CN101642569A - Usage of active natural product B for preparing antifungal and antitumor product - Google Patents

Abstract

The invention relates to a usage of an ocean fungal extract-active natural product B. According to the result of an in vitro research trial, the active natural product B has certain inhibitory effecton common clinical pathogenic fungi such as white tritirachium album limber, cryptococcus neoformans, trichophyton rubrum and aspergillus fumigatus and the like, is able to inordinately inhibit the growth of tumor cell lines such as liver cancer, breast cancer and lung cancer and the like, and is able to have stronger protective effect on pathological mechanism damages caused by rat multi-infarctcerebral ischemia damage and the like, thus being capable of obviously improving the learning and memory functions of dementia rat, and being applied to prepare dementia product having anti-angiogeniceffect. The invention provides a new pharmaceutical source for the curing of clinical antifungal, antitumor and anti-vascular dementia and directly related diseases thereof, having important value for developing and utilizing the pharmaceutical resource of our country.

Description

Bioactive natural product B is used to prepare the purposes of antifungal and antitumor product

Technical field

The present invention relates to medicine and food technology field, specifically relating to a kind of Chinese medicine extract---bioactive natural product is used to prepare the purposes of antifungal, antitumor and anti-angiogenic property senile dementia product, more particularly relate to a kind of marine fungi extract---bioactive natural product B, promptly 4,6-Dihydroxy-3-methylene-10-propenyl-2-oxa-spiro[4,5] dec-8-ene-1,7-dione is used to prepare the purposes of antifungal, antitumor and anti-angiogenic property senile dementia product.

Background technology

(1) research overview of marine fungi

1, general introduction

Marine microorganism is survived in special biotic environment with it and is had the great potential that produces the novel bioactive material.During the last ten years several, the secondary metabolite of nearly 600 novel structures of isolation identification from marine fungi, chemical compound lot has shown good antitumor, antibiotic and antiviral activity; Other has some compound exhibits to go out unique physiologically active, as anti-senile dementia.

Therefore, Marine microorganism is a research focus in recent years.---especially there is the microorganism of relations such as symbiosis or parasitism in those with marine animal or sea-plant---can produce the unique also metabolite of the strong physiologically active of tool of structure more and more to studies show that Marine microorganism.And the Marine microorganism breeding is fast, can utilize modern microbial technique, and the large-scale industry fermentation has broad application prospects.Scientists is estimated, is developed new marine drug and will place hope on this new field of Marine microorganism.

2, marine fungi

Marine fungi (Marine Fungi) is to live in can form spore and the microorganism of eucaryon structure is arranged in the ocean, is the fungus monoid that can breed and finish life cycle in sea water, can well grow on sea water medium again, claims the sea water fungus again.That is: have the eucaryon structure, can form marine organisms (1. J.Kohlmeyerand E.Kohlmeyer, Marine Mycology, Academic Press, New York, 1979 of spore, the saprophytic or parasitic life of battalion; 2. T.W.Johnson, F.K.Sparrow, Fungi in Oceans and Estuaries, J.Cramer, Weinheim, 1961).

Marine fungi is generally mycelioid and many cells, has only yeast that unicellular appearance was arranged in the stage of development.Nutritional mode except that Acarasiales for the formula of ingesting, mostly be absorption.Some derive from the ocean and can grow and the beginner in the habitat, ocean, are called the obligate marine fungi; Other derive from land or fresh water, but can grow and the beginner in the habitat, ocean, are called facultative marine fungi.

They adapt to the acid-base value of sea water, and the osmophilic strain ability is stronger, and great majority are dwelt and lived in certain basic thing, for example parasitize Sargassum and marine animal, and are perhaps saprophytic on the timber that soaks in sea water; The minority free living for example can be grown in saliniferous wetland and the evergreen chinquapin marsh.Therefore, the distribution of fungus in the ocean depends primarily on host's distribution.

French M.C.de Deere waited approximately and at first find marine fungi from zostera marina 1869 brief histories of research.1934, F.K. Si Paro was studied the algal fungi class in Denmark marine site.Nineteen forty-four, E.S. Ba Hong and D.H. woods moral are delivered the paper of the living fungus of research ocean wood.1961, " ocean and the river mouth fungus " of T.W. Claes Johanson and F.K. Si Paro discussed classification and the biological characteristics of marine fungi.E.B.G. Jones chief editor's in 1978 " aquatic fungi is learned progress " discussed the progress of marine fungi form, physiology and ecological aspect." marine mycology-higher fungus " of J. Cole mayers in 1979 etc. systematically discussed the achievement in research of high marine fungi.

Classification is present, and most scholars think that fungus should independently become boundary (King-dom mycota).But, go back the scheme that neither one is comparatively generally acknowledged so far to the division of affiliated door, guiding principle etc.According to existing record, marine fungi is 500 kinds of less thaies still, only are equivalent to 1% of the true strain number in land (about 50,000 kinds).Comprise 209 kinds of thread high marine fungis, 177 kinds of marine yeast mushrooms, low marine fungi algal fungi class (Phycomycetes) about 70 kinds that wait.In high marine fungi, 56 kinds of 149 kinds of ascomycetes (Ascomycetes), 4 kinds of basidiomycetes (Basidiomycetes) and Fungi Imperfectis (Deuteromycetes) are arranged.

Marine fungi can be divided into Sargassum bacterial parasite, the saprophytic sea water bacterium of timber and spoon sporangiocyst order 3 classes:

1. the parasitic kind on the Sargassum has knee Pseudomonas, genus thraustochytrium, chain Chytridium, Saprolegnia, hat spore shell to belong to, belong to and the mould genus of change spore etc. every sorosphere shell genuss, ball seat Pseudomonas, proximal branch chain spore;

2. the saprophytic sea water bacterium of common timber has hat spore shell to belong to, the shell genus is given birth in the sea, wood is given birth to shell genus, oar spore shell genus, Leucosporidium, plan Corallium Japonicum Kishinouye spore genus, Humicola and do not have stalk spore genus etc.;

3. spoon sporangiocyst order colonizes on the red algae, and the ability of decomposing halogen is stronger.The evidence that provides mushroom to originate from red algae is provided the spoon sporangiocyst, and the someone thinks that it is that timber living ascomycetous direct ancestors in the sea water.

The distribution marine fungi is widely distributed, to the deep-sea, to halmeic deposit, its member is arranged all from the sandy beach, shallow sea from Intertidal zone high-water mark or river mouth.Marine yeast and lower fungus grow nonparasitically upon another plant on plankton and animal body, thereby at big midocean distribution are arranged also; The growth of high marine fungi requires suitable basic thing as dwelling living place, therefore focuses mostly on and is distributed in littoral sea.Because the saprophytic or parasitic life of marine fungi battalion is so the characteristics of its geographical distribution are the geographic ranges that depends on the host.But the concentration of dissolved oxygen and sea water water temperature in the sea water also are to influence marine fungi to exist and the important factor that develops.Nearly all fungus all can be in less than sea water grows under the condition of sodium oxide concentration, so salt tolerance can not be as the sign of distinguishing marine fungi and land fungus.

Derive from the depth of water and surpass fungus in 500 meters marine environment, have the ability that adapts to high pressure, low temperature and grow significantly.The deep-sea fungus of now knowing has only 5 kinds, and the maximum water depth of collected specimens is 5315 meters.

The most of marine fungis dependences of ecological habit are dwelt certain basic thing and are lived, and have only the minority fungus not rely on basic thing and free living.

According to its living habit of dwelling, marine fungi can be divided into 5 kinds of basic ecotypes:

1. wooden living fungus.The widest with the distribution at most higher fungus of quantity in ocean water body, battalion's saprogenesis, kind decomposition of cellulose.It is more extensive to distribute in tropical marine site and neritic environment.Known have 76 kinds of ascomycetes, 29 kinds of Fungi Imperfectis, 2 kinds of basidiomycetes.

One class quantity at most, the widest high marine fungi distributes.Can decompose timber and other fibrous matters consumingly.They are extensive than temperate zone and polar region in the distribution in tropical marine site, and are extensive than the deep-sea in the distribution of neritic environment.In the living fungus of known 107 kinds of oceans wood, ascomycetes accounts for 76 kinds, and Fungi Imperfecti accounts for 29 kinds, and basidiomycetes has only 2 kinds.

2. parasitic frond fungus.Account for 1/3 of marine fungi kind number, wherein in the majority with ascomycetes.Types such as saprophytic, parasitism and symbiosis are arranged.

In marine fungi, there are 1/3 kind and algae to be related approximately, wherein in the majority with ascomycetes.Types such as saprophytic, parasitism and symbiosis are arranged.The distribution of different Sargassums can directly influence the distribution of such fungus, as in the Sargasso Sea of the North Atlantic Ocean, kind of fungus and quantity find seldom also by the Sargassum of fungal infection that all seldom this is because Alga Sgrgassi Enerves (Sar as-sum spp) has the tannin material of antibacterial action.In general, rot saccharomycetic quantity on the Sargassum to be higher than on the frond alive and sea water in quantity; Be attached to the yeast quantity on red algae and the chlorella, be higher than on the Brown algae.Because the excretory phenolic material mass-energy of Brown algae suppresses the growth of marine fungi.Some marine fungis combine the coalition that forms obligate symbiosis with specific Sargassum, be the ocean lichens.

3. mangrove fungi.Mostly be saprophytic bacteria, wherein ascomycetes is 23 kinds, 17 kinds of Fungi Imperfectis, 2 kinds of basidiomycetes.

Rhizophora apiculata Blume is the plant (show woods biome) of tropical and subtropical zone Intertidal zone salt moorland.The marine fungi that gives birth in Rhizophora apiculata Blume that dwells is saprophytic bacteria mostly, 23 kinds of ascomycetes is wherein arranged, 17 kinds of Fungi Imperfectis, 2 kinds of basidiomycetes.Be immersed in that Rhizophora apiculata Blume pivot in the sea water is done, after the epidermis of branch and root is perforated animal, stormy waves or artificial damage, very easily invaded and cause rotten by fungus.But be embedded in the root in the mud, then be not subject to the fungus infringement.Marine fungi can be decomposed the Rhizophora apiculata Blume blade, for the ocean provides a large amount of organic debris.

4. zostera marina fungus.Quantity seldom, many perch are in leaf portion.

Negligible amounts, wherein the fungus of the perch leaf portion root person of dwelling is many.Contain the material of tannin and other inhibition biological growths in the zostera marina root, have only those marine fungis that can resist this class material could on the zostera marina root, adapt to growth.

5. zooparasite body fungus.Only limit colonizes in ectoskeleton and shell portion place.

Being confined to colonize in ectoskeleton, shell etc. locates.In the processes such as cellulose, chitin, protein and calcium carbonate of marine fungi in decomposing animal body, play an important role.Low marine fungi such as grade is the important pathogenic bacteria that causes marine fishes and invertebrates disease.

Marine fungi often has following characteristics:

If 1. belong to Ascomycotina, then be mainly the member of the gang pyrenomycetes of decomposition of cellulose vigor, shell of ascus black, ascospore often have the appendage that contains acidic polysaccharose, help sticking on the new substrate.Usually no conidium from generation to generation.

2. if imperfect fungi, then spore is a roof spore type, mostly is dark-coloured.

Marine fungi and marine bacteria are all participated in the regenerative process of organic decomposition in ocean and inorganic nutrients thing, constantly provide effective nutrition for sea-plant; But marine fungi is bacterial parasite and the pathogenic bacterium of marine animal, and the sea-plant that can make that has is caused a disease, even the wooden structures in the port equipment is rotted; Some marine fungi can be destroyed Polymer Synthesizing materials such as polyurethanes.

The meaning marine fungi is the same with marine bacteria, participates in the decomposition of ocean organic substance and the regenerative process of inorganic nutrients thing, for sea-plant constantly provides effective nutrition, occupies critical positions in marine food chain.Particularly fungal mycelium in marine sediment and yeast thalline provide the source of bait for ocean protozoacide, zoobenthos etc.Some marine fungi can produce antibiotics and other biological active substanceies, is the huge treasure-house of new drug development, and is also significant in ecology and application facet, and the pollutant as in the degraded marine environment promote ocean self-cleaning etc.; Utilize marine fungi processing Testa Tritici, bagasse, Caulis et Folium Oryzae etc., make cheap microorganism crumb mixture, as the feedstuff in the aquaculture.

Marine fungi is bacterial parasite and the pathogenic bacterium of some marine animal, once sustains losses severely because of the infringement of fungus as U.S. Concha Ostreae production area.Can cause the disease of some sea-plant, once seriously be infected by net Acarasiales genus fungus as the zostera marina in the North Atlantic Ocean, pacific rim and Europe.The living fungus of ocean wood can rot wooden structures and other fibrous materials in port facilities, breakwater and the weir dike.Some marine fungi can be destroyed the synthetic material in the sea water, and can expanding after being penetrated by them as polyurethane material, it is bad to split.

3, the chemical constitution of bioactive natural product B and preparation method

The metabolite B of marine fungi Massarina tunicata obtains for therefrom separating in the marine fungi of state sea first.Activated product B and mannitol all have higher content in the fungus, if its condition of culture is carried out more deep research, Massarina tunicata can be developed as a kind of resource bacterium, further strengthen the research aspect raising immunologic function, anticancer, the anti-ageing effect of waiting for a long time.

In the metabolite of marine fungi #K26, the choline sulfuric ester obtains for separating from southern China sea marine fungi first; 3,4, the 5-trihydroxybenzoic acid is to find from marine fungi first.Choline sulfuric ester and mannitol all have higher content among the fungus #K26, if its condition of culture is carried out more deep research, #K26 can be developed as a kind of resource bacterium.The choline sulfuric ester (is called for short: COS) extensively be present in occurring in nature, play the part of important role aspect the microorganism conversion of its sulfur in soil.Various plants, soil fungi and antibacterial can both utilize choline and PA (3 '-phosphoadenosine-5 '-phosphosulphate) as substrate, synthetic COS under sulfur conversion enzyme catalysis, hydrolysis can take place in COS under the effect of choline sulfuric ester hydrolytic enzyme on the other hand, becomes one of source of interior choline of organism and sulfate group.Key player of the row of COS is as the impermeabilisation chemical compound.3,4,5-trihydroxybenzoic acid (being gallic acid) is a kind of polyphenol compound, owing in the molecule a certain amount of ROH base is arranged, can be formed with the hydroperoxyl radical (H) of antioxidation, and superoxide anion (O is arranged ₂ ^-) and the activity of hydroxyl radical free radical free radicals such as (OH), thereby protective tissue is avoided the infringement of Oxidation, and improve immunologic function, anticancer, the anti-ageing effect of waiting for a long time.

By to marine fungi Massarina sp., the further investigation of strain CNT-016, found at present spiro lactone chemical compound 6-epi-5 '-hydroxy-mycosporulone (referring to: M.A.Abdel-Wahab et al./Phytochemistry 68 (2007) 1212-1218), but do not carry out deep bioactivity research as yet, the chemical compound that research of the present invention relates to has similar parent nucleus with it, and has carried out the active and anti-angiogenic property senile dementia research of antifungal and antitumor.

(2) extraction separation method of conventional effective ingredient

1, solvent extraction method

(1) principle: solvent extraction method is according to the extract raw material dissolution properties of various compositions in solvent in the Chinese herbal medicine for example, select for use the active component dissolubility big, to not needing the little solvent of stripping composition dissolubility, and the method that effective ingredient is dissolved out in the medical material tissue.When solvent is added to extract raw material (Chinese herbal medicine for example, suitably pulverizing) in the time of in, solvent is owing to diffusion, osmosis penetrate in the cell by cell wall gradually, dissolved solable matter, and cause concentration difference inside and outside the cell, so intracellular concentrated solution is constantly to external diffusion, solvent constantly enters in the medical material histiocyte again, so repeatedly come and go, when solution concentration reaches dynamic equilibrium inside and outside cell, this saturated solution is leached, continue repeatedly to add novel solvent, just can be bordering on complete stripping or the stripping of big portion to desirable ingredients.The dissolubility of extract material composition in solvent is directly relevant with solvent property.Solvent can be divided into hydrophilic organic solvent and lipotropy organic solvent, and dissolved material also has hydrophilic and lipophilic difference.Hydrophilic radical is many in the organic compound molecule structure, and its polarity is negligent of oil greatly; The hydrophilic radical that has is few, and its polarity is little and be negligent of water.The character of each kind solvent, equally also relevant with its molecular structure.Like this, the inventor just can remove to estimate their this type of character and the solvent of selecting for use by to for example medicinal herb components structural analysis of extract raw material.Generally speaking,, bigger dissolubility will be arranged therein, i.e. the rule of so-called " similar mixing " as long as this character of the hydrophilic of extract material composition and lipotropy and solvent is suitable.This is to select appropriate solvent to extract one of foundation of required composition in the extract raw material.

