CN102293767B - Application of active natural product B in preparing anti-vascular dementia products - Google Patents

Application of active natural product B in preparing anti-vascular dementia products Download PDF

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CN102293767B
CN102293767B CN201110193628.4A CN201110193628A CN102293767B CN 102293767 B CN102293767 B CN 102293767B CN 201110193628 A CN201110193628 A CN 201110193628A CN 102293767 B CN102293767 B CN 102293767B
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natural product
bioactive natural
dementia
product
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CN102293767A (en
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秦路平
游飞
冯建平
徐丽莉
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Hangzhou Polytron Technologies Inc
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Hangzhou Twin-Horse Bioengineering Co Ltd
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Abstract

The invention relates to application of a marine fungus extract-active natural product B. The in-vitro study test indicates that the active natural product B has certain inhibiting actions on candida albicans, Cryptococcus neoformans, Trichophyton rubrum, aspergillus fumigatus and other common clinical pathogenic fungi, can inhibit growth of liver cancer, mammary cancer, lung cancer and other tumor cell strains to different degrees, can have strong protective actions on pathogenic injuries caused by rat multiple infarctional cerebral ischemia injuries and the like, can obviously improve the learning and memory functions of rats with dementia, and can be used for preparing anti-vascular dementia products. The invention provides a new medicine source for clinically resisting fungi, tumors and vascular dementia and treating directly related diseases, and has an important value for developing and utilizing medicine resources in China.

Description

Bioactive natural product B is for the preparation of the purposes of anti-angiogenic dementia product
The application is the divisional application of original application " bioactive natural product B is for the preparation of the purposes of antifungal and antitumor product " (application number: 200910194849.6, the applying date: on 08 31st, 2009).
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 for the preparation of the purposes of anti-angiogenic dementia product, more particularly relate to a kind of marine fungi extract---bioactive natural product B, 4,6-Dihydroxy-3-methylene-10-propenyl-2-oxa-spiro[4,5] dec-8-ene-1,7-dione, for the preparation of the purposes of anti-angiogenic dementia product.
Background technology
(1) research overview of marine fungi
1, general introduction
Marine microorganism survives in special biotic environment with it and has a great potential that produces 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, antibacterial and antiviral activity; Separately there are some compounds to demonstrate unique physiologically active, as anti-senile dementia.
Therefore, Marine microorganism is a study hotspot in recent years.Increasing research shows, Marine microorganism, and---especially those exist the microorganism of the relation such as symbiosis or parasitism with marine animal or sea-plant---can produce the metabolite of structure uniqueness the strong physiologically active of tool.And Marine microorganism breeding is fast, can utilize modern microbial technique, 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 having the microorganism of eucaryon structure in ocean, be one and can in sea water, breed and complete life cycle, the fungus monoid that can well grow again on sea water medium, claim again sea water fungus.That is: there is eucaryon structure, can form spore, seek marine organisms (1. J.Kohlmeyer and E.Kohlmeyer, Marine Mycology, Academic Press, New York, 1979 of saprophytic or parasitic life; 2. T.W.Johnson, F.K.Sparrow, Fungi in Oceans and Estuaries, J.Cramer, Weinheim, 1961).
Marine fungi is generally mycelioid and many cells, only has yeast in the stage of development, to have unicellular appearance.Nutritional mode, except Acarasiales is for the formula of ingesting, mostly is absorption.Some derive from growth and reproduction person in habitat, Bing Neng ocean, ocean, are called obligate marine fungi; Other derive from land or fresh water, but can be in habitat, ocean growth and reproduction person, be called facultative marine fungi.
The acid-base value of their adaptation sea water, osmophilic strain ability is stronger, and great majority are dwelt and are lived in certain base thing, for example, parasitize Sargassum and marine animal, or on the saprophytic timber in soaking in sea water; Minority free living, for example, can be grown in the wetland and evergreen chinquapin marsh of saliferous.Therefore, the distribution of fungus in ocean depends primarily on host's distribution.
Study History French M.C.de Deere in 1869 approximately waits and from zostera marina, first finds marine fungi.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 raw 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., has systematically discussed the achievement in research of high marine fungi.
At present, most scholars think that fungus should independently become boundary (King-dom mycota) in classification.But to the division of affiliated door, guiding principle etc., go back so far the scheme that neither one is comparatively generally acknowledged.According to existing record, still 500 kinds of less thaies of marine fungi, are only equivalent to 1% of the true strain number in land (approximately 50,000 kinds).Comprise 209 kinds of thread high marine fungis, 177 kinds of marine yeast mushrooms, low marine fungi algal fungi class (Phycomycetes) approximately 70 kinds that wait.In high marine fungi, there are 56 kinds of 149 kinds of ascomycetes (Ascomycetes), 4 kinds of basidiomycetes (Basidiomycetes) and Fungi Imperfectis (Deuteromycetes).
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 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 have that hat spore shell belongs to, the raw shell in sea belongs to, raw shell genuss of wood, oar spore shell genus, Leucosporidium, plan Corallium Japonicum Kishinouye spore genus, Humicola and without obstructing spore genus etc.;
3. spoon sporangiocyst order colonizes on red algae, and the ability of decomposing halogen rope is stronger.The evidence that provides mushroom to originate from red algae is provided spoon sporangiocyst, and someone thinks that it is that in sea water, timber raw ascomycetous direct ancestors.
Distribution ocean Distribution of fungi is extensive, from Intertidal zone high-water mark or river mouth to deep-sea, to halmeic deposit, all has its member from sandy beach, shallow sea.Marine yeast and lower fungus grow nonparasitically upon another plant on plankton and animal body, thereby also have distribution at large midocean; The growth of high marine fungi requires suitable base thing as the raw place of dwelling, and therefore focuses mostly on and is distributed in littoral sea.Due to the saprophytic or parasitic life of marine fungi battalion, so the feature of its geographical distribution is the geographic range that depends on host.But the concentration of Dissolved Oxygen in Seawater and sea water water temperature are also to affect marine fungi to exist and the important factor developing.Nearly all fungus all can grow under the condition that is less than sodium oxide concentration in sea water, and therefore salt tolerance can not be served as the mark of distinguishing marine fungi and land fungus.
Derive from the depth of water and exceed 500 meters of funguses in marine environment, there is significantly the ability that adapts to high pressure, low temperature and grow.The deep-sea fungus of now knowing only has 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 base thing and live, and only have minority fungus not rely on base thing and free living.
According to its raw habit of dwelling, marine fungi can be divided into 5 kinds of basic ecotypes:
1. the raw fungus of wood.The widest with the distribution at most higher fungus of quantity in ocean water body, battalion's saprogenesis, kind decomposition of cellulose.In Tropical Ocean Area and neritic environment, distribute more extensive.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 consumingly timber and other fibrous matters.They are extensive compared with temperate zone and polar region in the distribution of Tropical Ocean Area, extensive compared with deep-sea in the distribution of neritic environment.In 107 kinds of known raw funguses of ocean wood, ascomycetes accounts for 76 kinds, and Fungi Imperfecti accounts for 29 kinds, and basidiomycetes only has 2 kinds.
2. parasitic frond fungus.Account for 1/3 of marine fungi kind number, wherein in the majority with ascomycetes.There are the types such as saprophytic, parasitism and symbiosis.
In marine fungi, approximately there are 1/3 kind and algae to be related, wherein in the majority with ascomycetes.There are the types such as saprophytic, parasitism and symbiosis.The distribution of different Sargassums can directly affect the distribution of such fungus, as in the Sargasso Sea of the North Atlantic Ocean, the kind of fungus and quantity are all little, also seldom find by the Sargassum of fungal infection, and this is the tannin material due to Alga Sgrgassi Enerves (Sar as-sum spp) with antibacterial action.In general, on rotten Sargassum, saccharomycetic quantity will be higher than the quantity on the frond of living and in sea water; Be attached to the yeast quantity on red algae and chlorella, be higher than on Brown algae.Because the phenolic material mass-energy of Brown algae secretion suppresses the growth of marine fungi.Some marine fungis are combined with specific Sargassum and form the coalition of obligate symbiosis, are ocean lichens.
3. mangrove fungi.Mostly be saprophytic bacteria, wherein 23 kinds of ascomycetes, 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 life of dwelling is saprophytic bacteria the marine fungi of Rhizophora apiculata Blume mostly, wherein has 23 kinds of ascomycetes, 17 kinds of Fungi Imperfectis, 2 kinds of basidiomycetes.Be immersed in that Rhizophora apiculata Blume pivot in sea water is dry, the epidermis of branch and root is perforated after animal, stormy waves or artificial damage, very easily invaded by fungus and cause rotten.But be embedded in the root in mud, be not subject to fungus infringement.Marine fungi can be decomposed Rhizophora apiculata Blume blade, for ocean provides a large amount of organic debris.
4. zostera marina fungus.Quantity is little, and many perch are in leaf portion.
Negligible amounts, wherein the fungus of the perch leaf portion root person of dwelling is many.In zostera marina root, contain the material that tannin and other suppress biological growths, only have those marine fungis that can resist this class material could be on zostera marina root Adaptable growth.
5. zooparasite body fungus.Only limit colonizes in ectoskeleton and place of shell portion.
Being confined to colonize in ectoskeleton, shell etc. locates.In the processes such as cellulose, chitin, protein and the calcium carbonate of marine fungi in decomposition 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 features:
If 1. belong to Ascomycotina, be mainly the member of the gang pyrenomycetes of decomposition of cellulose vigor, shell of ascus black, ascospore often has the appendage containing acidic polysaccharose, is conducive to stick in new substrate.Conventionally without conidium from generation to generation.
2. if imperfect fungi, spore is roof spore type, mostly is dark-coloured.
Marine fungi and marine bacteria are all participated in the regenerative process of the organic decomposition in ocean and inorganic nutrients thing, constantly for sea-plant provides effective nutrition; But marine fungi is bacterial parasite and the pathogenic bacterium of marine animal, the sea-plant that can make having is caused a disease, and even makes the wooden structures in port equipment rot; Some marine fungi can be destroyed the high molecular synthetic materials such as polyurethanes.
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.The particularly fungal mycelium in marine sediment and yeast thalline, for ocean protozoacide, zoobenthos etc. provide the source of bait.Some marine fungi can produce antibiotics and other biological active substanceies, is the huge treasure-house of new drug development, also significant in ecology and application aspect, as the pollutant in degraded marine environment, promotes 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 aquaculture.
Marine fungi is bacterial parasite and the pathogenic bacterium of some marine animal, if U.S. Concha Ostreae production area is once because the infringement of fungus sustains losses severely.Can cause the disease of some sea-plant, seriously infect as the zostera marina in the North Atlantic Ocean, pacific rim and Europe is once belonged to fungus by net Acarasiales.Wooden structures and other fibrous materials that the ocean raw fungus of wood can be rotted in port facilities, breakwater and weir dike.Some marine fungi can be destroyed the synthetic material in sea water, as polyurethane material penetrated by them after can expand split bad.
3, the chemical constitution of bioactive natural product B and preparation method
The metabolite B of marine fungi Massarina tunicata is for therefrom separating in the marine fungi of state sea and obtain first.In fungus, activated product B and mannitol all have higher content, if its condition of culture is carried out to more deep research, Massarina tunicata can be developed as to 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, choline sulfuric ester for separating and obtain first from southern China sea marine fungi; Gallic Acid is from marine fungi, to find first.In fungus #K26, choline sulfuric ester and mannitol all have higher content, if its condition of culture is carried out to more deep research, #K26 can be developed as to a kind of resource bacterium.Choline sulfuric ester (is called for short: COS) be extensively 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 utilize choline and PA (3 '-phosphoadenosine-5 '-phosphosulphate) as substrate, synthetic COS under the catalytic action of sulfur converting Enzyme, under the effect of choline sulfuric ester hydrolytic enzyme, COS can be hydrolyzed on the other hand, becomes one of source of the interior choline of organism and sulfate group.Another key player of COS is as impermeabilisation compound.Gallic Acid (being gallic acid) is a kind of polyphenol compound, owing to there being a certain amount of ROH base in molecule, can be formed with the hydroperoxyl radical (H) of antioxidation, has superoxide anion (O 2 -) and the activity of the free radical such as hydroxyl radical free radical (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 compound 6-epi-5 '-hydroxy-mycosporulone (referring to: M.A.Abdel-Wahab et al./Phytochemistry 68 (2007) 1212-1218), but not yet carry out deep bioactivity research, the 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 for example dissolution properties of various compositions in solvent in Chinese herbal medicine of extract raw material, select active component dissolubility large, to not needing the solvent that dissolved element dissolubility is little, and by effective ingredient the method from being dissolved out in medical material tissue.For example, when solvent is added to extract raw material (Chinese herbal medicine, need suitably to pulverize) in time, solvent is because diffusion, osmosis penetrate in cell by cell wall gradually, dissolve solable matter, and cause the concentration difference inside and outside cell, so intracellular concentrated solution is constantly to external diffusion, solvent constantly enters again in medical material histiocyte, so repeatedly come and go, until the inside and outside solution concentration of cell is while reaching dynamic equilibrium, this saturated solution is leached, continue repeatedly to add novel solvent, just needed composition can be bordering on to complete stripping or the stripping of large portion.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.In organic compound molecule structure, hydrophilic radical is many, and its polarity is negligent of greatly oil; Some hydrophilic radicals are 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, inventor just can, by for example medicinal herb components structural analysis of extract raw material, remove to estimate their this type of character and the solvent of selecting.Generally speaking, as long as the hydrophilic of extract material composition is suitable with this character of lipotropy and solvent, will there is therein larger dissolubility, i.e. the rule of so-called " similar mixing ".This be select appropriate solvent in extract raw material, extract required composition according to one of.
