CN103768583A - Application of extract composition of natural variform arsenite - Google Patents
Application of extract composition of natural variform arsenite Download PDFInfo
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Abstract
The invention provides application of an extract composition of natural variform arsenite. The extract composition comprises extract of natural variform arsenite and at least one of the following anti-tumor medicines: camptothecin, vincaleukoblastinum, vincristine, taxol, tumor necrosis factor, interleukin, interferon, transforming growth factor-beta, lipopolysaccharide, phorbol ester, adenylate cyclase activating agent andbone morphogenetic protein 4, wherein the weight of the extract of the natural variform arsenite accounts for 0.001-99.9 percent of the total weight of the prepared medicine. The extract composition of natural variform arsenite can be used for preparing anti-tumor stem cells, anti-hepatitis B and anti-AIDS (acquired immune deficiency syndrome) virus medicines, can be used for inhibiting hepatitis B and resisting AIDS and other viruses, and expands the application range to development and utilization of natural arsenite preparations.
Description
[technical field]
The present invention relates to a kind of application of extracting solution compositions of natural variform arsenic.
[background technology]
Tumor is the common cancer that threatens human health, although chemicotherapy and operation method constantly make progress, case fatality rate is still higher.For example,, as the total still less than 15% of 5 years survival rates of common type nonsmall-cell lung cancer (non-small cell lung cancer, NSCLC).Based on tumor stem cell (cancer stem cells, CSC) theory is thought, the tumor stem cell with self renewal and unlimited multiplication capacity be tumor occur and recurrence " root " (open source literature is: Yang Ming. the apoptosis-induced effect .[D of nanometer-size realgar to pulmonary carcinoma and hepatoma carcinoma cell and tumor stem cell thereof]. Lanzhou University's doctorate paper: Lanzhou .2010, P10～15).For this reason, for the drug screening strategy that suppresses tumor stem cell, by a new way that is current tumor treatment.
Along with going deep into of research, arsenical (comprises As
2o
3and Realgar) receive publicity again, once because toxicity compared with being reused As greatly and not
2o
3be found to eliminate leukemic stem cells LICs by reducing cancer protein PML level, it may comprise that (open source literature is: Zhang XW for a system, lymphoid malignant tumor by eliminating LICs healing, Yan XJ, Zhou ZR, et al.Arsenic Trioxide Controls the Fate of the PML-RAR Oncoprotein by Directly Binding PML.Science[J], 2010,328:240-243), also find recently, nanometer-size realgar can effectively be induced pulmonary carcinoma and liver-cancer stem cell generation apoptosis.With As
2o
3compare, natural arsenical have aboundresources, cheap, the feature such as can orally use, but on many target spots, the effect that suppresses tumor stem cell, scope and effect range, As
2o
3all show stronger drug influence.This gap, also causes domestic relevant scholar, is the arguement of coarse former powder or internal metabolism arsenic for the effective substance of natural arsenical.Modern analysis proves: arsenic metabolite in animal body, may there are multiple arsenic metabolism species, comprise: (open source literature is as follows: Xu Juhui for iAsIII, iAsV, MMAV, DMAV etc., Wang Hongwei, Cui Rong etc. Realgar and the morphological analysis [J] containing arsenic in Realgar Chinese patent medicine. pharmaceutical analysis magazine, 2007,27 (3): 395～396, Zhang Jinghong, Li Hongyu. high performance capillary electrophoresis is measured realgar microorganism and is concocted arsenic morphology composition [J] in liquid. time precious traditional Chinese medical science traditional Chinese medicines, 2011.22(2): 409～410), for this reason, utilize modern biotechnology, the natural arsenic minerals powder of external bionical metabolism, produce natural solubilized the arsenic multiple and form that internal metabolism is identical or different, composition compositions, replace expensive, clinical use Liver and kidney toxicity strengthens, be difficult to universal arsenic trioxide injection, as supplementing or succedaneum of the anticancer arsenical arsenic trioxide of an existing line, by an important directions that is natural arsenical research.
