CN103638054B - The preparation of mercerising brown woods ant antifungal effective site and medical usage thereof - Google Patents
The preparation of mercerising brown woods ant antifungal effective site and medical usage thereof Download PDFInfo
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Abstract
The present invention relates to preparation and the medical usage thereof of mercerising brown woods ant antifungal effective site.Specifically, the present invention relates to and a kind of there is Chinese medicine Formica fusca mercerising brown woods ant antifungal effective site of control fungal infection class function of diseases and preparation method thereof and the medical usage for the preparation of antifungal drug.Does is mercerising of the present invention brown woods ant effective site the brown woods ant of medical ant mercerising (Formica? fusca) through Ultrafiltration Membrane enrichment, refining and obtain.Specifically, brown for mercerising woods ant polypide is carried out through the ultrafilter membrane that the extract that alcohol merceration, heat raise is 5KDa through molecular cut off again ultrafiltration is refining to be formed, it is active that this effective site has the significant albicans growth that suppresses, tablet, granule, capsule, oral liquid, drop pill, varnish, externally-applied liniment, membrane, unguentum, spray, injection, transdermal patch, aerosol form can be prepared into, for the preparation of the medicine of prevention and therapy fungal infection, cosmetics of everyday use or medical apparatus and instruments.
Description
Technical field
The present invention relates to medical art, specifically, the present invention relates to a kind of mercerising brown woods ant antifungal effective site with antifungal activity and preparation method thereof, pharmaceutical composition containing this extract and preparing the purposes in anti-fungal infection medicine.
Background technology
Along with medical science applied development, various novel drugs and new technique widely using as medical procedure such as broad ectrum antibiotic, hormone, various intubation catheter technology, chemotherapy, transplanting, and HIV and tumor, cause human immune system to be increased year by year by the case destroyed gradually, thus the mycotic sickness rate causing deep fungal infection to cause is more and more high; And on the other hand, along with widely using of mankind's abuse of antibiotics and clinical antifungal drug, cause conventional antibiotic effectively can not resist some serious fungal infections and bacteriological infection, the drug resistance phenomenon of fungus is day by day serious.The effect of conventional antibiotic has met with remarkable bottleneck, and most of antibacterial, fungus create significant Drug resistance to common antibiotic; The concurrent fungal infections such as HIV sufferers, patient with severe symptoms, fire victim more make sufferer hang by a hair.Therefore, find wide spectrum, antifungal new drug that is efficient, low toxicity has become the focus of drug research.
Extremely urgent to the research of fastbacteria/intractable fungus.The disease that fungal infection causes can be divided into following four classes substantially: studies of invasive fungal infections and systemic mycosis (as aspergillosis and candidiasis etc.), mucosal pattern mycosis (as " thrush " etc.), shallow phenotype dermatomycosis (as " tinea pedis " or " tinea capitis ", " tinea unguium " etc.) and anaphylactic type mycosis (as asthma and chronic inflammatory disease etc.).In above four class mycosises, endanger maximum with the first kind to the mankind, then three class mycosises are relatively light.According to from external clinical statistics data, in the crowd dying from infectious disease, have 4% to be die from general aspergillosis mushroom fungal infection, the people of about 2% dies from systemic Candida infections.Clinical statistics data shows, patient once suffer from general aspergillosis, its mortality rate up to 85%, as suffer from blood borne candidiasis then its mortality rate reach 40%.Utilizing existing antifungal drug to treat systemic mycosis or the mycotic effect of blood infection type so far still cannot be satisfactory.Therefore, pharmacy circle is finding the newtype drug that can suppress systemic fungal infection.
At present, the antifungal drug having developed both at home and abroad listing mainly contains four large classes, i.e. echinocandin (echinocandins) class (as Caspofungin and meter Ka Min etc.) of polyenoid class (as amphotericin B), triazole type (as fluconazol, miconazole, econazole, Itraconazole and ketoconazole etc.), alkyl amine (as terbinafine) and listing newly developed.Front three major types antifungal agent is the old medicine of 20th century exploitation listing, and echinocandin class is then develop the new drug of listing at the beginning of 21 century.The health azole of old kind is as the onset by lanosterol 14 demethylase in Antifungi ergosterol biosynthesis such as fluconazol, itraconazole, voriconazole, posaconazole, and this type of old brand health triazole antifungal agent thing antimicrobial spectrum is narrower and resistance to pressure grows with each passing day.Though new varieties antifungal drug antimicrobial spectrum expand metabolisming property and physicochemical property not good, water solublity is low, bioavailability is poor; Needs of patients high fat diet is in order to drug absorption, or the extraordinary dosage form taking high price can onset; The cyclodextrin etc. added for increasing water solublity also brings larger threat to renal insufficiency person.
