7 in the preparation of the medicine for improving embryonic nervous system dysplasia caused by the intake of heterocyclic amine in pregnant women, which is characterized in that:

the heterocyclic amine is at least one of 2-amino-1-methyl-6-phenyl-imidazole [4,5-b ] pyridine, 2-amino-1, 6-dimethyl imidazole [4,5-b ] pyridine and 2-amino-1, 5, 6-trimethyl imidazole [4,5-b ] pyridine.

9. The application of the sulforaphane or the nanoparticles thereof according to any one of claims 1 to 7 in the preparation of the medicine for improving embryonic nervous system dysplasia caused by the intake of heterocyclic amine in pregnant women, which is characterized in that:

the embryonic nervous system dysplasia comprises neural tube malformation, neural tube maldifferentiation, generation inhibition of neural crest cells, migration inhibition of neural crest cells, proliferation inhibition of neural tube cells and neural tube apoptosis.

10. The application of the sulforaphane or the nanoparticles thereof according to any one of claims 1 to 7 in the preparation of the medicine for improving embryonic nervous system dysplasia caused by the intake of heterocyclic amine in pregnant women, which is characterized in that:

the dosage form of the medicine is capsule, pill, tablet, oral liquid, granule, tincture or injection;

the medicine also contains one or more pharmaceutically acceptable auxiliary materials or carriers.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of sulforaphane or nanoparticles thereof in preparation of a medicine for improving embryonic nervous system dysplasia caused by intake of heterocyclic amine by pregnant women.

Background

The neural tube develops into the central nervous system of the body. The Henry's segment is induced to form spinal cord and neural plate, and the neural plate is induced to differentiate and fold and fuse to the dorsiflexion to form neural tube. Malformation caused by abnormal neural tube development, called neural tube malformation, is a serious malformation disease, including anencephaly, brain expansion, brain exposure, cranial spine fissure, etc. Neural Tube Defects (NTDs) are the most common birth defects, accounting for about 0.5-2 per mill of the worldwide birth rate (Sanna et al, Nat Genet 2019). With the closing of the nerve canal, a group of special cells, namely neural crest cells, are arranged on the back side of the nerve canal and are differentiated into peripheral nervous system, pigment cells, most cartilage and skeleton of the head and face, endocrine cells, partial heart tissues, smooth muscles and tendons and the like through a series of processes such as induction (Specification), Epithelial-to-mesenchymal transition (EMT), Migration (Differentiation) and Differentiation (Differentiation). Neural crest dysplasia is associated with a variety of congenital diseases, including congenital megacolon, neurofibromas, diseased craniofacial skeletal defects, and the like (Kalcheim, Genesis 2018; Prasad et al, Genesis 2019).

3-8 weeks of embryo development is the teratogenic phase, during which the cells of the embryo move and assemble to form organ primordia, which are most sensitive to teratogenic factors. For example, teratogenic factor PhIP (2-amino-1-methyl-6-phenyl-imidazo [4,5-b ] pyridine, a relatively high content of heterocyclic amines in food products) can affect neuronal development, leading to abnormal development of the nervous system. However, at present, no medicine with high safety aiming at protecting embryonic neurodevelopment induced by heterocyclic amine during pregnancy exists.

The liver of the fetus is not developed perfectly, the excretion of the drugs and degradation products is delayed, and the discharged partial metabolites can be reabsorbed by the fetus due to the 'amniotic fluid intestinal circulation', and are easy to accumulate in the body, thereby influencing the development of tissues and organs of the fetus. Therefore, even drugs that do not have serious adverse effects on the mother may have toxic effects on the fetus during pregnancy. The application of the drug to embryo protection not only needs to consider the treatment effect, but also needs to consider the influence on the development of the fetus or the newborn, so that natural small molecule drugs are screened from green foods, and the development of the protection effect on the embryo development is particularly feasible.

Sulforaphane (1-isothiocyanato-4-methanesulfonylbutane, Sulforaphane, SFN), also known as Sulforaphane or Sulforaphane, is a derivative of glucosinolate, belonging to the isothiocyanate group of substances, which is abundant in cruciferous vegetables such as broccoli sprouts (Keum et al, Drug News variety 2005). Sulforaphane is easily soluble in water, has a relative molecular mass of 177.29 and a molecular formula of C₆H₁₁S₂NO, structural formula:

