CN103768583B - A kind of application of the extracting solution compositions of natural variform arsenic - Google Patents
A kind of application of the extracting solution compositions of natural variform arsenic Download PDFInfo
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Abstract
The present invention provides the application of the extracting solution compositions of a kind of natural variform arsenic, the extracting solution compositions of described natural variform arsenic includes the extracting solution of natural variform arsenic and at least one antitumor drug following, described antitumor drug includes: camptothecine, vinblastine, vincristine, paclitaxel, tumor necrosis factor, interleukin, interferon, transforming growth factor β, lipopolysaccharide, i.e. phorbol exters, adenyl cyclase activator and bone morphogenetic protein 4, the extracting solution weight of the most natural variform arsenic accounts for the 0.001～99.9% of made medicine gross weight, the extracting solution compositions of described natural variform arsenic can be used to prepare resisting tumour stem cells, anti-hepatitis B and the medicine of AIDS virus resisting.The present invention can suppress the virus such as hepatitis B and anti-AIDS, and the exploitation for natural arsenical extend range of application.
Description
[technical field]
The present invention relates to the application of the extracting solution compositions of a kind of natural variform arsenic.
[background technology]
Tumor is the most common malignant tumor threatening human health, although chemicotherapy and operation method are continuous
Progress, but case fatality rate is the highest.Such as, as common type nonsmall-cell lung cancer (non-small
Cell lung cancer, NSCLC) total 5 years survival rates are still less than 15%.Based on tumor stem cell
(cancer stem cells, CSC) theory is thought, the tumor with self renewal and unlimited multiplication capacity is done
Cell be tumor occur and recurrence " root " (open source literature is: Yang Ming. nanometer-size realgar is to pulmonary carcinoma regulating liver-QI
Cancerous cell and the apoptosis-induced effect of tumor stem cell thereof. [D]. Lanzhou University Ph.D. Dissertation: Lanzhou.
2010, P10～15).To this end, for the drug screening strategy of suppression tumor stem cell, will be current
One new way of tumor treatment.
Along with going deep into of research, arsenical (includes As2O3And Realgar) receive publicity again, once because
Toxicity is not reused As2O3, it is found to eliminate white blood by reducing cancer protein PML level
Sick stem cell LICs, it may include a system, lymphoid malignant tumor by eliminating LICs healing
(open source literature is: Zhang XW, Yan XJ, Zhou ZR, et al.Arsenic Trioxide Controls
the Fate of the PML-RAR Oncoprotein by Directly Binding PML.Science[J],201
0,328:240-243), it is also recently found that, nanometer-size realgar can effectively induce pulmonary carcinoma and liver-cancer stem cell to occur
Apoptosis.With As2O3Compare, natural arsenical have aboundresources, cheap, the spy such as can orally use
Point, but on Mutiple Targets, the suppression effect of tumor stem cell, scope and effect range, As2O3
All show higher drug influence.This gap, also causes domestic relevant scholar, for natural arsenic
The effective substance of agent is coarse former powder or the arguement of internal metabolism arsenic.Modern analysis proves: arsenic is dynamic
Metabolite in object, it is understood that there may be multiple arsenic metabolism species, including: iAsIII, iAsV, MMAV,
DMAV etc. (open source literature is as follows: Xu Juhui, Wang Hongwei, Cui Rong etc. Realgar and containing in Realgar Chinese patent medicine
The morphological analysis [J] of arsenic. pharmaceutical analysis magazine, 2007,27 (3): 395～396；Zhang Jinghong, Li Hongyu. high
Effect Capillary Electrophoresis realgar microorganism concocts arsenic morphology composition [J] in liquid. time treasure's traditional Chinese medical science traditional Chinese medicines,
2011.22(2): 409～410), to this end, utilize modern biotechnology, the external natural arsenic of bionical metabolism
Breeze, produces the natural soluble solution arsenic of the multiple form identical or different with internal metabolism, is grouped compound,
Replacing expensive, Clinical practice Liver and kidney toxicity strengthens, it is difficult to universal arsenic trioxide injection, makees
Supplementing or succedaneum for an existing line anticancer arsenical arsenic trioxide, will be one of the research of natural arsenical
Important directions.
The research of the present inventor seminar finds, Marine microorganism metabolism the various form arsenic dissolved, its
In animal tissue, arsenic cumulative toxicity is greatly lowered, in animal body almost without distribution (open source literature
As follows: Zhang Jinghong, Fan Qin, Li Hongyu. liquid arsenic morphology and oxicity analysis [J] are concocted in Realgar and microorganism.
Chinese crude drug, 2010,11 (5): 283-285).Meanwhile, inside and outside pharmacological activity test shows, sea
Ocean microorganism arsenic compound of polymorphic generation, is possible not only to induce multidrug resistance apoptosis of leukemia, and
Can lower MDR1/P-gp drug resistant gene, (open source literature is such as to reach the effect of its reverse multidrug drug resistance
Under: Zhang Jinghong, Li Hongyu. realgar microorganism extracting solution is to K562/ADM cell P-pg albumen and MDRI
The impact [J] of gene expression. Chinese Clinical pharmacology and therapeutics, 2009,14 (8): 855～861).And
And the more important thing is, its inductive effect is better than similar arsenical arsenious acid.So, arsenious acid (As2O3?
The principal mode dissolved in water) it is not the sole active agent of reverse multiple drug resistance of tumor effect, from sky
So mineral resources develop the metabolism arsenic of variform as As2O3Important supplement, will be that pole has application
The drug development direction of prospect.