Antifungal drug in triazole class no matter the first generation or the second filial generation all to there being the fungus-caused systemic infection unsatisfactory curative effect of silk; And although echinocandin class antifungal new drug has certain curative effect to this kind of disease, but its greatest problem faced is: there is no peroral dosage form, be only limited to injection (and being only limitted to hospital's use), therefore patient uses rather inconvenience.Secondly, although echinocandin waits fungal infection curative effect pretty good to Candida spp disease, it is expensive, so limit the sales volume in their market beyond developed country.It is reported, in China market, the retail price of often propping up Caspofungin injection (dosage is 50 milligrams) is 3,148 yuan, and the meter Ka Min price that Japan produces is slightly lower, but often props up (dosage is 50 milligrams) and also want 650 yuan.Therefore, the systemic antifungal medicine that China's hospital clinical is the most frequently used is at present still Fluconazole and terbinafine.
In sum, finding novel antifungal drug and active substance source as early as possible, is that many decades medicine workers will need the task of top priority of promptly development from now on.Untoward reaction is low in the treatment not easily produces the advantages such as drug resistance because of it for Chinese medicine and natural drug, in widespread attention; The various folk prescription be made up of Chinese medicine or natural product or compound preparation and Chinese medicine and western medicine compatibility agent, be applied to clinical more and more, show good effect.Because Chinese medicine and natural drug have the features such as aboundresources, toxic and side effects be low, sight is just aimed at this field by domestic expert, excavate Chinese medicine treasure-house, try hard to find and effectively can infect the medicine of the various diseases caused by Antifungi, and our early-stage Study sees hope.
The insecticide earth recorded and has named, its kind is more than 1,000,000 kinds, and account for more than 80% of whole regnum animale kind, wherein China about has 150,000 kinds.In very long evolutionary process, insecticide has become the animal species of advantage on the earth.It is estimated, the total biomass of insecticide exceedes all animal total biomasses on the earth, is one of untapped biological group in the world today.And pharmaceutical insects is the ingredient in Chinese pools of traditional Chinese medicine, according to records, have the insecticide of medical value about to have more than 800 to plant, 14 orders 35 being under the jurisdiction of Insecta are above section level.The extensive use clinically because of determined curative effect of many insects Chinese medicine.In recent years, to excavation and the original effect of pharmaceutical insects in some difficult miscellaneous diseases for the treatment of of insecticide source medicinal ingredient, cause the great interest of numerous medical personal and bughunter, the research of pharmaceutical insects has had considerable progress.The exploitation research of the present inventor team long campaigns pharmaceutical insects, and successfully to have developed with Blatta seu periplaneta be the medicine such as " Kangfuxin Liquid ", " Ganglong capsule ", " Xinmailing Solution " of raw material, cure mainly: the ischemic cardiovascular and cerebral vascular diseases etc. such as traumatic surface, hepatitis B, acute and chronic heart failure and ventricular premature contraction such as burn and scald.
