CN110036947A - A method of induction coral Metamorphore improves adhesive ability - Google Patents

A method of induction coral Metamorphore improves adhesive ability Download PDF

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CN110036947A
CN110036947A CN201910330824.8A CN201910330824A CN110036947A CN 110036947 A CN110036947 A CN 110036947A CN 201910330824 A CN201910330824 A CN 201910330824A CN 110036947 A CN110036947 A CN 110036947A
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coral
metamorphore
shelly
bromine
pyrrolidine alkaloid
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CN110036947B (en
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聂磊
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Shenzhen Institute of Information Technology
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K61/00—Culture of aquatic animals
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A10/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE at coastal zones; at river basins
    • Y02A10/26—Artificial reefs or seaweed; Restoration or protection of coral reefs
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
    • Y02A40/81—Aquaculture, e.g. of fish

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  • Life Sciences & Earth Sciences (AREA)
  • Environmental Sciences (AREA)
  • Marine Sciences & Fisheries (AREA)
  • Zoology (AREA)
  • Animal Husbandry (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Farming Of Fish And Shellfish (AREA)

Abstract

The present invention relates to a kind of methods that induction coral Metamorphore improves adhesive ability, method includes the following steps: adhering to culture medium bottom by larva of shelly-shaped coralline algae living body；Bromine pyrrolidine alkaloid (-)-Agelastatin A polycaprolactone microballoon sphere is uniformly sprayed on shelly-shaped coralline algae surface；Or the organic solvent solution of bromine pyrrolidine alkaloid (-)-Agelastatin A is smeared on shelly-shaped coralline algae living body；Coral Metamorphore is placed in the substrate for the culture vessel that the above processing is formed, is cultivated in the seawater.The present invention also provides bromine pyrrolidine alkaloid polycaprolactone microballoon spheres and preparation method thereof.The present invention is by adhering to culture medium bottom by larva of shelly-shaped coralline algae living body, addition has bromine pyrrolidine alkaloid (-)-Agelastatin A polycaprolactone microballoon sphere, it can promote attachment and development of the coral Metamorphore in substrate well, and then improve the ecological nursing level of coral and the ecological reestablishment level of coral reef.

Description

A method of induction coral Metamorphore improves adhesive ability
Technical field
The invention belongs to marine biotechnologies and ecological environmental protection field, are specifically related to a kind of induction coral floating wave children The method of worm raising adhesive ability.
Background technique
It is well known that coral reef ecologic system is that bio-diversity is most complicated on the earth, the highest ecosystem of production capacity One of system, is pregnant with a variety of marine organisms, and small to single celled protozoan is big to move to huge vertebrate and without vertebra Object, a variety of sea-plants are also using coral reef as habitat.Zingber corallinum hance has not only bred biology abundant, while It is considerable fishery resources and tourist resources.Coral reef as Global Ecological and the highest ecosystem of economic value it One, huge economy, social and cultural value and Ecosystem Service are provided for human society.However in the past 20 years, global coral Coral reef experienced unprecedented destruction under the influence of mankind's activity and whole world change, and global coral all suffers from degeneration Trend, protection coral reef bio-diversity are extremely urgent.The bioremediation technology requirement of coral reef ecologic system is quite high, and Period is long, understands ecological functions of coral during Coral reef development, is the conservative management of coral reef, restoration of the ecosystem and thereafter The core work that continuous living marine resources utilize.
Cnidaria (Cnidaria) was also referred to as coelenterate (Coelenterata) in the past, was a kind of more original Multicellular animals occupies important position in zoogeny history, is the beginning of real metazoa, represents animal system hair One important feature of exhibition.As other cnidarias, there are two kinds of forms of sexual propagation and vegetative propagation for coral, wherein having Have the characteristics that during sexual reproduction one it is common: early development process requires experience this particular stage of Metamorphore.
Since early 20th century, external many scholars just to the sexual reproduction process of project cost consulting item, sexual propagation mode and Sexual propagation condition etc. has carried out research extensively and profoundly.The sexual propagation mode of cave coral can be divided into 4 big classifications: male and female Androgynous ovulation type, hermaphroditic arrange young type, gonochorism ovulation type and gonochorism and arrange young type (Baird et al, 2009). Baird etc. (2009) has counted the sexual propagation mode of 400 kinds of cave corals, wherein ovulation type accounts for 84.5%, and male and female are same Body type accounts for 68.9%.In the Ecological Restoration Project of coral reef, the mankind have carried out coral transplanting, horticulture type cultivation coral And the associated restorations technology such as artificial reef.But compared with vegetative propagation, protection and the sustainable development of coral reef be the key is to By anthozoic sexual propagation.Reef-building coral is the frame biology of coral reef ecologic system, and the survival and success of larva are attached It is most important to the restoring force of healthy coral reef and the recovery of degeneration coral reef.Nearly 80% project cost consulting item belongs to In ovulation type coral, the Metamorphore reached maturity is spread with surface seawater, undergoes a longer pelagic period until encountering Suitable substrate, which is just adhered to, becomes the young.The key for realizing coral reef restoration of the ecosystem is that floating larvae will add to coral reef Area.