Though having natural arsenic such as in China, arsenic disulfide (As2S3), Orpimentum (Auripigmentum) (As2S3) and arsenic disulfide
(As2O3, realgar) equal size enrich, and as clinical medicine treatment tumor, parasitism
The aspect such as worm, herpesvirus obtains significant curative effect.But the preparation process of modern report include water fly+
The methods such as vinegar system, nanorize and micronization, obtain only suspension, it is impossible to meet present pharmaceutical preparation
Requirement, open Chinese invention 200610200067.5 patent of seminar of the present invention, the most successfully utilize many
Plant metallurgy acidophilic thiobacillus, extract Realgar and produce containing comprising arsenic acid (III, V), methyl MMA
(V) and substantial amounts of auxiliary element (ferrum and the change of ferrum and thing) mixed extract (open source literature is as follows:
Li Hongyu, Zhang Jinghong, Zhi Dejuan. the heavy metals removal method in mineral drug. Chinese patent [P]: 200610
200067.5,2006-1-02.), although these methods can obtain the mixed liquor containing portion forms arsenic,
But owing to being mixed with other compositions of part, its definite active component cannot be determined at present, it is impossible to and its
His medicine or adjuvant composition complex, the in addition work of its tumor cell such as vitro inhibition pulmonary carcinoma and hepatocarcinoma
With, hence it is evident that less than As2O3, and the more important thing is, have not yet to see it and can suppress tumor simultaneously
The similar effect of stem cell, hepatitis B and HIV (human immunodeficiency virus).
In order to obtain multiple natural variform, the compositions of solubilized arsenic, applicant is in Application No.
201010580193.4 Chinese invention patent disclose a kind of Marine microorganism extract natural, polymorphic,
The preparation method and applications of the arsenical extracting solution of suppression tumor, wherein utilize applicant independently to divide
From multiple Marine microorganism, metabolism arsenic disulfide (As2O3、As4S4) and or arsenic trisulfide (As2S3)
Obtain containing arsenic acid (AsIII, AsV), arsenious acid (V), monomethyl arsenic acid MMA(III, V), two
Methylarsonic acid DMA(III, V) (DMA), monomethyl monothio arsenic acid MMTA, Methyl disulfide
For arsenic acid DMDTA, TMAO (TMAO), AsB (AsB, arsenobetaine) and
The extracting solution of the multiple natural polymorphic arsenic of arsenocholine (AsC) etc..
[summary of the invention]
The technical problem to be solved in the present invention, is that the extracting solution providing a kind of natural variform arsenic combines
The application of thing, it is possible to the suppression virus such as hepatitis B and anti-AIDS, the exploitation for natural arsenical extend
Range of application.
The present invention is achieved in that
A kind of application of the extracting solution compositions of natural variform arsenic, carrying of described natural variform arsenic
Take liquid compositions and include extracting solution and at least one antitumor drug following, the institute of natural variform arsenic
State antitumor drug to include: camptothecine, vinblastine, vincristine, paclitaxel, tumor necrosis factor,
Interleukin, interferon, transforming growth factor-β, lipopolysaccharide, i.e. phorbol exters, adenyl cyclase
Activator and bone morphogenetic protein 4, the extracting solution weight of the most natural variform arsenic accounts for made
The 0.001～99.9% of medicine gross weight, the extracting solution compositions of described natural variform arsenic can be used to make
Standby resisting tumour stem cells, anti-hepatitis B and the medicine of AIDS virus resisting.
Further, in the extracting solution compositions of described natural variform arsenic containing arsenic acid (AsIII),
Arsenic acid (AsV) and or monomethyl arsenic acid MMA(III) monomethyl arsenic acid MMA(V) and or diformazan
Base arsenic acid DMA(III), dimethyl arsenate DMA(V) and or monomethyl monothio arsenic acid MMTA
With or Methyl disulfide for arsenic acid DMDTA and or TMAO (TMAO) and or AsB
And or arsenocholine (AsC) (AsB)；And the weight sum of above-claimed cpd accounts for medicine gross weight
0.001～99.9%.
Further, the extracting solution compositions of described natural variform arsenic can be added and pharmaceutically can be connect
The adjuvant being subject to be prepared as soft capsule, hard capsule, drop pill, tablet, granule, injection or injectable powder,
Oral liquid, microcapsule, drop pill, aerosol and suppository formulations, oral administration, injection or rectal give
Medicine.
Present invention have the advantage that
(1) present invention utilizes the special Marine microorganism with bionical metabolic capacity to natural drug containing arsenic
Mineral carry out microorganism extraction, thus obtain multiple identical from internal metabolism or different, have attenuation,
The extracting solution of the multiple natural form arsenic combination of potentiation, adds camptothecine (camptothecin), length
Spring alkali (VLB), vincristine (VCR), paclitaxel (Taxol), tumor necrosis factor (TNF), white
Cytokine (interleukin, IL-6), interferon (IFN), transforming growth factor-β (TGF-β),
Lipopolysaccharide (LPS), i.e. phorbol exters (TPA), adenyl cyclase activator (Forskolin) and bone
Morphogenetic proteins 4(BMP-4) etc. compatibility be grouped compound, said composition shows potent suppression lung
The medicine effect of cancer stem cell, hepatitis B and HIV (human immunodeficiency virus), its drug influence is apparently higher than positive drug
As2O3Solution, this advantage, novel for screening, As can be substituted2O3Efficient, the arsenical of low toxicity
Provide excellent basis.