The research of inventor seminar is found, Marine microorganism metabolism the various form arsenic that dissolve, its arsenic cumulative toxicity in animal tissue significantly reduces, almost not distributing in animal body, (open source literature is 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, Marine microorganism arsenic compound of polymorphic generation, not only can induce multidrug resistance apoptosis of leukemia, and can lower MDR1/P-gp drug resistant gene, reach its reverse multidrug resistance effect (open source literature is as follows: Zhang Jinghong, Li Hongyu. the impact [J] of realgar microorganism extracting solution on K562/ADM cell P-pg albumen and MDRI gene expression. Chinese Clinical pharmacology and therapeutics, 2009,14 (8): 855～861).And the more important thing is, its inductive effect is better than similar arsenical arsenious acid.So, arsenious acid (As
2o
3the principal mode dissolving in water) be not unique active substance of reverse multiple drug resistance of tumor effect, from natural mineral resource, develop the metabolism arsenic of variform as As
2o
3important supplement, will be the drug development direction that the utmost point has application prospect.
Though for example have natural arsenic in China, arsenic disulfide (As
2s
3), Orpimentum (Auripigmentum) (As
2s
3) and arsenic disulfide (As
2o
3, realgar) equal size is abundant, and obtained significant curative effect as clinical medicine at aspects such as treatment tumor, parasite, herpesviruss.But the preparation process of modern report comprises fly+vinegar system of water, the method such as nanorize and micronization, obtain is only suspension, cannot meet the requirement of present pharmaceutical preparation, open China of seminar of the present invention invention 200610200067.5 patents, also successfully utilize multiple metallurgy to use acidophilia Thiobacillus, extraction Realgar produces to contain and comprises arsenic acid (III, V), methyl MMA(V) and the mixed extract of a large amount of auxiliary element (change of ferrum and ferrum and thing) (open source literature is as follows: Li Hongyu, Zhang Jinghong, Zhi Dejuan. the heavy metal removal method in mineral drug. Chinese patent [P]: 200610200067.5, 2006-1-02.), although these methods can obtain the mixed liquor that contains part form arsenic, but owing to being mixed with other compositions of part, cannot determine at present the active component that it is definite, cannot form complex with other drug or adjuvant, in addition the effect of the tumor cell such as its vitro inhibition pulmonary carcinoma and hepatocarcinoma, be starkly lower than As
2o
3, and the more important thing is, have not yet to see the similar effect that it can suppress tumor stem cell, hepatitis B and HIV (human immunodeficiency virus) simultaneously.
In order to obtain the compositions of multiple natural variform, solubilized arsenic, the Chinese invention patent that applicant is 201010580193.4 at application number discloses a kind of Marine microorganism and has extracted natural, polymorphic, to suppress the arsenical extracting solution of tumor preparation method and application thereof, wherein utilize the autonomous multiple Marine microorganism separating of applicant, metabolism arsenic disulfide (As
2o
3, As
4s
4) and or arsenic trisulfide (As
2s
3) obtain 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, Methyl disulfide be for the extracting solution of the multiple natural polymorphic arsenic of arsenic acid DMDTA, TMAO (TMAO), AsB (AsB, arsenobetaine) and arsenocholine (AsC) etc.
[summary of the invention]
The technical problem to be solved in the present invention, is the application of the extracting solution compositions that a kind of natural variform arsenic is provided, and can suppress the virus such as hepatitis B and anti-AIDS, for range of application has been expanded in the exploitation of natural arsenical.
The present invention is achieved in that
A kind of application of extracting solution compositions of natural variform arsenic, the extracting solution compositions of described natural variform arsenic comprises extracting solution and following at least one the antitumor drug of natural variform arsenic, described antitumor drug comprises: camptothecine, vinblastine, vincristine, paclitaxel, tumor necrosis factor, interleukin, interferon, transforming growth factor-beta, lipopolysaccharide, it is Buddhist ripple ester, adenylate cyclase activating agent and bone morphogenetic protein 4, wherein the extracting solution weight of natural variform arsenic accounts for 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, the medicine of anti-hepatitis B and AIDS virus resisting.
Further, in the extracting solution compositions of described natural variform arsenic, contain 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); And the weight sum of above-claimed cpd accounts for 0.001～99.9% of medicine gross weight.