there is reliable evidence that broccoli sprouts and other sources of edible sulforaphane, which are rich in sulforaphane, induce the production of biphasic detoxification and antioxidant enzymes by activating the Nrf2 signaling pathway and contribute to the prevention of diseases such as cancer (Dinkova-Kostova et al, Proc Natl Acad Sci U S a 2002). In the application of sulforaphane, patents for resisting adult protection effects such as oxidation, cancer and the like are mostly focused, for example, drugs for inhibiting skin carcinogenesis induced by ultraviolet light (200680022993.6; 101208079B), liver protection effect of sulforaphane and application thereof in non-alcoholic fatty liver, obvious protection effect of 10.0 mu mol/L of sulforaphane (201610938091.2) and the like. The patent of the nano sulforaphane (nano SFN) mostly focuses on the preparation and anticancer effects, such as a sulforaphane-loaded drug-loading system and a preparation method thereof (201811510938.2), and a preparation method and application of self-assembled nanoparticles loaded with docetaxel and sulforaphane (201410060679.3).

Nanotechnology is a new technology developed rapidly in recent years, and nanoparticles in pharmacy, or nano carriers and nano drugs, have a size limited between 1-1000 nm. The nanometer medicine is prepared through adsorbing medicine particle or medicine coated in carrier to form nanometer size particle and preparing different kinds of preparation forms based on the nanometer size particle. The nano-drug has unique advantages in improving drug stability, reducing stimulation to gastrointestinal tract, eliminating toxic and side effects, improving drug efficacy, realizing targeted drug delivery, providing sustained release effect and the like (Gaojie et al, chemical industry and engineering 2012; Xiehua, pharmaceutical chemistry 2018). Common nano-drug carriers include nano-magnetic particles, polymer nano-drug carriers, and the like. Common nanoparticle types in the medicine include nanospheres, nanocapsules, nanoliposomes, solid lipid nanoparticles, nano micelles, nano emulsions and the like. The particle size, surface charge and surface properties of nanoparticles are the main factors affecting the distribution and pharmacokinetic characteristics of nanoparticles in vivo, and changes in pharmacokinetic and in vivo distribution will in turn affect their efficacy and toxicity (everbright, monograph research and evaluation monograph treatise on nano-material genotoxicity, proceedings of the national toxicological Committee: the national toxicological Committee 2018).

Disclosure of Invention

Although sulforaphane has good antioxidant capacity and wide antitumor effect, the embryo developmental toxicity of sulforaphane and the possibility of being used for preparing embryo nervous system developmental protection medicaments are not clear. The invention aims to overcome the defects in the prior art and provide the application of sulforaphane or nanoparticles thereof in preparing a medicine for improving embryonic nervous system dysplasia caused by the intake of heterocyclic amine in pregnant women.

The purpose of the invention is realized by the following technical scheme:

application of sulforaphane or nanoparticles thereof in preparing a medicine for improving embryonic nervous system dysplasia caused by intake of heterocyclic amine in pregnant women.

The effective concentration of the sulforaphane or the nanoparticles thereof is preferably 0-10 mu mol/L (not equal to 0); more preferably 5. mu. mol/L.

The medicine comprises at least one of sulforaphane and a pharmaceutically acceptable modifier synthesized by the sulforaphane as a lead compound.

The modification comprises at least one of salification modification, esterification modification, amidation modification, cyclization modification and ring-opening modification.

The sulforaphane nanoparticles comprise a nano-carrier and sulforaphane encapsulated in the nano-carrier.

The nano-carrier is preferably a liposome or a polymer capable of forming micelles; more preferably methoxypolyethylene glycol-polyglutamic acid; wherein the molecular weight of the methoxy polyethylene glycol segment is 3000-7000, and the molecular weight of the polyglutamic acid segment is 8000-12000; most preferably methoxypolyethylene glycol-polyglutamic acid; wherein the molecular weight of the methoxy polyethylene glycol segment is 5000, and the molecular weight of the polyglutamic acid segment is 10000.

Methoxy polyethylene glycol-polyglutamic acid belongs to a macromolecule nano-drug carrier, mPEG-PGA can be automatically assembled into a corolla-shaped structure with stable physical properties in water, has hydrophilic and lipophilic groups, and spontaneously forms a macromolecule micelle after being dissolved in water. Since sulforaphane is a fat-soluble drug, it is wrapped around the inner core of mPEG-PGA. mPEG-PGA polymers can prolong the half-life of macromolecular organisms (Kataoka et al, Adv Drug Deliv Rev2001), and have good cell membrane penetrating effect (Hubbell, Science 2003).

