CN103520187B - The application of ginkgoic acid in anti-Cryptosporidium - Google Patents
The application of ginkgoic acid in anti-Cryptosporidium Download PDFInfo
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Abstract
The present invention relates to the application of ginkgoic acid in anti-Cryptosporidium.Particularly, the invention discloses and a kind ofly comprise the pharmaceutical composition of monomer C13:0, C15:1 and C17:2 and the application in anti-Cryptosporidium spp medicine thereof, monomer C13:0, C15:1 and C17:2 structure is as defined in description.
Description
Technical field
The invention belongs to field of biological pharmacy, be specifically related to the application of a kind of ginkgoic acid in anti-Cryptosporidium.
Background technology
Cryptosporidium is that a kind of infecting both domestic animals and human newly sends out parasite, is to cause the mankind to suffer from diarrhoea one of six large pathogen.Cause extensive outbreak of epidemic in multiple developed countries such as America and Europes at present.The popular worm strain in different regions, different Susceptible population of this disease is different, shows as gene pleiomorphism.At present still using microscopy in feces to Cryptosporidium parvum oocysts suspended as diagnosis goldstandard.
So far, Cryptosporidium has 26 effective worm kinds, more than 60 genotype; That infects people has 13 kinds, comprises people Cryptosporidium, Cryptosporidum parvum, Cryptosporidium meleagridis, cryptosporidium andersoni, dog Cryptosporidium, cat Cryptosporidium, rabbit Cryptosporidium, Cryptosporidium muris etc.Infect mainly Cryptosporidum parvum and the people Cryptosporidium of people, seminar's research finds, cryptosporidium andersoni may be the worm kind of the fourth-largest infection people after people Cryptosporidium, Cryptosporidum parvum, rabbit Cryptosporidium.Age and the immune state of the clinical symptoms after human infection Cryptosporidium and polypide kind, quantity and patient have in close relations, and particularly the HIV positive/AIDS patient and underfed child, can cause choleraic diarrhea serious for a long time, even dead.But cryptosporidiosis is so far still without effective medicine and vaccine prevention.
Nitazoxanide (NTZ) is used for the treatment of the normal child of immunologic function and adult as the appointment medicine of U.S. FDA, but invalid to immunodeficiency patient.There is in the experiment of NTZ cell culture and the animal model such as newborn mice, aseptic porkling in vitro the activity of appropriate anti-Cryptosporidium.For some immunodeficiency patient, the tumor patient as organ transplantation or experience chemotherapy can not use this medicine due to its serious side effect.HAART partly can reduce AIDS patient infection Cryptosporidium.
Therefore, Cryptosporidium constant threat and lack effective medicine and all pointed out the effective medicine of searching to be of great immediate significance.
Summary of the invention:
An object of the present invention is to provide the pharmaceutical composition infected for anti-cryptosporidium andersoni.
In a first aspect of the present invention, provide a kind of pharmaceutical composition, containing active component: monomer C13:0, C15:1 and C17:2:
And described pharmaceutical composition is not containing monomer C15:0 and C17:1:
In another preference, described pharmaceutical composition is also containing pharmaceutically acceptable carrier.
In another preference, the application concentration of described pharmaceutical composition is 5 μ g/ml ~ 0.05 μ g/ml; Preferably, be 3.125 μ g/ml ~ 0.5 μ g/ml; More preferably, be 3.125 μ g/ml ~ 0.78 μ g/ml.
In another preference, in described pharmaceutical composition, C13:0, C15:1 and C17:2 are with the mixing of any content ratio.
In another preference, in described pharmaceutical composition, the content of C13:0, C15:1 and C17:2 is than being 1-10:20-40:1.
In another preference, in described pharmaceutical composition, the content of C13:0, C15:1 and C17:2 is than being 4-8:25-36:1; Preferably, the content of C13:0, C15:1 and C17:2 is than being 5.5-6.5:28-33:1.
A kind of pharmaceutical composition is provided, containing component in second aspect present invention:
(a) active component: monomer C13:0, C15:1 and C17:2:
(b) monomer C15:0 and C17:1:
And in described pharmaceutical composition, the weight ratio of component (a) and component (b) is 0.8-1.2:1.2-0.8.
In another preference, described pharmaceutical composition is also containing pharmaceutically acceptable carrier.