Further, the extracting solution compositions of described natural variform arsenic can be added pharmaceutically acceptable adjuvant and is prepared into 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.
Tool of the present invention has the following advantages:
(1) the special Marine microorganism that utilization of the present invention has a bionical metabolic capacity carries out microorganism extraction to natural drug arsenic-containing ores, thereby obtain multiple identical from internal metabolism or different, there is attenuation, the extracting solution of the multiple natural form arsenic combination of potentiation, add camptothecine (camptothecin), vinblastine (VLB), vincristine (VCR), paclitaxel (Taxol), tumor necrosis factor (TNF), interleukin (interleukin, IL-6), interferon (IFN), transforming growth factor-beta (TGF-β), lipopolysaccharide (LPS), it is Buddhist ripple ester (TPA), the compatibility composition compositionss such as adenylate cyclase activating agent (Forskolin) and bone morphogenetic protein 4 (BMP-4), said composition has shown potent inhibition pulmonary carcinoma stem cell, the drug effect of hepatitis B and HIV (human immunodeficiency virus), its drug influence is apparently higher than positive drug As
2o
3solution, this advantage, novel for screening, can substitute As
2o
3arsenical efficient, low toxicity good basis is provided.
(2) extracting solution compositions of the present invention can contain containing arsenic acid (AsIII simultaneously, AsV), arsenious acid (V) and or monomethyl arsenic acid MMA(III, V) and or dimethyl arsenate DMA(III, V) (DMA) and or monomethyl monothio arsenic acid MMTA and or Methyl disulfide for arsenic acid DMDTA and or TMAO (TMAO) and or AsB (AsB, arsenobetaine) and or the multiple Soluble Arsenic anatomic element such as arsenocholine (AsC), making realgar microorganism mixing leachate with the multiple metallurgy of employing with acidophilia Thiobacillus with nanometer-size realgar compares, it has unique inhibition tumor stem cell effect, and extracting solution of the present invention is in suppressing tumor stem cell, there is again good inhibition hepatitis B and the effect of acquired immune deficiency syndrome (AIDS), show good drug development prospect.
[specific embodiment]
The present invention relates to a kind of application of extracting solution compositions of natural variform arsenic, the extracting solution compositions of described natural variform arsenic comprises extracting solution and following at least one the antitumor drug of natural variform arsenic, described antitumor drug comprises: camptothecine, vinblastine, vincristine, paclitaxel, tumor necrosis factor, interleukin, interferon, transforming growth factor-beta, lipopolysaccharide, it is Buddhist ripple ester, adenylate cyclase activating agent and bone morphogenetic protein 4, wherein the extracting solution weight of natural variform arsenic accounts for 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, the medicine of anti-hepatitis B and AIDS virus resisting.
In the extracting solution compositions of described natural variform arsenic, contain 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); And the weight sum of above-claimed cpd accounts for 0.001～99.9% of medicine gross weight.
The extracting solution compositions of described natural variform arsenic can be added pharmaceutically acceptable adjuvant and is prepared into 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 mono-, prepare the extracting solution of natural variform arsenic
Being 201010580193.4 according to application number, open day be that the disclosed a kind of Marine microorganism of patent of invention of 2011-04-20 extracts the preparation method processing arsenic disulfide (As described in preparation method and the application thereof of natural, polymorphic, to suppress tumor arsenical extracting solution
2o
3, content 36%, As
4s
4, content 62%) and or arsenic trisulfide (As
2s
3, 72%, AsC, 25%) and breeze, embodiment specifically comprises the steps:
Step 1: by arsenic breeze 20g, be crushed to after 200 orders, according to 1:100(weight and mixeding liquid volume ratio) add in the mixed liquor (volume ratio 8:2) of the sulphuric acid of 1mol/L and the composition of 15% ferric chloride, then be that 180r/min, temperature are to cultivate 15d in the constant-temperature table of 30 ℃ at rotating speed, obtain extracting solution; Be the membrane filtration of 0.45 μ m by extracting solution with aperture, obtain filtrate; Described filtrate is to carry out reaction precipitation under 20 ℃, the magnetic agitation rotating speed condition that is 800～1000r/min in temperature, after 2min, filters, and is precipitated thing; Then precipitate is with after deionized water wash, dry at 60 ℃ of temperature, i.e. handy microbial metabolism ultrafine powder fine powder.