Develop novel antifungal natural drug to explore, the present invention with pharmaceutical insects Formica fusca for object of study.Formica fusca is also known as " Formica fusca ", " ant ", " elder brother Fu ", " ant ", " horse ant ", " ZHUANGYUANZI ", be a kind of social insect, Formica fusca is subordinate to Insecta (ClassInsecta), Hymenoptera (Hymenoptera), apocrita (Apocrita), Formicoidea (Formicoidea), Formicidae (Formicidae).Ant species is various, and the whole world has 260 genus, about has 16, kind more than 000, and that has named at present has 8, kind more than 800.China estimates to reach 2, more than 000 kind, nearly 600 kinds that have wherein named.What Formica fusca was used as medicine is recited as supplement to the Herbal that CHEN Zang-Qi the is shown Tang Dynasty the earliest, wherein just has the description of " unipods ant cures mainly furuncle and to swell deep carbuncle, smash painting "; Formica fusca is also included as Chinese medicine by Ming Dynasty's Li Shizhen (1518-1593 A.D.) Compendium of Material Medica, Qing Dynasty's ZHAO Xue-Min supplementary Amplifications of the Compendium of Materia Medica etc., effects such as " lactogenic juice, damp colors " of thinking that ant larva has, and can close " strong medicine (i.e. tonic) ".The Formica fusca of current China clinical practice, less than 20 kinds, wherein have been named and conventional what be used as medicine is the entirety comprising mercerising brown woods ant, intend 10 kinds of nontoxic Formica fuscas of Formicidae animal such as black many wings ant.Cure mainly dizzy tinnitus of suffering from a deficiency of the kidney, insomnia and dreamful sleep, impotence and seminal emission, rheumatic arthralgia, apoplectic hemiplegia, numb hand and foot, lupus erythematosus, scleroderma, dermatomyositis, carbuncle furuncle, venom.Recent researches finds, Formica fusca has antiinflammatory, analgesia, raising immunity, reduces gastric acid secretion, pre-ulcer generation, epilepsy, resists and shy effects such as sticking up; Medical ant has the pharmacological action regulating human body hormonal system aspect, particularly all there is good therapeutic effect (" the development and utilization overview of China's medical ant " to rheumatoid, diabetes, cancer etc., Institutes Of Chifeng's journal (natural science edition), 2011,27 (7): 22-24).
Formica fusca, except being used as medicine, goes back edible, and it contains 70 multiple nutritional components of needed by human; Wherein rich in protein, accounts for 42% ~ 67%, and the adult of Formica fusca reaches 55% containing protein, and ovum can reach 67%; Crude protein content reaches more than 42%; Containing 26 kinds of free amino acids, add aminoacid totally 27 seed amino acids of proteolysis, the total amount of free amino acid can reach 1, and 868 milligrams/100 grams, hydrolysis amino acid reaches 3,994 milligrams/100 grams, wherein has 8 kinds to be the aminoacid of needed by human; Meanwhile, also containing 28 kinds of free fatties, wherein oleic acid 62.44% in Formica fusca body, Palmic acid 21.14%, palmitoleic acid 11.03%.Also contain multiple hormone in Formica fusca body, (Formica fusca body includes 19 kinds of enzymes and coenzyme to organized enzyme, as FAD, SOD etc., these materials are to the energy metabolism of body, the renewal of brain cell and improve cerebral anoxia situation and play an important role), high-energy phosphate compound and formaldehyde (mainly cursive script formaldehyde C
10h
16o
2, be terpenes derivant, have the function of good relaxing muscles and tendons to promote blood circulation, its nourishing ability can compare favourably with wild ginseng), formic acid (formic acid, some content is up to 20% of body weight, and concentration reaches 70%).In addition, also containing abundant vitamin in Formica fusca body, comparatively outstanding has vitamin A, C, D, E, B1, B2, B12, for human health provides abundant nutrient substance; In addition, according to surveying and determination, Formica fusca body includes 28 kinds of trace element, as: manganese, zinc, selenium, magnesium, calcium, phosphorus, chromium, ferrum, iodine, molybdenum, fluorine etc., wherein the content of zinc is the highest, therefore wind resistance damp disease (" progress of medical ant ", food and pharmaceutical evident in efficacy, 2006,8 (01A): 26-28).
As above-mentioned, the health due to Formica fusca is exactly a nutrition library and pharmaceutical factory, and therefore it is medicinal high with edibility.Leaf cutting ant (the Attini race in America is originated in scientist's research of the U.S., Apterostigmadentigerum), finding that the actinomycetes Pseudonocardiaspp. of this kind of Formica fusca health body surface symbiosis can produce can a kind of cyclic peptidal antibiotics Dentigerumycin(" Dentigerumycin:abacterialmediatorofanant-fungussymbiosis " of Antifungi Escovopsissp. and part candidiasis bacterial strain, NatChemBiol.2009,5 (6): 391-393).But, leaf cutting ant Apterostigmadentigerum is only distributed in the some areas such as America Costa Rica, and also do not find in other place with the actinomycetes Pseudonocardiaspp. of its symbiosis, therefore Dentigerumycin cannot prepare to develop further in a large number.Australia scientist then originate in Australia Bulldog ant (Bulldogant) Myrmecianigriscapa metapleura gland (metapleuralgland) secretions in find can anti-bacteria, conk metapleura parathyrine (metapleurins) (" Antsandantibiotics ", ECOS, 1989,61:27-28).But Bulldog ant (Myrmecianigriscapa and sibling species) is Australian extraordinary insecticide, and other countries and area do not distribute; And metapleurins be collected as stimulating living Bulldog ant, make its metapleura glandular secretion go out the antibiotic active substance of class, therefore, be difficult to meet a large amount of samples further needed for research and development.