(2) selection of solvent: using the key of solvent extraction method, is to select suitable solvent.Solvent is selected suitably, just can more successfully the composition of needs be extracted.Selective solvent will be noted following 3 points: 1. solvent is large to effective ingredient dissolubility, little to impurity dissolubility; 2. solvent can not play chemical change with the composition of Chinese medicine; 3. solvent is wanted economical, is easy to get, uses safety etc.Common extraction solvent can be divided into following three classes:
1. water: water is a kind of strong polar solvent.Hydrophilic composition in extract raw material, as the not too large polysaccharide of inorganic salt, saccharide, molecule, tannin, aminoacid, protein, acylate, alkaloid salt and glycoside etc. can by water-soluble go out.In order to increase the dissolubility of some composition, also often adopt sour water and aqueous alkali as extracting solvent.
2. hydrophilic organic solvent: namely general said and the miscible organic solvent of water, as ethanol (claim not only: ethanol), methanol (but also claiming: another name for), acetone etc., the most frequently used with ethanol.The solubility property of ethanol is relatively good, stronger to the penetration capacity of extract raw cell.Outside hydrophilic composition isolating protein, phlegmatic temperament, pectin, starch and part polysaccharide etc., large multipotency dissolves in ethanol.Be insoluble in the low-polarity component of water, the dissolubility in ethanol is also larger.Can also be according to the character that is extracted material, adopt the ethanol of variable concentrations to extract.More less than water consumption with ethanol extraction, extraction time is short, dissolves the water-solubility impurity also few.Ethanol is organic solvent, though inflammable, toxicity is little, low price, and convenient sources, have a locking equipment can reclaim Reusability, and the extracting solution of ethanol is difficult for moldy metamorphism.Due to these reasons, be one of always the most frequently used method by the method for ethanol extraction.The character of methanol is similar with ethanol, boiling point lower (64 DEG C), but toxic, when use, should note.
3. lipophilic organic solvent: the namely general said and not miscible organic solvent of water, as petroleum ether, benzene, chloroform, ether, ethyl acetate, dichloroethanes etc.The selectivity of these solvents is strong, can not or be not easy to propose hydrophilic impurities.But this kind solvent volatility is large, how inflammable (except chloroform), generally poisonous, price is more expensive, equipment requirements is higher, and they penetrate plant tissue ability a little less than, often need repeatedly to extract for a long time and could extract completely.If contain more moisture in medical material, be just difficult to leach its effective ingredient with this kind solvent, therefore, while extracting in a large number extract raw material, directly applying this kind solvent has certain limitation.
(3) extracting method: with solvent extraction extract material composition, conventional infusion process, percolation, decocting method, reflux extraction and continuous circumfluence extraction method etc.Meanwhile, the factors such as the degree of grinding of raw material, extraction time, extraction temperature, appointed condition also can affect extraction efficiency, must take in.
1. flood extraction method (being called for short: infusion process): the dipping genealogy of law packs extract material powder or fragment in suitable container into, adds suitable solvent (as ethanol, rare alcohol or water), dipping medical material is with the wherein method of composition of stripping.This law is relatively simple, but leaching rate is poor, and as water be solvent, the easy moldy metamorphism of its extracting solution, must note adding suitable antiseptic.
2. permeating extraction (being called for short: percolation): percolation is that extract material powder is contained in percolator, constantly adds novel solvent, makes it penetrate medical material, flows out a kind of leaching method of leachate from top to bottom from percolator bottom.When solvent infilters that medicated powder, dissolved element proportion strengthen and while moving down, 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, therefore leaching effect is better than infusion process.But answer coutroi velocity, on powder, supplement at any time novel solvent oozing in transient, make in medical material till effective ingredient fully leaches.Maybe extremely shallow or when oozing the volume that gushes liquid and being equivalent to heavy 10 times of crude drug when oozing dropping liquid color, just can think and substantially extract completely.The normal use using rare leachate of collecting as the solvent of another batch of new raw material in a large amount of production.
3. decoct extraction method (being called for short: decocting method): decocting method is traditional leaching method that China is used the earliest.Container used is generally pottery, sand tank or copper, enamel ware, should not use iron pan, in order to avoid medicinal liquid variable color.When straight fire heating, preferably often stir, in order to avoid that local medical material is heated is too high, be easily charred.There is the pharmaceutical factory of steam-heating apparatus, adopt large reaction pot, large copper pot, barrel more, or pass into Steam Heating in the pond of cement block.Also several decocting devices can be connected to each other by pipeline, decoct and soak continuously.
4. heating and refluxing extraction method: application organic solvent heating extraction, needs to adopt reflux device, in order to avoid solvent evaporates loss.While operation in a small amount, can on round-bottomed flask, connect reflux condenser.The in-built medical material of bottle is about 20%～60% of capacity, and solvent soaked approximately 1～2cm of medical material surface.Reflux in water-bath, general keeps boiling 3～6 hours, lets cool filtration, then in medicinal residues solubilizer, do second and third reflux about half an hour respectively, or to till substantially carrying most effective ingredient.This method extraction efficiency is high compared with cold-maceration, the continuous extractions that adopt in a large amount of production more.
5. continuous circumfluence extraction method: application volatile organic solvent extracts extract material effective component, no matter small test or large-scale production, all with continuous extraction for well, and need by quantity of solvent lessly, extraction composition is also more complete.The conventional extraction equipment for fat of laboratory or title apparatus,Soxhlet's.Continuous extraction, generally needs a few hours could extract completely.Extract composition heated time longer, the labile composition of case of thermal instability should not adopt this method.
2, Isolation and purification method:
Extract raw material extracting solution or extract that said extracted method obtains remain mixture, need further remove impurity, separate and refine.
(1) solvent segregation: be generally by above-mentioned total extract, select three, the solvent of four kind of opposed polarity, extract and separate to high polarity proceed step by step by low polarity.Aqueous extract or ethanol extract are often jelly, be difficult to be dispersed in low polar solvent, therefore can not extract completely, can admix appropriate inert filler, as kieselguhr or fiber powder etc., then low temperature or natural drying, after pulverizing, to select solvent to extract successively, make each constituent in total extract again, separated according to the difference of its dissolubility in opposed polarity solvent.Utilize extract material chemical component, the dissolubility in opposed polarity solvent carries out separation and purification, is the most frequently used method.
(2) solvent extraction:
1. extraction: solvent extraction extraction is called for short again extraction is to utilize the difference of each composition partition coefficient in two kinds of immiscible solvents in mixture and the method that reaches separation.If each composition partition coefficient in solvent differs larger when extraction, separation efficiency is higher; If the effective ingredient in aqueous extract is lipophilic material, general multiplex lipotropy organic solvent, as benzene, chloroform or ether extract, if effective ingredient is to be partial to hydrophilic material, indissoluble solution in lipophilic solvent, just need to use weak lipophilic solvent, such as ethyl acetate, butanols etc. instead.Can also in chloroform, ether, add appropriate ethanol or methanol to increase its hydrophilic.While extracting flavones ingredient, multiplex ethyl acetate and water extraction.Extract the strong saponin of hydrophilic for multiselect n-butyl alcohol, isoamyl alcohol and water extract.But, common organic solvents hydrophilic is larger, and to make the effect that extracts just more bad with water, because can make more hydrophilic impurities follow, on effective ingredient, further refining impact is very large.
2. counter current continuous extraction method: be a kind of continuous solvent extraction.Its device can have one, several or more extracting tube.In pipe with little porcelain circle or the filling of little rustless steel wire ring, the contact surface when increasing solvent extraction.If a kind of infusion of extract raw material need to extract by benzene, the ethyl acetate etc. lighter than water, need water extracting liquid to be contained in extracting tube, and benzene, ethyl acetate are stored in high-level container.Extract whether complete, desirable for sample thin layer chromatography, paper chromatography and chromogenic reaction or precipitation check.
3. counter-current distribution method: counter-current distribution method claims again CCD method, counter-current distribution or countercurrent distribution.Counter-current distribution method is consistent with solvent counter-current extraction principle, but application of sample amount is certain, and continuous in the solvent of certain capacity, reaches the separation of mixture through multi-shift extraction distribution.
4. drop counter-current distribution method: drop counter-current distribution method claims again droplet countercurrent chromatography method.For improved solvent extraction on counter-current distribution method basis in recent years.To the substantially same counter-current distribution method of the selection of solvent system, but requirement can be separated at short notice, and can generate effective drop.Form mutually drop due to mobile, in thin distribution extracting tube, effectively contacting with immobile phase, rubbing constantly forms new surface, promotes the distribution of solute in solvent, therefore its separating effect is often good than counter-current distribution method.
(3) Flavonoids by Macroporous Adsorption Resin: macroporous adsorbent resin is the class organic polymer adsorbent growing up the sixties in 20th century, there is good absorption property, be applied to gradually in the recent decade the extraction separation of extract material chemical component and developing of new Chinese medicine.
Macroporous adsorbent resin is the parting material that absorption and screening principle combine.Its adsorptivity is the result due to Van der Waals force or generation hydrogen bond.Screening principle is because itself cellular structure determines.Due to absorption and screening principle, organic compound, according to the size of the difference of absorption affinity and molecular weight, separates through certain solvent elution on macroporous adsorbent resin.This make organic compound especially the purification of water soluble compound greatly simplified.The skeleton of macroporous adsorbent resin is by styrene and divinylbenzene polycondensation and generate, and due to adding of modifier, the polarity of macroporous adsorbent resin changes, and according to the surface nature of resin, that adsorbent resin is generally divided into is nonpolar, Semi-polarity and polarity three classes.
Nonpolar adsorption resin is the not adsorbent resin with any function base being made by the very little monomer-polymer of dipole moment.Typical example is the adsorbent resin of styrene-divinylbenzene system, as D101, XAD-1, DiaionHP-10 macroporous adsorbent resin.
Semi-polarity adsorbent resin refers to the adsorbent resin containing ester group, as a crosslinked analog copolymer such as acrylate or methacrylate and double methyl methacrylate.It is on the basis of nonpolar macroporous adsorption resin, adds acrylic acid methyl ester. or acrylonitrile polycondensation to form, as the often AB-8 macroporous adsorbent resin of use of China.
Polar Adsorbent Resin refers to 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 the ligand groups such as nitrogenous, oxygen, sulfur is called to 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 sulfoxide type polycondensation, as the XAD-10 of the Diaion HP 2MG of Mitsubishi chemical industry, Rohm-hass company of the U.S., XAD-9 macroporous adsorbent resin.
Compare with other adsorbent with active carbon, macroporous adsorbent resin has advantages of a lot, as higher in the adsorptive selectivity to certain material; Physical and chemical stability and mechanical strength are better; Description is more, can change as required resin physics or chemical constitution; Adsorbent resin is generally spherical particle, and fluid resistance is less etc.Thereby be widely used in chemical industry, medicine and other fields, about macroporous adsorbent resin, the report of the applied research in natural product extraction separates is more and more in recent years.Macroporous adsorbent resin has certain adsorption to extract material chemical component as alkaloid, flavone, saponin, coumarin and some other methods of glycosides.Very poor to sugared absorbability, stronger to the absorbability of pigment.