Invertebrate (including coral polyp) history of life will pass through the floating larvae phase mostly, and larva is to inhabiting bottom environment Selection often the attachment of larva later period, abnormal and adult distribution situation are had a very big impact；Transplant the sexual of coral It is the main breeding side of coral that breeding, which generates the coral offspring sexual propagation with strong accommodative ability of environment and asexual budding breeding, Formula, sexual propagation are that mature coral is ovulated in vitro fertilization as Metamorphore.Metamorphore drifts about through water flow and ciliary movement, The suitable regional apposition growth of selection, develops into coral.It can be lured by the chemistry of dispersion of flow, larva attachment and abnormal behavior It leads, the help that mima type microrelief adheres to larva etc. can promote the propagation and survival of coral Metamorphore.Transplant the sexual propagation of coral Generating the coral offspring sexual propagation with strong accommodative ability of environment and asexual budding breeding is the main modes of reproduction of coral, is had Sexual reproduction is that mature coral is ovulated in vitro fertilization as Metamorphore.The supplement for completing larva includes 3 continuous stages: children Worm is drifted about, larva attachment and abnormal, larva are grown, and wherein the deformation and attachment of Metamorphore are the key that coral history of life ranks Section.Metamorphore has certain travelling ability, and gradually shows as purposive activity, and larva is by selection, attachment, change The processes such as state, grow tentacle, develop into coral larva.The early development of reef-building coral can generally be divided into 5 stages of development: by Smart ovum, the spilting of an egg, blastaea, primitive gut, Metamorphore.Metamorphore can also be divided into 4 periods: Metamorphore early stage, Metamorphore Mid-term, Metamorphore advanced stage, Metamorphore latter stage.Ellipse or circular Metamorphore occur after fertilization about 48h, the period Metamorphore has mobility, but does not have apparent purpose, only turn-takes or random walk.About 97h after oviposition can be seen It has observed coral larva to be attached on attachment base bottom, but deformation occurs, slightly having water flow to break through will fall off from substrate, It is not really to adhere to, it may be that souning out whether substrate properly adheres to.By microscope it can be seen that larva is soundd out close to substrate The process abandoned again afterwards.It with basinal lake close to substrate when really adhering to, and expands, has mouthful end upward, subsequent entire floating wave Larva is become dish and is attached in attachment base by ellipse or circle, and mouth disk is upward.Until the complete deformation of larva, tentacle is grown simultaneously It extends, entire shape is rendered as flower shape, and the attachment of larva is just properly completed at this time.If substrate is suitable for that larva is attached , then larva comparatively fast sinks close and completes to adhere to, if substrate is not suitable for, larva constantly floats at random, keeps floating wave shape State, and finally melt dead.
Project cost consulting item mainly by sexual propagation generate new individual carry out population supplement, the survival of coral Metamorphore and Success attachment is two crucial ecological processes that population is successfully supplemented.Ovulation type coral usually simultaneously smart ovum is discharged, the water surface by The Metamorphore for developing into just tool adhesive ability after essence by 1~6d, later with surface layer dispersion of flow, therefore in pelagic period larva It is highly susceptible to the influence of external factor.The attachment of coral larva is coral larva identification attachment base surface chemistry signal and carries out The response process of abnormal fixation, is directed to series of genes expression, and the processes such as cell differentiation and rearrangement are one and expend very much The process of energy.Metamorphore can be made to drift about to suitable place and adhered to by the attraction of the various chemical informations in ocean.So And how to increase the attachment of coral larva and metamorphosis by inductive technology, floating larvae is replenished in time to Coral Reef Region, and Promote the attachment of project cost consulting item larva and abnormal Related Mechanism, the crucial skill of this coral restoration of the ecosystem is adhered to for larva Art, there are still a large amount of technological gaps at present.
Chemical signal induction has a major impact the attachment and metamorphosis of marine animal larva.Studies have found that by sea grass It is immersed in water and tests the selection of fish and the growth attachment of coral larva in water.Compared with the water without sea grass chemical signal, often The signal that the sea grass Sargassum polycystum (Sargassum polycystum) seen issues can reduce water to the attraction of the fish young Up to 86%, the chemical signal that sea grass issues can be reduced to coral larva attraction up to 81%.The georgia ,U.S.A Institute of Technology and Ah La Bai joint emirate Zha Yede university the it was discovered by researchers that chemical signal as expressed by healthy coral can attract coral young Worm and the coral reef fish of childhood, and the signal issued by seaweed can then enable these aquatic floating species retreat.Related article hair Table is on August 22nd, 2014 " Science " magazine.Existing research at present is by being added allogenic material such as algae, lamina affixad Equal promotions attachment occurs, and artificially controls environmental factor such as temperature etc., accelerates coral larvae development speed.
Coralline algae (Coralline algae) belongs to Rhodophyta (Rhodophyta), Rhodophyceae (Rhodophyceae), true Red algae subclass (Florideophycidae), coral Cutleriales (Corallinales), be a group cell wall contain a large amount of calcium carbonate, Thallus is hard, rich biodiversity special plant monoid, and frond is broadly divided into without saving and having section two major classes, usually there is shell Shape, dendritic, 3 kinds of different forms of tumor mass shape.It is built as large ocean calcification algae, coralline algae by providing coral reefs Calcareous material resource constructs the functions such as coral reef physique frame, the hard substrate for providing coral polyp settlement and metamorphosis with strong cementation, in coral Coral reef plays important central role during developing and making reef lithogenesis.Coralline algae, especially shelly-shaped coralline algae are that ocean bottom is dwelt generation The abnormal important role but obviously ignored in the principal component and marine ecology on boundary.Coralline algae is a kind of large ocean calcium Alga, by providing the calcareous material resource of coral reefs construction, constructing coral reef physique frame, offer coral polyp with strong cementation The functions such as the hard substrate of settlement and metamorphosis play important central role with during making reef lithogenesis in Coral reef development.To coral The further investigation of coral algae will be helpful to disclose its multiple mechanisms in coral reef ecologic system, understand it in Coral reef development mistake Ecological functions in journey preferably serve conservative management, restoration of the ecosystem and its subsequent living marine resources benefit of coral reef With.Coralline algae belongs to cell wall and contains that a large amount of calcium carbonate, thallus is hard, special plant monoid of rich biodiversity, The organic reef ecosystem and reefs play important function affect in building, and are indispensable in coral reef ecologic system One of biological group.Generally approve that coralline algae plays two critical important works in the marine organisms reef ecosystem at present With dwelling zoobenthos larva the other is can induce many reefs, including coral first is that huge to the contribution of the calcification of reefs Attachment and abnormal, especially shelly-shaped coralline algae (Crustose Coralline Algae, CCA), in chemical induction coral larva Attachment and abnormal growth aspect, have important promotion functions.However at present about the photosynthetic carbon fixation of coralline algae, calcification and Organic matter secretes the mechanism of cementation mechanism, the especially attachment of chemical induction coral larva and abnormal growth and biological prosthetic The relevant technologies of coral reef ecologic system are worldwide still research blank.The bioremediation technology of coral reef ecologic system It is required that quite high, the period is also very long, successfully promotes at present and the coral reef restoration of the ecosystem case for obtaining effect is all seldom, wherein one A major reason is that status and effect of the coralline algae in reparation, especially and helps the mechanism of Coral reef development still very not It is clear.