In another preference, the weight ratio of component (a) and component (b) is 0.9-1.1:1.1-0.9; Preferably, the weight ratio of component (a) and component (b) is 1:1.
In another preference, in described pharmaceutical composition,
In composition (a), the content (as wt% or HPLC content) of C13:0, C15:1 and C17:2 is than being 1-10:20-40:1; And/or
In composition (b), the content (as wt% or HPLC content) of C15:0 and C17:1 is than being 1:1-20.
In another preference, in composition (a), the content of C13:0, C15:1 and C17:2 is than being 4-8:25-36:1; Preferably, the content of C13:0, C15:1 and C17:2 is than being 5.5-6.5:28-33:1.
In another preference, in composition (b), the content of C15:0 and C17:1 is than being 1:5-15; More preferably, the content of C15:0 and C17:1 is than being 1:8-12.
In another preference, the application concentration of described pharmaceutical composition is 0.05 μ g/ml ~ 0.9 μ g/ml; Preferably, the application concentration of described pharmaceutical composition is 0.05 μ g/ml ~ 0.78 μ g/ml.
In third aspect present invention, provide a kind of purposes of pharmaceutical composition as described in first aspect present invention or second aspect, for the preparation of the medicine that anti-cryptosporidium andersoni infects; And/or infect for anti-cryptosporidium andersoni.
In fourth aspect present invention, provide a kind of method of vitro inhibition cryptosporidium andersoni, comprise step: have in the cell culture system of cryptosporidium andersoni in infection, the pharmaceutical composition added as described in first aspect present invention or second aspect is cultivated, thus suppresses the propagation of cryptosporidium andersoni.
In another preference, described cell is HCT-8.
In another preference, when adding pharmaceutical composition as claimed in claim 1, its application concentration is that 5 μ g/ml ~ 0.05 μ g/ml (preferably, are 3.125 μ g/ml ~ 0.5 μ g/ml; More preferably, be 3.125 μ g/ml ~ 0.78 μ g/ml); Maybe when adding pharmaceutical composition as claimed in claim 4, its application concentration 0.05 μ g/ml ~ 0.9 μ g/ml (preferably, being 0.05 μ g/ml ~ 0.78 μ g/ml).
Should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and can combining mutually between specifically described each technical characteristic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tiredly no longer one by one to state at this.
Accompanying drawing explanation
Fig. 1 obtains the electrophoretogram of COWP genetic fragment under showing different annealing temperatures.
Fig. 2 shows COWP gene fragment clone to the electrophoretogram on plasmid.
Fig. 3 shows has the plasmid of COWP fragment as the standard curve constructed by real-timePCR standard substance using the clone of embodiment 1 structure.
Fig. 4 shows the proliferative effect of medicine variable concentrations to HCT-8 cell.
Fig. 5 shows different pharmaceutical external multiplication effect affecting cryptosporidium andersoni under variable concentrations.
Detailed description of the invention
The present inventor, through extensive and deep research, finds that the combination of part ginkgoic acid monomer can effectively anti-Cryptosporidium spp.And the combination of different ginkgoic acid monomer must use the effect of the good anti-Cryptosporidium spp of competence exertion under variable concentrations.On this basis, inventor completes the present invention.
Ginkgoic acid monomer
Pharmaceutical composition
Pharmaceutical composition of the present invention has excellent anti-cryptosporidium andersoni infection activity.On this basis, the invention provides the pharmaceutical composition of effective anti-cryptosporidium andersoni.
In a preference, pharmaceutical composition of the present invention comprises: the active component in the safe and effective weight range of component (i): monomer C13:0, C15:1 and C17:2; And component (ii) pharmaceutically acceptable carrier; And described pharmaceutical composition is not containing monomer C15:0 and C17:1.
In another preference, pharmaceutical composition of the present invention comprises: component (a) active component in safe and effective weight range: monomer C13:0, C15:1 and C17:2 and component (b) monomer C15:0 and C17:1; And the pharmaceutically acceptable carrier of component (c).
Wherein, " safe and effective amount " refers to: the amount of active component is enough to obviously improve the state of an illness, and is unlikely to produce serious side effect.Usually, pharmaceutical composition contains 1-2000mg active component/agent, more preferably, containing 10-200mg active component/agent.Preferably, described " potion " is a capsule or tablet.