(4) sedimentation method: be to add some reagent to make to produce precipitation in extract raw material extracting solution, with the method that obtains effective ingredient or remove impurity.As lead salt precipitation: lead salt precipitation is one of classical way separating some extract material composition.Because lead acetate and Lead monosubacetate are in water and alcoholic solution, can generate with multiple extract material composition lead salt or the complex salt precipitation of indissoluble, therefore can utilize this character that effective ingredient is separated with impurity.Then lead salt precipitation is suspended in novel solvent, passes to hydrogen sulfide gas, make to decompose and transfer insoluble sulfuration lead to and precipitate.
(5) salting out method: salting out method is in the water extraction liquid of extract raw material, adds inorganic salt to finite concentration, or the state that reaches capacity, can make the dissolubility of some composition in water reduce Precipitation, and the impurity large with water solublity separates.Be commonly used for the inorganic salt of saltouing and have sodium chloride, sodium sulfate, magnesium sulfate, ammonium sulfate etc.
(6) dialysis: dialysis is to utilize small-molecule substance can pass through semipermeable membrane in solution, and macromolecular substances can not be by the character of semipermeable membrane, reaches the method for separation.Otherwise also macromolecular impurity can be stayed in 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: qualification extract material chemical component, study its chemical constitution, must first extract material composition be prepared into monomer sterling.At normal temperatures, material nature is the compound of liquid, can carry out separation and purification by fractionating process or chromatography respectively.In general, extract material chemical component is the material of solid at normal temperatures mostly, all has the general character of crystalline solid, can reach according to the difference of dissolubility the object of separation and purification 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. namely operation at low temperatures of operation under vacuum, can avoid at high temperature nutritional labeling as the destruction of vitamin etc., to 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.Conventional form mainly contains box type vacuum exsiccator, double-cone type vacuum desiccator, belt vacuum desiccator etc.These traditional Minton dryers mainly adopt hot blast, and the heating such as steam or electricity, utilize conduction of heat, convection current or radiation theory heat to be passed to material inside from outside.It is low that vacuum drying has baking temperature, and relatively hypoxia in hothouse, can avoid fat oxidation, and the series of advantages such as pigment brown stain are suitable for the dry of heat sensitivity food material, and equipment cost, dry expense are also relatively low in addition.
(2) spraying is dry: be that fluidization technique is for a kind of dry method of liquid material.Because being wink-dry, be specially adapted to heat sensitive material, therefore products obtained therefrom quality is good, keep original color, smell and taste, and soluble.The research that utilizes spraying to be dried to prepare microcapsule is carried out, it is that heart material is suspended in the solution of dress material, sprayed in thermal current through centrifugal atomizer, 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 dry liquid material is frozen into solid, utilize the distillation performance of ice under low-temperature reduced-pressure condition, low-temperature material is dewatered and reach a kind of dry method.Because material is dry under high vacuum and cryogenic conditions, therefore the dry of some extremely thermo-labile article is well suited for.Wang great Lin has reported a kind of spraying ventilation lyophilizing new technique, to utilize cold air or nitrogen as medium, the scars of flowing through rapidly make water sublimate, and the product microgranule that makes of spraying lyophilizing is little, fast drying, time are short, even, good fluidity, and the good instant capacity of tool.In recent years, plaster material and the dry research of sticky material have been obtained to greater advance, fluidization technology, spraying technique, inert carrier technology grow up on this Research foundation.Rotatingandflashstreamingdrier, thermojet pneumatic drier, inert carrier drying machine are all applicable to the dry of heat sensitive material and plaster material.These new achievements in research are produced for Chinese medicine preparation, by greatly improving the technical merit of Chinese medicine processing, 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 vibration and the rotation of molecule and atom, cause object heating, through thermal diffusion, evaporation and chemical change, finally reach dry object.Far-infrared ray drying can be saved electric energy 20%～50%, and effect is better.
(5) micro-wave drying method: be the new technique developing rapidly a sixties in 20th century, microwave drying is actually by eddy-current heating and dielectric heating, the moisture and the fat microwave energy absorbing to some extent that make to be dried in thing, thus and it is changed into heat reach dry object.Microwave drying can killing microorganisms and mycete, and has disinfective action.The microwave heating installation of domestic product has 915mhz and two frequencies of 2450mhz at present.
(3) research overview of vascular dementia
Dull-witted (Dementia) is the acquired and persistence disturbance of intelligence syndrome producing due to disordered brain function, and disturbance of intelligence comprises that memory, language, visual space function, abnormality of personality and cognitive competence in various degree reduce.
Dull-witted main Types is to comprise vascular dementia (vascular dementia, or claim: vascular senile dementia, be called for short: senile dementia VD), in senile dementia (Alzheimer ' s Disease, or claim: alzheimer's disease, or claim: Alzheimer's disease, is 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 Types in senile dementia.Because AD falls ill in more than 60 years old old people well, so custom is called as degenerative brain disorder.
Vascular dementia is caused by cerebrovascular, is mainly caused by cerebral hypoxia ischemia.The type such as common multi-infarct dementia, massive cerebral infarction dementia, thalamic dementia and the concurrent dementia of hemorrhagic cerebrovaseular and Mixed dementia clinically.Clinical manifestation is depression, delirium, aphasia etc.Pathological change is that multiple lacuna sexually transmitted disease (STD) becomes or large area infarct and atherosclerotic lesion (referring to neurological, the 4th edition, People's Health Publisher).Clinical treatment can use cholinergic inhibitor, blood vessel dilating, increase cerebral blood flow, or 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.
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 one progression (Guo Mingying slowly, Korea S's tinkling of pieces of jade, Deng. the comparative study of alzheimer disease and vascular dementia disturbance of intelligence. the journal .2007 of Qinghai Medicine College, 28 (2): 125-127.).
Vascular risk factor is all being played the part of key player 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, Imageologies.
Vascular dementia is the carrying out property decline property disease by the acquired Premium Features of cerebral cortex due to repeatedly apoplexy or long-term chronic cerebral ischemia.
Show in America and Europe's investigation, dementia incidence rate approximately 1.1% in 55 years old above old men, wherein alzheimer's disease accounts for top priority (incidence rate approximately 7.7 ‰), be about 50%～60% of dull-witted total number of persons, and VD occupies time (incidence rate approximately 1.5 ‰), account for 10%～20%, and with age, VD proportion significantly increases (1. Ott A, Breteler MMB, Harskamp F, 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, AD incidence rate approximately 3.5% in the old man of over-65s, VD incidence rate 1.1% (Zhang ZX, 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 lacking at present the cognitive impairment pattern that is applicable to diagnose VD, for discriminating and " mixed type " dull-witted (AD+ cerebrovascular of the diagnosis of AD and VD, be called for short: diagnosis AD+CVD) still has certain challenge (1. fourth element chrysanthemum, 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.Int Psychogeriatr.1997; 9:51-58.), so someone thinks that vascular dementia may be dull-witted type the most common in old people, mainly because a lot " mixed type " dull-witted (AD+CVD) may be diagnosed as AD (1. Rom á n GC.Vascular dementia may be the most common form of dementia in the elderly.J Neurol Sci.2002,203-204:7-10; 2. fourth element chrysanthemum. vascular dementia. Aged in China is learned 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.Gender differences 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 Canadian study of health and aging.Stroke, 2000; 31 (7): 1487-1493.],, according to Epidemiological study, in the crowd who is greater than 60 years old, 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 apoplexy and ischemic heart desease (ischemic heart disease, abbreviation: IHD), and the two is all commonly encountered diseases in old people.Only with regard to apoplexy, the now annual whole world newly increases apoplexy patient 1,000 ten thousand, wherein just there are 2,000,000 (1. Feigin VL in China, Lawes CMM, Bennett DA, et al.Stroke epidemiology:a review of population-based studies of incidence, prevalence, and case-fatality in the late 20th century.Lancet Neurol., 2003,2 (1): 43-53; 2. Wu Zhao Soviet Union, Yao Chonghua, Zhao Dong. the epidemiological study of Chinese population Cerebral Haemorrhage Invasion Rate, mortality rate. Chinese epidemiology magazine, 2003; 24 (3): 236-239.), in old people, sickness rate is especially high, thereby causes the concurrent VD of a lot of patients.The national dull-witted epidemiology statistics data of China also shows along with the aging of 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 grievous injury patient health not only, brings long-term misery to patient, affects patient's quality of life, brings white elephant also to society and family, has caused the common concern of countries in the world, and it is an important topic in geriatrics field.Generally believe that the age affects by many-side the effect of VD, the aging change of the self-regulation of for example brain, the metabolism of cell, blood brain barrier and autonomic function aspect makes cerebrovascular be easy to suffer damage, and encephaloclastic accumulative total effect is also one of reason.
Dull-witted diagnosis must meet three conditions: 1. dull-witted; 2. cerebrovascular; 3. more than, the two is closely related.Dull-witted and apoplexy is maintained close ties with in time, conventionally after apoplexy, in 3 months, occurs dull-witted.Cardinal symptom is: 1. histopathology form changes; 2. hypomnesis.
About the treatment of VD, there is no at present sure Therapeutic Method and can change the whole course of disease, because brain cell downright bad after cerebral infarction can not reverse, be mainly that the treatment of the brain cell to blood supply insufficiency is to alleviate stage of attack symptom, to prevent infringement again etc.The method etc. that normal employing actively improves brain cell oxygen supply, improve microcirculation, prevent new thrombosis and re-infarction.Improve cerebral circulation treatment conventional have 9,10-Dihydroergotoxine class, calcium ion antagonist, nicotinic acid class, other medicament for expanding vascellum and a medicament for resisting platelet aggregation.Cholinesterase inhibitor is developed recently dementia treatment medicine comparatively rapidly, example hydrochloric acid donepezil and rivastigmine-hydrogentartrate.Also often apply clinically Cerebral protection medicine as calcium ion antagonist (nimodipine and western pyrrole spirit); Free radical scavenger (vitamin E, vitamin C and gingko leaf preparation) etc.But from present clinical effect of said medicine, curative effect is general, and major part is only all anti symptom treatment, can not improve mortality rate.Some Chinese medicine preparation, as the preparations such as Folium Ginkgo, Herba Erigerontis, Radix Et Caulis Acanthopanacis Senticosi exist the weak points such as active component is unclear, mechanism of action not clear, quality is unstable, toxic and side effects is large mostly, are difficult to meet clinical needs at present.
Therefore, at present countries in the world there is no effective ways and medicine to controlling this sick course advancement, the drug research and development of prevention, treatment and the prognosis of VD become the research topic paid much attention to various countries (1. fourth element chrysanthemum. vascular dementia. Aged in China is learned magazine .2003; 23 (4): 200-202; 2. the old thunder of pounding, Qiu Zhihui, Su Shixin, Zheng Zhongcheng, Chen Peifen, Ye Zhiping. the therapeutic advance of vascular dementia. the new medical science .2007 of forum of China, 7 (7): 59-62.).
Therefore; working out a kind of effective Therapeutic Method, to stop potential pathogenic process be very necessary; in conjunction with Chinese traditional extract raw material; therefrom excavate effectively, low toxicity, inexpensive treatment vascular dementia and the medicine of alzheimer disease; for the product that prevents, diagnose, detect, protect and treat the aspects such as VD particularly medicine have very important significance, can produce significant Social benefit and economic benefit.
(4) research overview of antifungal drug
Fungal infection has gradually the trend increasing in recent years, and it comprises superficial part and deep mycosis, and the latter's polyphyly body's immunity of falling ill is impaired, is often in a bad way, and threat to life, needs whole body to apply potent antifungal drug treatment.Such medicine can be divided into external and system applies two classes, and external used medicine is only for mycotic infection of superficial part.
The mankind's fungal infection can involve epidermis, subcutaneous and whole body; Generally can be divided into superficial part and infect (being confined to angleplied laminate, squamous mucosa or cornea) and the large class of deep infection (subcutaneous and whole body) two.Their medicine is also different, infects, can be used for again deep infection and there is some drugs 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 46% of department of dermatologry outpatients, and wherein tinea unguium has just accounted for approximately 16%; Infect for these, mainly adopt local application's treatment, because it infects, 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 treatment tinea unguium.
Deep fungal infection comprises that subcutaneous tissue and whole body system infect, and comprise candidiasis, cryptococcosis, aspergillosis and mucormycosis etc.Increasing due to extensive use broad ectrum antibiotic, 17-hydroxy-11-dehydrocorticosterone, immunosuppressant, radiotherapy, chemotherapy, organ transplantation, operative catheter and acquired immune deficiency syndrome (AIDS) in recent years, making to occur invasive infections with fungi chance increases, 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.Infect and need systemic administration, the drug combination of sometimes still needing for whole body system.Due to the mechanism of action difference of medicine, should be noted that especially their safety and drug interaction problem.