The brown woods ant (FormicafuscaLinnaeus) of mercerising, also known as Black Hills ant, polyrhachis, ant (Yi nationality's medicine), refer to that Formicidae (Formicidae) ant belongs to (Formicaspp.) insecticide, be distributed in Asia, Europe, be widely distributed in various places within Chinese territory.Mercerising brown woods ant is the one in China nearly 20 kinds " Formica fuscas " of applying clinically, long 13 millimeter of body, black, level and smooth, glossy.Pronotary base is flourishing, and mesonotum is less, and handle abdomen has 1 piece of scale upwards.Matter is crisp, frangible, often has cephalopodium defect, and that licks has tart flavour.The present inventor is used as medicine to China the large quantity research Late Cambrian of Formica fusca: the brown woods ant of mercerising has antifungal activity, and the present inventor according to lot of documents investigation and related experiment, devises the preparation technology of mercerising brown woods ant antifungal effective site again.Look into new discovery through the present inventor, all carry out relevant report or the patent of the pharmaceutical preparation of preparing in the past without the mercerising identical with the present invention brown woods ant effective part extract as principal agent, more without using above-mentioned preparation for preventing and treating the bibliographical information of fungal infection disease.Antifungal result of the test finds, mercerising brown woods ant effective site has significant antifungal activity, completes the present invention thus.
Summary of the invention
The object of this invention is to provide a kind of mercerising brown woods ant effective part extract with antifungal activity.
Present invention also offers the preparation method of mercerising brown woods ant antifungal effective site, specifically adopt following steps:
A) extract mercerising brown woods ant polypide by the 75% alcoholic solution soaking at room temperature that weight ratio is 30-300 times amount, the soak extraction time is 10-24 hour, concentrating under reduced pressure recycling design, obtains mercerising brown woods ant crude extract; Polypide after lixiviate is dried naturally;
B) polypide of drying after step a soak extraction tissue refiner is ground, homogenate, polypide slurry is extracted by the 90% alcoholic solution soaking at room temperature that weight ratio is 30-300 times amount, the soak extraction time is 10-24 hour, concentrating under reduced pressure recycling design, obtains mercerising brown woods ant 90% ethanol extraction position; Polypide slurry residue after lixiviate dries naturally;
C) be the polypide slurry residue dried after soak extraction in 50% methanol solution heating extraction step b of 10-100 times amount by weight ratio, extraction time is 2-12 hour, concentrating under reduced pressure recycling design, obtains mercerising brown woods ant 50% methanol extraction position; Polypide residue after lixiviate dries naturally;
D) after the dissolution with solvents of mercerising step c obtained brown woods ant 50% methanol extraction position, ultrafiltration is carried out with the ultrafilter membrane that molecular cut off (MWCO) is 5 kilodaltons (5KDa), collect the permeate through ultrafilter membrane, after concentrating under reduced pressure, dry, pulverize to obtain product.
Wherein, temperature during concentrating under reduced pressure is no more than 80 DEG C; In step c, the temperature of heating extraction is 60 DEG C-80 DEG C; The preferred vacuum decompression drying of drying means or lyophilization.
Another object of the present invention is to provide mercerising brown woods ant antifungal effective site and is preparing the application in prevention and therapy antifungal medicine; Application preferably in the medicine preparing prevention and therapy monilial infection disease.
Another object of the present invention is to provide the drug excipient that allows containing mercerising brown woods ant antifungal effective site and preparation or carrier is prepared into pharmaceutical composition, and prepared pharmaceutical composition can also contain other antibacteriums and/or antifungal drug.Described medicine can make multiple dosage form by means known in the art, comprise tablet, granule, capsule, oral liquid, drop pill, varnish, externally-applied liniment, membrane, unguentum, spray, injection, transdermal patch, aerosol, controlled release or slow releasing agent, and nanometer formulation.
Through the Literature Consult that the present inventor is detailed, up to the present, there is no about mercerising brown woods ant antifungal effective site is for the preparation of the report of antifungal drug.Mercerising brown woods ant antifungal effective site belongs to beyond thought discovery for the potent suppression of conk, has definite originality, completes the present invention accordingly.