Wherein, " pharmaceutically acceptable carrier " refers to: one or more biocompatible solid or liquid filler or gelatinous mass, and they are suitable for people and use, and must have enough purity and enough low toxicity." compatibility " referred to herein as in compositions and mutually admixes between each component, and the drug effect of not obvious reduction active component.Pharmaceutically acceptable carrier part example have cellulose and its derivates (as sodium carboxymethyl cellulose, ethyl cellulose sodium, cellulose ethanoate etc.), gelatin, Talcum, kollag (as stearic acid, magnesium stearate), calcium sulfate, vegetable oil (as Oleum Glycines, Oleum sesami, Oleum Arachidis hypogaeae semen, Fructus Canarii albi wet goods), polyhydric alcohol (as propylene glycol, glycerol, mannitol, sorbitol etc.), emulsifying agent (as
), wetting agent (as sodium lauryl sulphate), coloring agent, flavoring agent, stabilizing agent, antioxidant, antiseptic, apirogen water etc.
The method of application of pharmaceutical composition of the present invention is not particularly limited, and representational method of application comprises (but being not limited to): oral, parenteral (intravenous, intramuscular or subcutaneous) etc.
Solid dosage forms for oral administration comprises capsule, tablet, pill, powder and granule.In these solid dosage formss, active component mixes with at least one conventional inert excipients (or carrier), as sodium citrate or dicalcium phosphate, or mix with following compositions: (a) filler or solubilizing agent, such as, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binding agent, such as, hydroxy methocel, alginate, gelatin, polyvinyl pyrrolidone, sucrose and arabic gum; (c) wetting agent, such as, glycerol; (d) disintegrating agent, such as, agar, calcium carbonate, potato starch or tapioca, alginic acid, some composition silicate and sodium carbonate; (e) retarding solvent, such as paraffin; F () absorbs accelerator, such as, and quaternary ammonium compound; (g) wetting agent, such as spermol and glyceryl monostearate; (h) adsorbent, such as, Kaolin; (i) lubricant, such as, Talcum, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulphate, or its mixture.In capsule, tablet and pill, dosage form also can comprise buffer agent.
Solid dosage forms such as tablet, sugar pill, capsule, pill and granule can adopt coating and the preparation of shell material, as casing and other material well known in the art.They can comprise opacifying agent, and in this compositions, the release of active component can discharge in certain part in a delayed fashion in digestive tract.The example of adoptable embedding component is polymeric material and Wax.If desired, active component also can form microencapsulation form with one or more in above-mentioned excipient.
Liquid dosage form for oral administration comprises pharmaceutically acceptable emulsion, solution, suspension, syrup or tincture.Except active component, liquid dosage form can comprise the conventional inert diluent adopted in this area, as water or other solvent, solubilizing agent and emulsifying agent, example is known, the mixture etc. of ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3 butylene glycol, dimethyl formamide and oil, particularly Oleum Gossypii semen, Oleum Arachidis hypogaeae semen, maize embryo oil, olive oil, Oleum Ricini and Oleum sesami or these materials.
Compositions for parenteral injection can comprise physiologically acceptable sterilized water or anhydrous solution, dispersion liquid, suspension or emulsion, and for being again dissolved into aseptic Injectable solution or the sterilized powder of dispersion liquid.Suitable moisture and nonaqueous carrier, diluent, solvent or excipient comprise water, ethanol, polyhydric alcohol and suitable mixture thereof.
Inventive compound can be individually dosed, or with other pharmaceutically acceptable compound administering drug combinations.
When making pharmaceutical composition, it is the mammal (as people) being applicable to the inventive compound of safe and effective amount need treatment, when wherein using, dosage is the effective dosage pharmaceutically thought, for the people of 60kg body weight, day dosage is generally 1 ~ 2000mg, preferably 20 ~ 500mg.Certainly, concrete dosage also should consider the factor such as route of administration, patient health situation, and these are all within skilled practitioners skill.
The present invention mainly has the following advantages:
(1) provide a kind of pharmaceutical composition only containing three kinds of ginkgoic acid monomers and the pharmaceutical composition of five kinds of ginkgoic acid monomers that exists with special ratios, two kinds of compositionss have excellent anti-Cryptosporidium spp effect under specific application concentration.