Step 2: take step 1 gained micropowder 0.1g, in 250mL conical flask after sterilizing, add LA fluid medium 90mL, the inoculation volume fraction combinatorial optimization Marine microorganism strain that is 20%, then 30 ℃, pH be 5.0 hunting speeds while being 180r/min shaking flask leach 15d, timing is carried 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, Methyl disulfide is for arsenic acid DMDTA, TMAO (TMAO), AsB (AsB, and the detection such as arsenocholine (AsC) arsenobetaine), after the total content of above-mentioned Multiple components reaches requirement, stopping concussion cultivating, after 13000r/min is centrifugal, get supernatant, refilter after degerming, gained supernatant is exactly the extracting solution that contains natural variform arsenic.
Described detection method of content is: capillary electrophoresis separation method: condition is: BGE:PDC/10mmol.L
-1and CTAOH/1mmol.L
-1, voltage 25KV, 25 ℃ of temperature, pH11.0, ultraviolet detection wavelength set be at 216nm.
The extracting solution A of described natural variform arsenic: in per unit preparation, arsenical content composition obtains 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 is prepared according to embodiment 1, and the arsenic breeze of employing is arsenic disulfide (As
2o
3, content 36%, As
4s
4, content 62%), preferably Marine microorganism is combined as: A(alcaligenes aquamarinus): O(brevibacterium flavum): B (Bacillus foecalis alkaligenes) is 5:2:3(volume ratio).
The extracting solution B of described natural variform arsenic: in per unit preparation, arsenical content composition obtains 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%; Methyl disulfide is for arsenic acid DMDTA4%; Dimethyl arsenate (DMA) 3%; TMAO (TMAO) 7.5%; AsB AsB (AsB) 2%; Arsenocholine (AsC) 5%.Its preparation method is prepared according to embodiment 1, and the arsenic breeze of employing is arsenic disulfide (As
2o
3, content 36%, As
4s
4, content 62%), preferably Marine microorganism is combined as: A(alcaligenes aquamarinus): O(brevibacterium flavum): B (Cunninghamella sp) is 8:3:5(volume ratio).
The extracting solution C of described natural variform arsenic: in per unit preparation, arsenical content composition obtains 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 is prepared according to embodiment 1, and the arsenic breeze of employing is arsenic disulfide (As
2o
3, content 36%, As
4s
4, content 62%), preferably Marine microorganism is combined as: A(alcaligenes aquamarinus): O(brevibacterium flavum): B (crescent handle bacillus) is 7:1:6(volume ratio).
Embodiment bis-: the extracting solution compositions of preparing natural variform arsenic
The thing of getting it filled comprises: 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-beta (TGF-β, 2.5ng/ml), lipopolysaccharide (LPS, 1ug/mL), be Buddhist ripple ester (TPA, 20ng/ml), adenylate cyclase activating agent (Forskolin, 20ng/ml) and bone morphogenetic protein 4 (BMP-4, any one drug solution 100ul 10ng/ml) etc., add 0.05mol/L Tri(Hydroxymethyl) Amino Methane Hydrochloride buffer (Tris-HCL) to 1mL, 80 ℃ of water-baths were boiled after 1 hour, after centrifugal filtration, according to drug extract: the extracting solution (1:2 of natural variform arsenic, volume ratio), add extracting solution A or B or the C of natural variform arsenic made in embodiment 1, it is 0.0167u/mL that filtrate adds lyases 50ul(concentration), 37 ℃, enzymatic reaction 2 hours, collecting filtrate filters with the filter membrane of 0.45um, must contain the compositions of the extracting solution of natural variform arsenic.
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 diphosphate glucuronate transferring enzyme (UGT), Epoxide hydrolase (EH), sulfate transferase, Acetylase and transmethylase, catechol-O-methyl transferase, COMT, esterase, hydroxylase flavin monooxygenase (FMO), any one of monoamine oxidase, MAO (MAO) and ketoreductase.