(2) choice of Solvent: the key of utilization solvent extraction method is to select appropriate solvent.Solvent is selected suitably, just can be more successfully the composition of needs be extracted.Selective solvent will be noted following 3 points: 1. solvent is big to the effective ingredient dissolubility, and is little to the impurity dissolubility; 2. solvent can not play chemical change with the composition of Chinese medicine; 3. solvent want economical, be easy to get, safe in utilization etc.Common extraction solvent can be divided into following three classes:

1. water: water is a kind of strong polar solvent.Hydrophilic composition in the extract raw material can both be gone out by water-soluble as the not too big polysaccharide of inorganic salt, saccharide, molecule, tannin, aminoacid, protein, acylate, alkaloid salt and glycoside etc.In order to increase the dissolubility of some composition, also often adopt sour water and aqueous alkali as extracting solvent.

Ethanol), methanol (but also claims: another name for), acetone etc., the most frequently used with ethanol 2. hydrophilic organic solvent: just general said and the miscible organic solvent of water (claim not only: as ethanol.Alcoholic acid solubility property is relatively good, and is stronger to the penetration capacity of extract raw cell.Outside hydrophilic composition isolating protein, phlegmatic temperament, pectin, starch and the part polysaccharide etc., big multipotency dissolves in ethanol.Be insoluble in the low-polarity component of water, the dissolubility in ethanol is also bigger.Can also adopt Different concentrations of alcohol to extract according to the character that is extracted material.More less than water consumption with ethanol extraction, extraction time is short, and it is also few to dissolve the water-solubility impurity that.Ethanol is organic solvent, though inflammable, toxicity is little, low price, and convenient sources has a locking equipment can reclaim repeatedly and use, and alcoholic acid extracting solution is difficult for moldy metamorphism.Owing to these reasons, be one of always the most frequently used method with the method for ethanol extraction.The character of methanol is similar with ethanol, boiling point lower (64 ℃), but toxic, should note during use.

3. lipophilic organic solvent: the organic solvent that just general said and water can not be miscible, as petroleum ether, benzene, chloroform, ether, ethyl acetate, dichloroethanes etc.These choice of Solvent performances are strong, can not or be not easy to propose hydrophilic impurities.But this kind solvent volatility is big, how inflammable (except the chloroform), generally poisonous, price is more expensive, equipment requirements is higher, and they penetrate plant tissue ability a little less than, often need to extract repeatedly for a long time and could extract fully.If contain more water in the medical material, just be difficult to leach its effective ingredient with this kind solvent, therefore, when extracting the extract raw material in a large number, directly using this kind solvent has certain limitation.

(3) extracting method: with solvent extraction extract material composition, infusion process commonly used, percolation, decocting method, reflux extraction and continuous backflow extraction method etc.Simultaneously, factors such as the degree of grinding of raw material, extraction time, extraction temperature, appointed condition also can both influence extraction efficiency, must take in.

1. (be called for short: infusion process): the dipping genealogy of law is packed extract material powder or fragment in the proper container, adds The suitable solvent (as ethanol, rare alcohol or water), and the dipping medical material is with the stripping method of composition wherein to flood extraction method.This law is relatively simple, but leaching rate is relatively poor, and is solvent as water, and the easy moldy metamorphism of its extracting solution must note adding suitable preservatives.

2. (be called for short: percolation): percolation is that extract raw material powder art is contained in the percolator to the percolation extraction method, constantly adds novel solvent, makes it penetrate medical material, flows out a kind of leaching method of leachate from top to bottom from the percolator bottom.When moving down when solvent infilters medicated powder, stripping composition proportion strengthens, its position is just replaced in the solution on upper strata or rare immersion, causes good concentration difference, and diffusion energy is carried out preferably, so leaching effect is better than infusion process.But should control flow velocity, in oozing transient, on powder, replenish novel solvent at any time, make till effective ingredient fully leaches in the medical material.Maybe extremely shallow or when oozing the volume that gushes liquid and being equivalent to heavy 10 times of crude drug when oozing the dropping liquid color, just can think and extract basically fully.The rare leachate that often will collect in mass production is as the usefulness of the solvent of another batch new raw material.

3. (be called for short: decocting method): decocting method is traditional leaching method that China uses the earliest to decoct extraction method.Used container is generally pottery, sand jar or copper, enamel ware, should not use iron pan, in order to avoid the medicinal liquid variable color.Preferably stir often during straight fire heating, in order to avoid that local medical material is heated is too high, burnt easily the paste.Big reaction pot, big copper pot, barrel are adopted in the pharmaceutical factory that steam-heating apparatus is arranged more, or feed Steam Heating in the pond of cement block.It is interconnection by pipeline also several can be decocted device, fries in shallow oil continuously and soaks.

4. heating and refluxing extraction method: use the organic solvent heating extraction, need to adopt the reflux device, in order to avoid the solvent evaporates loss.When operating in a small amount, can on round-bottomed flask, connect reflux condenser.The powder charge material is about 20%～60% of capacity in the bottle, and solvent soaked the about 1～2cm in medical material surface.Reflux in water-bath, the general maintenance, seethed with excitement 3～6 hours, puts cold filtration, and solubilizer in medicinal residues is made second and third time reflux and is made an appointment with half an hour respectively again, or to carrying till the most effective ingredient substantially.This method extraction efficiency adopts continuous extractions than the cold-maceration height more in the mass production.

5. continuous backflow extraction method: use volatile organic solvent and extract the extract material effective component, no matter small test or large-scale production, all with continuous extraction for well, and need with quantity of solvent lessly, the extraction composition is also more complete.Laboratory fat-extraction device commonly used or title apparatus,Soxhlet's.Continuous extraction generally needs a few hours could extract fully.It is longer to extract the composition heated time, and the labile composition of case of thermal instability should not adopt this method.

2, separation and purification process:

Said extracted method resulting extract raw material extracting solution or extract remain mixture, need further remove impurity, separate and make with extra care.

(1) solvent segregation: generally be with above-mentioned total extract, select three for use, the solvent of four kind of opposed polarity, by low polarity to high polarity proceed step by step extraction separation.Aqueous extract or ethanol extract often are jelly, be difficult to be dispersed in the low polar solvent, so can not extract fully, can admix an amount of inert filler, as kieselguhr or fiber powder etc., low temperature or natural drying then, after the pulverizing, to select for use solvent to extract successively, make each constituent in the total extract again, obtain according to the difference of its dissolubility in the opposed polarity solvent separating.Utilize the extract material chemical component, the dissolubility in the opposed polarity solvent carries out separation and purification, is the most frequently used method.

(2) solvent extraction:

1. extraction: the solvent extraction extraction is called for short extraction again, is to utilize the difference of each composition partition coefficient in two kinds of immiscible solvents in the mixture and reach isolating method.If each composition partition coefficient in solvent differs big more during extraction, then separation efficiency is high more; If the effective ingredient in aqueous extract is lipophilic material, the general lipotropy organic solvent of using more, extract as benzene, chloroform or ether, if effective ingredient is to be partial to hydrophilic material, indissoluble is separated in lipophilic solvent, just need use weak lipophilic solvent, for example ethyl acetate, butanols etc. instead.Can also in chloroform, ether, add an amount of ethanol or methanol to increase its hydrophilic.When extracting flavones ingredient, how to extract with ethyl acetate and water.Then multiselect n-butyl alcohol, isoamyl alcohol and water extract to extract the strong saponin of hydrophilic.But, the common organic solvents hydrophilic is big more, and the effect of doing extraction with water is just bad more, and is because more hydrophilic impurities is followed, very big to the further refining influence of effective ingredient.

2. counter current continuous extraction method: be a kind of successive solvent extraction.Its device can have one, several or more extracting tube.Fill the contact surface during with increase solvent extraction in the pipe with little porcelain circle or little rustless steel wire ring.If a kind of infusion of extract raw material need extract with the benzene lighter than water, ethyl acetate etc., then need water extracting liquid is contained in the extracting tube, and benzene, ethyl acetate are stored in the high-level container.Extract whether complete, but sample thief is analysed with thin layer chromatography, ply of paper and chromogenic reaction or precipitation are checked.

3. counter-current distribution method: counter-current distribution method claims CCD method, counter-current distribution or countercurrent distribution again.Counter-current distribution method is consistent with solvent counter-current extraction principle, but the application of sample amount is certain, and continuous in the solvent of a constant volume, reaches the separation of mixture through repeatedly being shifted the extraction distribution.

4. drop counter-current distribution method: the drop counter-current distribution method claims the droplet countercurrent chromatography method again.Be improved solvent extraction on the counter-current distribution method basis in recent years.To the same substantially counter-current distribution method of the selection of solvent system, but requirement can be separated at short notice, and can generate effective drop.Because mobile phase forms drop, contacting effectively with immobile phase in thin distribution extracting tube, rubbing constantly forms new surface, promotes the distribution of solute in solvent, so its separating effect is often good than counter-current distribution method.

(3) macroporous adsorbent resin method: macroporous adsorbent resin is a class organic polymer adsorbent that grows up the sixties in 20th century, have the good adsorption performance, be applied to the development of the extraction separation and the new Chinese medicine of extract material chemical component surplus in the of nearly ten over year gradually.

Macroporous adsorbent resin is for absorption and screen the parting material that principle combines.Its adsorptivity is because the result of Van der Waals force or generation hydrogen bond.The screening principle is because itself cellular structure determines.Because absorption and screening principle, organic compound separates through certain solvent elution on macroporous adsorbent resin according to the difference of absorption affinity and the size of molecular weight.This make organic compound especially the purification of water soluble compound simplified greatly.The skeleton of macroporous adsorbent resin is generated by styrene and divinylbenzene polycondensation, because the adding of modifier, the polarity of macroporous adsorbent resin changes, and according to the surface nature of resin, that adsorbent resin generally is divided into is nonpolar, Semi-polarity and polarity three classes.

Nonpolar adsorption resin be by the very little monomer-polymer of dipole moment make not with the adsorbent resin of any functional group.Typical example is the adsorbent resin of styrene-divinylbenzene system, as D101, XAD-1, DiaionHP-10 macroporous adsorbent resin.

The Semi-polarity adsorbent resin refers to contain the adsorbent resin of ester group, as an acrylate or a crosslinked analog copolymer such as methacrylate and double methyl methacrylate.It is on the basis of nonpolar macroporous adsorption resin, adds acrylic acid methyl ester. or acrylonitrile polycondensation and forms, as the frequent AB-8 macroporous adsorbent resin that uses of China China.

Polar Adsorbent Resin is meant that amide-containing, itrile group, phenolic hydroxyl group etc. are nitrogenous, the adsorbent resin of oxygen, sulfur polar functionalities base.In addition, sometimes the ion exchange resin of ligand groups such as nitrogenous, oxygen, sulfur is called strong Polar Adsorbent Resin, the boundary of strong Polar Adsorbent Resin and ion exchange resin is difficult to difference.Polar macroporous adsorption resin can be formed by methyl methacrylate, acrylamide or the polycondensation of sulfoxide class, as the Diaion HP 2MG of Mitsubishi chemical industry, the XAD-10 of U.S. Rohm-hass company, XAD-9 macroporous adsorbent resin.

Compare with other adsorbent with active carbon, macroporous adsorbent resin has a lot of advantages, and is higher as the adsorptive selectivity to certain material; Physical and chemical stability and mechanical strength are better; Description is more, can change resin physics or chemical constitution as required; Adsorbent resin is generally spherical particle, and fluid resistance is less or the like.Thereby be widely used in chemical industry, medicine and other fields, more and more about the applied research report of macroporous adsorbent resin in natural product extraction is separated in recent years.Macroporous adsorbent resin all has certain adsorption to extract material chemical component such as alkaloid, flavone, saponin, coumarin and some other glycoside compositions.Absorbability to sugar is very poor, and is stronger to the absorbability of pigment.

(4) sedimentation method: be in extract raw material extracting solution, to add some reagent to make the generation precipitation, with the method that obtains effective ingredient or remove impurity.As lead salt precipitation: lead salt precipitation is one of classical way of separating some extract material composition.Because lead acetate and Lead monosubacetate in water and alcoholic solution, can generate the lead salt or the complex salt precipitation of indissoluble with multiple extract material composition, so can utilize this character that effective ingredient is separated with impurity.Then lead salt precipitation is suspended in the novel solvent, passes to hydrogen sulfide gas, make and decompose and transfer insoluble vulcanized lead to and precipitate.

(5) salting out method: salting out method is in the water extract of extract raw material, adds inorganic salt to finite concentration, or the state that reaches capacity, and can make the dissolubility of some composition in water reduce precipitation and separate out, and separate with the big impurity of water solublity.Be commonly used for the inorganic salt of saltouing sodium chloride, sodium sulfate, magnesium sulfate, ammonium sulfate etc. are arranged.

(6) dialysis: dialysis is to utilize small-molecule substance can pass through semipermeable membrane in solution, and macromolecular substances can not reach isolating method by the character of semipermeable membrane.Otherwise also macromolecular impurity can be stayed in the semipermeable membrane, and micromolecular material is entered in the outer solution of film by semipermeable membrane, and separation and purification in addition.

(7) crystallization, recrystallization and Steppecd crystallization: identify the extract material chemical component, study its chemical constitution, must at first the extract material composition be prepared into the pure product of monomer.At normal temperatures, the character of material own is the chemical compound of liquid, can carry out separation and purification with fractionating process or chromatography respectively.In general, the extract material chemical component is solid material at normal temperatures mostly, all has the general character of crystalline solid, can reach the purpose of separation and purification according to the difference of dissolubility with crystallization process.

3, conventional drying method

(1) vacuum drying: be based on such ultimate principle: water saturation vapour pressure and temperature are closely related, under vacuum state, the boiling point lowering of water, i.e. operation operation at low temperatures just under vacuum, can avoid the destruction of nutritional labeling such as vitamin etc. at high temperature, improve rate of drying simultaneously.Vacuum drying is widely used in industries such as food, pharmacy, chemical industry, and China also develops and introduced various vacuum dryers, and its version is varied.Form commonly used mainly contains box type vacuum exsiccator, bipyramid formula vacuum desiccator, belt vacuum desiccator etc.These traditional Minton dryers mainly adopt heating such as hot blast, steam or electricity, utilize conduction of heat, convection current or radiation theory that heat is passed to material inside from the outside.It is low that vacuum drying has a baking temperature, and anoxia relatively in the hothouse can be avoided fat oxidation, and series of advantages such as pigment brown stain are suitable for the drying of heat sensitivity food material, and equipment cost, dry expense are also relatively low in addition.

(2) spray drying: be that fluidization technique is used for the exsiccant a kind of method of liquid material.Because of being wink-dry, be specially adapted to heat sensitive material, so the products obtained therefrom quality is good, keep original color, smell and taste, and easily dissolving.The research that utilizes spray drying to prepare microcapsule is carried out, it is that heart material is suspended in the solution of dress material, through centrifugal atomizer it is sprayed in the thermal current, the product of gained is the microcapsule that dress material bag heart material forms, this microcapsule powder can be used in direct compression, also can prepare capsule, syrup or suspensoid.