In treatment application aspect, deep fungal infection is mainly selected both sexes enzyme element B, itraconazole, ketoconazole, fluconazol, voriconazole, posaconazole, Caspofungin and flucytosine, other drug is used for mycotic infection of superficial part, but most have more serious toxic and side effects, as amphotericin has Toxicity of Kidney.In the process of long-term medicinal application, pathogen also produces drug resistance in various degree gradually, in the urgent need to us development of new good effect and the less antifungal drug of toxic and side effects.
(5) research overview of antitumor drug
Malignant tumor serious harm people's health, according to RST statistics, the more dies from every year on average malignant tumor person in hundred million populations in the whole world 50 and reaches 6,900,000 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 at present major progress.The development of molecular weight tumor in recent years,, molecular pharmacology is progressively illustrated tumor essence; The invention of the advanced technologies such as extensive rapid screening, combinatorial chemistry, genetic engineering and application acceleration drug development process; The research and development of antitumor drug have entered brand-new epoch.Antitumor drug is just from traditional cytotoxic drug, to the new type antineoplastic medicine development of the too many levels effect for machine-processed.
Therefore, research and develop novel antitumor drug and become exigence.The data that medicine is sold show, global pharmacy corporation the last 50 is 2.8% at the prescription drugs sale growth of 2007, and wherein antitumor class medicine growth is the fastest, reaches 14%.2006 annual datas show equally, and in 10 class medicines, the sales growth of antitumor drug ranks first, and reaches 20.5%.This has reflected the continuing of antitumor drug, the market demand of rapid growth.On the basis of China's long-term scientific development planning in formulation, propose to set up a collection of great special project." great new drug initiative " be from Eleventh Five-Year Plan to the year two thousand twenty between one of the major project supported of country.This project, by the major disease for the multiple serious harm people such as tumor, cardiovascular diseases, is developed original new drug.Clearly, the research and development of anti-cancer agent, are needs, the sustainable development of Ye Shi China and the vital task of building innovation-oriented country of control serious harm people disease.What " great new drug initiative " was special sets up, and will the process of China's anti-cancer agent research and development be produced to active influence.
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 ".Compared with advanced international standard, China is still having big gap aspect 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 work without single devotion for a long time and accumulate.Therefore, need to provide stable working environment and condition for researcher; Need each side professional and associated mechanisms to work in concert; Need to be conducive to promote Guaranteeing measures of policy and the organizational coordination mechanism of cooperation; Need to there be larger amt funds to comprise the support energetically of the funds of preclinical study and clinical research.
Current, anti-cancer agent research and development are faced with good opportunity.The development of biomedical science, for development of new antitumor drug provides important theoretical basis and new technical support.The development of Health Care in China cause, has higher requirement to the therapy of serious disease medicine including cancer, and the great demand of Chinese market and world market brings considerable economic benefit can to 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; not yet find that there is bioactive natural product B (polydatin, piceid) with and compositions at the report being directly used in aspect prevention, diagnosis, detection, protection, treatment and Effect of Anti fungus, antitumor, vascular dementia and alzheimer disease and 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 active Chinese drug component natural product B antifungal, antitumor and anti-angiogenic dementia, this extract is for the preparation of antifungal products, antitumor product and anti-angiogenic dementia product, the above-mentioned defect existing to overcome prior art.
That is to say, the present invention, by the research such as cell and zoopery and theory study, is intended to the activity of clearly a kind of bioactive natural product B in antifungal, antitumor and anti-angiogenic dementia application aspect, the present invention relates to the new purposes of a kind of bioactive natural product B.
Another object of the present invention is to provide the pharmaceutical composition that contains above-mentioned bioactive natural product B as the application of preparing antifungal and antitumor product and anti-angiogenic dementia product.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 refers to 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 refers to white coccus, neogenesis cryptococcus, trichophyton and the Aspergillus fumigatus read.
Antitumor product of the present invention refers to 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 refers to hepatocarcinoma, breast carcinoma and pulmonary carcinoma.
Anti-angiogenic dementia product of the present invention refers to 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 are all the one that comprise in the product of the field such as medicine and food, are to comprise one or more in medicine, reagent or food etc., preferred agents.
The medicinal raw material that bioactive natural product B of the present invention uses refers to marine fungi or marine fungi crude extract, preferably 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 Effect of Anti fungus, antitumor, anti-angiogenic dementia; its occupation mode is to comprise independent use or combine the one in use etc. with other chemical substances; preferably use separately, all can be used in and prepare antifungal products, antitumor product and anti-angiogenic dementia product.
Test sample is all to take conventional preparation method to obtain, the general < 60% of bioactive natural product B content obtaining, but by purification, the purity that can obtain the bioactive natural product B after 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 directly employing contains bioactive natural product B is raw material, or directly adopting the marine fungi crude extract that contains bioactive natural product B is raw material, can both be directly or indirectly for the preparation of 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 substantially pure form, as purity >=95% of bioactive natural product B.
(2) technical conceive
Independent development original new drug is a current urgent task of China, China's Chinese medicine and pharmacy has a long history, aspect prevention and treatment disease, also accumulating rich experience, therefore from Chinese medicine, finding effective active component or find that its new purposes is all effectively quick approach, is also the place of the advantage of the quick original new drug development of China.
Lu Sheng microbial fermentation product is one of valuable source of pharmaceuticals industry always, but continually developing along with land resources in recent years, find new microorganism or screen the difficulty of new active metabolite increasing, and Drug resistance problem is day by day serious, make the exploitation of new drug be faced with severe challenge.
Marine microorganism is a study hotspot in recent years.Ocean account for earth surface long-pending 71%, microbial resources are richly stored with..Increasing research also shows, Marine microorganism---especially those exist the microorganism of the relation such as symbiosis or parasitism with marine animal or sea-plant---can produce the unique also metabolite of the strong physiologically active of tool of structure.And Marine microorganism breeding is fast, can utilize modern microbial technique, 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 Marine microorganism secondary metabolite, find new anti-tumor activity metabolite, for 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 compound 6-epi-5 '-hydroxy-mycosporulone (referring to: M.A.Abdel-Wahab et al./Phytochemistry 68 (2007) 1212-1218), but not yet carry out bioactivity research, the present invention studies the compound relating to similar parent nucleus with it, and carry out antifungal and antitumor activity research, cell and animal experiment study are carried out.
Inventor is by carrying out the chemical constitution study of system to marine fungi extract, screen and prove activity and the purposes of this marine fungi extract bioactive natural product B, thereby inventor infers that bioactive natural product B is in prevention, diagnosis, detect, protection, treatment and Effect of Anti fungus, antitumor, the clinical drug effect of the aspect activity such as anti-angiogenic dementia, also should be mainly 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 there is significant pharmacologically active.
Inventor through the latest find of research is: bioactive natural product B can resist amyloid-beta and cause the effect that brain cell damages, can resist hydrogen peroxide and cause brain cell damaging action, improve the learning and memory function that scopolamine causes dementia mice model, improve A β and cause dementia mice model learning memory ability, suppress the peroxidating of cerebral ischemia-reperfusion in mice model cerebrum lipid, strengthen cerebral ischemia-reperfusion in mice model cerebral tissue superoxide dismutase activity and lower cerebral ischemia-reperfusion in mice model brain sheet intracellular calcium concentration.
Dull-witted particularly vascular dementia and alzheimer disease have a strong impact on health and the life quality of Chinese population; the product of the aspects such as development prevention, diagnosis, detection, protection, treatment and research vascular dementia and alzheimer disease; particularly medicine, diagnostic reagent and health product etc., have significant 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 compound, bioactive natural product B structural formula is as follows:
The experimental data of bioactive natural product B:
Colorless solid; Fusing point 203-205 DEG C, 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) .NMR data are in 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 the aspects such as prevention, diagnosis, detection, protection, treatment and Effect of Anti vascular dementia.
Cardinal symptom that now there are some researches show vascular dementia is: 1. histopathology form changes; 2. hypomnesis.Inventor's research shows: bioactive natural product B can obviously improve vascular dementia rat models brain ischemic pathology damage, the learning and memory function of dementia rats is had to stronger protection and facilitation.Therefore, bioactive natural product B and compositions thereof can be used in and prepare 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, by following experiment, illustrates that bioactive natural product B has the effect for the treatment of of vascular dementia.
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 1, vascular ischemic injuries (multiple local embolization modeling) being formed
Learning and memory is the Premium Features of brain, is to form 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 using the improvement of learning and memory as a main standard evaluating intelligence raising.
(1) animal: 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 domestic analytical reagent.
(3) compound method: medicine of the present invention and Ginaton, milling evenly with 0.5% sodium carboxymethyl cellulose before use, 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 DEG C of freeze-day with constant temperature become blood clot, after grinding, through 200～300 mesh sieve hole sieving for standby, add blood clot 1mg make suspension when application with the every 1ml of 6% dextran Glucose Liquid, under microscope, measuring diameter is 50～100 μ m.Then by chloral hydrate intraperitoneal anesthesia (35mg/100g), cervical region medisection exposure separation right carotid and external carotid artery for experimental rat.Interim folder closes external carotid artery, injects embolus dextran suspension from common carotid artery, and in injecting embolus, open common carotid artery, utilizes Carotid blood flow that embolus is sent into intracranial to the each tremulous pulse of brain by internal carotid artery, causes many kitchen ranges cerebral infarction.Within second day, by successful modeling animal grouping, drug component of the present invention is high dose group (100mg/kg), low dose group (50mg/kg), 16 every group.With medicinal liquid of the present invention gastric infusion respectively, every Mus 10ml/kg every day, the every Mus of positive drug control group gavage every day Ginaton suspension (2.5mg/ml) 10ml/kg, 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 10 days, Morris water maze laboratory:
1. experiment starts the previous day, first allows rat free swimming 2min in the pond of not containing platform, to be familiar with environment.
2. orientation navigation experiment: train continuously every day 4 times 4 days.Each time all random order never 4 of equal diversion basin work the rats of naming a person for a particular job and drop in pond, while putting into, make rat towards pool wall, record rat from entering water to the time of finding and climb up platform, this time is called escape latency.Be allowed to condition on platform, have a rest 10 seconds after again row detect next time.If rat is not found platform yet in 120 seconds, be 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: train the 5th day, after directed swimming test finishes, immediately platform is removed, put into rat for that still chooses at random in 4 place of entry, the time of staying of meter record rat in the quadrant of original platform place.
(6) experimental result: (seeing: table 2 and table 3).In table: after the swimming instruction of Morris water maze, the swimming of the each dosage group of medicine significantly reduces (P < 0.05) compared with model group incubation period; In space exploration experiment, medicine treated animal in the platform place quadrant time of staying obviously more than model group (P < 0.05).The above results shows, bioactive natural product B has protective effect to the learning and memory function of rat.
Table 2, Morris water maze laboratory animal swimming incubation period (second)
With model group comparison, *p < 0.05; *p < 0.01; * *p < 0.005; * * *p < 0.001
Table 3, space exploration experiment (training the 5th day)
With model group comparison, P < 0.05; *p < 0.01;
(7) morphological examination
Postoperative 14 days, by rat sacrificed by decapitation, separate and take out prefrontal cortex, Hippocampus rapidly, fixing in neutral formalin liquid, HE dyeing is done in section.
Except sham operated rats, lamellar brain essence softening necrosis region in each region under the cortex that each group is all shown in various degree, the each group of scope is not etc.
1. sham operated rats: the each portion of central nervous system is showed no obvious pathological changes, cerebral cortex: neurocyte core shows no obvious abnormalities, and Distribution of chromatin is even, vascular endothelial cell is without extremely.Hippocampus: pyramidal cell core shows no obvious abnormalities, and Distribution of chromatin is even, vascular endothelial cell is without extremely.
2. model group: cerebral cortex: solid area lamellar is softened kitchen range, part liquefies, and forms not shaping blister cavities, and scope is wide, and a large amount of neuron cavity samples become, nuclear membrane thickening, chromosome reduces or disappears, some neurocyte swelling and degeneration, nuclear membrane is unclear.Blood vessel endothelium swelling phenomenon.Hippocampus: many places neurocyte cavity sample becomes, nuclear membrane thickening, chromosome reduces or disappears, some neurocyte swelling and degeneration, nuclear membrane is unclear.