Usefulness of the present invention is: Late Cambrian mercerising brown woods ant antifungal effective site has Antifungi growth, prepares the patent medicine potentiality of anti-fungal infection aspect, provides new material base for exploitation becomes treatment fungal infection original new drug.There is potential huge Social benefit and economic benefit.A present invention again feature is: the Medical insect resources of the present invention enriches, and extraction preparation method is simple, raw material sources are conveniently easy to get, cost is low, it is little to pollute, and are beneficial to the large-scale production under energy-saving and emission-reduction condition, and industrialization prospect is very clear and definite.
embodiment 1: one of preparation process of mercerising brown woods ant antifungal effective site
The preparation of 1.1 mercerising brown woods ant extract
The present embodiment crude drug used, purchased from Baoshan, Yunnan city Chinese Medicinal Materials Markets, is accredited as the brown dried forest ant powder product of mercerising through the college of traditional Chinese medicine of Yunnan Province professor Zhao Ronghua.
1.1.1 get the brown woods ant of dry mercerising of 400.0 milligrams, to be soaked in 75% alcoholic solution of 60 milliliters 18 hours under room temperature, to leach soak, concentrating under reduced pressure, recycling design, obtain mercerising brown woods ant crude extract (A0) 43.2 milligrams.Polypide after lixiviate is dried naturally.
1.1.2 the polypide of drying after soak extraction under 1.1.1 item tissue refiner is ground, homogenate, and with 40 milliliters 90% alcoholic solution soaking at room temperature extract polypide slurry, extraction time is 18 hours; Leach solution, through concentrating under reduced pressure recycling design, obtain mercerising brown woods ant 90% ethanol extraction position (A1) 28.7 milligrams; Polypide slurry residue after lixiviate dries naturally.
1.1.3 with the polypide slurry residue dried after soak extraction under the 50% methanol solution heating extraction 1.1.2 item of 20 milliliters, extraction time is 6 hours, Extracting temperature is 70 DEG C, concentrating under reduced pressure recycling design, obtains mercerising brown woods ant 50% methanol extraction position (A2) 42.5 milligrams.
The preparation of 1.2 mercerising brown woods ant antifungal effective site (A3)
Get the 30.0 milligrams of mercerising prepared by the 1.1.3 of embodiment 1 brown woods ant 50% methanol extraction positions, after methanol aqueous solution concussion with 70% is dissolved, be the ultrafiltration membrance filter of 5KDa with MWCO, collect the permeate through ultrafilter membrane, concentrating under reduced pressure postlyophilization, weigh after pulverizing, obtain mercerising brown woods ant antifungal effective site (A3) 3.3 milligrams.
embodiment 2: the preparation process two of mercerising brown woods ant antifungal effective site
The preparation of 2.1 mercerising brown woods ant extract
The present embodiment crude drug used, purchased from Baoshan, Yunnan city Chinese Medicinal Materials Markets, is accredited as the brown dried forest ant powder product of mercerising through the college of traditional Chinese medicine of Yunnan Province professor Zhao Ronghua.
2.1.1 get the brown woods ant of dry mercerising of 400.0 milligrams, to be soaked in 75% alcoholic solution of 80 milliliters 24 hours under room temperature, to leach soak, concentrating under reduced pressure, recycling design, obtain mercerising brown woods ant crude extract (B0) 37.5 milligrams.Polypide after lixiviate is dried naturally.
2.1.2 the polypide of drying after soak extraction under 2.1.1 item tissue refiner is ground, homogenate, and with 20 milliliters 90% alcoholic solution soaking at room temperature extract polypide slurry, extraction time is 12 hours; Leach solution, through concentrating under reduced pressure recycling design, obtain mercerising brown woods ant 90% ethanol extraction position (B1) 24.1 milligrams; Polypide slurry residue after lixiviate dries naturally.
2.1.3 with the polypide slurry residue dried after soak extraction under the 50% methanol solution heating extraction 2.1.2 item of 10 milliliters, extraction time is 10 hours, Extracting temperature is 65 DEG C, concentrating under reduced pressure recycling design, obtains mercerising brown woods ant 50% methanol extraction position (B2) 38.0 milligrams.