(3) lyophilization: be that the dry liquid material is frozen into solid, under the low-temperature reduced-pressure condition, utilize the distillation performance of icing, make the low-temperature material dehydration and reach exsiccant a kind of method.Because material is dry under high vacuum and cryogenic conditions, so the drying of some extremely thermo-labile article is well suited for.Wang Dalin has reported a kind of spraying ventilation lyophilizing new technique, be to utilize cold air or nitrogen as medium, the scars of flowing through rapidly make water sublimate, the product microgranule that makes of spraying lyophilizing is little, fast drying, time are short, evenly, good fluidity, and the good instant capacity of tool.In recent years, plaster material and the exsiccant research of sticky material have been obtained bigger progress, fluidization technology, spraying technique, inert carrier technology then grow up on this research basis.Rotatingandflashstreamingdrier, thermojet pneumatic drier, inert carrier drying machine all are fit to the drying of heat sensitive material and plaster material.These new achievements in research are used for Chinese medicine preparation production, with improving the technical merit of Chinese medicine processing greatly, enhance productivity.

(4) far infrared heating drying method: be a new dry technology, its drying principles is to change electric energy into far infrared radiation, thereby by the molecule absorption of medical material, produce resonance, cause the vibration and the rotation of molecule and atom, cause the object heating,, finally reach exsiccant purpose through thermal diffusion, evaporation and chemical change.Far-infrared ray drying can be saved electric energy 20%～50%, and effect is better.

(5) micro-wave drying method: be the new technique that develops rapidly a sixties in 20th century, microwave drying is actually by eddy-current heating and medium heating, make moisture and fat in the thing that is dried absorb microwave energy to some extent, thereby and it is changed into heat reach exsiccant purpose.But microwave drying killing microorganisms and mycete, and has disinfective action.The microwave heating installation of China's production at present has 915mhz and two frequencies of 2450mhz.

(3) research overview of vascular dementia

Dull-witted (Dementia) is the acquired and persistence disturbance of intelligence syndrome that produces owing to disordered brain function, and disturbance of intelligence comprises that in various degree memory, language, visual space function, abnormality of personality and cognitive competence reduce.

Dull-witted main type is to comprise vascular dementia (vascular dementia, or claim: the vascular senile dementia, be called for short: VD), the senile dementia in the senile dementia (Alzheimer ' s Disease, or claim: alzheimer's disease, or claim: Alzheimer's disease, be called for short: AD) etc.

Senile dementia comprises senile dementia, multiple infraction type dementia (Multimfarct Dementia), alcoholic dementia disease (Alcoholic Dementia) and normal brain activity setting-out brain disease (Normal Pressure Hychocephalus), and wherein senile dementia is the main type in the senile dementia.Because AD falls ill in old people more than 60 years old well, so custom is called as degenerative brain disorder.

Vascular dementia is caused by cerebrovascular, is mainly caused by cerebral hypoxia ischemia.Common clinically multi-infarct dementia, large tracts of land cerebral infarction dementia, thalamic dementia and types such as concurrent dementia of hemorrhagic cerebrovaseular and Combination dementia.Clinical manifestation is depression, delirium, aphasia etc.Pathological change is that multiple lacuna sexually transmitted disease (STD) becomes or large tracts of land infarction kitchen range and atherosclerotic lesion (referring to the neurological, the 4th edition, People's Health Publisher).Clinical treatment can use the cholinergic inhibitor, and blood vessel dilating, cerebral blood flow increasing amount perhaps utilize cerebral protective agent-antioxidant or free radical scavenger can improve ischemia, the caused pathology damage minimizing brain cell necrosis of anoxia and apoptosis, protection cerebral tissue.

The vascular dementia person only shows nearly dysmnesia in early days, and memory far away keeps better relatively; With the increase of the course of disease, memory ability far away is lost gradually, and hypophrenia is gradual and increases the weight of.

And alzheimer disease just shows far and near dysmnesia in early days, hypophrenia presents a kind of (Guo Mingying of progression slowly, Korea S's tinkling of pieces of jade, etc. the comparative study of alzheimer disease and vascular dementia disturbance of intelligence. the journal .2007 of Qinghai Medicine College, 28 (2): 125-127.).

The vascular risk factor is all being played the part of important role in the morbidity of vascular dementia and alzheimer's disease, so there is theory to think that alzheimer's disease and vascular dementia may not be two independent illness.The general character research of alzheimer's disease and vascular dementia is carried out widely in various aspects such as risk factor, pathogenesis, Pathophysiology, iconography changes.

Vascular dementia is the carrying out property decline disease by the acquired Premium Features of cerebral cortex due to apoplexy repeatedly or the long-term chronic cerebral ischemia.

Show in America and Europe's investigation, dementia incidence rate about 1.1% among the old man more than 55 years old, wherein alzheimer's disease accounts for top priority (incidence rate about 7.7 ‰), be about 50%～60% of dull-witted total number of persons, and VD occupies time (incidence rate about 1.5 ‰), account for 10%～20%, and with age, the VD proportion significantly increases (1. Ott A, Breteler MMB, HarskampF, Stijnen T, Hofman A.Incidence and risk of dementia:the Rotterdam study.Am.J.Epidemiol., 1998,147 (6): 574-580.; 2. Di Carlo A, Baldereschi M, Amaducci L, et al.Incidence of dementia, Alzheimer ' s disease, and vascular dementia in Italy.The ILSA Study.J.Am.Geriatr.Soc., 2002; 50:41-48.).

The nearest research of China also shows, the AD incidence rate about 3.5% among the old man of over-65s, VD incidence rate 1.1% (ZhangZX, Zahner GE, Roman GC, et al.Dementia subtypes in China:prevalence in Beijing, Xian, Shanghai, and Chengdu.Arch Neurol.2005; 62 (3): 447-453.).

Owing to also lack the cognitive impairment pattern that is applicable to diagnosis VD at present, discriminating and " mixed type " dull-witted (AD+ cerebrovascular for the diagnosis of AD and VD, be called for short: diagnosis AD+CVD) still has certain challenge (1. plain chrysanthemum of fourth, Li Yunxia. the clinical diagnosis of vascular dementia and treatment. foreign medical science: cerebrovascular disease fascicle .2005,13 (9): 676-680; 2. Erkinjuntti T:Vascular dementia:challenge of clinical diagnosis.Iht Psychogeriatr.1997; 9:51-58.), so someone thinks that vascular dementia may be a dull-witted type the most common among the old people, mainly be because a lot " mixed types " dull-witted (AD+CVD) may be diagnosed as AD (1. Rom á m GC.Vascular dementiamay be the most common form of dementia in the elderly.J Neurol Sci.2002,203-204:7-10; 2. the plain chrysanthemum of fourth. vascular dementia. Chinese gerontology magazine .2003; 23 (4): 200-202.).

The incidence rate of VD is at men and women's no significant difference (Anderson K, Launer LJ, Dewey ME, et al.Genderdifferences in the incidence of AD and vascular dementia:the EURODEM studies.Neurology.1999; 53:1992-1997.), its the most significant risk factor is age [Hebert R., Lindsay J., Verreault R., Rockwood K., Hill G., Dubois MF.Vascular dementia:incidence and risk factors in the Canadianstudy of health and aging.Stroke, 2000; 31 (7): 1487-1493.], according to Epidemiological study, in greater than 60 years old crowd, the sickness rate of 5 years old VD of every increase of age just increases by 1 times.

Two main causes that cause VD are that (ischemic heart disease, be called for short: IHD), and the two all is a commonly encountered diseases in the old people for apoplexy and ischemic heart desease.Only with regard to apoplexy, the present annual whole world increases apoplexy patient 1,000 ten thousand newly, China (the 1. Feigin VL that just has 2,000,000 wherein, Lawes CMM, Bennett DA, et al.Strokeepidemiology:a review of population-based studies of incidence, prevalence, and case-fatality inthe late 20th century.Lancet Neurol., 2003,2 (1): 43-53; 2. Wu Zhao revives, Yao Chonghua, Zhao's winter. the epidemiological study of Chinese population stroke onset rate, mortality rate. and Chinese epidemiology magazine, 2003; 24 (3): 236-239.), sickness rate is high especially in the old people, thereby causes the concurrent VD of a lot of patients.The national dull-witted epidemiology statistics data of China also shows the aging along with population, the change of dietary structure, and the morbidity of VD is and increases progressively trend.

Along with the aging of population, the sickness rate of cardiovascular and cerebrovascular disease raises year by year, and concurrent VD patient also increases gradually.VD is the grievous injury patient health not only, brings long-term misery to the patient, influences patient's quality of life, brings white elephant also for society and family, has caused the common concern of countries in the world, and it is an important topic in the geriatrics field.Generally believe that the age influences by many-side the effect of VD, for example the aging change of metabolism, blood brain barrier and the autonomic function aspect of self the regulating of brain, cell makes cerebrovascular be easy to suffer damage, and encephaloclastic accumulative total effect also is one of reason.

Dementia diagnosis must meet three conditions: 1. dull-witted; 2. cerebrovascular; 3. the two is closely related more than.Dull-witted and apoplexy is maintained close ties with in time, takes place dull-witted usually after the apoplexy in 3 months.Cardinal symptom is: 1. the cerebral tissue pathomorphism changes; 2. hypomnesis.

About the treatment of VD, at present still do not have sure Therapeutic Method and can change the whole course of disease, because downright bad brain cell can not reverse behind the cerebral infarction, mainly be to alleviate stage of attack symptom, prevention infringement etc. again to the treatment of the brain cell of blood supply insufficiency.Normal employing actively improves the method for brain cell oxygen supply, microcirculation improvement, the new thrombosis of prevention and re-infarction etc.Improve cerebral circulation treatment commonly used 9,10-Dihydroergotoxine class, calcium ion antagonist, nicotinic acid class, other medicament for expanding vascellum and medicament for resisting platelet aggregation arranged.Cholinesterase inhibitor is a developed recently dementia treatment medicine comparatively rapidly, example hydrochloric acid donepezil and rivastigmine-hydrogentartrate.Also often should require mental skill clinically and protect medicine such as calcium ion antagonist (nimodipine and western pyrrole spirit); Free radical scavenger (vitamin E, vitamin C and gingko leaf preparation) etc.But from the present Clinical Application effect of said medicine, curative effect is general, and major part all only is anti symptom treatment, can not improve mortality rate.There are weak points such as active component is unclear, mechanism of action not clear, quality is unstable, toxic and side effects is big mostly in preparations such as some Chinese medicine preparation such as Folium Ginkgo, Herba Erigerontis, Radix Et Caulis Acanthopanacis Senticosi at present, are difficult to meet clinical needs.

Therefore, at present countries in the world still do not have effective ways and medicine to controlling this course of disease progress, the drug research of the prevention of VD, treatment and prognosis and exploitation become the research topic paid much attention to various countries (the 1. plain chrysanthemum of fourth. vascular dementia. Chinese gerontology magazine .2003; 23 (4): 200-202; 2. Chen Chunlei, Qiu Zhihui, Su Shixin, Zheng Zhong subtracts, Chen Peifen, Ye Zhiping. the therapeutic advance of vascular dementia. the new .2007 of medical science forum of China, 7 (7): 59-62.).

Therefore; working out a kind of effective Therapeutic Method, to stop potential pathogenic process be very necessary; traditional extract raw material in conjunction with China; therefrom excavate effectively, low toxicity, inexpensive treatment vascular dementia and the medicine of alzheimer disease; the product that is used to prevent, diagnose, detect, protect and treats aspects such as VD particularly medicine has very important significance, and can produce remarkable social benefit and economic benefit.

(4) research overview of antifungal drug

Fungal infection has gradually the trend that increases in recent years, and it comprises superficial part and deep mycosis, and latter's polyphyly body's immunity of falling ill is impaired, often is in a bad way, and threat to life needs whole body use potent antifungal drug treatment.Such medicine can be divided into external and system applies two classes, and external used medicine only is used for mycotic infection of superficial part.

Human fungal infection can involve epidermis, subcutaneous and whole body; Generally can be divided into superficial part and infect (promptly being confined to angleplied laminate, squamous mucosa or cornea) and deep infection (subcutaneous and whole body) two big classes.Their medicine is also different, infects, can be used for deep infection again and there is the part medicine both to can be used for superficial part.Mycotic infection of superficial part comprises dermatomycosis, skin, refers to the infection of (toe) first, hair, as tinea corporis, tinea cruris, tinea manus and pedis, tinea versicolor, tinea unguium, skin mucosa candidiasis and fungal keratitis etc.Superficial mycosis accounts for all dermopathicly 1/4 according to statistics, and particularly foot fungus infection account for department of dermatologry prescription on individual diagnosis patient's 46%, and wherein tinea unguium has just accounted for about 16%; For these infection, mainly adopt local application's treatment, because its infection, medication effect is not often felt quite pleased, and in some situation, also needs oral medication.The local ciclopirox olamine fingernail varnish that uses is coated in the better method that nail surface is the treatment tinea unguium.

Deep fungal infection comprises that subcutaneous tissue and whole body system infect, and comprise candidiasis, cryptococcosis, aspergillosis and mucormycosis etc.In recent years because the increasing of extensive use broad ectrum antibiotic, 17-hydroxy-11-dehydrocorticosterone, immunosuppressant, radiotherapy, chemotherapy, organ transplantation, operative catheter and acquired immune deficiency syndrome (AIDS), make the increase of invasive infections with fungi chance takes place, and cause deep fungal infection increasing, according to incompletely statistics, closely during the last ten years, the sickness rate of deep fungal infection has increased 3～5 times.Infection then needs systemic administration, the drug combination of still needing sometimes for the whole body system.Owing to the mechanism of action difference of medicine, should be noted that their safety and drug interaction problem especially.

In the treatment application facet, deep fungal infection is mainly selected the plain B of both sexes enzyme, itraconazole, ketoconazole, fluconazol, voriconazole, posaconazole, Caspofungin and flucytosine for use, the then mycotic infection of superficial part that are used for of other drug more, but most have more serious toxic and side effects, as amphotericin Toxicity of Kidney arranged.Pathogen also produces drug resistance in various degree gradually in the process of long-term medicinal application, presses for our development of new good effect and the littler antifungal drug of toxic and side effects.

(5) research overview of antitumor drug

Malignant tumor serious harm people's health according to RST statistics, is the more died from malignant tumor person every year on average in hundred million populations in the whole world 50 and is reached 690 power people, and new cases are 8,700,000 examples, and numeral is also increasing year by year.Therefore, national governments, research institution and drugmaker are paid much attention to tumor research and antitumor drug for a long time always, in the research of antitumor drug, have obtained major progress at present.The development of molecular weight tumor in recent years,, molecular pharmacology is progressively illustrated tumor essence; The drug development process has been quickened in the invention and the application of advanced technologies such as extensive rapid screening, combinatorial chemistry, genetic engineering; The research and development of antitumor drug have entered a brand-new era.Antitumor drug is just from traditional cytotoxic drug, to the new type antineoplastic medicine development at the too many levels effect of mechanism.

Therefore, the novel antitumor drug of research and development becomes exigence.The data that medicine is sold show that global pharmacy corporation the last 50 is 2.8% at prescription drugs sale growth in 2007, and wherein the growth of antitumor class medicine is the fastest, reaches 14%.2006 annual datas show that equally the sales growth of antitumor drug ranks first in the 10 class medicines, reaches 20.5%.This has reflected the market demand that continues, increases fast to antitumor drug.China proposes to set up a collection of great special project on the basis of formulating medium-term and long-term scientific development planning." great new drug initiative " be from the Eleventh Five-Year Plan to the year two thousand twenty between one of the major project supported of country.This project will be at the multiple serious harm people's such as tumor, cardiovascular diseases major disease, the development original new drug.Clearly, the research and development of anti-cancer agent are the needs of control serious harm people disease, also are the vital tasks of China's sustainable development and construction innovation-oriented country.What " great new drug initiative " was special sets up, and will produce active influence to the process of China's anti-cancer agent research and development.