3. bioactive natural product B low dose group: cerebral cortex: neurocyte Distribution of chromatin is very uneven, but aixs cylinder shows no obvious abnormalities, and a small amount of neurocyte cavity sample becomes, blood vessel endothelium mild swelling.Hippocampus: have the softening stove liquefaction that scope is less.
4. bioactive natural product B high dose group: cerebral cortex: have lamellar to soften kitchen range, scope is larger.Hippocampus: have special mess to soften kitchen range (with slight calcification, belonging to downright bad rear reaction) around.
5. Ginaton group: cerebral cortex: see and have lamellar to soften kitchen range, but scope is less.Hippocampus: pyramidal cell Distribution of chromatin is even, 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: mild degree, the scope of ischemia injury are little.
Above result of the test explanation bioactive natural product B has stronger protective effect to vascular dementia rats ischemia injury, improves the learning and memory function of rat model.Therefore, bioactive natural product B can be used for the medicine of preparation 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 due to amyloid-beta (Beta-Amyloid Protein, be called for short: A β) deposit and the senile plaque of formation, increasing evidence 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 enhancing proinflammatory cytokine causes etc.This experiment purpose is observation bioactive natural product B causes dementia rats model learning memory ability impact on A β.
(2) experimental technique
Male Sprague-Dawley rat, body weight 250-300g, at calvarium portion medisection skin, exposes skull after anesthesia, with reference to " rat brain stereotaxic atlas " (bag new people, Shu Siyun. rat brain stereotaxic atlas. the 1st edition, Beijing: People's Health Publisher, 1991,44-45.), select bilateral hippocampus CA1 district for injection target area, with dental burr bore open skull, with microsyringe by the A β of state of aggregation 1-40slowly inject (totally 5 μ l, 1 μ l/min), let the acupuncture needle remain at a certain point fully spreads with guarantee solution for 10 minutes, then slowly removes pin, sew up wound.Normal saline group is injected the normal saline of same volume.Within second day, by successful modeling animal grouping, 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 gastric infusion respectively, every Mus 10ml/kg every day, the every Mus of positive drug control group gavage every day Ginaton suspension (2.5mg/ml) 10ml/kg, 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, Morris water maze laboratory:
1. experiment starts the previous day, first allows rat free swimming 2min in the pond of not containing platform, to be familiar with environment.
2. orientation navigation experiment: train continuously every day 4 times 4 days.Each time all random order never 4 of equal diversion basin work the rats of naming a person for a particular job and drop in pond, while putting into, make rat towards pool wall, record rat from entering water to the time of finding and climb up platform, this time is called escape latency.Be allowed to condition on platform, have a rest 10 seconds after again row detect next time.If rat is not found platform yet in 120 seconds, be 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: train the 5th day, after directed swimming test finishes, immediately platform is removed, put into rat for that still chooses at random in 4 place of entry, the time of staying of meter record rat in the quadrant of original platform place.
(3) experimental result: (seeing: table 4 and table 5).In table: after the swimming instruction of Morris water maze, the swimming of the each dosage group of medicine significantly reduces (P < 0.05) compared with model group incubation period; In space exploration experiment, medicine treated animal in the platform place quadrant time of staying obviously more than model group (P < 0.05).The above results shows, bioactive natural product B can improve A β and cause the ability of learning and memory of dementia rats model.
Table 4, Morris water maze laboratory animal swimming incubation period (second)
With model group comparison, *p < 0.05; *p < 0.01; * *p < 0.005; * * *p < 0.001
Table 5, space exploration experiment (training the 5th day)
With model group comparison, P < 0.05; *p < 0.01;
3, bioactive natural product B opposing hydrogen peroxide causes neural cell injury effect
(1) experiment purpose
In the evolution of vascular dementia and alzheimer disease, free radical and active oxygen play very large 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, causes a series of pathological changes, causes long-term damage.This is in zoopery and may affect clinically cognitive function, develops into dementia.The object of this experiment is to observe bioactive natural product B to active oxygen hydrogen peroxide (H 2o 2) cause neural cell injury and there is resistant function.
(2) experimental technique
Use into neuroblastoma SK-N-SH 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, adds H 2o 2hatch 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 neural cell injury model, find obviously antagonism H of bioactive natural product B 2o 2the cell survival rate decline causing and LDH spill and increase, and lower H 2o 2neurotoxicity, and be dose-dependence, prompting bioactive natural product B has neuroprotective cytosis, vascular dementia is had to preventive and therapeutic effect (seeing: table 6, table 7).
Table 6, bioactive natural product B preincubate 12 hours are to H 2o 2cause the impact of SK-N-SH neural cell injury
M ± SD; *p < 0.05, with H 2o 2model group is compared.
M ± SD; *p < 0.05, with H 2o 2model group is compared.
Table 7, bioactive natural product B preincubate 24 hours are to H 2o 2cause the impact of SK-N-SH neural cell injury
M ± SD; *p < 0.05, 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 cholinergic neurons of basal forebrain, (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 acetylcholinesterase m receptor blocker, and ACh that can 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 group, model group, bioactive natural product B low dose group (50mg/kg) and high dose group (100mg/kg).The continuous gastric infusion of each group modeling after 10 days before 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.After 15 minutes, carry out Morris water maze test, one day twice, continuous three days.Date processing completes by image automatic monitoring and processing system.
2. keep away dark test: 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 passive avoidance test, and mice has the habit of liking dark, enters darkroom and is shocked by electricity.2nd, within 3,4 days, enter that incubation period in darkroom is longer, to enter number of times fewer, show that learning and memory function is better.
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), if show, bioactive natural product B can improve the good alkali model mice space learning memory ability in east.Keep away dark test and find that bioactive natural product B high dose group can obviously extend incubation period, reduce the number of times (table 9) that enters 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 dementia mice model water maze and swim out of the impact of time and swimming distance on scopolamine
M ± SD; *p < 0.05, compares with model group.
Table 9, bioactive natural product B scopolamine cause dementia mice model and keep away the dark impact of testing incubation period and errors number
M ± SD; *p < 0.05, compares with model group.
5, bioactive natural product B suppresses the peroxidating of cerebral ischemia-reperfusion in mice model cerebrum lipid
(1) experiment purpose
In Neurons Against Cerebral Ischemia, 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.The lipid peroxy and the oxidative stress that after cerebral ischemia, produce the generation of a large amount of free radicals and cause, cause lipid peroxidation.Lipid peroxide be in biomembrane and cell the contained polybasic unsaturated fatty acid of phosphorus matter by radical damage, oxidation and the Peroxidation Product forming, can cause the reactions such as membrane damage, enzyme inhibition, lysosome release, protein-crosslinking, DNA and RNA structural deterioration, thereby destroy the normal physiological function of cell, finally can cause cell death.This experiment is intended to observe bioactive natural product B cerebrum lipid Petoxidation product malonaldehyde (is called for short: the MDA) impact of increased content.
(2) experimental technique
Kunming mice, male, 22～28 grams of body weight, random packet, the continuous gastric infusion of each group 7 days before experiment, matched group (sham operated rats) and ischemia model group give respectively tap water.Operation same day is 1h after administration on an empty stomach, after mouse anesthesia, closes incline total tremulous pulse 15 minutes of bilateral with bulldog clamp folder, then pours into after 15 minutes broken end rapidly and get brain, removes cerebellum.Cerebral tissue is weighed, and adds the homogenate of 0.2M phosphate buffer at 1: 10,3000 revs/min centrifugal 10 minutes, get supernatant 0.5ml, (be called for short: TAB) method is measured MDA content in cerebral tissue with thiobarbituricacidα-.
(3) experimental result
Bioactive natural product B gavages and can reduce reperfusion after acute cerebral ischemia mouse brain and organize MDA content (seeing: table 10), shows that this medicine can suppress lipid peroxidation, and its mechanism may generate or strengthen free radical scavenging for reducing free radical.The lipoid peroxidization resistant of bioactive natural product B is conducive to treatment of vascular dementia and alzheimer disease.
Table 10, bioactive natural product B organize the impact of MDA content on reperfusion after acute cerebral ischemia mouse brain
M ± SD; *p < 0.05, *p < 0.01, compares with ischemia model group.
6, strengthen cerebral ischemia-reperfusion in mice model cerebral tissue superoxide dismutase activity
(1) experiment purpose
Oxidation and the Hangzhoupro oxidation balance of superoxide dismutase (SOD) to body plays vital effect.Too much superoxide anion can cause lipid oxidation in body, and infringement cell is relevant to various diseases and the pathological process such as old and feeble.This enzyme can catalysis superoxide anion generation dismutation reaction, makes it to be converted into hydrogen peroxide and oxygen, and Cell protection is avoided damage.This experiment is intended to observe the impact of bioactive natural product B on cerebral ischemia-reperfusion in mice model cerebral tissue SOD activity decreased, inquires into the mechanism that bioactive natural product B suppresses lipid peroxidation.
(2) experimental technique
Kunming mice, male, 22～28 grams of body weight, random packet, the continuous gastric infusion of each group 7 days before experiment, sham operated rats and ischemia model group give respectively tap water.1h after the administration of operation empty stomach on the same day, after mouse anesthesia, presss from both sides and closes bilateral common carotid arteries 15 minutes with bulldog clamp, then pour into after 15 minutes and break end and get brain rapidly, removes cerebellum.Cerebral tissue is weighed, and adds the homogenate of 0.2M phosphate buffer at 1: 10,3000 revs/min centrifugal 10 minutes, get supernatant 0.1ml, with Asia slightly hydrochlorate method measure SOD activity in cerebral tissue.
(3) real face result
The oral reperfusion after acute cerebral ischemia mouse brain tissue SOD activity (seeing: table 11) that increases of bioactive natural product B gavage, shows 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 be conducive to treatment of vascular dementia and alzheimer disease.
Table 11, the impact of bioactive natural product B on reperfusion after acute cerebral ischemia mouse brain tissue SOD activity
M ± SD; *p < 0.05, *p < 0.01, compares with ischemia model group.
7, lower cerebral ischemia-reperfusion in mice model brain sheet intracellular calcium concentration
(1) experiment purpose
In the pathogeny of vascular dementia and alzheimer disease, Cytosolic Free Calcium Concentration in Brain Nerve Cells increases, calcium overload plays an important role.After cerebral ischemia re-pouring, there are a series of variations, comprise especially extremely the increasing of calcium concentration, calcium overload in neurocyte of calcium ion, play an important role at ischemic brain injury, finally can make neuronal cell death.This experiment is intended to observe the impact that bioactive natural product B increases animal pattern brain sheet intracellular calcium concentration.
(2) experimental technique
Male mouse of kunming, 22～28 grams of body weight, animal random packet, the continuous gastric infusion of each group 7 days before experiment, sham operated rats and ischemia model group give respectively tap water.Operation same day is 1h after administration on an empty stomach, after mouse anesthesia, closes bilateral common carotid arteries 15 minutes with bulldog clamp folder, then pours into and after 15 minutes, break end rapidly and take out full brain, immerses rapidly 4 DEG C and is connected with 95% CO 2and 5%O 2artificial cerebrospinal fluid (artificial cerebrospinal fluid solution, is called for short ACSF, and ACSF formula is (mmol/L): NaCl 124, KCl 3.3, KH 2pO 41.2, NaHCO 326, CaCl 22.5, MgSO 42.4, glucose 10), on vibratome, cutting out brain sheet, thickness 400 μ m, put into CO 2in incubator, hatch 30min for 37 DEG C, add the Fluo-3AM 500ul of 5uM, at CO 2in incubator, after load 1h, ACSF rinses for several times, carries out optical section on laser scanning co-focusing microscope, observes fluorescence intensity.
(laser scanning confocal microscope is called for short: LSCM) can be used for cellularity (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, dynamically observation.Optical section is one of characteristic of laser scanning co-focusing microscope, can realize in vitro brain sheet alive is carried out continuously to harmless cutting, light is cut sample surfaces and information deep layer of can obtaining, and by three-dimensional reconstruction, can obtain three-dimensional image and stereochemical structure information.Calcium fluorescence indicator Fluo-3AM itself is without fluorescence, but it have can with [Ca in living cells 2+] send the characteristic of fluorescence after specific binding, utilize laser scanning co-focusing microscope to measure fluorescence intensity, can detect the level of intracellular free calcium.
Image processing: utilize LaserSharp image processing system, measure area or volume and total glorious intensity of left and right hippocampus, cortical area, (be Mean with unit are or unit volume fluorescence intensity and fluorescence intensity distribution intermediate value, 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) represent, the significance of group difference is checked with t.