Wherein, in the time that the extracting solution compositions of natural variform arsenic contains extracting solution A, the lyases of employing is B-glucosidase, arsenic acid in compositions (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 arsenical total amount accounts for 50% (percentage by weight) of extracting solution combination of Chinese medicine thing total amount.Wherein, in the time that the extracting solution compositions of natural variform arsenic contains extracting solution B, the lyases of employing is B-glucosidase and CYP3A4, contains 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% (percentage by weight).(percentage by weight).
Wherein, in the time that the extracting solution compositions of natural variform arsenic contains extracting solution C, the lyases of employing is B-glucosidase and CYP3A4, contains 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% (percentage by weight).(percentage by weight).
The extracting solution compositions pharmacodynamic evaluation experimental example of embodiment tri-, natural polymorphic arsenic
1, in following experimental example, given the test agent: the extracting solution compositions of the natural variform arsenic that contains extracting solution B:
Compositions 1: in per unit preparation, arsenical content composition obtains after testing as 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); In per unit preparation, the content of other drug composition obtains after testing as tumor necrosis factor (TNF, 25ng/ml), and other drug and arsenical combination be than being 1:1;
Compositions 2: in per unit preparation, arsenical content composition obtains after testing as 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); In per unit preparation, the content of other drug composition obtains after testing as interleukin (interleukin, IL-6,10ng/ml), and other drug and arsenical combination be than being 1:1;
Compositions 3: in per unit preparation, arsenical content composition obtains after testing as 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); In per unit preparation, the content of other drug composition obtains after testing as camptothecine (camptothecin, 3umol/L), and other drug and arsenical combination be than being 1:1;
Positive control compositions 1 ': in per unit preparation, arsenical content composition obtains after testing as As
2o
3(arsenic acid V, 5nM); In per unit preparation, the content of other drug composition obtains after testing as tumor necrosis factor (TNF, 25ng/ml), and other drug and arsenical combination be than being 1:1;
Positive control compositions 2 ': in per unit preparation, arsenical content composition obtains after testing as As
2o
3(arsenic acid V, 5nM); In per unit preparation, the content of other drug composition obtains after testing as interleukin (interleukin, IL-6,10ng/ml), and other drug and arsenical combination be than being 1:1;
Positive control compositions 3 ': As
2o
3(arsenic acid V, 5nM); In per unit preparation, the content of other drug composition obtains after testing as camptothecine (camptothecin, 3umol/L), and other drug and arsenical combination be than being 1:1;
Independent extracting solution B group: in per unit preparation, arsenical content composition obtains after testing as 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);
The growth inhibited effect of the human lung cancer cell A549 cell of the extracting solution compositions of the natural polymorphic arsenic that 1.1, contains extracting solution B to In vitro culture
A. common A549 cell culture: common A549 cell, in the RPMI1640 culture medium that contains 10% hyclone, at 37 ℃, is cultured to exponential phase under saturated humidity condition in 5%CO2 incubator.
The tumor stem cell of b.A549 cell ball is cultivated: by after the A549 cell dissociation cell of common logarithm trophophase, change into and contain 20l/ml B27, 20ng/ml EGF, the DMEM/F12 culture medium of 20ng/ml bFGF continues to be cultured to logarithmic growth after date peptic cell again, and be made into single cell suspension and plant in 6 orifice plates with the density in 20000/hole, every hole 2ml culture fluid, change liquid every 2～3d, half amount, at 37 ℃, under 5%CO2 condition, cultivate after 4-5 days, observation of cell balling-up situation and form under microscope, continuous passage 3 times, get the 3rd generation cell ball, stem cell transcription factor Sox2 and Oct4 expression in the tumor stem cell of utilization RT-PCR detection A549 cell ball, the tumor stem cell that screening Sox2 and Oct4 express higher A549 cell ball enters cell proliferation experiment.