(2) extracting solution compositions of the present invention can contain containing arsenic acid (AsIII, AsV), arsenious acid simultaneously
(V) and or monomethyl arsenic acid MMA(III, V) and or dimethyl arsenate DMA(III, V) (DMA)
With or monomethyl monothio arsenic acid MMTA and or Methyl disulfide for arsenic acid DMDTA and or trimethyl
Arsinoxide (TMAO) and or AsB (AsB, arsenobetaine) and or arsenocholine (AsC) etc. many
Plant Soluble Arsenic anatomic element, prepare Realgar with nanometer-size realgar and the multiple metallurgy acidophilic thiobacillus of employing
Microorganism mixing leachate is compared, and it has the suppression tumor stem cell effect of uniqueness, and the present invention carries
Take liquid suppression tumor stem cell while, there is again good suppression hepatitis B and the effect of acquired immune deficiency syndrome (AIDS), show
Show good drug development prospect.
[detailed description of the invention]
The present invention relates to the application of the extracting solution compositions of a kind of natural variform arsenic, described natural multiple
The extracting solution compositions of form arsenic includes the extracting solution of natural variform arsenic and at least one anti-following
Tumour medicine, described antitumor drug includes: camptothecine, vinblastine, vincristine, paclitaxel, swollen
Tumor necrosis factor, interleukin, interferon, transforming growth factor-β, lipopolysaccharide, i.e. phorbol exters,
Adenyl cyclase activator and bone morphogenetic protein 4, the extracting solution weight of the most natural variform arsenic
Amount accounts for the 0.001～99.9% of made medicine gross weight, the extracting solution group of described natural variform arsenic
Compound can be used to prepare the medicine of resisting tumour stem cells, anti-hepatitis B and AIDS virus resisting.
Containing arsenic acid (AsIII), arsenic acid (AsV) in the extracting solution compositions of described natural variform arsenic
With or monomethyl arsenic acid MMA(III) monomethyl arsenic acid MMA(V) and or dimethyl arsenate DMA
(III), dimethyl arsenate DMA(V) and or monomethyl monothio arsenic acid MMTA and or dimethyl
Dithio arsenic acid DMDTA and or TMAO (TMAO) and or AsB (AsB) and or arsenic
Choline (AsC)；And the weight sum of above-claimed cpd accounts for the 0.001～99.9% of medicine gross weight.
The extracting solution compositions of described natural variform arsenic can add pharmaceutically acceptable adjuvant
Be prepared as soft capsule, hard capsule, drop pill, tablet, granule, injection or injectable powder, oral liquid,
Microcapsule, drop pill, aerosol and suppository formulations, oral administration, injection or rectal administration.
Below in conjunction with specific embodiment, the present invention is further illustrated.
Embodiment one, prepare the extracting solution of natural variform arsenic
According to Application No. 201010580193.4, publication date is that the patent of invention of 2011-04-20 is open
A kind of Marine microorganism extract natural, polymorphic, the preparation of arsenical extracting solution of suppression tumor
Preparation method described in method and application thereof processes arsenic disulfide (As2O3, content 36%, As4S4, contain
Amount 62%) and or arsenic trisulfide (As2S3, 72%, AsC, 25%) breeze, embodiment is specifically wrapped
Include following steps:
Step 1: by arsenic breeze 20g, after being crushed to 200 mesh, according to 1:100(weight and mixing
Liquid volume ratio) add the sulphuric acid of 1mol/L and the mixed liquor (volume ratio 8:2) of the composition of 15% ferric chloride
In, then rotating speed be 180r/min, temperature be the constant-temperature table of 30 DEG C in cultivate 15d, carried
Take liquid；By the membrane filtration that extracting solution aperture is 0.45 μm, obtain filtrate；Described filtrate is in temperature
Be 20 DEG C, magnetic agitation rotating speed be to carry out reaction precipitation, after 2min under conditions of 800～1000r/min
Filter, be precipitated thing；Then, after precipitate is washed with deionized, it is dried at a temperature of 60 DEG C,
The most handy microbial metabolism ultrafine powder fine powder.
Step 2: weigh step 1 gained micropowder 0.1g, in the 250mL conical flask after sterilizing,
Adding LA fluid medium 90mL, inoculum fraction is the combination preferred Marine microorganism bacterium of 20%
Kind, then 30 DEG C, pH be 5.0 hunting speeds be 180r/min time shaking flask leach 15d；Regularly
Carry out in treatment fluid containing arsenic acid (AsIII, AsV), arsenious acid (V), monomethyl arsenic acid MMA(III,
V), dimethyl arsenate DMA(III, V) (DMA), monomethyl monothio arsenic acid MMTA, two
Methyl dithio arsenic acid DMDTA, TMAO (TMAO), AsB (AsB, arsenobetaine)
Detect with arsenocholine (AsC) etc., after the total content of above-mentioned Multiple components reaches requirement, stop concussion training
Support, after 13000r/min is centrifugal, take supernatant, refilter degerming after, gained supernatant is exactly containing natural
The extracting solution of variform arsenic.
Described detection method of content is: capillary electrophoresis separation method: condition is: BGE:PDC/10
mmol.L-1And CTAOH/1mmol.L-1, voltage 25KV, temperature 25 DEG C, pH11.0, ultraviolet inspection
Survey wavelength and be set in 216nm.