The attachment of coral Metamorphore is coral larva identification attachment base surface chemistry signal and the sound for carrying out abnormal fixation Process is answered, series of genes expression, the processes such as cell differentiation and rearrangement are directed to.As coral reef reefs " adhesive " Shelly-shaped coralline algae can play the part of the key player of " inducer " in the attaching process of coral larva.The attachment of coral larva is one double The process just interacted, it is more related with the inducing action of coralline algae not only with the physiological status of coral larva itself.
Invertebrate (including coral polyp) history of life will pass through the floating larvae phase mostly, and larva is to inhabiting bottom environment Selection often the attachment of larva later period, abnormal and adult distribution situation are had a very big impact；Shelly-shaped coralline algae can Special hard surface is formed, and coralline algae can usually generate attachment and abnormal signal to oceanic invertebrate larva, because This, coral frond just becomes them in the preferable attaching substratum selection in Coral Reef Region.Either there is section coralline algae, or without section coral Coral algae can generate preferable inducing action to invertebrate larva.Williams etc. has found knuckle Trentepohlia Amphiroa's Type induces clearly the attachment and metamorphosis of ear Bao (Haliotis asinine) larva, and the distortion ratio after inducing 48h can Up to 92.8%~100%；The researchs such as Neo find shelly-shaped coralline algae to large-scale shellfish-squama giant clam (Tridacna of Coral Reef Region Squamosal) attachment early period of the young has very strong inducing action；Desk research finds a kind of alcynorian soft coral The attachment of (Alcyonium Siderium) larva and metamorphosis be due to the inducement signal that it is contacted with coralline algae surface and is generated, Not dissolve the induction of frond chemistry inside in the seawater.Different types of coralline algae adheres to invertebrate larva There is otherness with abnormal inducing action.Johnson etc. has studied three kinds of coralline algaes to crown of thorns (Acanthaster Planci) the inducing action of larva, as a result, it has been found that, helictite algae (Lithothanmium pseudosorum) compares Kong Shizao The inducing action of (Porolithon onkodes) and string born of the same parents' Neogoniolithon frutescens (Neogoniolithon fosliei) is strong. Ritson-Williams etc. compares three kinds of coralline algaes to the otherness of two kinds of Acropora larvas attachments and metamorphosis, hair Existing Bu Shi water stone algae (Hydrolithon boergesenii) and two kinds of original Pi Shizao (Titanoderma prototypum) Inducing effect of the class all than pachydermia Kong Shizao (Porolithon pachydermum) is good, between difference be more than 10%.This A result carries out project cost consulting item artificial propagation research for current and future and provides effective information.Micro- life on coralline algae surface Object is formed by biomembrane (biofilms) and focuses on to the successful attachment of oceanic invertebrate larva with abnormal also the same play It acts on, and studies have found that the biomembrane that different types of bacterium is formed also has specifically the type adhered on its surface Property.
The nitrogenous alkalinity of one kind that alkaloid is present in nature (predominantly plant, but some exists in animal) has Machine compound has the property like alkali.Most of alkaloids have complicated cyclic structure, and nitrogen is included in ring more, have significant Bioactivity has optical activity.Some derive from the organic compounds containing nitrogen of plant without alkalinity, have apparent biology living Property, therefore be included in the range of alkaloid.There are many known alkaloid type, about at 10,000 kinds or so, there is some structural formulas It is determined there are no complete.Their structures are more complicated, can be divided into 59 seed types.With the discovery of new alkaloid, classification also will It updates therewith.Since there are many type of alkaloid, respectively there is different structural formulas, therefore property to each other can be poor It is different.But alkaloid is nitrogenous organic compound, total somewhat like property, because of the amino in the approach of its biosynthesis Acid is starting material, mainly there is ornithine, lysine, phenylalanine, histidine, tryptophan etc., mainly undergoes two kinds of reaction class classes Type: the cracking of cyclization reaction and carbon-nitrogen bond, so some total properties are similar.Alkaloid has cyclic structure, water is insoluble in, with acid Can there are certain optical activity and absorption spectrum with forming salt, there is bitter taste mostly.In colorless crystals, minority is liquid.Alkaloid There are thousands of kinds, is one of secondary metabolites from different amino acid or its direct derivative synthesis, it is toxic to living organism Property or strong physiological action.According to the basic structure of alkaloid, 60 classes or so can be divided into.Main Types have: organic amine, Pyrrolidines, pyridines, iloquinoline derivative, indoles, henbane alkanes, imidazoles, quinazolone class, purines, steroid, Diterpenes Deng.
Ocean is the cradle of life, wherein containing many compounds with pharmacological activity, wherein having many unknown Compound is just waiting for the mankind and is going to explore and develop.Pyrroles's Alkaloid is one of the important branch of alkaloid chemistry.Sea Midocean is richly stored with the pyrrolidine alkaloid with unique bioactive since the 1970s, and a large amount of pyrroles is raw Alkaloids are separated from the sponge of the types such as Agelasi-dae, Axinellidae and Hymeniacidonidae.These chemical combination Object Chang Yidan or dibromo pyrroles -2- formic acid part pass through aliphatic chain and are connected these marine natural products of with imidazole ring because of its chemistry Novelty, diversity and a variety of physiological activity such as antitumor, antiviral, antibacterial, anti-inflammatory of structure, and cause organic chemist With the great interest of Pharmaceutical Chemist.