The sulforaphane nanoparticles are preferably prepared by the following method: mixing methoxypolyethylene glycol-polyglutamic acid solution and sulforaphane solution, performing ultrasonic treatment, and standing; then centrifuging and washing the precipitate; preferably, the method comprises the following steps:

1) dissolving sulforaphane in dimethyl sulfoxide (DMSO) to obtain sulforaphane solution;

2) dissolving methoxypolyethylene glycol-polyglutamic acid in sodium lactate ringer's solution (LR) to obtain methoxypolyethylene glycol-polyglutamic acid solution;

3) mixing the methoxypolyethylene glycol-polyglutamic acid solution obtained in the step 2) with the sulforaphane solution obtained in the step 1), performing ultrasonic treatment, and standing; then centrifuging, washing the precipitate, and obtaining the precipitate which is the sulforaphane nano-particle encapsulated by the methoxypolyethylene glycol-polyglutamic acid.

DMSO is used as surfactant and organic solvent for acting on sulforaphane. DMSO also helps to increase the stability of the formed nanoparticles and to avoid rapid aggregation of the nanoparticles. A ringer's solution of sodium lactate is used as a solvent for mPEG-PGA and as a buffer solution for forming nanoparticles; by maintaining a stable pH value, the rapid aggregation of nanoparticles and the change in zeta potential can be slowed down compared to pure water.

The preferable concentration of the sulforaphane solution in the step 1) is 10-100 mmol/L; more preferably 40 to 60mmol/L, most preferably 56.4 mmol/L.

The concentration of the methoxypolyethylene glycol-polyglutamic acid solution in the step 2) is preferably 0.01-0.1 mu g/mu L; more preferably 0.04-0.06 μ g/μ L; most preferably 0.05. mu.g/. mu.L.

The preferred ratio of the methoxypolyethylene glycol-polyglutamic acid solution to the sulforaphane solution in the step 3) is 1: 5-20 by volume; more preferably 1: 10.

the specific operation of mixing in step 3) is preferably to add the methoxypolyethylene glycol-polyglutamic acid solution dropwise to the sulforaphane solution.

The specific operation of the ultrasonic treatment in the step 3) is as follows: carrying out ultrasonic treatment on the obtained mixed solution for 30-40 minutes, and then adding the mixed solution into the mixed solution according to the following ratio: adding the sodium lactate ringer 'solution into the sodium lactate ringer' solution in a volume ratio of 1: 10-15, shaking for 5-8 minutes, and performing ultrasonic treatment again for 30-40 minutes.

The conditions for standing as described in step 3) are preferably overnight in a dark room at 4 ℃.

The condition of the centrifugal treatment in the step 3) is preferably 15000-20000 g for 10-30 minutes; more preferably 18000g for 20 minutes.

The operation of centrifugation and washing of the precipitate in step 3) is preferably repeated 5 or more times.

The heterocyclic amine preferably refers to pyridine heterocyclic amines including 2-amino-1-methyl-6-phenyl-imidazo [4,5-b ] pyridine (PhIP), 2-amino-1, 6-dimethylimidazo [4,5-b ] pyridine (DMIP), 2-amino-1, 5, 6-trimethylimidazo [4,5-b ] pyridine (TMIP); in particular 2-amino-1-methyl-6-phenyl-imidazo [4,5-b ] pyridine (PhIP).

The embryonic nervous system dysplasia comprises neural tube malformation, neural tube maldifferentiation, generation inhibition of neural crest cells, migration inhibition of neural crest cells, proliferation inhibition of neural tube cells and neural tube apoptosis.

The dosage form of the medicine is capsule, pill, tablet, oral liquid, granule, tincture or injection.

The medicine also contains one or more pharmaceutically acceptable auxiliary materials or carriers.

The auxiliary material is at least one of a sustained release agent, an excipient, a filler, an adhesive, a wetting agent, a disintegrating agent, an absorption enhancer, a surfactant or a lubricant.

In our earlier studies, the development of neural tube cells and neural crests was observed by directly exposing chick embryos to teratogenic factors capable of passing through the placental barrier, and a model of the development defect of the nervous system was established (Cheng et al, toxicologic sciences 2017; Cheng et al, neurooxology 2012; Jin et al, Mol Reprod Dev 2015; Liu et al, Toxicol Sci 2016; Ma et al, PLoS One 2012; Zhang et al, toxicolett 2017), which provided an in vivo study model of a good protection drug for the development defect of the nervous system.