(2) a kind of method of In Vitro Anti Cryptosporidium spp is additionally provided.
Below in conjunction with concrete enforcement, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition, the people such as such as Sambrook, molecular cloning: laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise percentage ratio and number calculate by weight.
Embodiment 1: build iS-One hidden spore COWP gene plasmid
1 reagent
RPMI1640 is purchased from HycloneInc., USA.;
Trypsin is purchased from GIBCO, USA;
DMSO (DMSO) is purchased from AmrescoInc., USA.;
Mycillin (Penicillin – Streptomycin) is purchased from Gibco, USA;
Cell counting Kit (CCK-8) is purchased from the DojindoMolecularTechnologies of Japan;
People's colon cell (HCT-8 cell) is purchased from Chinese Academy of Sciences's Shanghai cell biological institute;
DNA extraction agent box is purchased from Shanghai Jierui Biology Engineering Co., Ltd;
DNA glue reclaims test kit purchased from vast Tyke, Beijing;
Sodium taurocholate (Sodiumtaurocholate) is purchased from Sigma.
2 experiment Cryptosporidium Species
Cattle source Cryptosporidium parvum oocysts suspended picks up from cow manure, is stored in 2.5% potassium dichromate (K after 60 orders sieve
2cr
2o
7) in conserving liquid, 4 DEG C for subsequent use.
3 experimental techniques
3.1 egg capsules are purified
With reference to saturated sucrose floating poly-method (Arrowood, M.J., Sterling, C.R., 1987.JParasitol73 (2), 314-319.) purification cattle source Cryptosporidium parvum oocysts suspended.Experimental procedure is as follows:
From 2.5%K
2cr
2o
7draw fecal suspension 10ml in conserving liquid, wash 2 times (the centrifugal 10min of 1500g) with the distilled water of 3 times of volumes, then make fecal suspension with 2 times of volume distilled waters, purification egg capsule.In 50ml centrifuge tube, add saturated sucrose solution 30ml, slowly drip feces re-suspension liquid 10ml thereon, 4 DEG C of centrifugal 20min of 1500g, adsorption surface liquid 5ml, add 3 times amount distilled waters and wash 2 times, the centrifugal 10min of 1500g, precipitation is used for egg capsule Genomic DNA Purification.
The asepticize process of 3.2 egg capsules
From 2.5%K
2cr
2o
7the cryptosporidium andersoni egg capsule 2x10 of purification is drawn in conserving liquid
5individual egg capsule, washs 2 times to remove K with distilled water
2cr
2o
7, by resuspended to 900 μ l with sterilizing distilled water for the egg capsule after washing in 1.5mlEP pipe, after being placed in cooled on ice, carry out disinfecting action process.In egg capsule suspension, add 1.05% liquor natrii hypochloritis 100 μ l of pre-cooling on ice during sterilizing, thermal agitation is placed on 5min on ice, washes twice with sterile PBS buffer, resuspended to 500 μ l.
3.3 cell culture
Human colon cancer cell (HCT-8) is incubated in the RPMI-1640 culture fluid containing 5% hyclone (FCS), is placed in 37 DEG C of 5%CO
2grow in cell culture incubator.With cell dissociation buffer digestion after at the bottom of cell covers with bottle, 1:5 sub-bottle transferred species is in new culture bottle.
3.4 asepticize cryptosporidium andersoni egg capsule skin lesion of the scrotums
Reference literature (Zhu, G., Marchewka, M.J., Keithly, J.S., 1999.FEMSMicrobiolLett176 (2), 367-372), in 500 aseptic μ l egg capsule suspensions, adding 500 μ l2 × egg capsule skin lesion of the scrotum liquid, (skin lesion of the scrotum liquid is prepared: 50mg pancreatin and 150mg cattle NaTDC, be dissolved in 10mlPBS, 0.22 μm of membrane filtration is degerming, be 2 × egg capsule skin lesion of the scrotum liquid (containing 0.5% pancreatin and 1.5% N of NaTDC), now join), be placed in 37 DEG C of water-bath skin lesion of the scrotum 40min.2 times are washed with sterile PBS buffer after skin lesion of the scrotum, resuspended to 1ml with the RPMI-1640 basic culture solution containing 10%FCS, for HCT-8 cell infection.