Large number of pyrrolidine alkaloid is separated as the metabolite of marine organisms, and shows good biology Activity.2000, Lindel etc. showed oroidin (1) and the like simple structure activity study carried out, such change Though the azoles moiety for closing object is that it shows that corresponding bioactivity is insufficient but necessary condition, and side chain part only serves influence The effect of activity intensity.Therefore, according to the structure feature of its azoles moiety, can be divided into three classes Study of Marine Bromopyrrole Alkaloids One kind is known as and ring class bromine pyrrolidine alkaloid, and structure feature is to form hexa-atomic or seven yuan and ring with pyrrole ring 2,3- or 1,2- (Chart1).Wherein, it is albumen as the Z-axinohy-dantoin (2) of the micro metabolin of Stylotella aurantium The inhibitor of kinase c.The hymenin (3a) being separated to from Hymeniacidon class sponge can impedance oc-adrenoreceptor. Spongiacidin A (4a) and spongiacidin B (4b), is equally the micro constitutent of Hymeniacidon class sponge, energy Inhibit c-erbB-2 kinases and cdk4 (cyclin dependent kinase4).It is separated from Phakellia mauritiana The dibromophakellstain (5) arrived has cellular toxicity, and the Xu Xiaohua group of Nankai University is also from China's Xisha Islands of South China Sea The guanidine radicals analog of the isolated compound in the sponge Phacellia fusca Schmidt in sea area dibromophakelin(6).The agelastatin A (8a) isolated from sponge Agelas dendromorpha has anti- Tumor promotion；Palau ' the amine series compound being separated to from Stylotella aurantium because of its cellular toxicity, exempt from Epidemic disease inhibiting effect, the structure feature of antibiosis and novelty and obtain widest concern.Second class can be described as simple chain type bromine Pyrrolidine alkaloid has been structurally characterized in that single bromine pyrrole ring is connected before (Chart2) by chain fatty chain with imidazole ring The oroidin (1) being previously mentioned, resists the important chemical substance of certain fish grazing eclipses as Agelas kind sponge and is widely distributed In secondary metabolites in, antiserum and can have the activity of anti-parasympathetic nerve physiological action.This kind of bromine pyrrolidine alkaloids In, further include be separated in Hymeniacidon class sponge can antagonism 5-hydroxytryptamine receptor hymenidin (10)；From In the Hymeniacidon class sponge of Okinawa the isolated taroacidin A (11a) that can inhibit tyrosine kinase and taroacidin B(11b)；Kobayashi etc. is separated to structure novel from the sponge Agelas nakamurai of Okinawa , secondary metabolites slagenin A (12a), B (12b) and C (12c) with cellular toxicity；From the Agelas class sea of Caribbean It is all separated in continuous A.conifera, A.clathrodes, A.dispar and A.longissima, with anti-histamine activity Dispaca-mide A (13a), dispacamide B (13b), dispacamide C (13c) and dispacamide D (13d)；Kobayashi etc. is also separated to the production of mukanain series natural from the sponge Agelas nakamurai of Okinawa Object, compound 13d is named as mukanandin A by them；And clathramide A (15a) from A.Clathrodes and Clathraide B (15b) also shows good antifungal activity.
In the growth course of coral Metamorphore, it can make to float by the attraction of the Marine Chemistries information such as bromine pyrrolidine alkaloid Unrestrained larva, which is drifted about to suitable place, to be adhered to.The larva that the coral of transplanting and coral hybridization in situ generate, in bacterium and coral Under the chemical substance of coral algae secretion attracts, attachment and metamorphosis are carried out.The bacterium of many biological surfaces all affects these larvas Attachment, such as Pseudoalteromonas on the surface shelly-shaped coralline algae (Neogoniolithon fosliei) (Pseudoalteromonas) tetrabromopyrrol (TBP) secreted can induce the attachment and metamorphosis of larva.In addition, shelly-shaped coral The bromine tyrosine derivative (11-deoxyfistularin-3) of algae secretion also can induce larval metamorphosis.Due to these alkaloids Substance belongs to compound not soluble in water, therefore how to solve the problems, such as seawater dissolution and these potential chemical inducers Key points and difficulties in practical applications.
Medicine microspheres (microspheres) refer to drug dispersion or are attracted in high molecular polymer matrix and are formed Tiny spherical solid, partial size is generally between 1-250 μm.It, can since microball preparation has long-acting slow-release or targeting It is a kind of dosage form of great potential to greatly promote convenience, the compliance of practical medication.In addition, microball preparation product is additional It is worth larger, wide market, has become the hot spot of medicament research and development in recent years.Microball preparation has slow release and long-term effect etc. special Point, currently, the common method for preparing medicine microspheres mainly has emulsion dispersion method, coacervation and three kinds of polymerization.Emulsion dispersion method After meaning that drug is mixed with carrier material solution, it is dispersed in immiscible medium and forms similar Water-In-Oil (W/O) or water Wrap oily (O/W) type emulsion, the method for then making solidifying in emulsion, separation prepare microballoon.Used in the microball preparation listed at present Carrier is mostly poly lactide-glycolide acid (PLGA), polycaprolactone (PCL), Poly L-lactide (PLLA) etc..
Bromine pyrrolidine alkaloid in the application is group sponge bromine Allercur alkaloid (-)-AgelastatinA, CAS Chemical classification number are as follows: 152406-28-5, molecular formula C12H13BrN4O3, molecular weight 341.16, natural products is from group's Spongiidae (Agelasidae) group's Sponge (Agelas) biology, genus polyporus animal door (Porifera), Demospongiae on taxology (Demospongiae), group sponge mesh (Agelasida).Molecular formula is as follows:
Summary of the invention
An object of the present invention is to provide a kind of method that induction coralline algae improves coral Metamorphore adhesive ability.
Realize that the technical solution of above-mentioned purpose is as follows:
A method of induction coral Metamorphore improves adhesive ability, comprising the following steps:
Adhere to culture medium bottom by coral larva of shelly-shaped coralline algae living body；
Bromine pyrrolidine alkaloid (-)-Agelastatin A polycaprolactone microballoon sphere is uniformly sprayed on shelly-shaped coralline algae surface；Or The organic solvent solution of bromine pyrrolidine alkaloid (-)-Agelastatin A is smeared on shelly-shaped coralline algae living body；
Coral Metamorphore is placed in the substrate for the culture vessel that the above processing is formed, is cultivated in the seawater.
The method that above-mentioned induction coral Metamorphore improves adhesive ability is that coral larva is attached by shelly-shaped coralline algae living body Culture medium bottom, addition have bromine pyrrolidine alkaloid (-)-Agelastatin A polycaprolactone microballoon sphere or bromine pyrrolidine alkaloid (-)- The organic solvent solution of Agelastatin A is especially added special bromine pyrrolidine alkaloid (-)-Agelastatin A and gathers oneself Interior ester microsphere can promote attachment and development of the coral Metamorphore in substrate well, and then the ecology for improving coral is protected It is horizontal to educate horizontal and coral reef ecological reestablishment.