Treatment of cancer is the great difficult problem of Med Biol.Development can be effected a radical cure the medicine of tumor, still " shoulders heavy responsibilities ".Compare with advanced international standard, China is still having big gap aspect the anti-cancer agent research.The research and development of anti-cancer agent are difficult tasks, are long-term, complicated processes, need scientific and technical personnel to work without single devotion for a long time and accumulate.Therefore, need provide stable working environment and condition for researcher; Need each side professional and associated mechanisms to work in concert; Need and be beneficial to the policy safeguard that promotes cooperation and organize and coordinate mechanism; Need the support energetically that the larger amt funds comprise the funds of preclinical study and clinical research.

Current, the anti-cancer agent research and development are faced with good opportunity.The development of biomedical science is for the development new type antineoplastic medicine provides important theoretical foundation and new technical support.The development of Chinese medical health service is had higher requirement to the therapy of serious disease medicine that comprises cancer, and the great demand of Chinese market and world market brings considerable economic can for the research and development unit.China sets up " great new drug initiative " great special project, will promote Chinese new drug to comprise the research and development of anti-cancer agent.

By literature search etc.; up to the present; still find no bioactive natural product B (polydatin, piceid) with and compositions at the report that is directly used in aspect prevention, diagnosis, detection, protection, treatment and research antifungal, antitumor, vascular dementia and alzheimer disease and the directly related disease thereof.

Summary of the invention

The technical problem that will solve required for the present invention is the new role that discloses a kind of Chinese medicine bioactive natural product B antifungal, antitumor and anti-angiogenic dementia, promptly this extract is used to prepare antifungal products, antitumor product and anti-angiogenic dementia product, to overcome the above-mentioned defective that prior art exists.

That is to say that the present invention is intended to the activity of clear and definite a kind of bioactive natural product B in antifungal, antitumor and anti-angiogenic dementia application facet by research and theory studies such as cell and zooperies, promptly the present invention relates to the new purposes of a kind of bioactive natural product B.

Another object of the present invention provides the application as preparation antifungal and antitumor product and anti-angiogenic dementia product of the pharmaceutical composition that contains above-mentioned bioactive natural product B.In said composition, bioactive natural product B of the present invention accounts for 10%～90% (percentage by weight), preferably accounts for 50%～90% (percentage by weight).

(1) definition of the present invention

Antifungal and antitumor product of the present invention is to comprise antifungal products and antitumor product;

Antifungal products of the present invention is meant one or more in the product that is directly used in prevention, diagnosis, detection, protection, treatment and research pathogenic fungi and directly related disease thereof, and described pathogenic fungi is meant white coccus, neogenesis cryptococcus, trichophyton and the Aspergillus fumigatus read.

Antitumor product of the present invention is meant one or more in the product that is directly used in prevention, diagnosis, detection, protection, treatment and research tumor and directly related disease thereof, and described tumor is meant hepatocarcinoma, breast carcinoma and pulmonary carcinoma.

Anti-angiogenic dementia product of the present invention is meant one or more in the product that is directly used in prevention, diagnosis, detection, protection, treatment and research vascular dementia and directly related disease thereof.

Antifungal and antitumor product of the present invention, anti-angiogenic dementia product all are to comprise a kind of in the field products such as medicine and food, are to comprise in medicine, reagent or the food etc. one or more, preferred agents.

Bioactive natural product B of the present invention be meant resveratrol-3-O-β-D-glucoside (piceid, polydatin).

The employed medicinal raw material of bioactive natural product B of the present invention is meant marine fungi or marine fungi crude extract, preferred marine fungi Massarina tunicata or marine fungi Massarina tunicata crude extract.

Bioactive natural product B is the active component of prevention, diagnosis, detection, protection, treatment and research antifungal, antitumor, anti-angiogenic dementia; its occupation mode is to comprise independent use or unite a kind of in use etc. with other chemical substances; preferred use separately all can be used in preparation antifungal products, antitumor product and anti-angiogenic dementia product.

For test agent all is to take conventional preparation method to obtain, resulting bioactive natural product B content generally＜60%, but by purification, the purity that can access the bioactive natural product B behind the purification can be more than 95%, i.e. the defined bioactive natural product B of the present invention.

That is to say, adopting bioactive natural product B is raw material, or the marine fungi that direct employing contains bioactive natural product B is a raw material, perhaps directly adopting the marine fungi crude extract that contains bioactive natural product B is raw material, can both directly or indirectly be used to prepare antifungal and antitumor product and anti-angiogenic dementia product.It is the defined bioactive natural product B of the present invention that bioactive natural product B preferably uses with pure substantially form, as purity 〉=95% of bioactive natural product B.

(2) technical conceive

The independent development original new drug is a present urgent task of China, China's Chinese medicine and pharmacy has a long history, also accumulating rich experience aspect prevention and the treatment disease, therefore seeking effective active component or find that its new purposes all is effectively quick approach from Chinese medicine, also is the place of the advantage of the quick original new drug development of China.

The Lu Sheng microbial fermentation product is one of valuable source of pharmaceuticals industry always, but continually developing along with land resources in recent years, seek new microorganism or screen the difficulty of new active metabolite increasing, and the Drug resistance problem is serious day by day, makes the exploitation of new drug be faced with severe challenge.

Marine microorganism is a research focus in recent years.The ocean account for earth surface long-pending 71%, microbial resources are richly stored with..Increasing research also shows Marine microorganism---especially there is the microorganism of relations such as symbiosis or parasitism in those with marine animal or sea-plant---can produce the unique also metabolite of the strong physiologically active of tool of structure.And the Marine microorganism breeding is fast, can utilize modern microbial technique, and the large-scale industry fermentation has broad application prospects.Scientists is estimated, is developed new marine drug and will place hope on this new field of Marine microorganism.

From the Marine microorganism secondary metabolite, seek new anti-tumor activity metabolite, for the development of new antitumor drug provides lead compound, by to marine fungi Massarina sp., the further investigation of strain CNT-016, found at present spiro lactone chemical compound 6-epi-5 '-hydroxy-mycosporulone (referring to: M.A.Abdel-Wahab et al./Phytochemistry 68 (2007) 1212-1218), but do not carry out bioactivity research as yet, the chemical compound that the present invention's research relates to has similar parent nucleus with it, and carried out the antifungal and antitumor activity research, carried out cell and animal experiment study.

The inventor is by carrying out the chemical constitution study of system to the marine fungi extract; screen and prove activity and the purposes of this marine fungi extract bioactive natural product B; thereby the inventor infers that bioactive natural product B is in prevention; diagnosis; detect; protection; treatment and research antifungal; antitumor; the active clinical drug effect in aspects such as anti-angiogenic dementia; also should mainly be by this marine fungi extract of active site particularly the drug effect of bioactive natural product B bring into play, result of study also prove and confirmed this marine fungi extract particularly bioactive natural product B have significant pharmacologically active.

The inventor through the latest find of research is: bioactive natural product B can resist the effect that amyloid-beta causes the brain cell damage, can resist hydrogen peroxide and cause the brain cell damaging action, improve the learning and memory function that scopolamine causes the dementia mice model, improve A β and cause dementia mice model learning memory ability, suppress cerebral ischemia re-pouring mouse model cerebral tissue lipid peroxidation, strengthen cerebral ischemia re-pouring mouse model cerebral tissue superoxide dismutase activity and lower cerebral ischemia re-pouring mouse model brain sheet intracellular calcium concentration.

Dull-witted particularly vascular dementia and alzheimer disease have a strong impact on the health and the life quality of Chinese population; the product of development prevention, diagnosis, detection, protection, treatment and aspects such as research vascular dementia and alzheimer disease; particularly medicine, diagnostic reagent and health product etc. have remarkable social benefit, economic benefit.

(3) structure and properties of bioactive natural product B

The object of the present invention is to provide a kind of noval chemical compound with potential medical value, illustrated the structure of this chemical compound, bioactive natural product B structural formula is as follows:

The experimental data of bioactive natural product B:

Colorless solid; Fusing point 203-205 ℃, IR (KBr) 3436,3402,2952,1815,1726,1679,1150,899cm ^-1[M+H] ⁺M/z 251.07, [M-H] ^-M/z 251.08, HRESITOFMS[M+H] ⁺M/z 251.0861 (C ₁₃H ₁₄O ₅), calcd.251.0874) the .NMR data see Table 1.

The NMR experimental data of table 1, bioactive natural product B

(4) pharmacologically active of bioactive natural product B

The present invention has carried out many-sided test to bioactive natural product B in the activity of preventing, diagnose, detect, protect, treat and study aspects such as anti-angiogenic dementia.

Have now and studies show that the cardinal symptom of vascular dementia is: 1. the cerebral tissue pathomorphism changes; 2. hypomnesis.The inventor studies show that: bioactive natural product B can obviously improve the vascular dementia model rat brain the ischemic pathology damage, the learning and memory function of dementia rats is had stronger protection and facilitation.Therefore, bioactive natural product B and compositions thereof can be used in preparation antifungal and antitumor product.

Effect and the in vitro tests thereof of bioactive natural product B aspect prevention, diagnosis, detection, protection, treatment and the research activity of vascular dementia

The present invention illustrates that by following experiment bioactive natural product B has the effect of treatment of vascular dementia.

1, the learning and memory protective effect (referring to the 1243rd page of Zhang Juntian chief editor " modern pharmacology experimental methodology " first volume) of the dementia rats that vascular ischemic injuries (multiple local embolization modeling) is formed

Learning and memory is the Premium Features of brain, is to constitute the highest function of human brain---the key element of intelligence.Learning memory disorder is to send out one of symptom the morning of MID.So the main standard of the improvement of learning and memory as the raising of evaluation intelligence.

(1) animal: the Sprague-Dawley rat, 80, male, body weight 250-300g is provided by Shanghai Branch of Chinese Academy of Sciences animal center, the animal quality certification number: SCXK (Shanghai) 2003-0003.

(2) instrument and reagent: Morris water maze video analytic system (Jiliang Software Sci-Tech Co., Ltd., Shanghai and " DigBehv animal behavior analytical system "); Ginaton (German Schwabe pharmaceutical factory); Sodium carboxymethyl cellulose, chloral hydrate are homemade analytical reagent.

(3) compound method: medicine of the present invention and Ginaton are milled evenly facing with preceding usefulness 0.5% sodium carboxymethyl cellulose, are made into desired concn.

(4) preparation of animal model and grouping:

This law is to get rat left ventricle blood sampling of the same race, aseptic 37 ℃ of freeze-day with constant temperature become blood clot, grind after 200～300 mesh sieve hole sieving for standby add blood clot 1mg with the every 1ml of 6% dextran Glucose Liquid during application and make suspension, it is 50～100 μ m that microscopically is measured diameter.With experimental rat chloral hydrate intraperitoneal anesthesia (35mg/100g), cervical region medisection exposes separates right carotid and external carotid artery then.Interim folder closes external carotid artery, injects embolus dextran suspension from common carotid artery, and open common carotid artery utilizes Carotid blood flow that embolus is sent into intracranial to each tremulous pulse of brain by internal carotid artery when injecting embolus, causes many kitchen ranges property cerebral infarction.Animal grouping with the modeling success in second day, drug component of the present invention is high dose group (100mg/kg), low dose group (50mg/kg), 16 every group.With medicinal liquid difference gastric infusion of the present invention, every Mus 10ml/kg every day, the every Mus of positive drug control group is irritated stomach Ginaton suspension (2.5mg/ml) 10ml/kg every day, and the every Mus of model control group and sham operated rats gives 0.5% sodium carboxymethyl cellulose 10ml/kg, all continuous 2 weeks every day.

(5) postoperative is 10 days, the Morris water maze laboratory:

1. experiment beginning the previous day allows rat free swimming 2min in the pond of not containing platform earlier, to be familiar with environment.

2. orientation navigation experiment: trained every day 4 times continuously 4 days.Equal each time random order never 4 rats of naming a person for a particular job of equal diversion basin drops in the ponds, makes rat towards pool wall when putting into, and to the time of finding and climb up platform, this time is called escape latency to the record rat from entry.Be allowed to condition at rest row detection next time again after 10 seconds on the platform.If rat is not found platform yet in 120 seconds, then it is directed to platform, be designated as best result incubation period 120 seconds.Every day, 4 preclinical arithmetic means were as the achievement of this time period, carried out statistical analysis.

3. space exploration experiment: trained the 5th day, and after directed swimming test finishes, immediately platform was removed, still in 4 place of entry of picked at random puts into rat, the meter record time of staying of rat in the quadrant of original platform place.

(6) experimental result: (seeing: table 2 and table 3).By the table in as seen: after the swimming instruction of Morris water maze, the swimming of each dosage group of medicine significantly reduces (P＜0.05) than model group incubation period; In space exploration experiment, the medicine treated animal the platform place quadrant time of staying obviously more than model group (P＜0.05).The above results shows that bioactive natural product B has protective effect to the learning and memory function of rat.

Table 2, Morris water maze laboratory animal are swum incubation period (second)

Compare with model group, ^*P＜0.05; ^*P＜0.01; ^{* *}P＜0.005; ^{* * *}P＜0.001

Table 3, space exploration experiment (training the 5th day)

Compare P＜0.05 with model group; ^*P＜0.01;

(7) morphological examination

Postoperative 14 days with the rat sacrificed by decapitation, separates rapidly and takes out prefrontal cortex, Hippocampus, and fixing in neutral formalin liquid, HE dyeing is done in section.

Except that sham operated rats, each organizes the softening necrosis region of each regional lamellar brain essence under the cortex of all seeing in various degree, and each group of scope does not wait.

1. sham operated rats: each one of central nervous system there is no obvious pathological changes, cerebral cortex: neurocyte nuclear shows no obvious abnormalities, and chromatin is evenly distributed, and vascular endothelial cell is no abnormal.Hippocampus: pyramidal cell nuclear shows no obvious abnormalities, and chromatin is evenly distributed, and vascular endothelial cell is no abnormal.

2. model group: cerebral cortex: the solid area lamellar is softened kitchen range, and part liquefies, and forms not shaping blister cavities, and scope is wide, and a large amount of neuron cavity samples become, and nuclear membrane thickening, chromosome reduce or disappear, the neurocyte swelling and degeneration that has, and nuclear membrane is unclear.Blood vessel endothelium swelling phenomenon.Hippocampus: many places neurocyte cavity sample becomes, and nuclear membrane thickening, chromosome reduce or disappear, the neurocyte swelling and degeneration that has, and nuclear membrane is unclear.

3. bioactive natural product B low dose group: cerebral cortex: the neurocyte chromatin distributes very uneven, but aixs cylinder shows no obvious abnormalities, and a small amount of neurocyte cavity sample becomes the blood vessel endothelium mild swelling.Hippocampus: the less softening stove liquefaction of scope is arranged.

4. bioactive natural product B high dose group: cerebral cortex: have lamellar to soften kitchen range, scope is bigger.Hippocampus: have special mess to soften kitchen range (, belonging to downright bad afterreaction) on every side with slight calcification.

5. Ginaton group: cerebral cortex: seeing has lamellar to soften kitchen range, but scope is less.Hippocampus: the pyramidal cell chromatin is evenly distributed, and aixs cylinder is obvious, visible minority neurocyte karyopycnosis, vascular endothelial cell mild swelling.

Two dosed administration groups and positive drug Ginaton group are compared with model group: the degree of ischemia injury is light, scope is little.

Above result of the test explanation bioactive natural product B has stronger protective effect to the vascular dementia rats ischemia injury, improves the learning and memory function of rat model.Therefore, bioactive natural product B can be used for preparing the medicine of prevention and treatment vascular dementia disease.

2, bioactive natural product B antagonism amyloid-beta causes dementia rats model learning memory ability

(1) experiment purpose

Feature pathological change of alzheimer disease is that cerebral cortex and Hippocampus occur because amyloid-beta (Beta-AmyloidProtein, be called for short: A β) deposit and the senile plaque of formation, more and more evidences shows that A β plays leading effect in the generation of AD, development, the neurotoxicity of A β relates to complicated molecular mechanism, mainly comprise and destroy intracellular calcium ion stable state, promote the formation of free radical, reduce the function of K ion channel, the inflammatory reaction that the enhancing proinflammatory cytokine causes etc.This experiment purpose is to observe bioactive natural product B causes dementia rats model learning memory ability to A β influence.