(3) experimental result
Oral cerebral ischemia-reperfusion in mice model cerebral cortex and the hippocampus Cytosolic Free Calcium Concentration in Brain Nerve Cells (seeing: table 12) of all can reducing of the each dosage group of bioactive natural product B gavage, this is significant for neuroprotective cell, treatment of vascular dementia and alzheimer disease.
Table 12, impact on cerebral ischemia-reperfusion in mice model Neuronal Calcium
M ± SD; *p < 0.05, *p < 0.01, compares with ischemia model group.
(5) bioactive natural product B antifungal activity
The present invention experimental results show that by In Vitro Anti fungus antitumor, bioactive natural product B has certain inhibitory action (in table 13) to four kinds of common clinical disease fungal pathogenses, and the growth of inhibition tumor cell strain in various degree, detect in anti-tumor activity test the MIC of bioactive natural product B in the MTT reducing process taking 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) as target cell 80as shown in table 14, show that bioactive natural product B can be used for preparing antitumor drug.
1, bacterial strain
ATCC type strain: Candida albicans (Candida albicans) ATCC76615, neogenesis cryptococcus (Cryptococcus neoformans) 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 qualification.
2, bacterium solution preparation
A small amount of with the inoculation circle spherical bacterium such as picking neogenesis cryptococcus, Candida albicans and Candida parapsilosis the SDA culture medium of preserving, be seeded to 1mlYEPD culture fluid, in 35 DEG C, 250rpm shaken cultivation, activation 16h, makes fungus in later stage exponential phase of growth.Get this bacterium liquid to 1mlYEPD culture fluid, again activate with said method, after 16h, with blood cell counting plate counting, adjust bacterial concentration to 1 × 10 with RPMI RPMI-1640 3～5 × 10 3individual/ml.
Aspergillus fumigatus is seeded to SDA inclined-plane, cultivates one week for 35 DEG C; Trichophyton, cultivates two weeks for 28 DEG C.Each bacterium, by this method activation twice, adds appropriate RPMI1640 culture fluid in SDA inclined-plane, blows and beats bacterium colony with suction pipe, and spore is free in RPMI1640 culture fluid, then filters through four layers of sterile gauze.Culture fluid, after blood cell counting plate counting, adds RPMI1640 culture fluid and adjusts spore concentration to 1 × 10 3～5 × 10 3individual/ml.
3, drug sensitive plate preparation
Get aseptic 96 orifice plates, add RPMI 1,640 100 μ l in No. 1 hole of every row and make blank; 3～No. 12 hole respectively adds freshly prepared bacterium liquid 120 μ l; No. 2 hole adds respectively bacterium liquid 160 μ l and test-compound solution 1.6 μ l.2～No. 11 4 times of dilutions in 10 grades, hole, 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, in each hole, DMSO content is all lower than 1%; Positive control, not containing medicine, is made in No. 12 holes, and each drug sensitive plate is in 35 DEG C of cultivations.
4, MIC 80value is judged:
Candidiasis, neogenesis cryptococcus and filamentous bacteria are cultivated 24h, 72h and after one week respectively at 35 DEG C, survey each hole OD value with enzyme micro-plate reader in 630nm.With positive control boring ratio, the drug level in the least concentration hole declining more than 80% taking OD value is as MIC 80(drug level when conk 80% is suppressed).
Table 13, the antibacterial activity (MIC of bioactive natural product B to 4 kinds of funguses 80, μ g/ml)
As the MIC of medicine 80value exceedes while measuring concentration range, adds up by the following method: MIC 80value is higher than maximum concentration 64mgL -1time, count " > 64mgL -1"; MIC 80while being worth for least concentration or below least concentration, do not distinguish, all count "≤0.00024mgL -1".The equal operation repetitive of above-mentioned experiment 2 to 3 times, works as MIC 80value is just accepted when can accurately repeating or only differing from a concentration, and using higher concentration as MIC 80value, works as MIC 80value differs two concentration when above, needs again to test, until meet the requirements.
There are four compounds to have antifungal activity (table 13) in various degree.
(6) anti-tumor activity of bioactive natural product B
The present invention experimental results show that by In Vitro Anti fungus antitumor, bioactive natural product B has certain inhibitory action (in table 13) to four kinds of common clinical disease fungal pathogenses, and the growth of inhibition tumor cell strain in various degree, detect in anti-tumor activity test the MIC of bioactive natural product B in the MTT reducing process taking 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) as 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 4 Cuo salt (MTT): with the phosphate buffer (abbreviation: PBS) dissolve MTT (3-(4 of 0.01mol/L, 5-dimethythiazol-z-yl) 2,5-diphenytetrazolium bromide, be called for short: SIGMA) final concentration 5mg/ml, filtration sterilization, after subpackage, 4 DEG C keep in Dark Place.
The dodecyl sodium sulfate of the preparation of 1.2MTT lysate: 80g (is called for short: SDS, Huamei Bio-Engrg Co.) be dissolved in the N-N-dimethyl methyl acid amide (traditional Chinese medicines) of 200ml, heating in water bath hydrotropy, add 200ml distilled water, mix with IN hydrochloric acid (1: 1) with 80% acetic acid and adjust pH to 4.7.
1.3 cell strain is selected: 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 RPMI-1640 basal medium by cell dilution to 3 × 10 4/ ml, the cell that every hole adds 20 μ l to dilute), 37 DEG C, 5%CO 2, under saturated humidity, cultivate 24 hours: every group of four parallel bars;
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, cultivate 48 hours;
C. every hole adds the MTT 20 μ l of 2mg/ml, hatches 4 hours;
D. culture fluid (as far as possible completely) in 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 cell viability curve chart, obtain IC 50value.
The cytotoxicity test result (μ g/ml) of table 14, bioactive natural product B
Result of the test is as shown in table 14.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 and is used as the potentiality of preparing antitumor drug.
(7) extracting method of bioactive natural product B
After stirring, PDB culture medium divides in the conical flask that installs to 250ml (100ml/ bottle), and through autoclaving, cooling for subsequent use after sealing.The bacterial strain of cultivating is transferred on PDA flat board, cultivate one week for 28 DEG C, in super-clean bench, there is the agar block of mycelia quantitatively by planting to 250m l conical flask (5 pieces/bottle) with card punch (d=3mm) by long, three bottles of each inoculation are to verify its repeatability, and three bottles of blank PDA culture medium in contrast.Then the shaking flask of having inoculated is placed in to 28 DEG C of concussions of constant-temperature table and cultivates one week (180 revs/min).
1, organic solvent extraction
The fermentation liquid that cultivation obtains, after filtration under diminished pressure, with isopyknic ethyl acetate extraction three times; Collecting organic facies (ethyl acetate layer) steams decompression in instrument and is spin-dried for to obtain extractum revolving.
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 completes,, 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 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 after loading, automatic reception instrument is collected automatically, 12 droplets/minute of flow velocitys.The each test tube solution of gained detects by TLC, processes by testing result.Owing to not losing sample, can be repeatedly upper prop repeatedly, and exchange mobile phase, with the separating effect having reached.
4, TLC prepares silica-gel plate
If compound sample is little and silicagel column and all well separation and purification of sephadex column, consider 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 TLC check-out console; Selection can make developing solvent that impact point and impure point significantly separate for preparing plate development agent, is advisable in Rf value 0.5 left and right.
In the specific implementation, need to, according to existing fund, technology and relevant requirement, adopt wherein suitable method, and the measure of choosing the necessity such as suitable condition be reaching the set goal.
And these methods are all also known technologies of this area, at needs further in confirmation, be to be all easy to find all technical data including ins and outs from relevant teaching material and relevant technical literature etc.
(8) purposes of bioactive natural product B
1, general introduction
The object of this invention is to provide a kind of for preventing, diagnose, detect, protect, the product for the treatment of and Effect of Anti fungus, antitumor, anti-angiogenic dementia disease and directly related disease thereof; comprise one or more in medicine, reagent, food etc., preferred agents.
Prove by Pharmacological Activity Screening, bioactive natural product B is the active site of its prevention, diagnosis, detection, protection, treatment and Effect of Anti fungus, 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 is levied bright, mouse stomach administration exceedes 1.0g/kg to the maximum tolerated dose of this active site, be equivalent to the more than 200 times of clinical recommended drug dosage, show that this effective site is safe and reliable, solved the problem that complicated component in Chinese medicine compound, active constituent content are low and contain toxic component.
In sum; inventor has carried out theory study to bioactive natural product B; through particularly long-term pharmacology test of a large amount of experimentation, find that the bioactive natural product B addressing has the activity of significant prevention, diagnosis, detection, protection, treatment and Effect of Anti fungus, 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, the medicine being preferably prepared from as raw material taking bioactive natural product B of the present invention.
2, the using method of bioactive natural product B and compositions thereof and requirement
Bioactive natural product B of the present invention can combine use separately or with other active component; comprise for the preparation of for preventing, diagnose, detect, protect, the product for the treatment of and Effect of Anti fungus, antitumor, anti-angiogenic dementia disease and directly related disease thereof; comprise medicine, reagent or food etc., especially medicine.
In concrete use, bioactive natural product B of the present invention can use separately, for example, with the form application of active site, its derivant or pharmaceutical salts, can also use together with other many chemical substances.No matter whether these chemical substances have biological activity or have the function for the treatment of disease, comprise miscellaneous function as collaborative amplification, antagonism or alleviate the side effect etc. of bioactive natural product B, these chemical substances are to comprise one or more in pharmaceutically acceptable carrier, food, natural product, chemical synthetic drug or mankind's medication etc.; Preferably include one or more in pharmaceutically acceptable carrier or food etc.; Further preferred pharmaceutically acceptable carrier.
" pharmaceutically acceptable carrier " used herein comprises one or more in the applicable solvent of any and all physiology, disperse medium, afterbirth, antibacterial and antifungal, isotonic agent or absorption delay agent etc.The example of pharmaceutically acceptable carrier comprises one or more in one or more water, saline, phosphate-buffered saline, glucose, glycerol or ethanol etc. and compositions thereof.In many cases, in said composition, preferably include isotonic agent, for example, sugar, such as one or more in polyhydric alcohol or the sodium chloride etc. of mannitol, sorbitol, sorbitol.Pharmaceutically acceptable carrier can also comprise a small amount of auxiliary substance, such as, in wetting agent or emulsifying agent, antiseptic or buffer etc. one or more, and they have strengthened effect duration or the effect of this bioactive natural product B.
From concrete classification, said pharmaceutically acceptable carrier refers to the pharmaceutical carrier of medicine and pharmacology field routine, comprises lubricant, as one or more in Pulvis Talci, Polyethylene Glycol or magnesium stearate etc.; Disintegrating agent, as micro-product fiber rope etc.; Filler, as one or more in starch, dextrin or lactose etc.; Binding agent, as one or more in pregelatinized Starch, cellulose derivative, alginate, gelatin or polyvinylpyrrolidone etc.; Osmotic pressure regulator, as one or more in sodium chloride, glucose, sucrose, sorbitol or mannitol etc.; PH adjusting agent, one or more in the acid such as example hydrochloric acid, sodium hydroxide or alkali; Solvent, as in water, buffer, ethanol or propylene glycol etc. one or more etc.; Antioxidant and chelating agent, as one or more in sodium sulfite, EDTA etc.; Surfactant, as quaternary ammonium compound, hexadecanol etc.; Absorption carrier, as one or more in Kaolin or soap clay etc.; Macromolecular scaffold agent, as one or more in cyclodextrin, Polyethylene Glycol, poloxamer etc.; In addition, can also in compositions, add other adjuvant, as one or more in flavouring agent, antiseptic or sweeting agent etc.
For example, active component bioactive natural product B is dissolved, suspendible or (be for example emulsifiable in suitable aqueous solvent, distilled water, one or more in normal saline or Green's solution etc.) or oil-based solvent in (for example, vegetable oil is olive oil such as, Oleum sesami, Oleum Gossypii semen, one or more in Semen Maydis oil or propylene glycol etc.) in, can make ejection preparation, wherein in solvent, (for example can contain solubilizing agent, polyoxyethylene sorbitan monoleate, polyoxyethylene hydrogenated Oleum Ricini, polyvidone, cyclodextrin, poloxamer, Polyethylene Glycol, benzyl alcohol, one or more in chlorobutanol or phenol etc.), osmotic pressure regulator (for example, sodium chloride, glycerol, D9-mannose, one or more in D-glucitol or glucose etc.).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 lignocaine etc.) etc.