The coral is white Acropora in one of the embodiments,.
The load medicine polycaprolactone microballoon sphere quantity on shelly-shaped coralline algae living body is set to reach 1- in one of the embodiments, 100mg.cm-2, or keep on shelly-shaped coralline algae living body or smearing bromine pyrrolidine alkaloid (-)-Agelastatin A organic solvent molten Liquid concentration is 1-100 μ g.ml-1。
It is organic in one of the embodiments, in the organic solvent solution of bromine pyrrolidine alkaloid (-)-Agelastatin A Solvent is methylene chloride；The concentration of bromine pyrrolidine alkaloid (-)-Agelastatin A is preferably 10-30 μ g.ml-1。
Another object of the present invention is to provide a kind of coral propagation methods.
A kind of coral propagation method includes the method that above-mentioned induction coral Metamorphore improves adhesive ability.
Another object of the present invention is to provide a kind of bromine pyrrolidine alkaloid polycaprolactone microballoon spheres and preparation method thereof.
Realize that the technical solution of above-mentioned purpose is as follows.
A kind of preparation method of bromine pyrrolidine alkaloid polycaprolactone microballoon sphere, comprising the following steps:
1) bromine pyrrolidine alkaloid (-)-Agelastatin A is weighed, polycaprolactone (PCL) fills after being codissolved in organic solvent Divide sonic oscillation to mix, obtains oily phase；Wherein, the mass ratio of bromine pyrrolidine alkaloid (-)-Agelastatin A and polycaprolactone For 40-50:280-320；
2) it draws above-mentioned oil mutually and is slowly dropped in polyvinyl alcohol water solution, high-speed stirred, closed emulsification；
3) lasting stirring vapors away organic solvent, solidifies microballoon, obtains lotion；
4) by lotion to be centrifugated, obtained solid is washed repeatedly, is centrifuged to distill water washing；It is dry to get.
It is in one of the embodiments, methylene chloride in the organic solvent.
The dosage of bromine pyrrolidine alkaloid (-)-Agelastatin A and methylene chloride in one of the embodiments, Than for 40-50mg:10-14mL.
The concentration of polyvinyl alcohol water solution is 1.5-2.5% (mass fraction) in one of the embodiments,.
In one of the embodiments, in step 2), emulsifying rate 250-350r.min-1, closed emulsification 50-70min Afterwards, 80-120r.min will be adjusted under speed-1。
The bromine pyrrolidine alkaloid polycaprolactone microballoon sphere being prepared by the above method.
Above-mentioned bromine pyrrolidine alkaloid polycaprolactone microballoon sphere using O/W emulsion-solvent evaporation method prepare, step of preparation process according to It is secondary are as follows: (-)-Agelastatin A mixes → be dissolved in methylene chloride → sonic oscillation → instillation polyvinyl alcohol with polycaprolactone → high-speed stirred → closed emulsification → microballoon solidification → centrifugation → distillation water washing → low temperature drying → product.Above-mentioned preparation method Resource utilization it is high, production equipment is simpler, high production efficiency, basic non-environmental-pollution.Preparation-obtained bromine pyrroles is raw Alkaloids polycaprolactone microballoon sphere had good sustained release effect can be effectively used for the adhesive ability that coralline algae induction improves coral Metamorphore.
Detailed description of the invention
Coral Metamorphore adhesive rate under the processing of Fig. 1 different base；
Coral Metamorphore adhesive rate under the processing of Fig. 2 bromine pyrrolidine alkaloid；
Coral Metamorphore under the processing of Fig. 3 different base adheres to selection；
Fig. 4 is that coral larva starts to adhere to；
Fig. 5 is that coral larva adheres in coralline algae substrate and differentiates histoorgan.
Specific embodiment
It to facilitate the understanding of the present invention, below will be to invention is more fully described.The present invention can be with many not With form realize, however it is not limited to embodiment described herein.On the contrary, purpose of providing these embodiments is makes to this The understanding of disclosure of the invention content is more thorough and comprehensive.
In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition, or according to manufacturer Proposed condition.Used various common agents, are commercial product in embodiment.
Unless otherwise defined, all technical and scientific terms used in the present invention and belong to technical field of the invention The normally understood meaning of technical staff it is identical.Term used in the description of the invention is intended merely to describe specific reality The purpose for applying example is not used in the limitation present invention.Term "and/or" used in the present invention includes one or more relevant listed Any and all combinations of project.
In one embodiment of the present of invention, bromine pyrrolidine alkaloid (-)-Agelastatin A polycaprolactone microballoon sphere is related to Preparation method, wherein since bromine pyrrolidine alkaloid is characteristic not soluble in water, design prepares sustained-release micro-spheres structure as bromine Pyrrolidine alkaloid container gathers oneself in this respect so as to play the effect of compound induction coral larva attachment in the seawater Lactone is the good carrier to form sustained-release micro-spheres structure, and methylene chloride has good solvent to imitate alkaloid compound It answers.
The preparation method comprises the following steps:
S1 bromine pyrrolidine alkaloid (-)-Agelastatin A) is weighed, after polycaprolactone (PCL) is codissolved in organic solvent Abundant sonic oscillation mixes, and obtains oily phase；Wherein, the quality of bromine pyrrolidine alkaloid (-)-Agelastatin A and polycaprolactone Than for 40-50:280-320, preferably 43-48:290-310, preferably 44-46:295-305, more preferably 45:300；It is described Organic solvent is preferably methylene chloride；
S2 it) draws above-mentioned oil mutually and is slowly dropped in polyvinyl alcohol water solution, high-speed stirred, closed emulsification, it is preferable that Emulsifying rate is 250-350r.min-1, after closed emulsification 50-70min, 80-120r.min will be adjusted under speed-1；
S3) lasting stirring vapors away organic solvent dichloromethane, solidifies microballoon, obtains lotion；
S4) by lotion to be centrifugated, obtained solid is washed repeatedly, is centrifuged to distill water washing；Drying is to get powder Last shape sustained-release micro-spheres solid.
Preferably, the amount ratio of bromine pyrrolidine alkaloid (-)-Agelastatin A and methylene chloride is 40-50mg: 10-14mL, further preferably 42-48mg:12-14mL, more preferably 40mg:12mL.