The preparation of 2.2 mercerising brown woods ant antifungal effective site
Get the 30.0 milligrams of mercerising prepared by the 2.1.3 of embodiment 2 brown woods ant lixiviate positions, after alcoholic solution concussion with 90% is dissolved, be the ultrafiltration membrance filter of 5KDa with MWCO, collect the permeate through ultrafilter membrane, concentrating under reduced pressure postlyophilization, weigh after pulverizing, obtain mercerising brown woods ant antifungal effective site (B3) 2.9 milligrams.
embodiment 3: mercerising brown woods ant antifungal effective site Antifungi Activity determination
With reference to the standardization antifungal sensitivity testing method that American National Clinical Laboratory Standard committee (NCCLS) proposes, the extracorporeal antifungal activity of each extract part of mercerising brown woods ant that testing example 1,2 prepares.
3.1 fungus type strains:
Candida albicans ATCC76615(CS3): the No2. University of Medical Sciences of PLA provides.
Candida albicans ATCC90029(5-FC Resistant strain): Beijing Hospital's Ministry of Public Health Clinical Laboratory center provides.
Candida albicans 98001: Wuhan DSMZ provides.
Candida albicans Y0119: Shanghai City Long March hospital provides.
3.2 reagent:
3.2.1 sabouraud's agar (sabourauddextroseagar): the triumphant microorganism Science and Technology Ltd. in Guangdong product.
3.2.2 yeast extract (yeastextract): Hai Shenggong biotechnology Services Co., Ltd subpackage.
3.2.3 three morpholino nitrogen quinolines propane sulfonic acid (3-N-morpholinopropanesulfonicacid, MOPS)
3.2.4 standard anti-fungal injection: fluconazol (Fluconazole, FCZ), company of Yangzijiang Pharmaceutical Group.Be configured to the doubling dilution liquid of variable concentrations gradient.
3.2.5 dimethyl sulfoxide (dimethylsulfoxide, DMSO) and dimethyl formamide (dimethylformamide, DMF): Shanghai Sangon Biological Engineering Technology And Service Co., Ltd's subpackage product.
3.3 instruments:
3.3.1 electronic balance JA1203N(AB204-5, METTLERTOLEDO): Shanghai precision scientific instrument Products.
3.3.2SW-CJ-2FD the double one side clean work station of type: Purifying Equipment Co., Ltd., Suzhou's product.
3.3.3 water isolation type constant incubator (GSP-9160MBE): Shanghai Yiheng Scientific Instruments Co., Ltd's product.
3.3.4 electro-heating standing-temperature cultivator (HZQ-F160A): Shanghai Yiheng Scientific Instruments Co., Ltd's product.
3.3.5 U.S. Pedicellus et Pericarpium Trapae electric refrigerator (BCD-221CHC): Hefei Meiling Co. Ltd.'s product.
3.3.6 micropipettor (ProlinePipette, DragonMedPipette): the vigorous Products of Finland's thunder.
3.3.7 cell counting count board (96wellcellculturecluster): Shanghai refinement instrument company product.
3.3.8 ordinary optical microscope (Olympus): Japanese Olympus optical instrument Co., Ltd. product.
3.3.9 hot air oven (101A-2): Shanghai Chongming experimental apparatus Products.
3.3.10 vertical autoclave sterilizer (YXQ-LS-50S II): Shanghai Bo Xun Industrial Co., Ltd. product.
3.3.11 magnetic force heating stirrer (ARE): Italian VELP Products.
3.3.12 filter membrane, filter (0.22 μm, SARTORIUS): Sartorius AG's product.
3.3.13 acidometer (PHSJ-4A): upper Nereid section thunder magnetic Products.
3.3.14 test tube oscillator (MS2): Guangzhou instrument section laboratory technique Products.
3.3.15 constant-temperature shaking incubator (THZ-18AB): the permanent Scientific Instruments Corporation in Shanghai one product.
3.3.16 cryogenic refrigerator (-20 DEG C, section dragon BCD-219WAK): Guangdong Ke Long electrical equipment Co., Ltd product.
3.3.1796 hole flat-bottom microplates: U.S. CorningIncorporated product.