(2) experimental technique

Male Sprague-Dawley rat, body weight 250-300g, the anesthesia back exposes skull at calvarium portion medisection skin, with reference to " rat brain stereotaxic atlas " (bag new people, Shu Siyun. the rat brain stereotaxic atlas. the 1st edition, Beijing: People's Health Publisher, 1991,44-45.), select bilateral Hippocampus CA1 district to be the injection target area, bore with dental burr and open skull, with the A β of microsyringe with state of aggregation _1-40Slowly inject (totally 5 μ l, 1 μ l/min), let the acupuncture needle remain at a certain point 10 minutes to guarantee that solution fully spreads, slowly remove pin, sew up wound then.The normal saline group is injected the normal saline of equal volume.Animal grouping with the modeling success in second day, drug component of the present invention is high dose group (100mg/kg), low dose group (50mg/kg), 16 every group.With bioactive natural product B medicinal liquid difference gastric infusion, every Mus 10ml/kg every day, the every Mus of positive drug control group is irritated stomach Ginaton suspension (2.5mg/ml) 10ml/kg every day, and the every Mus of model control group and sham operated rats gives 0.5% sodium carboxymethyl cellulose 10ml/kg, all continuous 2 weeks every day.Postoperative 10 days, the Morris water maze laboratory:

1. experiment beginning the previous day allows rat free swimming 2min in the pond of not containing platform earlier, to be familiar with environment.

2. orientation navigation experiment: trained every day 4 times continuously 4 days.Equal each time random order never 4 rats of naming a person for a particular job of equal diversion basin drops in the ponds, makes rat towards pool wall when putting into, and to the time of finding and climb up platform, this time is called escape latency to the record rat from entry.Be allowed to condition at rest row detection next time again after 10 seconds on the platform.If rat is not found platform yet in 120 seconds, then it is directed to platform, be designated as best result incubation period 120 seconds.Every day, 4 preclinical arithmetic means were as the achievement of this time period, carried out statistical analysis.

3. space exploration experiment: trained the 5th day, and after directed swimming test finishes, immediately platform was removed, still in 4 place of entry of picked at random puts into rat, the meter record time of staying of rat in the quadrant of original platform place.

(3) experimental result: (seeing: table 4 and table 5).By the table in as seen: after the swimming instruction of Morris water maze, the swimming of each dosage group of medicine significantly reduces (P＜0.05) than model group incubation period; In space exploration experiment, the medicine treated animal the platform place quadrant time of staying obviously more than model group (P＜0.05).The above results shows that bioactive natural product B can improve the ability of learning and memory that A β causes the dementia rats model.

Table 4, Morris water maze laboratory animal are swum incubation period (second)

Compare with model group, ^*P＜0.05; ^*P＜0.01; ^{* *}P＜0.005; ^{* * *}P＜0.001

Table 5, space exploration experiment (training the 5th day)

Compare P＜0.05 with model group; ^*P＜0.01;

3, bioactive natural product B opposing hydrogen peroxide causes the neural cell injury effect

(1) experiment purpose

In the evolution of vascular dementia and alzheimer disease, free radical and active oxygen play very big facilitation.Cerebral ischemia can cause that free radical and active oxygen increase, and free radical and active oxygen and then attack neurocyte cause cell death, cause a series of pathological changes, cause long-term damage.This is in zoopery and may influence cognitive function clinically, develops into dementia.The purpose of this experiment is to observe bioactive natural product B to active oxygen hydrogen peroxide (H ₂O ₂) cause neural cell injury and have resistant function.

(2) experimental technique

Use SK-N-SH human neuroblastoma cells cell line (form and function is similar to neuron), the cultivation of going down to posterity.Bioactive natural product B and neurocyte preincubate 12 hours or 24 hours change culture fluid, add H ₂O ₂Hatched 2 hours with cell.Get supernatant measure lactic acid dehydrogenase (be called for short: LDH) activity, measure cell survival rate with mtt assay.

(3) experimental result

At H ₂O ₂Cause on the neural cell injury model, find obviously antagonism H of bioactive natural product B ₂O ₂Cell survival rate decline that causes and LDH spill and increase, and lower H ₂O ₂Neurotoxicity, and be dose-dependence, prompting bioactive natural product B has the neuroprotective cytosis, promptly vascular dementia is had preventive and therapeutic effect (seeing: table 6, table 7).

Table 6, bioactive natural product B preincubate 12 hours are to H ₂O ₂Cause the influence of SK-N-SH neural cell injury

M ± SD; ^*P＜0.05 is with H ₂O ₂Model group is compared.

M ± SD; ^*P＜0.05 is with H ₂O ₂Model group is compared.

Table 7, bioactive natural product B preincubate 24 hours are to H ₂O ₂Cause the influence of SK-N-SH neural cell injury

M ± SD; ^*P＜0.05 is with H ₂O ₂Model group is compared.

4, bioactive natural product B raising scopolamine causes dementia mice model learning memory function

(1) experiment purpose

Alzheimer disease is lost the most serious with the basal forebrain cholinergic neuron, (acetylcholine is called for short: ACh) hyposecretion to cause acetylcholine.Vascular dementia has many similar pathology and symptom characteristic with alzheimer disease, comprises that cholinergic neurotransmitter ACh level reduces and relative cognitive disorder.Scopolamine is an acetylcholinesterase m receptor blocker, and ACh that can the antagonism nervus centralis causes hypomnesis, can simulate the result of ACh hyposecretion, is more effective dementia animal model.

(2) experimental technique

1. Morris water maze test: 50 of male mouse of kunming, body weight 22-28 gram, animal is divided 5 groups at random, is respectively normal control group, model group, bioactive natural product B low dose group (50mg/kg) and high dose group (100mg/kg).Respectively organize the modeling after 10 days of continuous gastric infusion before the experiment.Test administration on the same day after 1 hour, lumbar injection scopolamine hydrobromide injection 1mg/kg, the normal saline of matched group injection equivalent.Carry out the Morris water maze test after 15 minutes, one day twice, continuous three days.Date processing monitors automatically by image and processing system is finished.

2. keep away dark test: the mice administration is the same with injection scopolamine method, and scopolamine dosage is 20mg/kg.Inject and keep away dark test after 15 minutes.This method is the passive avoidance test, and mice has the habit of liking dark, enters the darkroom and is then shocked by electricity.2nd, entered in 3,4 days incubation period in darkroom long more, to enter number of times few more, shows that learning and memory function is good more.

3. experimental result

Test finds that bioactive natural product B high dose group group can obviously shorten mice and swim out of time and swimming distance (table 8), shows that bioactive natural product B can improve scopolamine model mice space learning memory ability.Keep away dark test and find that bioactive natural product B high dose group can obviously prolong incubation period, reduce the number of times (table 9) that enters the darkroom, show that bioactive natural product B can improve mice passive avoidance learning and memory function, its mechanism can function of nervous system strengthen relevant with ACh.

Table 8, bioactive natural product B cause the influence that dementia mice model water maze is swum out of time and swimming distance to scopolamine

M ± SD; ^*Compare with model group in P＜0.05.

Table 9, bioactive natural product B scopolamine cause the influence that the dementia mice model is kept away dark test incubation period and errors number

M ± SD; ^*Compare with model group in P＜0.05.

5, bioactive natural product B suppresses cerebral ischemia re-pouring mouse model cerebral tissue lipid peroxidation

(1) experiment purpose

The generation of free radical, the neuronal damage that oxidative stress causes are the key factors that alzheimer disease and vascular dementia cause cognitive impairment in the ischemia hindbrain.The lipid peroxy and the oxidative stress that produce the generation of a large amount of free radicals after the cerebral ischemia and cause cause lipid peroxidation.Lipid peroxide be in biomembrane and the cell the contained polybasic unsaturated fatty acid of phosphorus matter by radical damage, oxidation and the peroxidating product that forms, can cause reactions such as membrane damage, enzyme inhibition, lysosome release, protein-crosslinking, DNA and RNA structural deterioration, thereby destroy the cell normal physiological function, finally can cause cell death.This experiment is intended to observe bioactive natural product B cerebral tissue lipid peroxidation metabolite malonaldehyde (is called for short: MDA) the content influence of increasing.

(2) experimental technique

Kunming mice, male, body weight 22～28 grams, random packet is respectively organized continuous gastric infusion 7 days before the experiment, and matched group (sham operated rats) and ischemia model group give tap water respectively.1h after the administration of operation empty stomach on the same day behind mouse anesthesia, closed bilateral common carotid arteries 15 minutes with the bulldog clamp folder, and pouring into breaked end rapidly after 15 minutes again gets brain, removes cerebellum.Cerebral tissue is weighed, and adds the homogenate of 0.2M phosphate buffer at 1: 10,3000 rev/mins centrifugal 10 minutes, get supernatant 0.5ml, (be called for short: TAB) method is measured MDA content in the cerebral tissue with thiobarbituricacid.

(3) experimental result

Bioactive natural product B gavages and can reduce acute cerebral ischemia and pour into mouse brain again and organize MDA content (seeing: table 10), shows that this medicine can suppress lipid peroxidation, and its mechanism may be to reduce free radical to generate or strengthen free radical scavenging.The lipoid peroxidization resistant of bioactive natural product B helps treatment of vascular dementia and alzheimer disease.

Table 10, bioactive natural product B pour into the influence that mouse brain is organized MDA content again to acute cerebral ischemia

M ± SD; ^*P＜0.05, ^*Compare with the ischemia model group in P＜0.01.

6, strengthen cerebral ischemia re-pouring mouse model cerebral tissue superoxide dismutase activity

(1) experiment purpose

Superoxide dismutase (SOD) plays crucial effects to the oxidation and the Hangzhoupro oxidation balance of body.Too much superoxide anion can cause lipid oxidation in the body, and the infringement cell is relevant with pathological processes such as multiple disease and agings.But this enzyme catalysis superoxide anion generation dismutation reaction makes it to be converted into hydrogen peroxide and oxygen, and the protection cell is avoided damage.This experiment is intended to observe bioactive natural product B to the active influence that reduces of cerebral ischemia re-pouring mouse model cerebral tissue SOD, inquires into the mechanism that bioactive natural product B suppresses lipid peroxidation.

(2) experimental technique

Kunming mice, male, body weight 22～28 grams, random packet is respectively organized continuous gastric infusion 7 days before the experiment, and sham operated rats and ischemia model group give tap water respectively.1h after the administration of operation empty stomach on the same day behind mouse anesthesia, closed bilateral common carotid arteries 15 minutes with the bulldog clamp folder, and pouring into breaked end rapidly after 15 minutes again gets brain, removes cerebellum.Cerebral tissue is weighed, and adds the homogenate of 0.2M phosphate buffer at 1: 10,3000 rev/mins centrifugal 10 minutes, get supernatant 0.1ml, with the Asia slightly the hydrochlorate method measure SOD activity in the cerebral tissue.

(3) real face result

Bioactive natural product B filling appetite clothes can increase acute cerebral ischemia and pour into mouse brain tissue SOD activity (seeing: table 11) again, show that this medical instrument has enhancing body to remove the effect of free radical, and this may be its mechanism that suppresses lipid peroxidation.Bioactive natural product B promotes the effect of endogenous activities of antioxidant enzymes to help treatment of vascular dementia and alzheimer disease.

Table 11, bioactive natural product B pour into the active influence of mouse brain tissue SOD again to acute cerebral ischemia

M ± SD; ^*P＜0.05, ^*Compare with the ischemia model group in P＜0.01.

7, lower cerebral ischemia re-pouring mouse model brain sheet intracellular calcium concentration

(1) experiment purpose

In the pathogeny of vascular dementia and alzheimer disease, the interior calcium ion concentration of neurocyte increases, calcium overload plays an important role.A series of variations taking place behind the cerebral ischemia re-pouring, comprise calcium ion especially the increasing unusually of calcium concentration, calcium overload in the neurocyte, play an important role at ischemic brain injury, finally can make neuronal cell death.This experiment is intended to observe the influence that bioactive natural product B increases animal pattern brain sheet intracellular calcium concentration.

(2) experimental technique

Male mouse of kunming, body weight 22～28 grams, the animal random packet is respectively organized continuous gastric infusion 7 days before the experiment, and sham operated rats and ischemia model group give tap water respectively.Operation same day is 1h after the administration on an empty stomach, behind mouse anesthesia, closes bilateral common carotid arteries 15 minutes with the bulldog clamp folder, pours into to break end rapidly after 15 minutes and take out full brain again, immerses 4 ℃ rapidly and is connected with 95% CO ₂And 5%O ₂Artificial cerebrospinal fluid (artificial cerebrospinal fluid solution is called for short ACSF, and the ACSF prescription is (mmol/L): NaCl 124, and KCl 3.3, KH ₂PO ₄1.2, NaHCO ₃26, CaCl ₂2.5, MgSO ₄2.4, glucose 10), on vibratome, cut out the brain sheet, thickness 400 μ m put into CO ₂In the incubator, hatch 30min for 37 ℃, add the Fluo-3AM 500ul of 5uM, at CO ₂Behind the load 1h, ACSF washes for several times, carries out optical section on laser scanning co-focusing microscope in the incubator, observes fluorescence intensity.

(laser scanning confocal microscope is called for short: LSCM) can be used for pair cell structure (biomembrane, organelle, chromosome etc.) and molecule (nucleic acid, protein, lipid etc.), ion (Ca laser scanning co-focusing microscope ²⁺, K ⁺Deng) position, quantitatively, in real time, dynamic observation.Optical section is one of characteristic of laser scanning co-focusing microscope, can realize the brain sheet alive that exsomatizes is carried out continuously harmless cutting, light is cut sample surfaces and information deep layer of can obtaining, and by the three-dimensional reconstruction technology, can obtain three-dimensional image and stereochemical structure information.The no fluorescence of calcium fluorescence indicator Fluo-3AM itself, but it have can with [Ca in the living cells ²⁺] specificity in conjunction with after send the characteristic of fluorescence, utilize laser scanning co-focusing microscope to measure fluorescence intensity, can detect the level of intracellular free calcium.

Image processing: utilize the LaserSharp image processing system, measure area or volume and total glorious intensity of left and right sides hippocampus, cortical area, with unit are or unit volume fluorescence intensity and fluorescence intensity distribution intermediate value (is Mean, system provides) be index, the level of observing the intracellular free calcium of Hippocampus, cortex.

Statistical method: experimental data all adopts means standard deviation, and (x ± s) expression, the significance of group difference is checked with t.

(3) experimental result

Each dosage group of bioactive natural product B is irritated the appetite clothes all can reduce calcium ion concentration (seeing: table 12) in cerebral ischemia re-pouring mouse model cerebral cortex and the hippocampus neurocyte, and this is significant for neuroprotective cell, treatment of vascular dementia and alzheimer disease.

Table 12, to the influence of calcium ion in the cerebral ischemia re-pouring mouse model neurocyte

M ± SD; ^*P＜0.05, ^*Compare with the ischemia model group in P＜0.01.

(5) bioactive natural product B antifungal activity

The present invention experimental results show that by external antifungal and antitumor, bioactive natural product B has certain inhibitory action (seeing Table 13) to four kinds of common clinical disease fungal pathogenses, and can suppress the growth of tumor cell line in various degree, detect in the anti-tumor activity test MIC of bioactive natural product B in the MTT reducing process that with human normal cell line strain (L-02), human hepatoma cell strain (HepG-2), human breast cancer cell (MCF-7), human lung carcinoma cell line (A-549) is target cell ₈₀As shown in table 14, show that bioactive natural product B can be used for preparing antitumor drug.

1, bacterial strain

The ATCC type strain: Candida albicans (Candida albicans) ATCC76615, neogenesis cryptococcus (Cryptococcusneoformans) ATCC32609 give by Long March hospital strain preservation center.Clinical strain: trichophyton (Trichophyton rubrum) 0504656, Aspergillus fumigatus (Aspergillus fumigatus) 0504656, pick up from Shanghai Changhai Hospital, and through morphology and biochemical evaluation.