Preferably, the concentration of polyvinyl alcohol water solution be 1.5-2.5% (mass fraction), more preferably 2%.
The bromine pyrrolidine alkaloid polycaprolactone microballoon sphere being prepared by the above method is pulverulent solids, in the seawater Had good sustained release effect, in releasable 11.25% or so the alkaloid of 1d, the average release 3.19% daily of 1-7d is released to 30d It puts and carries 28% or so of medicine total amount.The bromine pyrrolidine alkaloid polycaprolactone microballoon sphere can be effectively used for coralline algae induction and improve coral The adhesive ability of Metamorphore.
Another embodiment of the present invention is related to a kind of method that induction coral Metamorphore improves adhesive ability, including following Step: using shelly-shaped coralline algae living body as culture medium bottom, bromine pyrrolidine alkaloid (-)-Agelastatin A is added wherein and gathers in oneself Ester microsphere smears bromine pyrrolidine alkaloid (-)-Agelastatin A organic solvent in shelly-shaped coralline algae living body, makes shelly-shaped coral Load medicine polycaprolactone microballoon sphere quantity on algae living body reaches 1-100mg.cm-2(preferably 2-90mg.cm-2, preferably 3-80mg.cm-2, preferably 4-70mg.cm-2, preferably 5-60mg.cm-2, preferably 5-50mg.cm-2, preferably 5-40mg.cm-2, preferably 5-30mg.cm-2, preferably 5-20mg.cm-2, more preferably 5-15mg.cm-2, most preferably 10mg.cm-2) or smearing bromine pyrroles Bromine pyrrolidine alkaloid (-)-AgelastatinA concentration of smearing of alkaloid (-)-AgelastatinA organic solvent solution reaches 1- 100μg.ml-1(preferably 5-90 μ g.cm-1, preferably 8-80 μ g.cm-1, preferably 10-70 μ g.cm-1, preferably 12-60 μ g.cm-1, preferably 15-50 μ g.cm-1, preferably 15-40 μ g.cm-1, more preferably 15-25 μ g.cm-1, most preferably 20 μ g.cm-1), it is uniformly smeared 2-3 times on shelly-shaped coralline algae living body surface；Coral Metamorphore is placed in the culture vessel that the above processing is formed It in substrate, is cultivated in the seawater of sand filtration and disinfection treatment, promotes attachment and development of the Metamorphore in substrate.
In experiment, the coral is selected as white Acropora (Acropora pruinosa), those skilled in the art, root According to the characteristic of coral, under design of the invention, the method which improves adhesive ability be can also be applied In coral of other types, such as expansion rose coral etc..
By above-mentioned bromine pyrrolidine alkaloid (-)-Agelastatin A polycaprolactone microballoon sphere being prepared, and with Shelly-shaped coralline algae living body is bromine pyrrolidine alkaloid (-)-Agelastatin A polycaprolactone microballoon sphere to be added in culture medium bottom or in shell Shape coralline algae living body smears bromine pyrrolidine alkaloid (-)-Agelastatin A organic solvent, so that coral Metamorphore be induced to mention The test that the method for high adhesive ability carries out, the results showed that, using shelly-shaped coralline algae living body as the bromine is added in culture medium bottom Pyrrolidine alkaloid (-)-Agelastatin A polycaprolactone microballoon sphere can obviously induce coral Metamorphore to improve attachment base bottom Ability, and then improve the ecological nursing level of coral and the ecological reestablishment level of coral reef.
Embodiment 1
The preparation method of bromine pyrrolidine alkaloid polycaprolactone microballoon sphere described in the present embodiment, comprising the following steps:
1) it is total that bromine pyrrolidine alkaloid (-)-Agelastatin A raw medicine 45mg, polycaprolactone (PCL) 300mg are accurately weighed It is dissolved in sufficiently sonic oscillation after 12mL methylene chloride to mix, obtains oily phase；
2) oil is drawn mutually with syringe and be slowly added dropwise in the polyvinyl alcohol water solution (2% mass fraction) of 100mL, it is high Speed stirring, emulsifying rate 300r.min-1；
3) after closed emulsification 60min, 100r.min will be adjusted under speed-1；Persistently stirring 3h volatilization methylene chloride, makes microballoon Solidification；
4) lotion is centrifugated with 1000r.min-1, obtained solid is to distill water washing, again with 1000r.min-1 Centrifuge separation is washed repeatedly, is centrifuged 3 times；It is placed in cryogenic freezing vacuum drier dry.
Obtained finished product load bromine pyrrolidine alkaloid polycaprolactone microballoon sphere particle is mellow and full, and there is micropore on surface, and size distribution is uniform, Average grain diameter is 50.25 ± 0.47 μm, drugloading rate is that 4.19 ± 0.02%, encapsulation rate is 42.05 ± 0.10%.
Microballoon alkaloidal drug is sustained efficiency by conventional determined by ultraviolet spectrophotometry total alkaloid concentration, in microballoon Start measurement on the day before immersing seawater for the first time, the starting original concentration as control.Opening for first day for seawater is immersed from microballoon Begin, in the seawater METHOD FOR CONTINUOUS DETERMINATION 30d, 12:00 sampling measures once after cleaning up at noon daily, remaining with the microballoon on the same day Alkaloid concentrations and the original concentration ratio of starting are as microballoon alkaloidal drug sustained release efficiency.The result shows that microballoon 1d is released 11.25% alkaloid is put, the average release 3.19% daily of 1-7d discharges to 30d and carries the 28.44% of medicine total amount.
Embodiment 2
Using shelly-shaped coralline algae living body as addition bromine pyrrolidine alkaloid (-)-Agelastatin A in culture medium bottom
Influence to coral Metamorphore adhesive ability.