3.5 cell infection
The RPMI-1640 culture fluid of HCT-8 cell containing 5% hyclone (FCS) is cultivated in 6 orifice plates, is placed in 37 DEG C of 5%CO
248h is grown in cell culture incubator.After changing liquid, according to 1 × 10
3quantity add skin lesion of the scrotum egg capsule continue cultivate 8h, change complete medium and hatch 48h.Collect culture fluid after peptic cell, be placed in 50ml centrifuge tube, for DNA extraction.
3.6 plasmid construction
48h after HCT-8 cell infection egg capsule, collects the cell infected.Proteinase-K pathway cell lysis is adopted to obtain DNA.
Primer is designed according to Cryptosporidium COWP gene order (GenBankno.DQ989570) in GenBank:
Forward primer (F): 5 '-CACCTCCCAACCCTGAATGTC-3 ' (SEQIDNO.:1)
Reverse primer (R): 5 '-TGCTGGCAAATACTGGA-3 ' (SEQIDNO.:2)
Adopt rTaq (TaKaRa, precious biological purchased from Dalian) to increase, product length is 134bp.
Pcr amplification condition: 95 DEG C, 30s; 95 DEG C, 10s, 52 DEG C, 20s, 72 DEG C, 30s, 35 circulations; 72 DEG C extend 7min.
Adopt cryptosporidium andersoni positive sample as plasmid construction template: Niu Yuan, laboratory gathers voluntarily and is accredited as the positive.
The electrophoretogram of the OWP genetic fragment obtained under different annealing temperature as shown in Figure 1.
Wherein, M represents molecular weight standard, swimming lane 1,6 represents 52.7 DEG C, swimming lane 2,7 represents 52.1 DEG C, swimming lane 3,8 represents 51.1 DEG C, swimming lane 4,9 represents 50.0 DEG C, and swimming lane 5,10 represents 49.0 DEG C, the COWP genetic fragment that swimming lane 1-10 obtains under being expressed as the different annealing temperature of cryptosporidium andersoni positive sample.
Result shows: the cryptosporidium andersoni positive sample that selective response specificity is high for we, object band is brighter, as the standard substance of subsequent experimental, adopts 51.1 DEG C of annealing temperatures as PCR.
Reclaiming through cutting glue, to be cloned on PGEMT-TEasy (purchased from Progma, USA) carrier and to be transformed into bacillus coli DH 5 alpha (purchased from vast Tyke, Beijing).Spend the night, from flat board, choose monoclonal bacterium, shake bacterium and take out plasmid.
The positive plasmid obtaining COWP is proved through order-checking.Electrophoretogram after COWP gene clone to plasmid as shown in Figure 2.Wherein, M represents molecular weight marker, and swimming lane 1-4 is expressed as the positive colony 1-4 of institute's picking.After order-checking, be correct COWP sequence.
There is the plasmid of COWP fragment as real-timePCR standard substance using the clone built, build standard curve (as shown in Figure 3).
The PCR primer that in Fig. 3, A figure obtains under showing the variable concentrations that the positive plasmid that builds carries out.Wherein, swimming lane 1-7 respectively indicated concentration be 10
7-10
1copy number/μ l, swimming lane 8 represents blank, and swimming lane 9 is molecular weight standard.
In Fig. 3, B figure shows the linear equation of standard substance.
Result illustrates: this linear equation may be used for the degree of the copy number reaction vitro detection cryptosporidium andersoni infection cell by COWP gene.
Embodiment 2: measure medicine maximum safe concentration
1. prepare the storing solution of nitazoxanide (NTZ group), GA group, GB group and GA+GB group (mixture of GA and GB) etc., storing solution composition and concentration as shown in the table.
| Composition (content ratio) | Total concentration | |
| NTZ group: | 10mg/ml |
| GA group: | The content (wt%) of C13:0:C15:1:C17:2 is than being 9.3:48.33:1.54 | 10mg/ml |
| GB group: | The content ratio (wt%) of C15:0:C17:1 is 3.42:37.42 | 10mg/ml |
| GA+GB group: | GA group and GB group mix with volume ratio 1:1 | 10mg/ml |
Each group with culture fluid doubling dilution extremely required experimental concentration.