2, bacterium liquid preparation

A small amount of with inoculation circle spherical bacterium such as picking neogenesis cryptococcus, Candida albicans and Candida parapsilosis on the SDA culture medium of preserving, be seeded to the 1mlYEPD culture fluid, in 35 ℃, the 250rpm shaken cultivation, activation 16h makes fungus be in later stage exponential phase of growth.Get this bacterium liquid to the 1mlYEPD culture fluid, activate once more, with the blood cell counting plate counting, adjust bacterial concentration to 1 * 10 behind the 16h with the RPMI1640 culture fluid with said method ³～5 * 10 ³Individual/ml.

Aspergillus fumigatus is seeded to the SDA inclined-plane, cultivates a week for 35 ℃; Trichophyton cultivated for two weeks for 28 ℃.Each bacterium adds an amount of RPMI1640 culture fluid in the SDA inclined-plane by this method activation twice, blows and beats bacterium colony with suction pipe, and spore is free in the RPMI1640 culture fluid, filters through four layers of sterile gauze then.Culture fluid adds the RPMI1640 culture fluid and adjusts spore concentration to 1 * 10 behind the blood cell counting plate counting ³～5 * 10 ³Individual/ml.

3, drug sensitive plate preparation

Get aseptic 96 orifice plates, add RPMI1640 100 μ l in No. 1 hole of every row and make blank; 3～No. 12 the hole respectively adds freshly prepared bacterium liquid 120 μ l; No. 2 the hole adds bacterium liquid 160 μ l and test-compound solution 1.6 μ l respectively.The 10 grades of 4 times of dilutions in 2～No. 11 holes make that medicine final concentration in each hole is respectively 64,16,4,1,0.25 and 0.0625,0.0156,0.0039,0.00097,0.00024mgL ^-1, DMSO content all is lower than 1% in each hole; No. 12 the hole does not contain medicine, makes positive control, and each drug sensitive plate is in 35 ℃ of cultivations.

4, MIC ₈₀Value is judged:

Candidiasis, neogenesis cryptococcus and filamentous bacteria are cultivated 24h, 72h and after the week, are surveyed each hole OD value with enzyme micro-plate reader in 630nm respectively at 35 ℃.With the positive control boring ratio, be MIC with the drug level in the least concentration hole of OD value decline more than 80% ₈₀(drug level when conk 80% is suppressed).

Table 13, bioactive natural product B are to the antibacterial activity (MIC of 4 kinds of funguses ₈₀. μ g/ml)

MIC when medicine ₈₀Value surpasses when measuring concentration range, adds up by the following method: MIC ₈₀Value is higher than maximum concentration 64mgL ^-1The time, count "＞64mgL ^-1"; MIC ₈₀Value is not distinguished for least concentration or when least concentration is following, all counts "≤0.00024mgL ^-1".The equal operation repetitive of above-mentioned experiment 2 to 3 times is worked as MIC ₈₀Value just is accepted in the time of accurately repeating or only differ from a concentration, and with higher concentration as MIC ₈₀Value is worked as MIC ₈₀Value differs two concentration when above, then needs experiment again, up to meeting the requirements.

There are four chemical compounds that in various degree antifungal activity (table 13) is arranged.

(6) anti-tumor activity of bioactive natural product B

The present invention experimental results show that by external antifungal and antitumor, bioactive natural product B has certain inhibitory action (seeing Table 13) to four kinds of common clinical disease fungal pathogenses, and can suppress the growth of tumor cell line in various degree, detect in the anti-tumor activity test MIC of bioactive natural product B in the MTT reducing process that with human normal cell line strain (L-02), human hepatoma cell strain (HepG-2), human breast cancer cell (MCF-7), human lung carcinoma cell line (A-549) is target cell ₈₀As shown in table 14, show that bioactive natural product B can be used for preparing antitumor drug.

MTT reducing process detection of active natural product B anti-tumor activity test is as follows:

1. material

1.1 four Cuo salt (MTT): (be called for short: PBS) (3-(4 for dissolving MTT for the phosphate buffer of usefulness 0.01mol/L, 5-dimethythiazol-z-yl) 2,5-diphenytetrazolium bromide is called for short: SIGMA) final concentration 5mg/ml, filtration sterilization, 4 ℃ keep in Dark Place after the packing.

1.2MTT the dodecyl sodium sulfate of the preparation of lysate: 80g (is called for short: SDS, Huamei Bio-Engrg Co.) is dissolved in the N-N-dimethyl methyl acid amide (traditional Chinese medicines) of 200ml, the heating in water bath hydrotropy adds the 200ml distilled water, mixes with IN hydrochloric acid (1: 1) with 80% acetic acid and transfers pH to 4.7.

1.3 cell strain is selected for use: human normal cell line strain (L-02), human hepatoma cell strain (hepg-2), human breast cancer cell (mlf-7) and human lung carcinoma cell line (a-549).

2. operating procedure

A. single cell suspension is inoculated in 96 orifice plates (with the RPMI-1640 basal medium with cell dilution to 3 * 10 ⁴/ ml, every hole adds 20 μ l and dilutes good cell), 37 ℃, 5%CO ₂, cultivated 24 hours under the saturated humidity: every group of four parallel samples;

B. remove culture medium, get new preparation culture medium and prepare cancer therapy drug (bioactive natural product B) solution by series concentration, every hole 200 μ l cultivated 48 hours;

C. every hole adds the MTT 20 μ l of 2mg/ml, hatches 4 hours;

D. culture fluid (as far as possible fully) in the sucking-off hole adds DMSO liquid (150 μ l/ hole), vibrates 10 minutes, and crystal is fully dissolved;

E. microplate reader detects each hole OD value (λ=570nm);

F. draw the cell viability curve chart, obtain IC ₅₀Value.

The cytotoxicity test result of table 14, bioactive natural product B (μ g/ml)

Result of the test is as shown in table 14.The result of the test explanation, bioactive natural product B has stronger cell toxicant, and its toxicity has certain selectivity to normal cell and cancerous cell; Show differential cytotoxicity for different tumor cells.Bioactive natural product B has the potentiality that are used as the preparation antitumor drug.

(7) extracting method of bioactive natural product B

Divide in the conical flask that installs to 250ml (100ml/ bottle) after the PDB culture medium stirs, seal after autoclaving cools off standby.The bacterial strain of cultivating is transferred on the PDA flat board, cultivate a week for 28 ℃, have the agar block of mycelia quantitatively to be seeded to (5 pieces/bottle) in the 250ml conical flask with card punch (d=3mm) with long in super-clean bench, three bottles of each inoculation are to verify its repeatability, and three bottles of blank PDA culture medium in contrast.To inoculate the good bottle that shakes then places 28 ℃ of concussions of constant temperature shaking table to cultivate a week (180 rev/mins).

1, organic solvent extraction

The fermentation liquid that cultivation obtains behind the filtration under diminished pressure, is used equal volume of ethyl acetate three times; Collect organic facies (ethyl acetate layer) revolve steam in the instrument decompression be spin-dried for extractum.

2, silicagel column gradient elution

The developing solvent of a series of gradients of configuration chloroform/methanol (100: 1,50: 1,30: 1,20: 1,10: 1,8: 1).After silica gel dress post is finished, on silica gel plate, in each ratio developing solvent, test one by one with sample solution point.Selection can make near the developing solvent eluting impact point of impact point Rf value 0.2, and at first the material of polarity minimum is eluted, by the eluting separation one by one of polarity size.

3, sephadex lh-20 purifying compounds

Mobile phase adopts methanol, two kinds of mobile phases of methanol/chloroform (1: 1), and the automatic reception instrument is collected automatically behind the last sample, 12 droplets/minute of flow velocitys.Each test tube solution of gained detects by TLC, handles by testing result.Owing to do not lose sample, can be repeatedly upper prop repeatedly, and transposing mobile phase is to reach good separating effect.

4, TLC prepares silica-gel plate

If compound sample seldom and silicagel column and all well separation and purification of sephadex column, considers then that preparing silica-gel plate with thin layer separates, with the suitableeest unfolding condition of TLC check-out console discussion.Sample launches with variant polarity developing solvent (the different proportioning developing solvents of chloroform/methanol) on the TLC check-out console; The developing solvent that selection can make impact point and impure point significantly separate is the agent of preparation plate development, and Rf value about 0.5 is advisable.

In the specific implementation, need adopt wherein suitable method, and choose measures necessary such as appropriate condition to reach the set goal according to existing fund, technology and relevant requirement.

And these methods also all are known technologies of this area, at needs further in the conclusive evidence, all are to be easy to find all technical data that comprise ins and outs from relevant teaching material and relevant technical literature etc.

(8) purposes of bioactive natural product B

1, general introduction

The purpose of this invention is to provide a kind of product that is used to prevent, diagnose, detect, protect, treat and study antifungal, antitumor, anti-angiogenic dementia disease and directly related disease thereof; comprise in medicine, reagent, the food etc. one or more, preferred agents.

By pharmacologically active screening proof, bioactive natural product B is the active site of its prevention, diagnosis, detection, protection, treatment and research antifungal, antitumor, anti-angiogenic dementia disease and directly related disease thereof.

Show the external development that can significantly delay relevant disease of bioactive natural product B through experimentation.Completed acute toxicity testing proves, the mouse stomach administration surpasses 1.0g/kg to the maximum tolerated dose of this active site, be equivalent to more than 200 times of clinical recommended drug dosage, show that this effective site is safe and reliable, solved complicated component in the Chinese medicine compound, active constituent content is low and has contained the problem of toxic component.

In sum; the inventor has carried out theory study to bioactive natural product B; through a large amount of particularly secular pharmacology tests of experimentation, find that the bioactive natural product B that is addressed has the activity of significant prevention, diagnosis, detection, protection, treatment and research antifungal, antitumor, anti-angiogenic dementia disease and directly related disease thereof.

Therefore, bioactive natural product B and compositions thereof can be used for preparing antifungal products, antitumor product and anti-angiogenic dementia product, are the medicine that feedstock production forms with bioactive natural product B of the present invention preferably.

2, the using method of bioactive natural product B and compositions thereof and requirement

Bioactive natural product B of the present invention can unite use separately or with other active component; comprise and be used to prepare the product that is used to prevent, diagnose, detect, protect, treat and study antifungal, antitumor, anti-angiogenic dementia disease and directly related disease thereof; comprise medicine, reagent or food etc., especially medicine.

Aspect concrete use, bioactive natural product B of the present invention can use separately, for example uses with the form of active site, its derivant or pharmaceutical salts, can also use with other many chemical substances.These chemical substances biologically active or have the function of treatment disease whether no matter, comprise miscellaneous function as collaborative amplification, antagonism or alleviate the side effect etc. of bioactive natural product B, these chemical substances are to comprise in pharmaceutically acceptable carrier, food, natural product, chemical synthetic drug or the human medication etc. one or more; Preferably include in pharmaceutically acceptable carrier or the food etc. one or more; Further preferred pharmaceutically acceptable carrier.

" pharmaceutically acceptable carrier " used herein comprises one or more in any He all physiology suitable solvent, disperse medium, afterbirth, antibacterial and antifungal, isotonic agent or the absorption delay agent etc.The example of pharmaceutically acceptable carrier comprises one or more water, saline, phosphate-buffered saline, glucose, glycerol or ethanol etc. and in the compositions one or more thereof.In many cases, in said composition, preferably include isotonic agent, for example, sugar, such as in the polyhydric alcohol of mannitol, sorbitol, sorbitol or the sodium chloride etc. one or more.Pharmaceutically acceptable carrier can also comprise a spot of auxiliary substance, one or more in wetting agent or emulsifying agent, antiseptic or the buffer etc. for example, and they have strengthened effect duration or the effectiveness of this bioactive natural product B.

From concrete classification, said pharmaceutically acceptable carrier is meant the pharmaceutical carrier of medicine and pharmacology field routine, comprises lubricant, as in Pulvis Talci, Polyethylene Glycol or the magnesium stearate etc. one or more; Disintegrating agent is as microcrystalline Cellulose etc.; Filler is as in starch, dextrin or the lactose etc. one or more; Binding agent is as in pregelatinized Starch, cellulose derivative, alginate, gelatin or the polyvinylpyrrolidone etc. one or more; Osmotic pressure regulator is as in sodium chloride, glucose, sucrose, sorbitol or the mannitol etc. one or more; The pH regulator agent, one or more in acid such as example hydrochloric acid, sodium hydroxide or the alkali; Solvent, as in water, buffer, ethanol or the propylene glycol etc. one or more etc.; Antioxidant and chelating agent are as among sodium sulfite, the EDTA etc. one or more; Surfactant is as quaternary ammonium compound, hexadecanol etc.; Absorption carrier is as in Kaolin or the soap clay etc. one or more; The macromolecular scaffold agent is as in cyclodextrin, Polyethylene Glycol, the poloxamer etc. one or more; In addition, can also in compositions, add other adjuvant, as in flavouring agent, antiseptic or the sweeting agent etc. one or more.

For example, active component bioactive natural product B is dissolved, suspendible or (for example be emulsifiable in the suitable aqueous solvent, distilled water, in normal saline or the Green's solution etc. one or more) or in the oil-based solvent (for example, vegetable oil is olive oil for example, Oleum sesami, Oleum Gossypii semen, in Semen Maydis oil or the propylene glycol etc. one or more) in, can make ejection preparation, wherein (for example can contain solubilizing agent in the solvent, polyoxyethylene sorbitan monoleate, polyoxyethylene hydrogenated Oleum Ricini, polyvidone, cyclodextrin, poloxamer, Polyethylene Glycol, benzyl alcohol, in chlorobutanol or the phenol etc. one or more), osmotic pressure regulator (for example, sodium chloride, glycerol, the D9-mannose, in D-sorbitol or the glucose etc. one or more).In this case, if necessary, can add additive, for example stabilizing agent (for example, human serum albumin etc.), analgesic (for example, one or more in procaine hydrochloride or the lignocaine etc.) etc.

Of the present invention and bioactive natural product B can also unite use with the form of compositions, particularly with other chemical substance such as medicine animal especially mammal is comprised that people or other animals treat compositions for use or similar compositions.Described mammal, comprise in people, mice, rat, sheep, monkey, cattle, pig, horse, rabbit, dog, chimpanzee, baboon, Adeps seu carnis Rhiopithecus roxellanae, macaque or the Rhesus Macacus etc. one or more, in preferred people, mice, rat, monkey, pig, rabbit or the dog etc. one or more, one or more in further preferred people, rat or the monkey etc.For example, bioactive natural product B of the present invention can be added be suitable for to curee's Pharmaceutical composition in.Usually, this Pharmaceutical composition comprises bioactive natural product B of the present invention and pharmaceutically acceptable carrier.

The compositions of bioactive natural product B particularly pharmaceutical composition can have various forms, comprises in the dosage forms such as liquid, semisolid and solid for example one or more; Wherein said pharmaceutical composition comprises that the bioactive natural product B that treats effective dose is an active component, and one or more pharmaceutically acceptable carriers.

Bioactive natural product B of the present invention or its pharmaceutical composition can adopt conventional production method well known in the art to make any dosage form that is suitable for testing, study or uses clinically, comprise solid preparation such as capsule, tablet, granular preparation etc., liquid preparation such as oral liquid or injection etc.

Active component is mixed with one or more carriers, be made into required dosage form then.Described dosage form comprises one or more in tablet, capsule, granule, suspensoid, Emulsion, solution, syrup or the injection etc., takes one or more route of administration in oral or injection (comprise in intravenous injection, intravenous drip, intramuscular injection or the subcutaneous injection etc. one or more), the mucosa dialysis etc. to carry out prevention, diagnosis, detection, protection, treatment or the scientific research of antifungal, antitumor, anti-angiogenic dementia disease and directly related disease thereof.