3.4 experimental techniques:
3.4.1 experimental procedure:
3.4.1.1RPMI-1640 the preparation of liquid: get 10.4 grams of RPMI-1640 powder (containing L glutamine, not containing sodium bicarbonate, GIBCO) be dissolved in 900 ml sterile waters, adding 34.53 gram of three morpholino nitrogen quinoline propane sulfonic acid (MOPS) is 0.165 mol/L to its endpoint concentration, with magnetic stirring apparatus mixing 2-3 hour under room temperature, makes it fully dissolve, by sodium hydroxide (1 mol/L) adjust ph to 7.0(25 DEG C), be settled to 1 liter with aquesterilisa, Entkeimung, after subpackage ,-20 DEG C save backup.
3.4.1.2 the preparation of storing solution: Stock concentrations should be 10 times of application liquid, 5-FC(5-fluorouracil) and FCZ(fluconazol) be 1,280 mcg/ml, Amb(amphotericin B) and KETO(ketoconazole) be 320 mcg/ml, each 10 milligrams of 5-FC, FCZ, KETO and Amb is taken respectively with electronic balance, 5-FC 1 milliliter of distilled water dissolves, FCZ dissolves with 1 milliliter of dimethyl formamide (DMF), Amb and KETO uses dimethyl sulfoxine (DMSO) to dissolve respectively, be diluted to storage liquid concentration with RPMI-1640 afterwards, after subpackage ,-70 DEG C save backup.
3.4.1.3 the preparation of liquid is applied: remake multiple proportions after storing solution being done 1:10 dilution with RPMI-1640 liquid rare, FCZ, 5-FC and Amb are that 128 mcg/ml-0.25 mcg/ml make 10 serial concentration, KETO is 32 mcg/ml-0.06 mcg/ml, 10 series concentration, is 2 times of final concentrations.
3.4.1.4 the preparation of micro-susceptibility culture plate: use disposable 96 orifice plates, 1-10 row add the application liquid of the testing drug of 10 Concentraton gradient respectively, from high concentration to low concentration.11st row add RPMI-1640, every hole 100 microlitre, and the 12nd row, as blank, put into-20 DEG C of refrigerators for subsequent use, through-4 DEG C, 4 DEG C and each 1 hour of room temperature during use.
3.4.1.5 the activation of candidiasis and dilution: by bacterial strain to be measured at YPD Agar culture medium (1% yeast extract, 1% peptone, 2% glucose) after upper activation twice, the bacterium colony that cut-off footpath is greater than 1 millimeter a little in 3 milliliters of physiological saline solution mixing make bacteria suspension, get a little, count the bacterial cell number in four block plaid with hemocyte technology plate, average X, and now bacterial cell number is X × 10
4cFU/ milliliter is 3 × 10 by RPMI-1640 liquid adjustment concentration
4cFU/ milliliter (twice final concentration).
3.4.1.6 application of sample: add candidiasis suspension on the above-mentioned culture plate containing testing drug prepared, every hole 100 microlitre blank does not add, and puts into 35 DEG C, wet box (prevent evaporating of micro plate and affect the concentration of medicine) and hatch observed result after 24-48 hour after concussion mixing.
3.4.1.7 naked eyes judged result: blank for foundation with growth control, suppresses (i.e. MIC with 80%
80) for observing terminal, growth obviously reduces, liquid little cloudy; Well-grown in Growth positive simultaneously, blank is limpid, without bacterial growth.The MIC value of Quality-control strains is in the scope of U.S. clinical Microbiological Lab standard C LSIM27-A2 scheme prescribes, and display experimental result is effective.
3.5 experimental results:
3.5.1 Quality Control strain is to the sensitivity of antifungal drug: four kinds of positive drugs meet the requirement of CLSIM27-A2 scheme for Quality Control strain type strain to Quality Control strains A TCC90029 and 98001 type strain drug sensitivity tests.That is: the MIC value change repeating to carry out for more than 3 times AmB, 5-FC, FCZ and KETO is no more than 2 Concentraton gradient.
3.5.2 the antifungal activity of positive control and testing drug sample the results are shown in Table 1.
Table 1 testing drug is to the inhibitory action of Candida albicans
3.6 conclusions:
By the vitro inhibition conk activity adopting " Herbs By Broth Microdilution " to study mercerising brown woods ant antifungal effective site, experimental study shows: ant belongs to mercerising brown woods ant antifungal effective site clear and definite anti-Candida albicans activity.Illustrate that it is potential Antifungi growth activity material, there is further Development volue.