NTZ group (Stock concentrations 10mg/ml): experimental concentration is respectively 100 μ g/ml, 50 μ g/ml, 25 μ g/ml, 12.5 μ g/ml, 6.25 μ g/ml, 3.125 μ g/ml, 1.56 μ g/ml, 0.78 μ g/ml;
GA group (Stock concentrations 10mg/ml): experimental concentration is respectively 100 μ g/ml, 50 μ g/ml, 25 μ g/ml, 12.5 μ g/ml, 6.25 μ g/ml, 3.125 μ g/ml, 1.56 μ g/ml, 0.78 μ g/ml;
GB group (Stock concentrations 10mg/ml): experimental concentration is respectively 100 μ g/ml, 50 μ g/ml, 25 μ g/ml, 12.5 μ g/ml, 6.25 μ g/ml, 3.125 μ g/ml, 1.56 μ g/ml, 0.78 μ g/ml;
GA+GB group (Stock concentrations 10mg/ml): experimental concentration is respectively 100 μ g/ml, 50 μ g/ml, 25 μ g/ml, 12.5 μ g/ml, 6.25 μ g/ml, 3.125 μ g/ml, 1.56 μ g/ml, 0.78 μ g/ml.
Matched group: concentration is the DMSO of 1%.
Each concentration group repeats 3 times.
2.MTT method measures maximum safety of medicine concentration:
Conventional mtt assay measures NTZ, GA and GB to HCT-8 cell safe concentration.
Digestion is in exponential phase HCT-8 cell, is inoculated in 96 porocyte culture plates, l × 10
5individual cells/well, is placed in 37 DEG C, 5%CO
2after growing 8h in cell culture incubator, abandon original fluid, cryptosporidium andersoni culture fluid 100 μ l is added after washing 2 times with sterile PBS buffer, and add NTZ, GA, GB, GA+GB (mixture of GA and GB) of different experiments concentration, supplement final volume most 200 μ l (DMSO concentration is not more than l%) with culture fluid.37 DEG C are continued at, 5%CO after dosing
2cultivate 48h in cell culture incubator, cultivation terminates each hole of front 4h and adds 20 μ l/ml1MTT working solutions (20mg/ml).After culture fluid is abandoned in cultivation end suction, each hole adds DMSO150 μ l/ml, and lucifuge vibration 10min, measures each hole OD value (dominant wavelength 490nm, commplementary wave length 630nm) in microplate reader.
Cell proliferation rate computing formula under different pharmaceutical concentration:
The rate of increase (%)=(OD
untreated-OD
after process)/OD
untreated.
Different pharmaceutical concentration to the proliferative effect of HCT-8 cell as shown in Figure 4.Choosing cell proliferation rate, to be greater than drug level corresponding to 95% be safe concentration.The maximum safe concentration of different pharmaceutical is as shown in table 1.
Table 1
Result shows: add after GA, GB, GA+GB of different pharmaceutical concentration and experiment contrast medicine NTZ cultivate 24h and 48h in HCT-8 cell, be presented in 24h cultivation from table 1, the maximum safe concentration of GA is the maximum safe concentration of 3.125 μ g/ml, GB is 2.5 μ g/ml; And after 48h cultivation, the maximum safe concentration of GB is reduced to 2.0 μ g/ml.
Therefore think that the safe concentration in GA use is stablized, and there is cytotoxic effect in the use of GB.
Embodiment 3: the in-vitro multiplication effect of Drug inhibition cryptosporidium andersoni
3.1 experimental technique
3.1.1 cell culture is with embodiment 1.
3.1.2 egg capsule collection and asepticize process are with embodiment 1.
3.1.3 cell infection is with embodiment 1.
3.1.4 the in-vitro multiplication experiment of Drug inhibition cryptosporidium andersoni
Cryptosporidium andersoni infects HCT-8, in 6 porocyte culture plates, inoculate polypide 1 × 10
3individual/hole, adds 5ml cryptosporidium andersoni culture fluid, is placed in 37 DEG C, 5%CO
2cultivate 8h in environment, inhale and abandon original fluid, wash 2 times with sterile PBS buffer, add the NTZ group of high, medium and low three concentration of doubling dilution, GA group, GB group and GA+GB group:
NTZ group (Stock concentrations 10mg/ml):
Experimental concentration is respectively 3.125 μ g/ml (high concentration group), 1.56 μ g/ml (middle concentration group), 0.78 μ g/ml (low concentration group);
GA group (Stock concentrations 10mg/ml):
Experimental concentration is respectively 3.125 μ g/ml, 1.56 μ g/ml, 0.78 μ g/ml;
GB group (Stock concentrations 10mg/ml):
Experimental concentration is respectively 3.125 μ g/ml, 1.56 μ g/ml, 0.78 μ g/ml;
GA+GB group (Stock concentrations 10mg/ml):
Experimental concentration is respectively 3.125 μ g/ml, 1.56 μ g/ml, 0.78 μ g/ml, in each concentration, and GA:GB=1:1.