The invention adopts a self-assembly method, uses methoxy polyethylene glycol 5000-b-polyglutamic acid 10000(mPEG5K-PGA10K) with good biocompatibility and biodegradability as a base material (Jiang et al., Adv Funct Mater 2018; Xiao et al., Chem Commun (Camb) 2013; Yang et al., Mater Sci Eng C Mater Biol Appl 2019; Zhang et al., J Colloid Interface Sci 2016), and prepares the nano sulforaphen (SFN-mPEG5K-PGA 10K).

We performed chick embryo experiments by inducing chick embryo nervous system dysplasia by PhIP. The result proves that the sulforaphane and the nano sulforaphane with certain concentrations can effectively relieve teratogenic factors such as embryonic nervous system dysplasia induced by PhIP:

(1) preparing and self-assembling nano sulforaphane;

(2) analyzing the drug loading efficiency of the nano sulforaphane;

(3) detecting the nanometer characteristics of the nanometer sulforaphane;

(4) sulforaphane ameliorates PhIP-induced embryonic death and neural tube malformations;

(5) sulforaphane improves the differentiation badness of embryo neural tube cells induced by PhIP;

(6) sulforaphane improves the production of PhIP-inhibited embryonic neural crest cells;

(7) sulforaphane improves migration of PhIP-inhibited embryonic neural crest cells;

(8) sulforaphane improves the proliferation of PhIP-inhibited embryonic neural tube cells;

(9) sulforaphane ameliorates PhIP-induced apoptosis of embryonic neural tube cells.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides a new application of sulforaphane, namely an embryonic development protection application, which not only expands the application range of the sulforaphane and improves the application value of the sulforaphane, but also is beneficial to further developing a new nano-medicament for improving embryonic nervous system dysplasia induced by heterocyclic amine intake of pregnant women.

Drawings

Fig. 1 is a schematic diagram of a nanoparticle formation process.

FIG. 2 is a graph showing the results of the analysis of the loading efficiency of the nano-sulforaphane drugs; wherein A is a chromatogram of a standard sample DMSO and DMSO + SFN; and B is a chromatogram of the supernatant after and before loading.

FIG. 3 is a Zeta potential, particle size and morphology analysis result chart of nano sulforaphane; wherein A is a Zeta potential diagram; b is a particle size distribution diagram; and C is a transmission electron microscope image.

FIG. 4 is a graph showing the results of experimental studies of sulforaphane in ameliorating PhIP-induced embryonic death and neural tube malformations; wherein A is a normal embryo and dead embryo detection map under white light and PAX7 fluorescence; b is a head and trunk photographing picture of the normal embryo and the neural tube malformed embryo; c is the statistics of neural tube defects and embryonic mortality.

FIG. 5 is a graph showing the results of experimental studies on the improvement of PhIP-induced embryonic neural tube cell differentiation by sulforaphane; wherein A is a detection result graph of NF immunofluorescence observation of different groups of chick embryo neural tube cell morphology; b is the statistics of the ratio of NF positive cells to neural tube cells.

FIG. 6 is a graph of the results of experimental studies of sulforaphane in improving the production of PhIP-inhibited embryonic neural crest cells; wherein A is a detection result graph of different groups of Ap2 alpha labeled neural crest cells; b is Ap2 α positive area statistics.

FIG. 7 is a graph of experimental findings of sulforaphane improving migration of PhIP-inhibited embryonic neural crest cells; wherein A is a detection result graph of different groups of HNK1 labeled migrating neural crest cells; b is the statistic result of positive area of HNK 1.

FIG. 8 is a graph showing the results of experimental studies of sulforaphane in improving PhIP-inhibited embryonic neural tube cell proliferation; wherein A is a detection result graph of different groups of PHIS3 labeled neural tube proliferation cells; b is the statistics of the number of positive cells of PHIS 3.

FIG. 9 is a graph showing the results of experimental studies of sulforaphane in ameliorating PhIP-induced apoptosis in embryonic neural tube cells; wherein A is a detection result graph of different groups of c-Caspase3 labeled and marked neural tube apoptotic cells; b is the statistical result of the number of positive cells of c-Caspase 3.

Detailed Description

The technical solution of the present invention is further explained by the following detailed description and the accompanying drawings. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

The experimental methods in the following examples are all conventional methods unless otherwise specified; the experimental materials used, unless otherwise specified, were purchased from conventional biochemical manufacturers.

Among the materials described in the examples below:

sulforaphane was purchased from Sigma (S6317) and has the molecular formula C₆H₁₁NOS₂Molecular weight is 177.29;

methoxy polyethylene glycol 5000-b-polyglutamic acid 10000(mPEG5K-PGA10K) was purchased from Saian Rexi Biotech Ltd (R-PL 1236-15K).