C. the extracting solution compositions of the natural polymorphic arsenic of the present invention suppresses the tumor stem cell proliferation function evaluation of A549 cell ball:
Different extracting solution suppress A549 cell and cell ball multiplication capacity detects: A549 cell and A549 cell ball are made to single cell suspension by 1 × 10
4the density in/hole adds respectively 96 orifice plates, and every hole 100ul adds respectively extracting solution B, contains extracting solution B compositions 1,2 and 3 and contain As
2o
3the positive compositions 1 ', 2 ' and 3 ' of solution, matched group only adds culture medium at 37 ℃, under 5%CO2 condition, cultivate after 72h, add 90uL basal medium and MTT10uL (5mg/ml), 37 ℃, 4h, remove culture fluid, every hole adds 150ul DMSO, rocks 10min microplate reader and measures optical density value ［ D (490) ］, adopt 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 both be to the growth of the tumor stem cell of the human lung cancer cell A549 of In vitro culture and A549 cell ball, there is good inhibition proliferation function, and under the condition identical containing arsenic total amount, with positive As
2o
3reference composition 1 ', 2 ' is compared with 3 ' group, and compositions 1,2 and 3 its inhibitory action are better than positive control compositions group.And on the other hand, compared with independent extracting solution B group, although 1,2 and 3 groups of compositionss, it has reduced by 50% containing arsenic total amount, but its drug influence is but higher than independent extracting solution B group, explanation accordingly, after extracting solution B and other drug combination, the present composition of formation, arsenic content reduces, its drug influence that suppresses tumor stem cell is better, apparently higher than the extracting solution of single natural polymorphic arsenic, and concrete outcome table 1:
The extracting solution compositions of the natural polymorphic arsenic that table 1 contains 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
The extracting solution compositions of the natural polymorphic arsenic that 1.2, contains extract B, the growth inhibited effect of the TZM-bl cell to Infection in Vitro acquired immune deficiency syndrome (AIDS) HIV-1IIIB virus
The take the logarithm TZM-BL cell of trophophase, by 2 × 10
4/ mL hole density is inoculated in 96 porocyte culture plates, and 100 μ L/ holes are placed in cell culture incubator (37 ℃, 5%CO2) and cultivate.The next day treat cell attachment and well-grown, suck culture fluid, then adding respectively extracting solution B, contain extracting solution B compositions 1,2 and 3 and contain As with serum-free medium dilution
2o
3the positive compositions 1 ', 2 ' and 3 ' of solution, and add respectively 96 well culture plates, three parallel holes are set, 37 ℃, 5%CO
2cultivate after 3h, add the HIV-1IIIB virus 100 μ L infection cells of 1000 × TCID50, then continue to cultivate after 24h, utilize Bright-Glo Luciferase Assay reagent (Promega) to measure the relative fluorescence unit (RLU) in every hole, according to the half-inhibition concentration IC50 of Prism Graphruan computed in software compound, result criterion: effectively IC50<10nM, invalid IC50>10nM.IC50 the results are shown in Table shown in 2:
The growth inhibited effect of the TZM-bl cell of the compositions of the extracting solution of the natural polymorphic arsenic that table 2 contains extracting solution B to Infection in Vitro acquired immune deficiency syndrome (AIDS) HIV-1IIIB virus
Result: in the extracting solution compositions of the natural polymorphic arsenic that contains extracting solution B, compositions 1,2 and 3 can both be to suppressing the growth of TZM-bl cell of Infection in Vitro acquired immune deficiency syndrome (AIDS) HIV-1IIIB virus, show that it has the effect of effective In Vitro Anti HIV (human immunodeficiency virus), and under the condition identical containing arsenic total amount, with positive As
2o
3reference composition 1 ', 2 ' is compared with 3 ' group, and compositions 1,2 and 3 its inhibitory action are better than positive control compositions group.And on the other hand, compared with independent extracting solution B group, although 1,2 and 3 groups of compositionss, it has reduced by 50% containing arsenic total amount, but its drug influence is but higher than independent extracting solution B group, explanation accordingly, after extracting solution B and other drug combination, the present composition of formation, arsenic content reduces, and its drug influence that suppresses HIV (human immunodeficiency virus) effect is better, apparently higher than the extracting solution of single natural polymorphic arsenic, concrete outcome table 2:
The extracting solution compositions of the natural polymorphic arsenic that 1.3, contains extracting solution B, the effect that the external HBV antigen of HepG2.2.15 cell is suppressed
The take the logarithm HepG2.2.15 cell of trophophase, by 2 × 10
4/ mL hole density is inoculated in 24 porocyte culture plates, 1000 μ L/ holes, and each concentration is established 3 multiple holes altogether, puts in cell culture incubator (37 ℃, 5%CO2) and cultivates.The next day treat cell attachment and well-grown, suck culture fluid, add respectively extracting solution B, contain extracting solution B compositions 1,2 and 3 and contain As
2o
3the positive compositions 1 ', 2 ' and 3 ' of solution, the complete culture solution 1000 μ L of each concentration, each concentration is established 3 multiple holes, puts in cell culture incubator (37 ℃, 5%CO2) and cultivates.The complete culture solution of equivalent is made blank, after continuous culture 72h, draws cell culture fluid and is added on respectively in the aseptic Eppnedorf pipe of 1.5mL, to be checked in-20 ℃ of preservations.The cell culture fluid of-20 ℃ of preservations is detected to tiring of HBsAg and HBeAg with the while with batch hepatitis B virus surface antigen, E antigen enzyme linked immunological kit.Adopt following formula calculate antigen inhibition percentage=[l-experimental port antigen OD value/control wells antigen OD value] × 100%.Result criterion: effectively: when compound concentration is 10nM, it suppresses the suppression ratio >50% of HBsAg, HBeAg; Invalid: when compound concentration is 10nM, it suppresses the suppression ratio <50% of HBsAg, HBeAg.It the results are shown in Table shown in 3:
The inhibitory action that the compositions of the extracting solution of the natural polymorphic arsenic that table 3 contains extracting solution B is expressed epG2.2.15 cell line HBsAg, HBeAg
Result: in the compositions of the extracting solution of the natural polymorphic arsenic that contains extracting solution B, compositions 1,2 and 3 all can have dose dependent and suppress the effect of the HBV antigen (HB sAg, HBeAg) of HepG2.2.15 cell, show that it has effective effect on hepatitics B virus in vitro effect, concrete outcome table 3.And under the condition identical containing arsenic total amount, with positive As
2o
3reference composition 1 ', 2 ' is compared with 3 ' group, and compositions 1,2 and 3 its inhibitory action are better than positive control compositions group.And on the other hand, compared with independent extracting solution B group, although 1 group of compositions, its containing arsenic total amount lower 50%, but its drug influence, but higher than independent extracting solution B group, illustrates extracting solution B and tumor necrosis factor (TNF accordingly, 25ng/ml) after combination, the compositions forming, compares with the extracting solution of other compositionss and single natural polymorphic arsenic, and arsenic content reduces, and it suppresses drug influence the best of hepatitis B virus effect, concrete outcome table 3:
The compositions of the extracting solution of the natural polymorphic arsenic that 1.4, contains extracting solution B, to the acute toxicity testing of mice
Extracting solution B is set, contains extracting solution B compositions 1,2 and 3 and contain As
2o
3positive compositions 1 ', 2 ' and the 3 ' test group separately of solution.Get 50 of mices for every group, male and female half and half, body weight 18-20g, is divided into 7 groups at random mice, 10 of every groups, before experiment, mice fasting (can't help water) is after 14 hours, and lumbar injection or gavage are to mice administration, and administration volume is every 20g body weight 0.2mL.After administration, under room temperature, observe animal behavior activity, record death condition, observe to 7 days, carry out gross anatomy, perusal.
Result: the compositions 1,2 and 3 of the extracting solution of the natural polymorphic arsenic that contains extracting solution B, do not observe obvious animal dead situation, illustrate that thus this dosage belongs to the dosage of a safety.