The ant prepared in the present invention belongs to insecticide mercerising brown woods ant antifungal effective site and can be combined with pharmaceutically conventional adjuvant or carrier, prepares and has prevention and therapy by the medicine of the fungus-caused infection such as Candida albicans and pharmaceutical composition or health product or cosmetics of everyday use.The dosage form that above-mentioned various kinds of drug compositions, health product or cosmetics of everyday use can adopt comprises tablet, granule, capsule, oral liquid, drop pill, varnish, externally-applied liniment, membrane, unguentum, spray, injection, transdermal patch, aerosol, controlled release or slow releasing agent, and nanometer formulation.
Ant of the present invention belongs to insecticide mercerising brown woods ant antifungal effective site can also infect the medicine of associated conditions and crude drug thereof as polyenoid class (as amphotericin B) with the treatment fungus bacterium now gone on the market, triazole type is (as fluconazol, miconazole, econazole, Itraconazole and ketoconazole etc.), the kind conbined usage such as the echinocandin (echinocandins) of alkyl amine (as terbinafine) and listing newly developed, prepare the compositions or compound preparation with treatment fungal infection associated conditions effect activity, can expect and become treatment fungal infection disease medicine/health product or cosmetics of everyday use.Above-mentioned various kinds of drug compositions, medicine/health product or cosmetics of everyday use can adopt the dosage forms such as tablet, granule, capsule, oral liquid, drop pill, varnish, membrane, unguentum, liniment, injection, transdermal patch, aerosol or spray, comprise the preparation of pharmaceutics general knowledge routine and the various slow release, controlled release form or the nanometer formulation that obtain that employing now generally acknowledged.
When above-mentioned description is set forth of the present invention, the object simultaneously providing embodiment illustrates actual mechanical process of the present invention and meaning of the present invention.When entering in the claims in the present invention and its equivalency range, practical application of the present invention comprises all general changes, cooperation, or improves.
Claims (5)
1. prepare a method for mercerising brown woods ant antifungal effective site, it is characterized in that:
A) extract mercerising brown woods ant polypide by the 75% alcoholic solution soaking at room temperature that weight ratio is 30-300 times amount, the soak extraction time is 10-24 hour, concentrating under reduced pressure recycling design, obtains mercerising brown woods ant crude extract; Polypide after lixiviate is dried naturally;
B) polypide of drying after step a soak extraction tissue refiner is ground, homogenate, polypide slurry is extracted by the 90% alcoholic solution soaking at room temperature that weight ratio is 30-300 times amount, the soak extraction time is 10-24 hour, concentrating under reduced pressure recycling design, obtains mercerising brown woods ant 90% ethanol extraction position; Polypide slurry residue after lixiviate dries naturally;
C) be the polypide slurry residue dried after soak extraction in 50% methanol solution heating extraction step b of 10-100 times amount by weight ratio, extraction time is 2-12 hour, concentrating under reduced pressure recycling design, obtains mercerising brown woods ant 50% methanol extraction position;
D), after the dissolution with solvents of mercerising step c obtained brown woods ant 50% methanol extraction position, carry out ultrafiltration with the ultrafilter membrane that molecular cut off is 5KDa, collect the permeate through ultrafilter membrane, after concentrating under reduced pressure, dry, pulverize to obtain product.
2. method according to claim 1, is characterized in that: temperature during concentrating under reduced pressure is no more than 80 DEG C; In step c, the temperature of heating extraction is 60 DEG C-80 DEG C; Drying refers to vacuum decompression drying or lyophilization.
3. the mercerising brown woods ant antifungal effective site that method prepares according to claim 1 prevents and treats the application in fungal infection medicine in preparation.
4. there is a pharmaceutical composition for anti-fungal infection, it is characterized in that: mercerising brown woods ant antifungal effective site, pharmaceutic adjuvant and pharmaceutical carrier that the method according to claim 1 that this pharmaceutical composition contains treatment effective dose prepares.
5. pharmaceutical composition according to claim 4, its dosage form is tablet, granule, capsule, oral liquid, drop pill, varnish, externally-applied liniment, membrane, unguentum, spray, injection, transdermal patch or aerosol.
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| CN1091312A (en) * | 1993-12-01 | 1994-08-31 | 吉林市康富制药有限公司 | Queen ant extrat oral liquid and preparation method thereof |
| CN101322752A (en) * | 2007-06-12 | 2008-12-17 | 杨思齐 | Spray using termite as principal raw material for treating fungal dermatopathy |
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