The acquisition and culture of 1.1 coral larvas
White Acropora (the Acropora of this test selection main hermatypic coral species in South China Sea sea area It pruinosa) is subjects.On May 15th, 2018 by the white Acropora sample strain of acquisition, temporarily supports in seawater pond, waits coral Coral oviposition.2d starts whether the white Acropora in close observation cultivating system ovulates before moon circle (on June 3rd, 2018) Phenomenon, and the conic net with plastic bottle is upside down on oviposition coral, using the buoyancy of coral ovum by egg follicle automatically into modeling Expect in bottle.Frost Acropora starts that egg follicle is discharged when 6 days 20 June in 2018, and discovery ovulates after phenomenon, at once goes to egg follicle It is stirred mixing in the experiment container of 20L, it is made sufficiently to be fertilized, until microscopy discovery fertilization egg division, in indoor plastic barrel Middle cultivation, cultivation temperature are 27 DEG C of natural waters scene water temperature, and continuous charge changes the sea of 1/10 volume in container every 1h Water keeps its water quality good and the stabilization of water temperature, DM500 biomicroscope and its digital analysis using German Leica company System is observed and is photographed to record to coral embryonic development.The strong Metamorphore of travelling ability is selected after 48h for subsequent Test.
The acquisition and processing of 1.2 coralline algaes
Test with shelly-shaped coralline algae be South China Sea common shelly-shaped coralline algae middle period algae (Mesophyllum mesomorphum).By the collected fresh and alive frond being grown on the granite of seabed go removal of impurities algae, select growth it is vigorous and Relatively complete frond washes away silt and other attachments with distilled water, is cut into the small pieces of 4cm × 4cm, temporarily after feeding 7d For coral larva adhiesion test.Dead mussel (Pernaviridis) shell after selecting seabed granite stone and cutting is made It is control without life base material, size is similarly 4cm × 4cm.
The adhiesion test of 1.3 coral Metamorphores designs
After development of fertilized ova 48h, early stage Metamorphore is transferred in 2000mL inflation beaker and carries out adhiesion test, beaker Mouth is sealed with 180 mesh thin,tough silk.Test beaker is put into having a size of 1200mm × 600mm × 600mm coral culturing jar, seawater in cylinder Sand filtration is directly introduced and passed through from Coral Reef Region, is changed daily water 1 time, quantity of exchanged water 30%, water process life-support system recirculated water flow velocity 4t/h is spent, water body is kept to stablize.Two groups are equipped at left and right sides of culturing jar and makes unrestrained pump, and intermittent operation makes wave.Stablize through air conditioner Water temperature makes in beaker and culturing jar water temperature be maintained at 27.5 DEG C by air conditioner.Each beaker selects 20 Metamorphores at random, The number (n) of larva in beaker is recorded daily.Because the larva that larva after death decomposes in for 24 hours, therefore lacks can assert it is in heaven It dies, calculates the pelagic period average time of Metamorphore in various processing schemes.When coral larva is tightly attached to attachment primary surface, change is flat And when differentiating tentacle and diaphragm, the coral larva number (n) adhered in each beaker is counted, while recording the attachment position of larva It sets.Calculate larva adhesive rate and attachment selection.The attachment base bottom of adhiesion test shares 7 seed types, i.e. granite, shell, shelly-shaped Shelly-shaped coralline algae, load bromine pyrrolidine alkaloid polycaprolactone microballoon sphere after coralline algae, high pressure sterilization are uniformly sprayed on shelly-shaped coralline algae Surface (makes the load medicine polycaprolactone microballoon sphere quantity on shelly-shaped coralline algae living body reach 10mg.cm-2), carry bromine pyrrolidine alkaloid gather oneself Interior ester microsphere is uniformly sprayed on granite face, and (microballoon additional amount is 10mg.cm-2), smear bromine pyrrolidine alkaloid shelly-shaped coral (alkaloid concentrations are 20 μ g.ml to algae-1), it is uniformly smeared 2-3 times on shelly-shaped coralline algae living body surface.Observe coral Metamorphore Adhesion condition, Fig. 4 are that coral larva starts to be attached in substrate, and Fig. 5 is that coral larva completes attachment simultaneously in coralline algae substrate Grow tentacle.
Larva adhesive rate (SR) is calculated according to following formula:
SR=(n/20) × 100%.
Adhere to Selection experiment equally using inflation beaker, beaker bottom places 4 kinds of substrates, i.e. granite, load bromine pyrroles altogether Alkaloid polycaprolactone microballoon sphere+granite, shelly-shaped coralline algae, load bromine pyrrolidine alkaloid polycaprolactone microballoon sphere+shelly-shaped coralline algae are (micro- Ball additional amount is 10mg.cm-2), four kinds of substrates respectively account for the 1/4 of beaker bottom area.
The calculation formula of attachment selection (SC) are as follows:
SC=(ni/ n) n in × 100% formulaiFor the larva number for being attached to different base.
Every kind of processing is repeated 3 times.
1.4 data processings: all test datas are analyzed using SPSS18.0, using one-way analysis of variance (one- WayANOVA the significance of difference) examining each processing and compareing.Numerical value P < 0.05 is to have significant difference.
1.4 interpretations of result:
As shown in Figure 1, compared with compareing abiotic hard substrate shell and granite, shelly-shaped coralline algae living body as Larva adheres under base treatment, and the Metamorphore of white Acropora is significantly raised for the adhesive rate of substrate, compared with shell and Hua Gang Rock rises 70.37-84.21% respectively, has reached significant difference (P < 0.05).
Induction coral floating wave children is fallen in order to inquire into biofilm (Biofilm) and the microbial bacteria on shelly-shaped coralline algae surface The possibility function of worm attachment aspect, the adhesive rate difference after analysis of experiments sterilization treatment.When shelly-shaped middle period algae through scrub and After high pressure sterilization processing, the Metamorphore adhesive rate on surface has dropped 39.59%, the adhesive rate after sterilization treatment and no life Rock and shell substrate be in that same numerical value is horizontal, the calcareous hardening structure for showing that shelly-shaped coralline algae is formed is mentioned for coral polyp For hard substrate function in terms of may have no significant difference with marine rock structure, and the biofilm on frond surface and micro- life Object bacterium colony important in inhibiting in terms of inducing larva attachment.