The extracting solution A of described natural variform arsenic: arsenical content composition in per unit preparation
Obtain after testing as arsenic acid (V) 15%；Arsenious acid (III) 3.5%；Methylarsonic acid (III) 1.5
%, methylarsonic acid (V) 12.5%；Dimethyl arsenate (DMA) 3%；TMAO (TMAO)
7.5%；AsB AsB (AsB) 2%；Arsenocholine (AsC) 5%.Its preparation method according to
Prepared by embodiment 1, the arsenic breeze of employing is arsenic disulfide (As2O3, content 36%, As4S4, content
62%), preferably Marine microorganism is combined as: A(alcaligenes aquamarinus): O(brevibacterium flavum): B (excrement
Bacillus alcaligenes) be 5:2:3(volume ratio).
The extracting solution B of described natural variform arsenic: arsenical content composition in per unit preparation
Obtain after testing as arsenic acid (V) 7.5%；Arsenious acid (III) 3.5%；Methylarsonic acid (III)
1.5%, methylarsonic acid (V) 12.5%；Monomethyl monothio arsenic acid MMTA3.5%；Dimethyl two
Sulfarsenic acid DMDTA4%；Dimethyl arsenate (DMA) 3%；TMAO (TMAO)
7.5%；AsB AsB (AsB) 2%；Arsenocholine (AsC) 5%.Its preparation method according to
Prepared by embodiment 1, the arsenic breeze of employing is arsenic disulfide (As2O3, content 36%, As4S4, content
62%), preferably Marine microorganism is combined as: A(alcaligenes aquamarinus): O(brevibacterium flavum): B is (little
Gram Mildy Way is mould) be 8:3:5(volume ratio).
The extracting solution C of described natural variform arsenic: arsenical content composition in per unit preparation
Obtain after testing as arsenic acid (V) 25%；；Arsenious acid (III) 12.5%；Methylarsonic acid (III)
12.5%, methylarsonic acid (V) 20%；Dimethyl arsenate (DMA) 10%；TMAO (TMAO)
5%；AsB AsB (AsB) 5%；Arsenocholine (AsC) 9.99%.Its preparation method according to
Prepared by embodiment 1, the arsenic breeze of employing is arsenic disulfide (As2O3, content 36%, As4S4, content
62%), preferably Marine microorganism is combined as: A(alcaligenes aquamarinus): O(brevibacterium flavum): B is (new
Month shank bacterium) be 7:1:6(volume ratio).
Embodiment two: prepare the extracting solution compositions of natural variform arsenic
Thing of getting it filled includes: camptothecine (camptothecin, 3umol/L), vinblastine (VLB, 0.2ug/mL),
Vincristine (VCR, 0.16ug/mL), paclitaxel (Taxol, 1umol/L), tumor necrosis factor
(TNF, 25ng/ml), interleukin (interleukin, IL-6,10ng/ml), interferon (IFN,
10ng/ml), transforming growth factor-β (TGF-β, 2.5ng/ml), lipopolysaccharide (LPS, 1ug/mL),
I.e. phorbol exters (TPA, 20ng/ml), adenyl cyclase activator (Forskolin, 20ng/ml) and bone
Morphogenetic proteins 4(BMP-4,10ng/ml) etc. in any one drug solution 100ul, add
0.05mol/L Tri(Hydroxymethyl) Amino Methane Hydrochloride buffer (Tris-HCL) to 1mL, 80 DEG C of water-baths
After boiling 1 hour, after centrifugal filtration, according to drug extract: the extracting solution of natural variform arsenic (1:
2, volume ratio), add natural variform arsenic made in embodiment 1 extracting solution A or B or
Person C, it is 0.0167u/mL that filtrate adds lyases 50ul(concentration), 37 DEG C, enzymatic reaction 2 hours,
The filter membrane collecting filtrate 0.45um filters, and obtains the combination of extracting solution containing natural variform arsenic
Thing.
Wherein said lyases is: B-glucosidase, P450 enzyme (CYP1A2, CYP2A6, CYP2B6,
CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP3A5),
Glutathione S one transferring enzyme (GST), N mono-acetyl transferase (NAT), uridine diphosphoglucose aldehyde
Acid transferring enzyme (UGT), Epoxide hydrolase (EH), sulfate transferase, Acetylase and methyl turn
Move enzyme, catechol-O-methyl transferase, COMT, esterase, hydroxylase flavin monooxygenase (FMO), monoamine oxygen
Change enzyme (MAO) and any one of ketoreductase.
Wherein, when the extracting solution compositions of natural variform arsenic contains extracting solution A, the cracking of employing
Enzyme is B-glucosidase, arsenic acid (V) 15% in compositions；Arsenious acid (III) 3.5%；First
Base arsenic acid (III) 1.5%, methylarsonic acid (V) 12.5%；Dimethyl arsenate (DMA) 3%；Three
Methyl arsinoxide (TMAO) 7.5%；AsB AsB (AsB) 2%；Arsenocholine (AsC) 5
%.Its arsenical total amount accounts for 50% (percentage by weight) of extracting solution combination of Chinese medicine thing total amount.