Infected group: represent not by the infection cryptosporidium andersoni group of any drug treating.
Establish 1%DMSO matched group simultaneously.
Each concentration group repeats 3 times, continues to be cultured to 48h.
With cell dissociation buffer peptic cell and polypide after experiment terminates, the centrifugal 10min of 1500g collects polypide, extracting DNA, preserves in-20 DEG C.
3.1.5real-timePCR Cryptosporidium parvum oocysts suspended COWP gene copy number is measured:
Protein nucleic acid analyser (purchased from Bole company) measures PGEM-TEasy/COWP recombinant plasmid dna concentration, calculates plasmid DNA copies number, through the doubling dilution of 10 times, obtains 10
7-10
2the Concentraton gradient of/μ l, adopts quantitative real time PCR Instrument (ABI7000) amplification, and reaction terminates the rear threshold value (Ct) according to setting, and with the logarithm of copy number for abscissa, Ct value is vertical coordinate, automatic Fitting drawing standard curve.
The DNA of different disposal group carries out fluorescence quantitative PCR detection COWP gene, calculates the copy number of Cryptosporidium parvum oocysts suspended COWP gene in each sample according to standard curve as shown in Figure 3.
3.2 result
GA, GB and GA and GB mixing group (GA+GB) is adopted to carry out the in-vitro multiplication of the anti-hidden spore assessed on HCT-8 cell.Result as shown in Figure 5.
As found from the results:
(1) anti-Cryptosporidium standard drug NTZ is under the concentration of 3.125 μ g/ml (high concentration group), significantly can reduce the COWP gene copy number of cryptosporidium andersoni, namely significantly suppress this worm propagation in vitro;
(2) GA more effectively reduces the copy number of COWP gene, and effect is much better than NTZ.As shown in Figure 5, in high concentration group, the relative rate of increase of the cryptosporidium andersoni of GA group is about 0.058 times of NTZ group; The suppression efficiency of the low concentration group of GA is almost suitable with the high concentration group of NTZ, even better.
(3) GB is conducive to external cryptosporidium andersoni propagation.After this cell that may infect in 48h process due to high concentration group 3.125 μ g/ml, cause certain cytotoxic effect (table 1), therefore the COWP gene of polypide is a little less than non-treated with medicaments group, and in middle concentration group 1.56 μ g/ml and low concentration group 0.78 μ g/ml, concentration is low, maximum safe drugs concentration 2.000 μ g/ml(table 1 in 48h cultured cell), this shows that GB is conducive to the invasion propagation of Cryptosporidium, and therefore the COWP gene of polypide is higher than simple infection group.
(4) experimental result of GA+GB group supports the conclusion of (3) too: GA has insecticidal action, and GB has the effect promoting polypide propagation.Specific as follows:
In high concentration and middle concentration group, the effect that GB is conducive to the hidden sporogony of external iS-One is greater than the inhibitory action of GA, therefore, than the high concentration and middle concentration group that are used alone GA polypide propagation obviously;
In low concentration group, the insecticidal action of GA just embodies, and therefore at low concentrations, GA+GB effectively can suppress the propagation of cryptosporidium andersoni.
3.3 discuss
(1) under higher concentration (as 3.125 μ g/ml), after GA acts on the cell infecting cryptosporidium andersoni, polypide propagation is subject to obvious suppression, the effect of the anti-Cryptosporidium propagation of GA is more better than NTZ, the Cryptosporidium of In vitro culture can be made to stop propagation, protection host cell is a kind of novel anti-Cryptosporidium medicine having development potentiality; And the cultivation effect of the anti-Cryptosporidium of GB is very low; Even if the superposition group of GA and GB
can not have effectground resists hidden sporogony.