Fig. 2 is the results show that the processing of bromine pyrrolidine alkaloid can significantly improve coral Metamorphore on shelly-shaped coralline algae surface Adhesive rate.There are significant differences for the adhesive rate of different bromine pyrrolidine alkaloid treatment conditions frost occurs Acropora Metamorphore, carry bromine Pyrrolidine alkaloid polycaprolactone microballoon sphere is uniformly sprayed on shelly-shaped coralline algae surface, and (microballoon additional amount is 10mg.cm-2) handle lower coral Coral Metamorphore adhesive rate is higher, has reached 84.23 ± 9.50, than the place for smearing bromine pyrrolidine alkaloid on shelly-shaped coralline algae surface (alkaloid concentrations are 20 μ g.ml to reason-1), larva adhesive rate improves 39.34%, compared with simple shelly-shaped coralline algae substrate, Adhesive rate has more been higher by 88.83%.It is floating under all shelly-shaped coralline algae base treatments compared with abiotic granite substrate Unrestrained larva adhesive rate has reached significant difference (P < 0.05).
Fig. 3 is the coral Metamorphore attachment selection result schematic diagram under different base processing.Due in true seabed Under habitat conditions, the patch of multiplicity is presented in the attachment environment that coral Metamorphore is faced.In order to distinguish Metamorphore to not Attachment with substrate selects, and test simulation devises four kinds of Distribution Patterns of base material.By Fig. 3 result it is found that in four kinds of bases In bottom material processing, carries bromine pyrrolidine alkaloid polycaprolactone microballoon sphere and be uniformly sprayed on shelly-shaped coralline algae surface (microballoon additional amount is 10mg.cm-2) processing frost occurs Acropora Metamorphore attachment selection rate highest, the larva selection rate of granite substrate is most Low, difference reaches 5.87 times between the two.Bromine pyrrolidine alkaloid polycaprolactone microballoon sphere is carried uniformly to be sprayed at granite surface The attachment selection rate of reason frost occurs Acropora Metamorphore is also significantly lower than shelly-shaped coralline algae and shelly-shaped coralline algae+bromine pyrroles's biology Alkali process just corresponds to the 49.36% and 20.75% of the two, is displayed without the participation of shelly-shaped coralline algae living body, only bromine pyrrole The chemical effect of alkaloid is coughed up, is also not enough to induce the attachment of coral Metamorphore.
In view of key player of the shelly-shaped coralline algae in coral Metamorphore attaching process, in view of bromine pyrrolidine alkaloid etc. Important function of the chemical substance in induction coral larva attaching process, induction coral Metamorphore of the present invention improve attachment The method of ability, the following highly chemical induction coral larva attachment under the Research scale of ocean, to shelly-shaped coralline algae The research of process spread more extensively and profoundly, biological prosthetic for current coral reef ecosystem provide most important theories guidance and technology Support.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.

Claims (10)

1. a kind of method that induction coral Metamorphore improves adhesive ability, which comprises the following steps:
Adhere to culture medium bottom by coral larva of shelly-shaped coralline algae living body；
Bromine pyrrolidine alkaloid (-)-Agelastatin A polycaprolactone microballoon sphere is uniformly sprayed on shelly-shaped coralline algae surface；Or in shell The organic solvent solution of bromine pyrrolidine alkaloid (-)-Agelastatin A is smeared on shape coralline algae living body；
Coral Metamorphore is placed in the substrate for the culture vessel that the above processing is formed, is cultivated in the seawater.
2. the method that induction coral Metamorphore according to claim 1 improves adhesive ability, which is characterized in that the coral Coral is white Acropora.
3. the method that induction coral Metamorphore according to claim 1 improves adhesive ability, which is characterized in that step 2) In so that bromine pyrrolidine alkaloid (-)-Agelastatin A polycaprolactone microballoon sphere on shelly-shaped coralline algae living body is reached 1- 100mg.cm-2；Or make bromine pyrrolidine alkaloid (-)-Agelastatin A organic solvent solution smeared on shelly-shaped coralline algae living body Concentration be 1-100 μ g.ml-1。
4. the method that induction coral Metamorphore according to claim 3 improves adhesive ability, which is characterized in that step 2) In so that bromine pyrrolidine alkaloid (-)-Agelastatin A polycaprolactone microballoon sphere on shelly-shaped coralline algae living body is reached 5-20mg.cm-2；Or make bromine pyrrolidine alkaloid (-)-Agelastatin A organic solvent concentration 15-25 μ smeared on shelly-shaped coralline algae living body g.ml-1.。
5. the method that induction coral Metamorphore according to claim 1-4 improves adhesive ability, feature exist In in the organic solvent solution of bromine pyrrolidine alkaloid (-)-AgelastatinA, the organic solvent is methylene chloride.
6. a kind of coral propagation method, which is characterized in that include that any induction coral Metamorphore of claim 1-5 mentions The method of high adhesive ability.
7. a kind of preparation method of bromine pyrrolidine alkaloid polycaprolactone microballoon sphere, which comprises the following steps:
1) bromine pyrrolidine alkaloid (-)-Agelastatin A is weighed, polycaprolactone is codissolved in organic solvent rear sufficiently ultrasound vibration Mixing is swung, oily phase is obtained；Wherein, the mass ratio of bromine pyrrolidine alkaloid (-)-Agelastatin A and polycaprolactone is 40-50: 280-320；
2) it draws above-mentioned oil mutually and is slowly dropped in polyvinyl alcohol water solution, high-speed stirred, closed emulsification；
3) lasting stirring vapors away organic solvent, solidifies microballoon, obtains lotion；
4) by lotion to be centrifugated, obtained solid is washed repeatedly, is centrifuged to distill water washing；It is dry to get.
8. the preparation method of bromine pyrrolidine alkaloid polycaprolactone microballoon sphere according to claim 7, which is characterized in that
The organic solvent is methylene chloride；And/or bromine pyrrolidine alkaloid (-)-AgelastatinA and organic solvent Amount ratio is 40-50mg:10-14mL.
9. according to the preparation method of any bromine pyrrolidine alkaloid polycaprolactone microballoon sphere of claim 7-8, which is characterized in that poly- The concentration of vinyl alcohol aqueous solution is 1.5-2.5% (mass fraction)；And/or in step 2), emulsifying rate 250- 350r.min-1, closed emulsification 50-70min；In step 3), 80-120r.min will be adjusted under speed-1, lasting stirring will be organic Solvent vapors away.
10. the bromine pyrrolidine alkaloid polycaprolactone microballoon sphere being prepared according to claim 7-9 the method.
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