Wherein, when the extracting solution compositions of natural variform arsenic contains extracting solution B, the lyases of employing is
B-glucosidase and CYP3A4, containing arsenic acid (V) 7.5% in compositions；Arsenious acid (III)
3.5%；Methylarsonic acid (III) 1.5%, methylarsonic acid (V) 12.5%；Monomethyl monothio arsenic acid
MMTA3.5%；Methyl disulfide is for arsenic acid DMDTA4%；Dimethyl arsenate (DMA) 3
%；TMAO (TMAO) 7.5%；AsB AsB (AsB) 2%；Arsenocholine (AsC)
5%.Its arsenical total amount account for extracting solution combination of Chinese medicine thing total amount 50% (weight percent
Than).(percentage by weight).
Wherein, when the extracting solution compositions of natural variform arsenic contains extracting solution C, the cracking of employing
Enzyme is B-glucosidase and CYP3A4, containing arsenic acid (V) 25% in compositions；Arsenious acid (III)
12.5%；Methylarsonic acid (III) 12.5%, methylarsonic acid (V) 20%；Dimethyl arsenate (DMA)
10%；TMAO (TMAO) 5%；AsB AsB (AsB) 5%；Arsenocholine (AsC)
9.99%.Its arsenical total amount account for extracting solution combination of Chinese medicine thing total amount 99.99% (weight
Percentage ratio).(percentage by weight).
Embodiment three, the extracting solution compositions pharmacodynamic evaluation experimental example of natural polymorphic arsenic
1, in following experimental example, given the test agent: the extracting solution of the natural variform arsenic containing extracting solution B
Compositions:
Compositions 1: in per unit preparation arsenical content composition obtain after testing for: arsenic acid (V,
1.5nM)；Arsenious acid (III, 0.7nM)；Methylarsonic acid (III, 0.3nM), and methylarsonic acid (V, 2.5
NM)；Monomethyl monothio arsenic acid (0.7nM)；Methyl disulfide is for arsenic acid DMDTA(0.8nM)；
Dimethyl arsenate (DMA, 0.6M)；TMAO (TMAO, 1.5nM)；AsB (AsB,
0.4nM)；Arsenocholine (AsC, 1nM)；In per unit preparation, the content of other drug forms after testing
Obtain for tumor necrosis factor (TNF, 25ng/ml), other drug and arsenical combination ratio be
1:1;
Compositions 2: in per unit preparation arsenical content composition obtain after testing for: arsenic acid (V,
1.5nM)；Arsenious acid (III, 0.7nM)；Methylarsonic acid (III, 0.3nM), and methylarsonic acid (V, 2.5
NM)；Monomethyl monothio arsenic acid (0.7nM)；Methyl disulfide is for arsenic acid DMDTA(0.8nM)；
Dimethyl arsenate (DMA, 0.6M)；TMAO (TMAO, 1.5nM)；AsB (AsB,
0.4nM)；Arsenocholine (AsC, 1nM)；In per unit preparation, the content of other drug forms after testing
Obtain as interleukin (interleukin, IL-6,10ng/ml), other drug and arsenical
Combination ratio is 1:1;
Compositions 3: in per unit preparation arsenical content composition obtain after testing for: arsenic acid (V,
1.5nM)；Arsenious acid (III, 0.7nM)；Methylarsonic acid (III, 0.3nM), and methylarsonic acid (V, 2.5
NM)；Monomethyl monothio arsenic acid (0.7nM)；Methyl disulfide is for arsenic acid DMDTA(0.8nM)；
Dimethyl arsenate (DMA, 0.6M)；TMAO (TMAO, 1.5nM)；AsB (AsB,
0.4nM)；Arsenocholine (AsC, 1nM)；In per unit preparation, the content of other drug forms after testing
Obtain as camptothecine (camptothecin, 3umol/L), other drug and arsenical combination ratio
For 1:1;
Positive control composition 1 ': in per unit preparation arsenical content composition obtain after testing for:
As2O3(arsenic acid V, 5nM)；In per unit preparation other drug content composition obtain after testing for:
Tumor necrosis factor (TNF, 25ng/ml), other drug and arsenical combination ratio are 1:1;
Positive control composition 2 ': in per unit preparation arsenical content composition obtain after testing for:
As2O3(arsenic acid V, 5nM)；In per unit preparation other drug content composition obtain after testing for:
Interleukin (interleukin, IL-6,10ng/ml), other drug and arsenical combination ratio are
1:1;
Positive control composition 3 ': As2O3(arsenic acid V, 5nM)；Other drug in per unit preparation
Content composition obtain as camptothecine (camptothecin, 3umol/L) after testing, other drug and
Arsenical combination ratio is 1:1;
Individually extracting solution B group: in per unit preparation, arsenical content composition obtains after testing
For: arsenic acid (V, 1.5nM)；Arsenious acid (III, 0.7nM)；Methylarsonic acid (III, 0.3nM),
Methylarsonic acid (V, 2.5nM)；Monomethyl monothio arsenic acid (0.7nM)；Methyl disulfide is for arsenic acid
DMDTA(0.8nM)；Dimethyl arsenate (DMA, 0.6M)；TMAO (TMAO,
1.5nM)；AsB (AsB, 0.4nM)；Arsenocholine (AsC, 1nM)；
1.1, the extracting solution compositions of the natural polymorphic arsenic containing extracting solution B people's pulmonary carcinoma to In vitro culture
The growth inhibited effect of cell A549 cell
A. common A549 cell is cultivated: by common A549 cell containing 10% hyclone
In RPMI1640 culture medium, at 37 DEG C, cultivate to right under the conditions of saturated humidity in 5%CO2 incubator
Number trophophase.