(2) under low concentration (as 0.78 μ g/ml), after GA acts on the cell infecting cryptosporidium andersoni, polypide propagation is subject to obvious suppression, and the effect of the anti-Cryptosporidium propagation of GA is more better than NTZ; And the cultivation effect of the anti-Cryptosporidium of GB is very low; But the superposition group of GA and GB
can be effectiveground resists hidden sporogony.
Visible, GA has the effect of excellent suppression cryptosporidium andersoni; And GA+GB need under low concentration, the effect of suppression cryptosporidium andersoni that could be good.
In addition, after a period of time placed by GA+GB group medicine, easily form flow-like, and be unfavorable for dissolving, and GA group medicine remains good character and dissolubility.
In addition, Cryptosporidium cell model is widely used in the screening of anti-Cryptosporidium medicine, the method is without the need to using childhood or immunodeficiency animal, very big reduction experimental work amount and cost, and large quantization compound can be screened within a short period of time, the high flux of drug screening can be realized, promote the development of anti-Cryptosporidium medicine.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. a pharmaceutical composition for anti-Cryptosporidium, is characterized in that, containing active component: monomer C13:0, C15:1 and C17:2:
And described pharmaceutical composition is not containing monomer C15:0 and C17:1:
In described pharmaceutical composition, the content of C13:0, C15:1 and C17:2 is than being 1-10:20-40:1.
2. pharmaceutical composition as claimed in claim 1, it is characterized in that, the application concentration of described pharmaceutical composition is 0.05 μ g/ml ~ 5 μ g/ml.
3. pharmaceutical composition as claimed in claim 1, is characterized in that, in described pharmaceutical composition, the content of C13:0, C15:1 and C17:2 is than being 4-8:25-36:1.
4. a pharmaceutical composition for anti-Cryptosporidium, is characterized in that, containing component:
(a) active component: monomer C13:0, C15:1 and C17:2:
(b) monomer C15:0 and C17:1:
And in described pharmaceutical composition, the weight ratio of component (a) and component (b) is 0.8-1.2:1.2-0.8;
In composition (a), the content of C13:0, C15:1 and C17:2 is than being 1-10:20-40:1;
Further, the application concentration of described pharmaceutical composition is 0.05 μ g/ml ~ 0.9 μ g/ml.
5. pharmaceutical composition as claimed in claim 4, is characterized in that, in described pharmaceutical composition,
In composition (b), the content of C15:0 and C17:1 is than being 1:1-20.
6. pharmaceutical composition as claimed in claim 4, it is characterized in that, the application concentration of described pharmaceutical composition is 0.05 μ g/ml ~ 0.78 μ g/ml.
7. the purposes of pharmaceutical composition as described in claim 1 or 4, is characterized in that, for the preparation of the medicine that anti-cryptosporidium andersoni infects.
8. the method for a vitro inhibition cryptosporidium andersoni, it is characterized in that, comprise step: have in the cell culture system of cryptosporidium andersoni in infection, the pharmaceutical composition added as described in claim 1 or 4 is cultivated, thus suppress the propagation of cryptosporidium andersoni
When adding pharmaceutical composition as claimed in claim 1, its application concentration is 0.05 μ g/ml ~ 5 μ g/ml; Or
When adding pharmaceutical composition as claimed in claim 4, its application concentration 0.05 μ g/ml ~ 0.9 μ g/ml.
9. method as claimed in claim 8, it is characterized in that, described cell is HCT-8.
10. method as claimed in claim 8, is characterized in that,
When adding pharmaceutical composition as claimed in claim 1, its application concentration is 0.5 μ g/ml ~ 3.125 μ g/ml; Or
When adding pharmaceutical composition as claimed in claim 4, its application concentration is 0.05 μ g/ml ~ 0.78 μ g/ml.
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|---|---|---|---|---|
| CN1806544A (en) * | 2006-02-21 | 2006-07-26 | 复旦大学 | Use of ginkgoic acid in preparation of biological pesticide for killing Oncomelania snail and preventing schistosomiasis |
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| CN1806544A (en) * | 2006-02-21 | 2006-07-26 | 复旦大学 | Use of ginkgoic acid in preparation of biological pesticide for killing Oncomelania snail and preventing schistosomiasis |
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