To sum up, the compositions of the extracting solution of natural polymorphic arsenic of the present invention all can significantly suppress the growth of tumor stem cell, acquired immune deficiency syndrome (AIDS) and the hepatitis B virus cell of In vitro culture, inhibitory action has obvious dose dependent, compared with single extracting solution, its arsenic content reduces, and relative drug influence significantly strengthens.Show that above-mentioned composition is more remarkable to tumor stem cell, acquired immune deficiency syndrome (AIDS) and hepatitis B effect.Single arsenical, particularly As2O
3the effect that suppresses tumor stem cell with nanometer-size realgar has obtained confirmation, but due to As
2o
3injection is expensive, and can cause liver, kidney toxic and side effects in strong body, although nanometer-size realgar and As
2o
3compare its toxicity and reduce, but its activity that suppresses mutually tumor stem cell is obviously weaker than As
2o
3and on the other hand, adopt multiple metallurgy to use acidophilia Thiobacillus, extraction Realgar produces to contain and comprises arsenic acid (III, V), methyl MMA(V) and the mixed extract (patent No. 200610200067.5) of a large amount of auxiliary element (change of ferrum and ferrum and thing), although its effect is better than nanometer-size realgar, its pharmacodynamics effect that suppresses tumor stem cell is also indefinite.
Tool of the present invention has the following advantages from the above:
(1) the special Marine microorganism that utilization of the present invention has a bionical metabolic capacity carries out microorganism extraction to natural drug arsenic-containing ores, thereby obtain multiple identical from internal metabolism or different, there is attenuation, the extracting solution of the multiple natural form arsenic combination of potentiation, add camptothecine (camptothecin), vinblastine (VLB), vincristine (VCR), paclitaxel (Taxol), tumor necrosis factor (TNF), interleukin (interleukin, IL-6), interferon (IFN), transforming growth factor-beta (TGF-β), lipopolysaccharide (LPS), it is Buddhist ripple ester (TPA), the compatibility composition compositionss such as adenylate cyclase activating agent (Forskolin) and bone morphogenetic protein 4 (BMP-4), said composition has shown potent inhibition pulmonary carcinoma stem cell, the drug effect of hepatitis B and HIV (human immunodeficiency virus), its drug influence is apparently higher than positive drug As
2o
3solution, this advantage, novel for screening, can substitute As
2o
3arsenical efficient, low toxicity good basis is provided.
(2) extracting solution compositions of the present invention can contain arsenic acid (AsIII) simultaneously, 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 the multiple Soluble Arsenic anatomic element such as arsenocholine (AsC), making realgar microorganism mixing leachate with the multiple metallurgy of employing with acidophilia Thiobacillus with nanometer-size realgar compares, it has unique inhibition tumor stem cell effect, and extracting solution of the present invention is in suppressing tumor stem cell, there is again good inhibition hepatitis B and the effect of acquired immune deficiency syndrome (AIDS), show good drug development prospect.
Although more than described the specific embodiment of the present invention; but being familiar with those skilled in the art is to be understood that; our described specific embodiment is illustrative; rather than for the restriction to scope of the present invention; those of ordinary skill in the art are in equivalent modification and the variation done according to spirit of the present invention, all should be encompassed in the scope that claim of the present invention protects.
Claims (3)
1. the application of the extracting solution compositions of a natural variform arsenic, it is characterized in that: the extracting solution compositions of described natural variform arsenic comprises extracting solution and following at least one the antitumor drug of natural variform arsenic, described antitumor drug comprises: camptothecine, vinblastine, vincristine, paclitaxel, tumor necrosis factor, interleukin, interferon, transforming growth factor-beta, lipopolysaccharide, it is Buddhist ripple ester, adenylate cyclase activating agent and bone morphogenetic protein 4, wherein the extracting solution weight of natural variform arsenic accounts for 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, the medicine of anti-hepatitis B and AIDS virus resisting.
2. the application of the extracting solution compositions of a kind of natural variform arsenic as claimed in claim 1, is characterized in that: in the extracting solution compositions of described natural variform arsenic, contain 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); And the weight sum of above-claimed cpd accounts for 0.001～99.9% of medicine gross weight.
3. the application of the extracting solution compositions of a kind of natural variform arsenic as claimed in claim 1, it is characterized in that: the extracting solution compositions of described natural variform arsenic can be added pharmaceutically acceptable adjuvant and is prepared into 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.
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