The tumor stem cell of b.A549 cell ball is cultivated: disappeared by the A549 cell of common logarithm trophophase
After changing cell, change into the DMEM/F containing 20l/ml B27,20ng/ml EGF, 20ng/ml bFGF
12 culture medium continue cultivate to logarithmic growth after date peptic cell again, and be made into single cell suspension with
The density in 20000/hole is planted in 6 orifice plates, every hole 2ml culture fluid, changes liquid every 2～3d half amounts,
At 37 DEG C, after cultivating 4-5 days under the conditions of 5%CO2, basis of microscopic observation cell balling-up situation and form,
Continuous passage 3 times, takes the 3rd generation cell ball, uses the Tumor Stem of RT-PCR detection A549 cell ball
Stem cell transcription factor Sox2 and Oct4 expression in cell, screening Sox2 and Oct4 expresses higher
A549 cell ball tumor stem cell enter cell proliferation experiment.
C. the Tumor Stem of the extracting solution compositions suppression A549 cell ball of the natural polymorphic arsenic of the present invention is thin
Born of the same parents' proliferation function is evaluated:
Different extracting solution suppression A549 cells and the detection of cell ball multiplication capacity: by A549 cell and
A549 cell ball makes single cell suspension by 1 × 104The density in/hole is separately added into 96 orifice plates, every hole
100ul, is separately added into extracting solution B, containing extracting solution composition B 1,2 and 3 and containing As2O3
The positive compositions 1 ', 2 ' and 3 ' of solution, matched group only adds culture medium at 37 DEG C, under the conditions of 5%CO2
Cultivate after 72h, add 90uL basal medium and MTT10uL (5mg/ml), 37 DEG C, 4h, removes
Removing culture fluid, every hole adds 150ul DMSO, rocks 10min microplate reader and measures optical density value ［ D (490)
, use logit method calculation composition half-inhibition concentration IC50, result criterion: effectively
IC50<10nM, effectively, IC50>10nM, invalid, IC50 the results are shown in Table shown in 1:
Result: in the extracting solution compositions of the natural polymorphic arsenic that the present invention contains extracting solution B, compositions 1,
2 and 3 can be to the human lung cancer cell A549 of In vitro culture and the tumor stem cell of A549 cell ball
Growth, there is good Inhibit proliferaton effect, and under conditions of the total amount containing arsenic is identical, with the positive
As2O3Reference composition 1 ', 2 ' is compared with 3 ' groups, and its inhibitory action of compositions 1,2 and 3 is better than sun
Property reference composition group.And on the other hand, compared with single extracting solution B group, although compositions 1,
2 and 3 groups, its total amount Han arsenic reduces 50%, but its drug influence is but higher than single extracting solution B
Group, illustrates accordingly, and after extracting solution B and other drug combination, the present composition of formation, containing arsenic
Amount reduces, and the drug influence of its suppression tumor stem cell is the most more preferably, hence it is evident that higher than single natural multiform
The extracting solution of state arsenic, concrete outcome table 1:
The extracting solution compositions of the table 1 natural polymorphic arsenic containing extracting solution B is to people's pulmonary carcinoma
The growth inhibited effect of the tumor stem cell of A549 cell and A549 cell ball
1.2, the extracting solution compositions of the natural polymorphic arsenic containing extract B, to Infection in Vitro acquired immune deficiency syndrome (AIDS)
The growth inhibited effect of the TZM-bl cell of HIV-1IIIB virus
Take the logarithm the TZM-BL cell of trophophase, by 2 × 104/ mL hole density is inoculated in 96 porocytes
In culture plate, 100 μ L/ holes, it is placed in cell culture incubator (37 DEG C, 5%CO2) and cultivates.The next day treat cell
Adherent and well-grown, sucks culture fluid, then is separately added into extracting solution with serum-free medium dilution
B, containing extracting solution composition B 1,2 and 3 and containing As2O3The positive compositions 1 ', 2 ' of solution
With 3 ', and it is separately added into 96 well culture plates, three parallel holes are set, 37 DEG C, 5%CO2Cultivate 3h
After, add the HIV-1IIIB virus 100 μ L infection cells of 1000 × TCID50, then proceed to training
After supporting 24h, Bright-Glo Luciferase Assay reagent (Promega) is utilized to measure the relative of every hole
Flat fluorescent (RLU), according to the half-inhibition concentration of Prism Graphruan computed in software compound
IC50, result criterion: effectively IC50<10nM, invalid IC50>10nM.IC50 the results are shown in Table
Shown in 2:
The compositions pair of the extracting solution of the table 2 natural polymorphic arsenic containing extracting solution B
The growth inhibited effect of the TZM-bl cell of Infection in Vitro acquired immune deficiency syndrome (AIDS) HIV-1IIIB virus
Result: containing in the extracting solution compositions of the natural polymorphic arsenic of extracting solution B, compositions 1,2
Can be to the life of the TZM-bl cell that can suppress Infection in Vitro acquired immune deficiency syndrome (AIDS) HIV-1IIIB virus with 3
Long, show that it has an effective In Vitro Anti HIV (human immunodeficiency virus) effect, and in the identical condition of the total amount containing arsenic
Under, with positive As2O3Reference composition 1 ', 2 ' is compared with 3 ' groups, its suppression of compositions 1,2 and 3
Effect is better than positive control composition group.And on the other hand, compared with single extracting solution B group,
Although compositions 1,2 and 3 groups, its total amount Han arsenic reduces 50%, but its drug influence is higher than
Individually extracting solution B group, illustrates accordingly, after extracting solution B and other drug combination, and this of formation