It is 0.5%～99% active component bioactive natural product B that pharmaceutical composition preferably contains weight ratio, further preferably contain weight ratio and be 1%～95% active component bioactive natural product B, most preferably contain weight ratio and be 5%～90% active component bioactive natural product B.

The pharmaceutical composition of bioactive natural product B generally must be aseptic and stable under the production condition of storage.Said composition can be mixed with solution, microemulsion, dispersion liquid, liposome or other is suitable for the ordered structure of high drug level.By with a kind of of this bioactive natural product B of aequum and required mentioned component or combine to add in the appropriate solvent and then carry out aseptic filtration and prepare aseptic parenteral solution.Generally speaking, prepare dispersion liquid by this bioactive natural product B being added in the aseptic solvent contain basic disperse medium and required above-mentioned other composition.Under the situation of the sterile powder that is used to prepare aseptic parenteral solution, the preparation method of recommendation is vacuum drying and lyophilized preparation.For example, by passing through to keep required granular size such as the coating of lecithin, under the situation of dispersion liquid and, can keeping the adequate liquidity of solution by using surfactant.

Can comprise the medicament that postpones absorption in the said composition, for example Monostearate or gelatin absorb with the prolongation that reaches injectable composition; Can comprise the high molecular polymer carrier,, discharge with the prolongation that reaches Orally administered composition as hydroxypropyl methylcellulose or polyoxyethylene.

When being used for the patient, bioactive natural product B dosage of the present invention is 5～20mg/kgd, can use one or more times, and this dosage or consumption decide according to the age of patient or user and the situation of body weight and health or patient's symptom usually.

Bioactive natural product B of the present invention and Pharmaceutical composition thereof can comprise the bioactive natural product B of the present invention of " treatment effective dose " or " prevention effective dose "." treatment effective dose " is meant at the dosage of necessity and effectively reaches the amount of required therapeutic effect under the time.The treatment effective dose of bioactive natural product B can cause that at this individuality the factors such as ability of required reaction change according to the patient's condition, age, sex and body weight and this bioactive natural product B such as individuality.The treatment effective dose also refers to that the useful therapeutic effect of this bioactive natural product B surpasses the amount of its any toxicity or harmful effect.

" prevention effective dose " is meant the amount that effectively reaches required preventive effect under necessary dosage and time.Because preventive dose is used for the ill preceding or early stage curee of disease, the prevention effective dose is usually less than the treatment effective dose.The typical non-limiting scope of the treatment of bioactive natural product B of the present invention or prevention effective dose is 5～20mg/kg, more preferably 5～10mg/kg.Should note, dose value will change according to disease type of desiring to alleviate and seriousness, that is to say when being used for the patient that bioactive natural product B dosage of the present invention or consumption decide according to the age of patient or user and the situation of body weight and health or patient's symptom usually.

In addition; should understand; for any specific curee; should along with the time according to individual need and give with or supervision give with the people's of described compositions professional judgement and adjust the given dose system; and the dosage range that this paper sets only be illustrative, the scope or the practice of the compositions of can't requirement for restriction protecting.

That is to say, need be according to object, route of administration, institute's disease for the treatment of and the situation etc. of treatment, change bioactive natural product B of the present invention at every turn and/or dosage or the consumption of every day.For example, give mammal through vein, adult (as body weight 60kg) especially, the single dose of described bioactive natural product B is about 50～1200mg, preferably about 100mg, preferred administration every day 1～3 time.Can adjust dosage unit, to propose the best required reaction of arch (for example, treatment or prevention are replied).

For example, can single heavy dose of administration can give several divided doses or reduce or increase dosage in proportion according to the urgency of treatment situation in a period of time.The non-intestinal compositions that preparation is easy to the unified dosage unit form of administration and dosage is especially favourable.Dosage unit form used herein refers to be suitable for the physical separation unit of dosage unit of the mammalian subject of desire treatment; The calculating that each unit contains scheduled volume is used for together producing with required pharmaceutical carrier the active matter bioactive natural product B of required therapeutic effect.The specification of dosage unit form of the present invention, determine and directly depend on the specific characteristic of following (a) this bioactive natural product B and the particular treatment of desiring to reach or preventive effect and (b) interior in mixing this technology that is used for the treatment of individual sensitivity bioactive natural product B by following in restriction.

3, the pharmaceutical dosage form of bioactive natural product B and compositions thereof and route of administration

The product that is used to prevent, diagnose, detect, protect, treat and study antifungal, antitumor, anti-angiogenic dementia disease and directly related disease thereof of bioactive natural product B of the present invention and preparation of compositions thereof, wherein the product according to the requirement of beverage, food technology field preparation can be used in prevention, protection and treats antifungal, antitumor, anti-angiogenic dementia disease and directly related disease thereof; Can be used in patient's treatment or health care according to the product of the requirement of medical technical field preparation, can either be directly used in the medicine of preparation treatment or health care separately, also can mix with many chemical substances or make up, directly or indirectly be used to prepare the medicine of treatment or health care.Chemical substance described here is above described identical with this section.

In the present invention, required material comprises raw material of the present invention, above-mentioned matching used chemical substance etc., all should adopt the material of food stage or pharmaceutical grade according to practical situation and needs.

Bioactive natural product B of the present invention and compositions thereof can be with the whole bag of tricks administration known in the art, although route of administration/administering mode of recommending in many therapeutic use is spray or oral administration.But the technical staff will appreciate that route of administration/administering mode changes with required result.In some concrete enforcement, the carrier that this reactive compound can avoid rapid release with this chemical compound of protection is preparation example such as empty release formulation together, comprises that graft transmission system, transdermal paste one or more in transmission system or the microcapsule transmission system etc.In addition, can also use biodegradable, biocompatible polymer, for example one or more in ethylene-ethyl acetate, polyanhydride, polyglycolic acid, collagen protein, polyorthoesters or the polylactic acid etc.The equal patent applied for of many methods or (the Sustainedand Controlled Release Drug Delivery Systems for example generally known to those skilled in the art that prepare this preparation, J.R.Robinson edits, Marcel Dekker, Inc., New York, 1978).

In bioactive natural product B of the present invention or its pharmaceutical composition, can contain pharmaceutically acceptable carrier well known in the art and other optional member.Carrier comprises carboxymethyl starch, starch, cellulose, gelatin, sodium bicarbonate, propylene glycol or Tween 80 etc.Optional member for example is coloring agent, sweeting agent, antioxidant etc.

Bioactive natural product B of the present invention and compositions thereof usually by one or more modes in oral, rectum or the parenteral etc., are applied to the patient who needs this treatment.

Bioactive natural product B of the present invention, its derivant or pharmaceutical salts, compositions particularly pharmaceutical composition can be made any dosage form that is suitable for using clinically, comprise solid preparation, as capsule, tablet, granular preparation etc., semi-solid preparation such as ointment etc., liquid preparation such as oral liquid, suspensoid, Emulsion etc., perhaps injection.Take one or more route of administration in oral or injection (comprise in intravenous injection, intravenous drip, intramuscular injection or the subcutaneous injection etc. one or more), the mucosa dialysis etc. to carry out prevention, diagnosis, detection, protection, treatment or the scientific research of vascular dementia and alzheimer disease and directly related disease thereof.

Be used for when oral, can be made into conventional solid preparation such as in tablet, powder, granule or the capsule etc. one or more.When implementing, bioactive natural product B of the present invention can be together oral with for example inert diluent or assimilable edible carrier.This bioactive natural product B (with its composition altogether, if desired) can also be wrapped in hard or soft shell gelatin capsules, is pressed into tablet or directly adds in curee's the meals.About oral therapeutic administration, described bioactive natural product B can be added with excipient and use with one or more forms in edible tablet, buccal tablet agent, lozenge, capsule, suspension, syrup or wafer or the like.

For to give bioactive natural product B of the present invention outside the parenterai administration, may need with preventing that the material of its inactivation from together giving to this bioactive natural product B coating or with this bioactive natural product B.The reactive compound that replenishes can also be added in the said composition.In the specific implementation, bioactive natural product B of the present invention and one or more other medicines that can be used for the treatment of disease are prepared altogether and/or given altogether.Thisly unite use, can utilize this medicine that gives primely, therefore avoid possible toxicity or the complication relevant with various monotherapies than low dosage.

Make in liquid preparation such as water preparation, oil-suspending agent or other liquid preparation one or more, as in syrup, tincture or the elixir etc. one or more; When being used for parenteral, can be made in solution, water preparation or the oiliness suspending agent etc. of injection one or more.

Above medicine or pharmaceutical composition can use various approach, in described type of service, preferred form is that oral formulations (as in tablet, coated tablet, capsule, solution or the suspension etc. one or more), non-intestinal give one or more in the dosage form (as in injection, ointment or the patch etc. one or more) etc., further one or more in preferred tablet, capsule or the injection etc., a kind of in special preferred tablet or the injection.

In addition; the employed medicinal raw material of bioactive natural product B comprises that marine fungi or marine fungi crude extract etc. also can be directly used in preparation in some cases separately and be used for prevention; diagnosis; detect; protection; treatment and research antifungal; antitumor; the product of anti-angiogenic dementia disease and directly related disease thereof; also can mix with many chemical substances or make up, directly or indirectly be used for preparation with the form of compositions and be used for prevention; diagnosis; detect; protection; treatment and research antifungal; antitumor; the product of anti-angiogenic dementia disease and directly related disease thereof.Chemical substance described here is above described identical with this section.

For example; the employed medicinal raw material of bioactive natural product B comprises that the powder of marine fungi or marine fungi crude extract etc. is used for preparation and is used for antifungal; antitumor; anti-angiogenic dementia disease prevention; diagnosis; protection and treatment product be the various dosage forms of medicine particularly; or the employed medicinal raw material of the bioactive natural product B powder that comprises marine fungi or marine fungi crude extract etc. is used for preparation with relevant adjuvant and is used for prevention; diagnosis; detect; protection; treatment and research antifungal; antitumor; the product of anti-angiogenic dementia disease and directly related disease thereof is the various dosage forms of medicine especially; or the employed medicinal raw material of the bioactive natural product B powder that comprises marine fungi or marine fungi crude extract etc. is used for prevention with relevant preparation; diagnosis; detect; protection; treatment and research antifungal; antitumor; the product of anti-angiogenic dementia disease and directly related disease thereof such as medicine one are used from preparation and are used for prevention; diagnosis; detect; protection; treatment and research antifungal; antitumor; the various dosage forms of the product of anti-angiogenic dementia disease and directly related disease thereof such as medicine; or the employed medicinal raw material of the bioactive natural product B powder that comprises marine fungi or marine fungi crude extract etc. is used from preparation with relevant ancillary drug one and is used for prevention; diagnosis; detect; protection; treatment and research antifungal; antitumor; the various dosage forms of the product of anti-angiogenic dementia disease and directly related disease thereof such as medicine; as tablet; in capsule or the suspensoid etc. one or more, preferred capsule.

One of described method is that the employed medicinal raw material of bioactive natural product B is comprised that the powder fill of marine fungi or marine fungi crude extract etc. is a capsule, to be powder that the employed medicinal raw material of bioactive natural product B is comprised marine fungi or marine fungi crude extract etc. be used for prevention with relevant preparation to two of method, diagnosis, detect, protection, treatment and research antifungal, antitumor, the product of anti-angiogenic dementia disease and directly related disease thereof such as medicine fill together are capsule, and three of method is powder that the employed medicinal raw material of bioactive natural product B is comprised marine fungi or marine fungi crude extract etc. with relevant ancillary drug fill together is capsule; Four of method is that the employed medicinal raw material of bioactive natural product B is comprised that it is tablet that the powder of marine fungi or marine fungi crude extract etc. is directly pressed together according to a conventional method with relevant adjuvant; five of method is the powder that the employed medicinal raw material of bioactive natural product B comprised marine fungi or marine fungi crude extract etc.; relevant preparation is used for prevention; diagnosis; detect; protection; treatment and research antifungal; antitumor; the product of anti-angiogenic dementia disease and directly related disease thereof such as medicine are directly pressed according to a conventional method with relevant adjuvant together and are tablet, and six of method is the powder that the employed medicinal raw material of bioactive natural product B comprised marine fungi or marine fungi crude extract etc.; it is tablet etc. that relevant ancillary drug is directly pressed according to a conventional method with relevant adjuvant together.

Except that six kinds of above-mentioned basic skills, can also select the employed medicinal raw material of bioactive natural product B to comprise other forms of marine fungi or marine fungi crude extract etc. or the employed medicinal raw material of bioactive natural product B is carried out after method well known in the art handles, prepare the product such as the medicine that contain the employed medicinal raw material of bioactive natural product B of various dosage forms.But, it should be noted that, when the employed medicinal raw material of above-mentioned direct use bioactive natural product B comprises marine fungi or marine fungi crude extract etc., should be earlier according to the dosage requirement of employed bioactive natural product B, the employed medicinal raw material of bioactive natural product B that obtains required use of converting comprises the consumption of marine fungi or marine fungi crude extract etc.

In sum; bioactive natural product B of the present invention and compositions thereof can be used for preventing, diagnose, detect, protect, treating and study the product of antifungal, antitumor, anti-angiogenic dementia disease and directly related disease thereof; preferred agents and food, further preferred agents.

(9) technology speciality

The present invention has expanded new medical usage to four or five spiro lactone class bioactive natural product B, also provides a kind of new medicament sources for prevention, diagnosis, detection, protection, treatment and research antifungal, antitumor, anti-angiogenic dementia disease and directly related disease thereof.Bioactive natural product B safety and low toxicity of the present invention; pharmacological action is stronger; its raw material sources are abundant, inexpensive; preparation technology is simple; the yield height, the product that can be used for preparing prevention, diagnosis, detection, protection, treatment and study antifungal, antitumor, anti-angiogenic dementia disease and directly related disease thereof.

The present invention studies bioactive natural product B targetedly, and bioactive natural product B pharmacological action is stronger, and it is safe in utilization, and one-object-many-purposes has been brought into play effect to greatest extent; Bioactive natural product B stable in properties uses the quality of the pharmaceutical preparations of preparation stable, is more suitable for the suitability for industrialized production of antifungal, antitumor, anti-angiogenic dementia disease product; The effect of prevention, diagnosis, detection, protection, treatment and research antifungal, antitumor, anti-angiogenic dementia disease and directly related disease thereof is obvious; and the scope of application is wide especially; therefore apply easily, can have a tremendous social and economic benefits in the short period of time.

In a word; active adaption of the present invention modern medical service and the job demand of scientific research field and the needs of human nature service; the present invention provides new source for researching and developing new antifungal, antitumor, anti-angiogenic dementia disease product; the resources of medicinal plant that develops China is had important value, is the safe raw material that is used to prevent, diagnose, detect, protect, treat and study aspects such as antifungal, antitumor, anti-angiogenic dementia disease and directly related disease thereof.

The specific embodiment

The present invention has studied the new pharmacological action of existing bioactive natural product B and new purposes; a kind of raw material that can be used in products such as preparation antifungal, antitumor, the prevention of anti-angiogenic dementia disease, diagnosis, protection and treatment is provided, has been convenient to the safe handling in medical industry and fields such as relevant industries such as food, beverage.

The preparation method of the common drug preparation of bioactive natural product B and compositions thereof

The present invention prepares injectable powder and generally adopts conventional freeze-drying, as solvent, the steps include: to get bioactive natural product B with water, adds excipient, be dissolved in water, regulate pH, add active carbon, filtration sterilization, plug is partly rolled in fill, and lyophilization, tamponade are rolled lid and got final product.Used excipient is selected from one or more in mannitol, gelatin hydrolysate, glucose, lactose, dextran, albumin, the pH regulator agent etc.Every bottle contains bioactive natural product B1～100mg.

The present invention prepares injectable powder also can adopt spray drying method, as solvent, the steps include: to get bioactive natural product B with water, adds or do not add excipient (excipient is the same), be dissolved in water, add active carbon, filtration sterilization, spray drying, aseptic subpackaged, tamponade is rolled lid and is got final product.Every bottle contains bioactive natural product B10～100mg.