Of the present invention and bioactive natural product B can also combine use with the form of compositions, particularly with by other chemical substance animal especially mammal is comprised to people or other animals treat compositions used or similar compositions as medicine.Described mammal, comprise one or more in people, mice, rat, sheep, monkey, cattle, pig, horse, rabbit, dog, chimpanzee, baboon, Adeps seu carnis Rhiopithecus roxellanae, macaque or Rhesus Macacus etc., preferably one or more in people, mice, rat, monkey, pig, rabbit or dog etc., one or more in further preferred people, rat or monkey etc.For example, bioactive natural product B of the present invention can be added be suitable for to curee's Pharmaceutical composition in.Conventionally, 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 one or more in the dosage forms such as such as liquid, semisolid and solid; Wherein said pharmaceutical composition comprises that the bioactive natural product B that treats effective dose is 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 experiment, research or applies clinically, comprise that solid preparation is as capsule, tablet, granular preparation etc., liquid preparation is as oral liquid or injection etc.
For example make active component mix with one or more carriers, be then made into required dosage form.Described dosage form comprises one or more in tablet, capsule, granule, suspensoid, Emulsion, solution, syrup or injection etc., takes one or more route of administration in oral or injection (comprise in intravenous injection, intravenous drip, intramuscular injection or subcutaneous injection etc. one or more), 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 production condition of storage.Said composition can be mixed with to the ordered structure that solution, microemulsion, dispersion liquid, liposome or other are suitable for high drug level.By being added in suitable solvent and then carry out aseptic filtration together with a kind of of required mentioned component or combination, this bioactive natural product B of aequum prepares aseptic parenteral solution.Generally speaking, prepare dispersion liquid by this bioactive natural product B being added in the aseptic solvent that contains basic disperse medium and required above-mentioned other composition.In the case of the sterile powder for the preparation of aseptic parenteral solution, the preparation method of recommendation is vacuum drying and lyophilized preparation.For example, by the coating such as lecithin, the dispersion liquid in the situation that by keeping required granular size and by using surfactant, can keeping the adequate liquidity of solution.
In said composition, can comprise the medicament that postpones absorption, for example Monostearate or gelatin, absorb with the prolongation that reaches injectable composition; Can comprise high molecular polymer carrier, as hydroxypropyl methylcellulose or polyoxyethylene, discharge with the prolongation that reaches Orally administered composition.
During for 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 situation of the age of patient or user and body weight and health or patient's symptom conventionally.
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 " refers to the amount that effectively reaches required therapeutic effect under necessary dosage and time.The treatment effective dose of bioactive natural product B can cause that at this individuality the factors such as the ability of required reaction change according to the patient's condition such as individual, age, sex and body weight and this bioactive natural product B.Treatment effective dose also refers to that the useful therapeutic effect of this bioactive natural product B exceedes the amount of its any toxicity or harmful effect.
" prevention effective dose " refers to the amount that effectively reaches required preventive effect under necessary dosage and time.Because preventive dose is for the ill front or early stage curee of disease, prevention effective dose is less than treatment effective dose conventionally.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 the disease type of wanting to alleviate and seriousness, while that is to say for patient, bioactive natural product B dosage of the present invention or consumption, decide according to the situation of the age of patient or user and body weight and health or patient's symptom conventionally.
In addition; should understand; for any specific curee; should along with the time according to individual need and give with or supervision to adjusting given dose system with the people's of described compositions professional judgement; and the dosage range of setting is herein only illustrative, can't limit scope or the practice of claimed compositions.
That is to say, need to be according to object, route of administration, disease and the situation etc. for the treatment of for the treatment of, change dosage or the consumption of the each and/or every day of bioactive natural product B of the present invention.For example, give mammal through vein, especially adult (as body weight 60kg), the single dose of described bioactive natural product B is about 50～1200mg, preferred about 100mg, preferably administration every day 1～3 time.Can adjust dosage unit, for example, to propose the best required reaction of arch (, treatment or prevention are replied).
For example, can single-bolus high-dose administration, can within a period of time, give several divided doses or reduce in proportion or increase dosage according to the urgency for the treatment of situation.The non-intestinal compositions that preparation is easy to administration and the unified dosage unit form of dosage is especially favourable.Dosage unit form used herein, refers to be suitable for the physical separation unit of the dosage unit of the mammalian subject of wish treatment; The calculating that each unit contains scheduled volume is for together producing the active matter bioactive natural product B of required therapeutic effect with required pharmaceutical carrier.The specification of dosage unit form of the present invention, be determined by the following and directly depend on the specific characteristic of following (a) this bioactive natural product B and particular treatment or the preventive effect of wanting to reach, and (b) interior restriction of mixing in this technology that is used for the treatment of individual sensitivity bioactive natural product B.
3, the pharmaceutical dosage form of bioactive natural product B and compositions thereof and route of administration
Bioactive natural product B of the present invention and compositions thereof prepare for preventing, diagnose, detect, protect, the product for the treatment of and Effect of Anti fungus, antitumor, anti-angiogenic dementia disease and directly related disease thereof, the product of wherein preparing according to the requirement of beverage, food technology field can be used in prevention, protection and treatment antifungal, antitumor, anti-angiogenic dementia disease and directly related disease thereof; The product of preparing according to the requirement of medical technical field can be used in patient's treatment or health care, can either be directly used in separately the medicine of preparation treatment or health care, also can mix or combine with many chemical substances, directly or indirectly for the preparation of the medicine for the treatment of or keeping healthy.Chemical substance described here and this section are above described identical.
In the present invention, required material comprises raw material of the present invention, above-mentioned matching used chemical substance etc., all should, according to practical situation and needs, adopt the material of food stage or pharmaceutical grade.
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 technical staff will appreciate that route of administration/administering mode changes with required result.In some concrete enforcement, this reactive compound can with this compound of protection avoid the carrier that discharges fast together preparation example as empty release formulation, comprise that graft transmission system, transdermal paste one or more in transmission system or microcapsule transmission system etc.In addition, can also use biodegradable, biocompatible polymer, such as, in ethylene-ethyl acetate, polyanhydride, polyglycolic acid, collagen protein, polyorthoesters or polylactic acid etc. one or more.Prepare the equal patent applied for of many methods of this preparation or general (for example Sustained and Controlled Release Drug Delivery Systems known to those skilled in the art, J.R.Robinson edits, Marcel Dekker, Inc., New York, 1978).
In bioactive natural product B of the present invention or tool 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 is for example coloring agent, sweeting agent, antioxidant etc.
Bioactive natural product B of the present invention and compositions thereof, conventionally by one or more modes in oral, rectum or parenteral etc., be 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 applying clinically, comprise solid preparation, as capsule, tablet, granular preparation etc., semi-solid preparation is as ointment etc., liquid preparation is as oral liquid, suspensoid, Emulsion etc., or injection.Take one or more route of administration in oral or injection (comprise in intravenous injection, intravenous drip, intramuscular injection or subcutaneous injection etc. one or more), 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.
When oral, can be made into conventional solid preparation as one or more in tablet, powder, granule or capsule etc.In the time 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 needed) can also be wrapped in hard or soft shell gelatin capsules, is pressed into tablet or directly adds in curee's meals.About oral therapeutic administration, described bioactive natural product B can be added together with excipient and use with one or more forms in edible tablet, buccal tablet agent, lozenge, capsule, suspension, syrup or wafer etc.
For to give bioactive natural product B of the present invention outside 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.Supplementary reactive compound can also be added in 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 combine use, can utilize primely this medicine giving compared with low dosage, therefore avoid possible toxicity or the complication relevant to various monotherapies.
Make liquid preparation as one or more in water preparation, oil-suspending agent or other liquid preparation, as one or more in syrup, tincture or elixir etc.; When the parenteral, can be made into one or more in solution, water preparation or the oiliness suspending agent etc. of injection.
Above medicine or pharmaceutical composition can use various approach, in described type of service, preferred form is that oral formulations (as one or more in tablet, coated tablet, capsule, solution or suspension etc.), non-intestinal give one or more in dosage form (as one or more in injection, ointment or patch etc.) etc., further one or more in preferred tablet, capsule or injection etc., the particularly preferably one in tablet or injection.
In addition, the medicinal raw material that bioactive natural product B uses comprises that marine fungi or marine fungi crude extract etc. also can be directly used in separately for the preparation of prevention in some cases, diagnosis, detect, protection, treatment and Effect of Anti fungus, antitumor, the product of anti-angiogenic dementia disease and directly related disease thereof, also can mix or combine with many chemical substances, with the form of compositions directly or indirectly for the preparation of for prevention, diagnosis, detect, protection, treatment and Effect of Anti fungus, antitumor, anti-angiogenic dementia disease and the good product that connects relevant disease thereof.Chemical substance described here and this section are above described identical.
For example, the powder that the medicinal raw material that bioactive natural product B uses comprises marine fungi or marine fungi crude extract etc. is for the preparation of for antifungal, antitumor, anti-angiogenic dementia disease prevention, diagnosis, protection and treatment product particularly various dosage forms of medicine, or the medicinal raw material that uses of the bioactive natural product B powder that comprises marine fungi or marine fungi crude extract etc. and relevant adjuvant are for the preparation of for preventing, diagnosis, detect, protection, treatment and Effect of Anti fungus, antitumor, the product of anti-angiogenic dementia disease and directly related disease thereof is the various dosage forms of medicine especially, or the medicinal raw material that uses of the bioactive natural product B powder that comprises marine fungi or marine fungi crude extract etc. with relevant for the preparation of prevention, diagnosis, detect, protection, treatment and Effect of Anti fungus, antitumor, the product of anti-angiogenic dementia disease and directly related disease thereof as medicine together for the preparation of for prevention, diagnosis, detect, protection, treatment and Effect of Anti fungus, antitumor, the product of anti-angiogenic dementia disease and directly related disease thereof is as the various dosage forms of medicine, or the medicinal raw material that uses of the bioactive natural product B powder that comprises marine fungi or marine fungi crude extract etc. and relevant ancillary drug are together for the preparation of for prevention, diagnosis, detect, protection, treatment and Effect of Anti fungus, antitumor, the product of anti-angiogenic dementia disease and directly related disease thereof is as the various dosage forms of medicine, as tablet, one or more in capsule or suspensoid etc., preferably capsule.
One of described method is that the medicinal raw material that bioactive natural product B is used comprises that the powder filling of marine fungi or marine fungi crude extract etc. is capsule, two of method is medicinal raw material that bioactive natural product B is used powder of comprising marine fungi or marine fungi crude extract etc. with relevant for the preparation of prevention, diagnosis, detect, protection, treatment and Effect of Anti fungus, antitumor, the product of anti-angiogenic dementia disease and directly related disease thereof as medicine together fill be capsule, three of method is medicinal raw material that bioactive natural product B is used powder of comprising marine fungi or marine fungi crude extract etc. is capsule with relevant ancillary drug together fill, four of method is that the medicinal raw material that bioactive natural product B is used comprises 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 powder that medicinal raw material that bioactive natural product B is used comprises marine fungi or marine fungi crude extract etc., relevant to prevention, diagnosis, detect, protection, treatment and Effect of Anti fungus, antitumor, it is tablet that the product of anti-angiogenic dementia disease and directly related disease thereof is directly pressed with relevant adjuvant together according to a conventional method as medicine, six of method is powder that medicinal raw material that bioactive natural product B is used comprises 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 six kinds of above-mentioned basic skills, the medicinal raw material that can also select bioactive natural product B to use comprises that other forms of marine fungi or marine fungi crude extract etc. or the medicinal raw material that bioactive natural product B is used carry out after method processing well known in the art, prepares the product that contains the medicinal raw material that bioactive natural product B uses of various dosage forms as medicine.But, it should be noted that, in the medicinal raw material using at above-mentioned direct use bioactive natural product B comprises marine fungi or marine fungi crude extract etc., should be first according to the dosage requirement of used bioactive natural product B, convert and obtain the consumption that medicinal raw material that the bioactive natural product B of required use uses comprises 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 the product of prevention, diagnosis, detection, protection, treatment and Effect of Anti fungus, 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 for prevention, diagnosis, detection, protection, treatment and Effect of Anti fungus, antitumor, anti-angiogenic dementia disease and directly related disease thereof provide a kind of new medicament sources.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; yield is high, can be used for preparing the product of prevention, diagnosis, detection, protection, treatment and Effect of Anti fungus, 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 uses safety, and one-object-many-purposes has been brought into play effect to greatest extent; Bioactive natural product B stable in properties, is used 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 successful of prevention, diagnosis, detection, protection, treatment and Effect of Anti fungus, antitumor, anti-angiogenic dementia disease and directly related disease thereof; and the scope of application is wide especially; therefore easily apply, 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; there is important value to developing Chinese resources of medicinal plant, be for preventing, diagnose, detect, protect, the safe raw material for the treatment of and the aspect such as Effect of Anti fungus, antitumor, anti-angiogenic dementia disease and directly related disease thereof.