Bright compositions, arsenic content reduces, and the drug influence of its suppression HIV (human immunodeficiency virus) effect is more preferably, hence it is evident that high
In the extracting solution of single natural polymorphic arsenic, concrete outcome table 2:
1.3, the extracting solution compositions of the natural polymorphic arsenic containing extracting solution B, thin to HepG2.2.15
The effect of the external HBV antigen suppression of born of the same parents
Take the logarithm the HepG2.2.15 cell of trophophase, by 2 × 104It is thin that/mL hole density is inoculated in 24 holes
In born of the same parents' culture plate, 1000 μ L/ holes, each concentration sets 3 multiple holes altogether, put cell culture incubator (37 DEG C, 5%
CO2) cultivate in.The next day treat cell attachment and well-grown, suck culture fluid, be separately added into extracting solution B,
Containing extracting solution composition B 1,2 and 3 and containing As2O3The positive compositions 1 ', 2 ' of solution and
3 ', the complete culture solution 1000 μ L of each concentration, each concentration sets 3 multiple holes, puts cell culture incubator
(37 DEG C, 5%CO2) are cultivated.The complete culture solution of equivalent makees blank, is cultivating 72h continuously
After, draw cell culture fluid and be added on respectively in 1.5mL aseptic Eppnedorf pipe, treat in-20 DEG C of preservations
Inspection.By-20 DEG C of cell culture fluids preserved with simultaneously with batch hepatitis B virus surface antigen, E antigen
The titer of enzyme linked immunological kit detection HBsAg and HBeAg.Employing following equation calculating antigen
Inhibition percentage=[l-experimental port antigen OD value/control wells antigen OD value] × 100%.Result judges mark
Accurate: effectively: when compound concentration is 10nM, the suppression ratio of its suppression HBsAg, HBeAg >
50%；Invalid: when compound concentration is 10nM, its suppression HBsAg, HBeAg suppression ratio <
50%.It the results are shown in Table shown in 3:
The compositions pair of the extracting solution of the table 3 natural polymorphic arsenic containing extracting solution B
The inhibitory action that epG2.2.15 cell line HBsAg, HBeAg express
Result: in the compositions of the extracting solution of the natural polymorphic arsenic containing extracting solution B, compositions
1,2 and 3 all can have the HBV antigen (HB dose-dependently suppressing HepG2.2.15 cell
SAg, HBeAg) effect, show that it has effective effect on hepatitics B virus in vitro effect, concrete outcome
Table 3.And under conditions of the total amount containing arsenic is identical, with positive As2O3Reference composition 1 ', 2 ' and 3 '
Group is compared, and its inhibitory action of compositions 1,2 and 3 is better than positive control composition group.And the opposing party
Face, compared with single extracting solution B group, although compositions 1 group, its total amount Han arsenic is relatively low 50%,
But its drug influence but higher than single extracting solution B group, illustrates accordingly, and extracting solution B and tumor are bad
After necrosis factor (TNF, 25ng/ml) combination, the compositions of formation, with other compositionss and single sky
The extracting solution of right polymorphic arsenic is compared, and arsenic content reduces, and the medicine effect of its suppression hepatitis B virus effect
Should be optimal, concrete outcome table 3:
1.4, the compositions of the extracting solution of the natural polymorphic arsenic containing extracting solution B, acute to mice
Toxicity test
Extracting solution B is set, containing extracting solution composition B 1,2 and 3 and containing As2O3Solution
The positive respective test group of compositions 1 ', 2 ' and 3 '.Often group takes mice 50, male and female half and half, body weight 18
-20g, is randomly divided into 7 groups, every group 10, mice fasting (can't help water) before experiment mice
After 14 hours, lumbar injection or gavage are administered to mice, are administered volume and are every 20g body weight 0.2mL.
Under room temperature, observe animal behavior activity after administration, record death condition, observe to 7 days, carry out big
Body is dissected, perusal.
Result: the compositions 1,2 and 3 of the extracting solution of the natural polymorphic arsenic containing extracting solution B, not
Observe obvious animal dead situation, thus illustrate that this dosage belongs to the dosage of a safety.
To sum up, the compositions of the extracting solution of the natural polymorphic arsenic of the present invention all can significantly inhibit In vitro culture
Tumor stem cell, acquired immune deficiency syndrome (AIDS) and the growth of hepatitis B virus cell, inhibitory action has obvious dosage and depends on
Lai Xing, compared with single extracting solution, its arsenic content reduces, and relative drug influence is obviously enhanced.Table
Bright above-mentioned composition is the most notable to tumor stem cell, acquired immune deficiency syndrome (AIDS) and hepatitis B effect.Single arsenical, especially
It is As2O3Effect with nanometer-size realgar suppression tumor stem cell has been obtained for confirming, but due to
As2O3Injection is expensive, and can cause strong internal liver, kidney toxic and side effects, and nanometer is male
Although yellow and As2O3Compare its toxicity to reduce, but the activity of its phase suppression tumor stem cell is the most weak
In As2O3, and on the other hand, use multiple metallurgy acidophilic thiobacillus, extract Realgar generation and contain
Have and comprise arsenic acid (III, V), methyl MMA(V) and substantial amounts of auxiliary element (ferrum and the change of ferrum
And thing) mixed extract (patent No. 200610200067.5), although its effect is better than nanometer-size realgar,
But the pharmacodynamics effect of its suppression tumor stem cell is the most indefinite.