When the present invention prepares small-volume injection, preparation gets final product as solvent with water for injection, also can add appropriate amount of auxiliary materials, adjuvant is selected from one or more in ethanol, propylene glycol, glycerol, Polyethylene Glycol, benzoic acid, dimethyl acetylamide, pH regulator agent, surfactant, cyclodextrin, antioxidant, complexing of metal ion agent, the antibacterial.Injection can be mixed with solution, microemulsion, emulsion, liposome, microsphere, microcapsule or other is suitable for the ordered structure of high drug level, wherein can comprise the medicament that postpones absorption, for example Monostearate, gelatin, ethylene-ethyl acetate, polyanhydride, polyglycolic acid, collagen protein, polyorthoesters or polylactic acid etc. absorb with the prolongation that reaches injectable composition.Every contains bioactive natural product B1～100mg.

The present invention prepares glucose infusion liquid or sodium chloride transfusion, with water for injection as solvent, adding the preparation of an amount of glucose or sodium chloride gets final product, also can add appropriate amount of auxiliary materials, adjuvant is selected from one or more in ethanol, propylene glycol, glycerol, Polyethylene Glycol, benzoic acid, dimethyl acetylamide, pH regulator agent, surfactant, antioxidant, cyclodextrin, complexing of metal ion agent, the antibacterial.Every bottle contains bioactive natural product B1～100mg.

The present invention prepares oral formulations such as tablet, capsule, granule, oral liquid, and adjuvant can be lactose, starch, dextrin, stearate etc., technology preparation routinely.Can comprise the high molecular polymer carrier,, discharge with the prolongation that reaches Orally administered composition as hydroxypropyl methylcellulose or polyoxyethylene etc.

In the present invention, the embodiment of the above-described specific embodiment and the following stated all is in order to set forth the present invention better, is not to be used for limiting the scope of the invention.

Below by embodiment the present invention is described in detail.

The preparation of embodiment 1, bioactive natural product B injectable powder

Get bioactive natural product B 10g, add dextran 30g, add 1500ml water for injection, regulate pH to 9.0, stir and make its dissolving; Add the injection water to 2000ml, add the 3.0g needle-use activated carbon, fully stirred 30 minutes; Decarbonization filtering; With 0.22 μ m filtering with microporous membrane; Plug is partly rolled in packing; Lyophilization, tamponade is rolled lid and is got final product again.

The preparation of embodiment 2, bioactive natural product B injectable powder

Get bioactive natural product B 50g, add mannitol 60g, add 5000ml water for injection, regulate pH to 8.0, stir and make its dissolving; Add the injection water to 6000ml, add the 1g needle-use activated carbon, fully stirred 30 minutes; Decarbonization filtering; With 0.22 μ m filtering with microporous membrane; Plug is partly rolled in packing; Lyophilization, tamponade is rolled lid and is got final product again.

The preparation of embodiment 3, bioactive natural product B injectable powder

Get bioactive natural product B 1g, add glucose 50g, add 900ml water for injection, regulate pH to 8.5, stir and make its dissolving; Add the injection water to 1000ml, add the 1.5g needle-use activated carbon, fully stirred 30 minutes; Decarbonization filtering; With 0.22 μ m filtering with microporous membrane; Plug is partly rolled in packing; Lyophilization, tamponade is rolled lid and is got final product again.

The preparation of embodiment 4, bioactive natural product B small-volume injection

Get bioactive natural product B 5g, add 900ml water for injection, regulate pH to 9.0, stir and make its dissolving; Add the injection water to 1000ml, add the 1.0g needle-use activated carbon, fully stirred 30 minutes; Decarbonization filtering; With 0.22 μ m filtering with microporous membrane; The packing embedding, every bottle of 10ml.

The preparation of embodiment 5, bioactive natural product B small-volume injection

Get bioactive natural product B 10g, add ethanol 600ml, stir and make its dissolving; Add the injection water to 1000ml, add the 0.5g needle-use activated carbon, fully stirred 30 minutes; Decarbonization filtering; With 0.22 μ m filtering with microporous membrane; The packing embedding, every bottle of 5ml.

The preparation of embodiment 6, bioactive natural product B glucose infusion liquid

Get bioactive natural product B 2g, add Polyethylene Glycol 10g, add glucose 500g, add 4500ml water for injection, stir and make its dissolving; Add the 5g needle-use activated carbon, fully stirred 30 minutes; Decarbonization filtering; Add the injection water to 5000ml; With 0.22 μ m filtering with microporous membrane; The packing embedding, every bottle of 500ml, pressure sterilizing.

The preparation of embodiment 7, the transfusion of bioactive natural product B sodium chloride

Get bioactive natural product B 1g, add sodium chloride 90g, add 9000ml water for injection, stir and make its dissolving; Add the 10g needle-use activated carbon, fully stirred 30 minutes; Decarbonization filtering; Add the injection water to 10000ml; With 0.22 μ m filtering with microporous membrane; The packing embedding, every bottle of 250ml, pressure sterilizing gets final product.

The preparation of embodiment 8, bioactive natural product B tablet

(1) prescription

Bioactive natural product B 1000.0g

Microcrystalline Cellulose 1170.0g

Pregelatinized Starch 690.0g

Lactose 125.0g

The 5%PVP dehydrated alcohol is an amount of

Magnesium stearate 15.0g

Make 10000 altogether

Coating fluid prescription: gastric solubleness thin film dress material: 85G61235, Shanghai Colorcon Coating Technology Co., Ltd

(2) preparation technology takes by weighing the principal agent and the adjuvant of recipe quantity respectively by above prescription, by the equivalent method mix homogeneously that progressively increases, 5%PVP dehydrated alcohol system soft material, drying, granulate, add the magnesium stearate mixing, measure content, calculate the heavy back of sheet tabletting, control nude film hardness 5～7kg, make 9698 in tablet altogether, yield rate is 96.98%.

Adopt conventional high-efficiency coating pan coating, technology is as follows: (hardness 5～7kg) is put into coating pan with nude film, start agitating device and air blast heater, when treating that the nude film temperature rises to 40 ℃, last 1/3 place that begins to open spray gun alignment tab bed sprays into the coating solution coating, 38～42 ℃ of control strip bed tempertaures, gas pound pressure 6kg, the coating solution flow velocity is 50mL/min, coating membrane heavily account for coated tablet heavy 3%.

The preparation of embodiment 9, bioactive natural product B tablet

(1) prescription

Bioactive natural product B 100.0g

Lactose 80.0g

Starch 60.0g

Carboxymethyl starch sodium 15.0g

The 10%PVP aqueous solution is an amount of

Magnesium stearate 3.0g

Make 1000 altogether

(2) preparation technology gets supplementary material and crosses 80 mesh sieves respectively, and mix homogeneously with an amount of 10%PVP solution system soft material, drying, adds magnesium stearate 3g, granulate, and tabletting is made 1000.

The preparation of embodiment 10, bioactive natural product B capsule

(1) prescription

Bioactive natural product B 1000.0g

Microcrystalline Cellulose 1000g

Starch 140g

Dehydrated alcohol is an amount of

Pulvis Talci 80g

Make 10000 capsules altogether

(2) preparation technology gets in crude drug bioactive natural product B and the prescription other adjuvant respectively and crosses 100 mesh sieves respectively, put 60 ℃ of oven dry, take by weighing recipe quantity bioactive natural product B and microcrystalline Cellulose, the starch equivalent method mix homogeneously that progressively increases, with an amount of dehydrated alcohol system soft material, 30 mesh sieves are granulated, and 50～60 ℃ of dryings 2 hours are with 30 mesh sieve granulate, the Pulvis Talci mix homogeneously that adds recipe quantity is filled.

The disease-resistant fungal pathogens activity of embodiment 11, detection of active natural product B

1. material

1.1. bacterial strain:

The ATCC type strain: Candida albicans (Candida albicans) ATCC76615, neogenesis cryptococcus (Cryptococcusneoformans) ATCC32609 give by Long March hospital strain preservation center.

Clinical strain: trichophyton (Trichophyton rubrum) 0504656, Aspergillus fumigatus (Aspergillus fumigatus) 0504656, pick up from the different section office of Shanghai Changhai Hospital clinical sample respectively, and through morphology and biochemical evaluation.

1.2. culture fluid:

RPMI1640 culture fluid: RPMI1640 (Gibco BRL) 10g, NaHCO ₃2.0g, morphine quinoline propane sulfonic acid (morpholinepropanesulfonic acid, MOPS) (Sigma) 34.5g (0.165M) add tri-distilled water 900ml dissolving, and NaOH transfers pH to 7.0 (25 ℃), is settled to 1000ml, filters sterilization, 4 ℃ of preservations.

Husky fort glucose agar medium (SDA): peptone 10g, glucose 40g, agar 18g adds tri-distilled water 900ml dissolving, adds 2mg/ml chloramphenicol solution 50ml, adjusts pH to 7.0, is settled to 1000ml, 4 ℃ of preservations behind the autoclaving.

The YEPD culture fluid: yeast extract 10g, peptone 20g, glucose 20g adds tri-distilled water 900ml dissolving, adds 2mg/ml chloramphenicol solution 50ml, is settled to 1000ml, 4 ℃ of preservations behind the autoclaving.

1.3. reagent:

It is homemade analytical pure that fluconazol (fluconazole) and ketoconazole (ketoconazole) provide .DMSO by the court's organic chemistry teaching and research room, heavily steams with preceding.

1.4. instrument:

Water isolation type electro-heating standing-temperature cultivator (Shanghai make a leapleap forward medical apparatus and instruments factory); The desk-top constant temperature oscillator of THZ-82A (Shanghai make a leapleap forward medical apparatus and instruments factory); Multiskan MK3 enzyme micro-plate reader (Labsystems Dragon);

2. operating procedure

2.1. bacterium liquid preparation:

Before the experiment, a small amount of with inoculation circle spherical bacterium such as picking neogenesis cryptococcus, Candida albicans and Candida parapsilosis on the SDA culture medium of 4 ℃ of preservations, be seeded to the 1mlYEPD culture fluid, in 35 ℃, the 250rpm shaken cultivation, activation 16h, make fungus be in later stage exponential phase of growth. get this bacterium liquid to the 1mlYEPD culture fluid, activate once more with said method, behind the 16h, with the blood cell counting plate counting, adjust bacterial concentration to 1 * 10 with the RPMI1640 culture fluid ³～5 * 10 ³Individual/ml.

Aspergillus fumigatus is seeded to the SDA inclined-plane, cultivates a week for 35 ℃; Trichophyton, cultivated for two weeks for 28 ℃. after each bacterium activates twice by this method, add an amount of RPMI1640 culture fluid in the SDA inclined-plane, blow and beat bacterium colony with suction pipe, fungal spore is free in the RPMI1640 culture fluid, filtering through four layers of sterile gauze then. culture fluid adds the RPMI1640 culture fluid and adjusts spore concentration to 1 * 10 behind the blood cell counting plate counting ³～5 * 10 ³Individual/ml.

2.2. medicinal liquid preparation:

Be subjected to the reagent thing to be made into 6.4gL with DMSO respectively ^-1Solution ,-20 ℃ of preservations before the experiment, are put 35 ℃ of incubators with the medicinal liquid taking-up and are melted standby.

2.3. drug sensitive plate preparation:

Get aseptic 96 orifice plates, add RPMI1640 100 μ l in No. 1 hole of every row and make blank; 3～No. 12 the hole respectively adds freshly prepared bacterium liquid 120 μ l; No. 2 the hole adds bacterium liquid 160 μ l and l.2～10 grades of 4 times of dilutions in No. 11 hole of test-compound solution 1.6 μ respectively, makes that medicine final concentration in each hole is respectively 64,16,4,1,0.25 and 0.0625,0.0156,0.0039,0.00097,0.00024mgL ^-1, DMSO content all is lower than 1% in each hole; No. 12 the hole does not contain medicine, makes positive control, and each drug sensitive plate is in 35 ℃ of cultivations.

2.4.MIC ₈₀Value is judged:

Candidiasis, neogenesis cryptococcus and filamentous bacteria are cultivated 24h, 72h and after the week, are surveyed each hole OD value with enzyme micro-plate reader in 630nm respectively at 35 ℃. and with the positive control boring ratio, be MIC with the drug level in the least concentration hole of OD value decline more than 80% ₈₀(drug level when conk 80% is suppressed).

MIC when medicine ₈₀Value surpasses when measuring concentration range, adds up by the following method: MIC ₈₀Value is higher than maximum concentration 64mgL ^-1The time, count "＞64mgL ^-1"; MIC ₈₀Value is not distinguished for least concentration or when least concentration is following, all counts "≤0.00024mgL ^-1".

The equal operation repetitive of above-mentioned experiment 2 to 3 times is worked as MIC ₈₀Value just is accepted in the time of accurately repeating or only differ from a concentration, and with higher concentration as MIC ₈₀Value is worked as MIC ₈₀Value differs two concentration when above, then needs experiment again, till meeting the requirements.The data show compd B is for Candida albicans in the table 13, and the effect of neogenesis cryptococcus is better than griseofulvin.

Claims (16)

1. the application of bioactive natural product B in preparation antifungal products, antitumor product and anti-angiogenic dementia product.

2. the application of the compositions of bioactive natural product B according to claim 1 in preparation antifungal products, antitumor product and anti-angiogenic dementia product.

3, the application of the medicinal raw material of bioactive natural product B according to claim 1 in preparation antifungal products, antitumor product and anti-angiogenic dementia product.

4, the application of bioactive natural product B according to claim 3 is characterized in that, the medicinal raw material of described bioactive natural product B is a kind of in marine fungi or the marine fungi crude extract.

5, the application of bioactive natural product B according to claim 4 is characterized in that, the medicinal raw material of described bioactive natural product B is a kind of in marine fungi Massarina tunicata or the marine fungi Massarina tunicata crude extract.

6, the application of the compositions of the medicinal raw material of bioactive natural product B according to claim 1 in preparation antifungal products, antitumor product and anti-angiogenic dementia product.

7, the application of bioactive natural product B according to claim 6 is characterized in that, the medicinal raw material of described bioactive natural product B is a kind of in marine fungi or the marine fungi crude extract.

8, the application of bioactive natural product B according to claim 7 is characterized in that, the medicinal raw material of described bioactive natural product B is a kind of in marine fungi Massarina tunicata or the marine fungi Massarina tunicata crude extract.

9, according to the application of each described bioactive natural product B of claim 1～8; it is characterized in that; described antifungal products is meant one or more in the product that is directly used in prevention, diagnosis, detection, protection, treatment and research pathogenic fungi and directly related disease thereof, and described pathogenic fungi is meant white coccus, neogenesis cryptococcus, trichophyton and the Aspergillus fumigatus read.

10, according to the application of each described bioactive natural product B of claim 1～8; it is characterized in that; described antitumor product is meant one or more in the product that is directly used in prevention, diagnosis, detection, protection, treatment and research tumor and directly related disease thereof, and described tumor is meant hepatocarcinoma, breast carcinoma and pulmonary carcinoma.

11. application according to each described bioactive natural product B of claim 1～8; it is characterized in that described anti-angiogenic dementia product is meant one or more in the product that is directly used in prevention, diagnosis, detection, protection, treatment and research vascular dementia and directly related disease thereof.

12. the application according to each described bioactive natural product B of claim 1～8 is characterized in that, described antifungal products, antitumor product and anti-angiogenic dementia product are to comprise a kind of in medicine, reagent or the food.

13. the application of bioactive natural product B according to claim 12 is characterized in that, described antifungal products, antitumor product and anti-angiogenic dementia product are medicines.

14. the application according to each described bioactive natural product B of claim 1～8 is characterized in that, this bioactive natural product B is meant resveratrol-3-O-β-D-glucoside, and structural formula is as follows:

15. the application of bioactive natural product B according to claim 14 is characterized in that, the purity of described bioactive natural product B 〉=95%.

16. the application of bioactive natural product B according to claim 14 is characterized in that, the occupation mode of described bioactive natural product B is to comprise independent use or unite a kind of in the use with other chemical substances.