Detailed description of the invention
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 preparing antifungal, antitumor, the prevention of anti-angiogenic dementia disease, diagnosis, protection and treatment is provided, has been convenient to medical industry and relevant industries as the safe handling in the field 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 the conventional dry method that is frozen in, and using water as solvent, the steps include: to get bioactive natural product B, adds excipient, be dissolved in water, regulate pH, add active carbon, filtration sterilization, fill, partly rolls plug, lyophilization, and lid is rolled in tamponade.Excipient used is selected from one or more in mannitol, gelatin hydrolysate, glucose, lactose, dextran, albumin, pH adjusting agent etc.Every bottle containing bioactive natural product B1～100mg.
The present invention prepares injectable powder also can adopt spray drying method, using water as solvent, the steps include: to get bioactive natural product B, adds or do not add excipient (excipient is the same), be dissolved in water, add active carbon, filtration sterilization, spraying is dry, and aseptic subpackaged, lid is rolled in tamponade.Every bottle containing bioactive natural product B10～100mg.
When the present invention prepares small-volume injection, prepare using water for injection as solvent, 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 adjusting agent, surfactant, cyclodextrin, antioxidant, complexing of metal ion agent, antibacterial.Injection can be mixed with the ordered structure that solution, microemulsion, emulsion, liposome, microsphere, microcapsule or other are suitable for high drug level, wherein can comprise the medicament that postpones absorption, such as Monostearate, gelatin, ethylene-ethyl acetate, polyanhydride, polyglycolic acid, collagen protein, polyorthoesters or polylactic acid etc., absorb with the prolongation that reaches injectable composition.Every containing bioactive natural product B1～100mg.
The present invention prepares glucose infusion liquid or sodium chloride transfusion, using water for injection as solvent, add appropriate glucose or sodium chloride preparation, 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 adjusting agent, surfactant, antioxidant, cyclodextrin, complexing of metal ion agent, antibacterial.Every bottle containing bioactive natural product B1～100mg.
The present invention prepares the oral formulations such as tablet, capsule, granule, oral liquid, and adjuvant can be lactose, starch, dextrin, stearate etc., technology preparation routinely.Can comprise high molecular polymer carrier, as hydroxypropyl methylcellulose or polyoxyethylene etc., discharge with the prolongation that reaches Orally administered composition.
In the present invention, the embodiment of above-described detailed description of the invention and the following stated is all in order to set forth better the present invention, is not 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; Inject water to 2000ml, add 3.0g needle-use activated carbon, fully stir 30 minutes; Decarbonization filtering; With 0.22 μ m filtering with microporous membrane; Subpackage, partly rolls plug; Lyophilization, then lid is rolled in tamponade.
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; Inject water to 6000ml, add 1g needle-use activated carbon, fully stir 30 minutes; Decarbonization filtering; With 0.22 μ m filtering with microporous membrane; Subpackage, partly rolls plug; Lyophilization, then lid is rolled in tamponade.
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; Inject water to 1000ml, add 1.5g needle-use activated carbon, fully stir 30 minutes; Decarbonization filtering; With 0.22 μ m filtering with microporous membrane; Subpackage, partly rolls plug; Lyophilization, then lid is rolled in tamponade.
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; Inject water to 1000ml, add 1.0g needle-use activated carbon, fully stir 30 minutes; Decarbonization filtering; With 0.22 μ m filtering with microporous membrane; Subpackage 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; Inject water to 1000ml, add 0.5g needle-use activated carbon, fully stir 30 minutes; Decarbonization filtering; With 0.22 μ m filtering with microporous membrane; Subpackage 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 5g needle-use activated carbon, fully stir 30 minutes; Decarbonization filtering; Inject water to 5000ml; With 0.22 μ m filtering with microporous membrane; Subpackage 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 10g needle-use activated carbon, fully stir 30 minutes; Decarbonization filtering; Inject water to 10000ml; With 0.22 μ m filtering with microporous membrane; Subpackage embedding, every bottle of 250ml, pressure sterilizing.
The preparation of embodiment 8, bioactive natural product B tablet
Coating fluid prescription: gastric solubleness thin film dress material: 85G61235, Shanghai Colorcon Coating Technology Co., Ltd
(2) preparation technology takes respectively principal agent and the adjuvant of recipe quantity by above prescription, by the equivalent method mix homogeneously that progressively increases, 5%PVP dehydrated alcohol soft material processed, dry, granulate, add magnesium stearate to mix, measure content, calculate the heavy rear tabletting of sheet, control nude film hardness 5～7kg, make altogether 9698, tablet, yield rate is 96.98%.
Adopt conventional high-efficiency coating pan coating, technique is as follows: nude film (hardness 5～7kg) is put into coating pan, start agitating device and air blowing heating device, in the time that nude film temperature rises to 40 DEG C, upper 1/3 place that starts to open spray gun alignment tab bed sprays into coating solution coating, 38～42 DEG C of control strip bed tempertaures, gas pound pressure 6kg, 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
(2) preparation technology gets supplementary material and crosses respectively 80 mesh sieves, and mix homogeneously is with appropriate 10%PVP solution soft material processed, dry, adds magnesium stearate 3g, granulate, and tabletting, makes 1000.
The preparation of embodiment 10, bioactive natural product B capsule
(1) prescription
(2) preparation technology gets respectively other adjuvant in crude drug bioactive natural product B and prescription and crosses respectively 100 mesh sieves, put 60 DEG C of oven dry, taking recipe quantity bioactive natural product B mixs homogeneously with microcrystalline Cellulose, the starch equivalent method of progressively increasing, with appropriate dehydrated alcohol soft material processed, 30 mesh sieves are granulated, and 50～60 DEG C are dried 2 hours, with 30 mesh sieve granulate, add the Pulvis Talci mix homogeneously of recipe quantity, fill.
The disease-resistant fungal pathogens activity of embodiment 11, detection of active natural product B
1. material
1.1. bacterial strain:
ATCC type strain: Candida albicans (Candida albicans) ATCC76615, neogenesis cryptococcus (Cryptococcus neoformans) ATCC32609 give by Long March hospital strain preservation center.
Clinical strain: trichophyton (Trichophyton rubrum) 0504656, Aspergillus fumigatus (Aspergillus fumigatus) 0504656, pick up from respectively Shanghai Changhai Hospital different department clinical sample, and through morphology and biochemical qualification.
1.2. culture fluid:
RPMI RPMI-1640: RPMI1640 (Gibco BRL) 10g, NaHCO 32.0g, (Sigma) 34.5g (0.165M) of morpholine propane sulfonic acid (morpholinepropanesulfonic acid, MOPS), add tri-distilled water 900ml and dissolve, NaOH adjusts pH to 7.0 (25 DEG C), is settled to 1000ml, filter sterilization, 4 DEG C of preservations.
Husky fort glucose agar medium (SDA): peptone 10g, glucose 40g, agar 18g, adds tri-distilled water 900ml and dissolves, and adds 2mg/ml chloramphenicol solution 50ml, adjusts pH to 7.0, is settled to 1000ml, 4 DEG C of preservations after autoclaving.
YEPD culture fluid: yeast extract 10g, peptone 20g, glucose 20g, adds tri-distilled water 900ml and dissolves, and adds 2mg/ml chloramphenicol solution 50ml, is settled to 1000ml, 4 DEG C of preservations after autoclaving.
1.3. reagent:
It is domestic analytical pure that fluconazol (fluconazole) and ketoconazole (ketoconazole) provide .DMSO by organic chemistry teaching and research room of the court, with front heavy steaming.
1.4. instrument:
Water isolation type electro-heating standing-temperature cultivator (Shanghai leap medical apparatus and instruments factory); The desk-top constant temperature oscillator of THZ-82A (Shanghai leap medical apparatus and instruments factory); Multiskan MK3 enzyme micro-plate reader (Labsystems Dragon);
2. operating procedure
2.1. bacterium solution preparation:
Before experiment, a small amount of with the inoculation circle spherical bacterium such as picking neogenesis cryptococcus, Candida albicans and Candida parapsilosis the SDA culture medium of 4 DEG C of preservations, be seeded to 1mlYEPD culture fluid, in 35 DEG C, 250rpm shaken cultivation, activation 16h, make fungus in later stage exponential phase of growth. get this bacterium liquid to 1mlYEPD culture fluid, again activate with said method, after 16h, with blood cell counting plate counting, adjust bacterial concentration to 1 × 10 with RPMI1640 culture fluid 3～5 × 10 3individual/ml.
Aspergillus fumigatus is seeded to SDA inclined-plane, cultivates one week for 35 DEG C; Trichophyton, to cultivate two weeks for 28 DEG C. each bacterium activates after twice by this method, add appropriate RPMI1640 culture fluid in SDA inclined-plane, blow and beat bacterium colony with suction pipe, fungal spore is free in RPMI1640 culture fluid, then filtering through four layers of sterile gauze. culture fluid, after blood cell counting plate counting, adds RPMI1640 culture fluid and adjusts spore concentration to 1 × 10 3～5 × 10 3individual/ml.
2.2. medicinal liquid preparation:
Tested medicine is made into 6.4gL with DMSO respectively -1solution ,-20 DEG C of preservations, before experiment, put 35 DEG C of incubators by medicinal liquid taking-up and melt for subsequent use.
2.3. drug sensitive plate preparation:
Get aseptic 96 orifice plates, add RPMI 1,640 100 μ l in No. 1 hole of every row and make blank; 3～No. 12 hole respectively adds freshly prepared bacterium liquid 120 μ l; No. 2 hole adds respectively bacterium liquid 160 μ l and test-compound solution 1.6 μ 4 times of dilutions in 10 grades, l.2～No. 11 holes, 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, in each hole, DMSO content is all lower than 1%; Positive control, not containing medicine, is made in No. 12 holes, and each drug sensitive plate is in 35 DEG C of cultivations.
2.4.MIC 80value is judged:
Candidiasis, neogenesis cryptococcus and filamentous bacteria are cultivated 24h, 72h and after one week respectively at 35 DEG C, survey each hole OD value with enzyme micro-plate reader in 630nm. and with positive control boring ratio, the drug level in the least concentration hole declining more than 80% taking OD value is as MIC 80(drug level when conk 80% is suppressed).
As the MIC of medicine 80value exceedes while measuring concentration range, adds up by the following method: MIC 80value is higher than maximum concentration 64mgL -1time, count " > 64mgL -1"; MIC 80while being worth for least concentration or below least concentration, do not distinguish, all count "≤0.00024mgL -1".
The equal operation repetitive of above-mentioned experiment 2 to 3 times, works as MIC 80value is just accepted when can accurately repeating or only differing from a concentration, and using higher concentration as MIC 80value, works as MIC 80value differs two concentration when above, needs again to test, until meet the requirements.In table 13, data show compd B is for Candida albicans, and the effect of neogenesis cryptococcus is better than griseofulvin.

Claims (7)

1. the application of bioactive natural product B in the anti-angiogenic dementia product of preparation, the structural formula of described this bioactive natural product B is as follows:
2. bioactive natural product B according to claim 1 application in anti-angiogenic dementia product in preparation, is characterized in that, by the compositions that contains bioactive natural product B for the preparation of anti-angiogenic dementia product.
3. the application of bioactive natural product B according to claim 1 and 2 in the anti-angiogenic dementia product of preparation, it is characterized in that, described anti-angiogenic dementia product refers to one or more in the product that is directly used in prevention, diagnosis, detection, treatment vascular dementia and directly related disease thereof.
4. the application of bioactive natural product B according to claim 1 and 2 in the anti-angiogenic dementia product of preparation, is characterized in that, described anti-angiogenic dementia product is the one in medicine, reagent or food.
5. the application of bioactive natural product B according to claim 4 in the anti-angiogenic dementia product of preparation, is characterized in that, described anti-angiogenic dementia product is medicine.
6. the application of bioactive natural product B according to claim 1 and 2 in the anti-angiogenic dementia product of preparation, is characterized in that purity >=95% of described bioactive natural product B.
7. the application of bioactive natural product B according to claim 1 and 2 in the anti-angiogenic dementia product of preparation, is characterized in that, the occupation mode of described bioactive natural product B is to comprise independent use or combine the one in use with other chemical substances.
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