Present invention have the advantage that from the above
(1) present invention utilizes the special Marine microorganism with bionical metabolic capacity to natural drug containing arsenic
Mineral carry out microorganism extraction, thus obtain multiple identical from internal metabolism or different, have attenuation,
The extracting solution of the multiple natural form arsenic combination of potentiation, adds camptothecine (camptothecin), length
Spring alkali (VLB), vincristine (VCR), paclitaxel (Taxol), tumor necrosis factor (TNF), white
Cytokine (interleukin, IL-6), interferon (IFN), transforming growth factor-β (TGF-β),
Lipopolysaccharide (LPS), i.e. phorbol exters (TPA), adenyl cyclase activator (Forskolin) and bone
Morphogenetic proteins 4(BMP-4) etc. compatibility be grouped compound, said composition shows potent suppression lung
The medicine effect of cancer stem cell, hepatitis B and HIV (human immunodeficiency virus), its drug influence is apparently higher than positive drug
As2O3Solution, this advantage, novel for screening, As can be substituted2O3Efficient, the arsenical of low toxicity
Provide excellent basis.
(2) extracting solution compositions of the present invention can contain simultaneously arsenic acid (AsIII), arsenic acid (AsV) and
Or monomethyl arsenic acid MMA(III) monomethyl arsenic acid MMA(V) and or dimethyl arsenate DMA(III),
Dimethyl arsenate DMA(V) and or monomethyl monothio arsenic acid MMTA and or Methyl disulfide for arsenic
Acid DMDTA and or TMAO (TMAO) and or AsB (AsB) and or arsenocholine (AsC)
Etc. multiple Soluble Arsenic anatomic element, prepare with nanometer-size realgar and the multiple metallurgy acidophilic thiobacillus of employing
Realgar microorganism mixing leachate is compared, its have uniqueness suppression tumor stem cell effect, and this
Bright extracting solution, while suppression tumor stem cell, has again good suppression hepatitis B and the work of acquired immune deficiency syndrome (AIDS)
With, it is shown that good drug development prospect.
Although the foregoing describing the detailed description of the invention of the present invention, but it is familiar with the technology people of the art
Member should be appreciated that our described specific embodiment is merely exemplary rather than for this
The restriction of bright scope, those of ordinary skill in the art are in the equivalence made according to the spirit of the present invention
Modify and change, all should contain in the scope of the claimed protection of the present invention.
Claims (3)
1. the extracting solution compositions of a natural variform arsenic is to prepare anti-lung tumor dry thin
The purposes of the medicine of born of the same parents, anti-hepatitis B and AIDS virus resisting, it is characterised in that:
The extracting solution compositions of described natural variform arsenic is: containing arsenic in per unit preparation
Compound is: arsenic acid V 1.5nM；Arsenious acid III 0.7nM；Methylarsonic acid III 0.3nM；First
Base arsenic acid V 2.5nM；Monomethyl monothio arsenic acid 0.7nM；Methyl disulfide is for arsenic acid
DMDTA 0.8nM；Dimethyl arsenate DMA 0.6M；Arsenic trimethide oxygen TMAO 1.5nM；
AsB AsB 0.4nM；Arsenocholine AsC 1nM；In per unit preparation, other drug is:
TNF 25ng/ml；Arsenical and other drug combination ratio are 1:1.
2. the extracting solution compositions of a natural variform arsenic is to prepare anti-lung tumor dry thin
The purposes of the medicine of born of the same parents, anti-hepatitis B and AIDS virus resisting, it is characterised in that:
The extracting solution compositions of described natural variform arsenic is: the chemical combination Han arsenic in per unit preparation
Thing is: arsenic acid V 1.5nM；Arsenious acid III 0.7nM；Methylarsonic acid III 0.3nM；Methyl arsenic
Acid V 2.5nM；Monomethyl monothio arsenic acid 0.7nM；Methyl disulfide is for arsenic acid DMDTA
0.8nM；Dimethyl arsenate DMA 0.6M；Arsenic trimethide oxygen TMAO 1.5nM；AsB
AsB 0.4nM；Arsenocholine AsC 1nM；In per unit preparation, other drug is: interleukin 8
Element interleukin IL-6 10ng/ml, arsenical and other drug combination ratio are 1:1.
3. the extracting solution compositions of a natural variform arsenic is to prepare anti-lung tumor dry thin
The purposes of the medicine of born of the same parents, anti-hepatitis B and AIDS virus resisting, it is characterised in that:
The extracting solution compositions of described natural variform arsenic is: the chemical combination Han arsenic in per unit preparation
Thing is: arsenic acid V 1.5nM；Arsenious acid III 0.7nM；Methylarsonic acid III 0.3nM；Methyl arsenic
Acid V 2.5nM；Monomethyl monothio arsenic acid 0.7nM；Methyl disulfide is for arsenic acid DMDTA
0.8nM；Dimethyl arsenate DMA 0.6M；Arsenic trimethide oxygen TMAO 1.5nM；AsB
AsB 0.4nM；Arsenocholine AsC 1nM；In per unit preparation, other drug is: camptothecine
Camptothecin 3umol/L, arsenical and other drug combination ratio are 1:1.
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| 三氧化二砷抗鸭乙型肝炎病毒的体内实验;付美丽;《中国优秀博硕士学位论文全文数据库 (硕士)医药卫生科技辑》;20061215;摘要 * |
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