CN105168248A - Application of sodium arsenite to preparation of medicine for treating echinococcosis - Google Patents
Application of sodium arsenite to preparation of medicine for treating echinococcosis Download PDFInfo
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- CN105168248A CN105168248A CN201510446010.2A CN201510446010A CN105168248A CN 105168248 A CN105168248 A CN 105168248A CN 201510446010 A CN201510446010 A CN 201510446010A CN 105168248 A CN105168248 A CN 105168248A
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- protoscolex
- sodium arsenite
- echinococcus granulosus
- naaso
- granulosus cysts
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Abstract
The invention discloses application of sodium arsenite to preparation of medicine for treating echinococcosis. The sodium arsenite is used for killing echinococcosis pathogen, namely echinoeococcus gransosus. The obvious effect of inhibiting and killing echinococcus protoscolex is achieved, so that the treatment on the echinococcosis is realized.
Description
Technical field
The present invention relates to the pharmaceutical applications of sodium arsenite, belong to medicinal chemistry art.
Background technology
Echinococcosis granulosa (echinococcosis), i.e. cystic echinococcosis (cysticechinococcosis, CE), that a kind of Major Epidemic is in the infecting both domestic animals and human parasitic disease in herding area, by Echinococcus granulosus (echinococcosisgranulosus, Eg) middle silk ribbon phase parasitized larvae is caused by the human body, each internal organs of whole body can be occurred in, the most common with echinococciasis of liver (echinococcosisoftheliver) clinically, accounting for 75%, is secondly echinococcosis pulmonum; This disease is global distribution, common with animal husbandry developed regions such as Xinjiang, Qinghai, Gansu, Ningxia, Tibet, the Inner Mongol, Shaanxi and western Sichuan in China.
At present, the first-selected surgical operation therapy of hepatic echinococcosis, is aided with Drug therapy.Traditional operation mainly contains Bile fistula excision of internal capsule, Bile fistula excision of internal capsule+external capsule partial enucleation art and partial hepatectomy, the complete enucleation of external capsule in Bile fistula adventitia adopted in recent years, although the relapse rate of echinococcosis greatly can be reduced, farthest reduce operation to hepatic parenchymal damage, but when hydatidoma volume is comparatively large or when pressing close to important organ or pipeline (hepatic portal or bile duct), then need to select Bile fistula intracapsular extraction or Bile fistula intracapsular extraction+external capsule ablation.This kind of art formula may have leaking outside of cephalomere and remain, biological characteristics due to echinococcus is different from general occupancy focus, recurrence after operation, send out transfer, not extract completely and anaphylactic shock etc. is difficult to avoid, thus in art and the postoperative medicine auxiliary treatment that needs prevent Echinococcus hydatid cyst recurrence.Therefore, the problem dealt with improperly of and cephalomere excessive for cephalomere in art, finds suitable local chemotherapeutic drug and oral anti-Echinococcus hydatid cyst medicine in art most important for the treatment of hepatic echinococcosis.
Based on this, this area has carried out trial and the screening of multi-medicament in vitro, and clinical conventional local chemotherapeutic drug has 20% hypertonic saline, and 15min 100% can kill Echinococcus Granulosus Cysts protoscolex, but there is certain destruction to surrounding normal hepatic tissue, be only limitted to lavation external capsule; Also there is the ethanol of research existing 95% effectively can kill protoscolex equally, but limit its application because a series of serious liver metabolism function can be caused; Presently, the defect of this type of medicine existence treatment aspect all in various degree generally.
Therefore, need badly and find the treatment of short, efficient, safe chemotherapeutics of a kind of used time for hepatic echinococcosis.
Summary of the invention
Sodium arsenite (sodiumarsenite, NaAsO is found in applicant's research process
2) can effectively suppress and kill the growth of protoscolex, thus can be used for the clinical treatment of echinococcosis granulosa.
In the present invention, sodium arsenite (NaAsO
2), be a kind of trivalent arsenical, its molecular weight is 129.91, white powdery solids, and micro-have hygroscopy, soluble in water, and solution is alkalescence, and sodium arsenite has the effect of inducing cell hypertrophy and apoptosis.
First, the invention discloses the application of sodium arsenite in preparation treatment Echinococcus Granulosus Cysts medicine.
Above-mentioned medicine, both can be simple using sodium arsenite as active raw materials, can also be the various preparations that sodium arsenite is made as active component and other adjuvants, such as common tablet, capsule, drop pill, transfusion, freeze-dried powder etc., for the selection of pharmaceutical dosage form and corresponding adjuvant thereof, those skilled in the art can adjust as required.
Above-mentioned echinococcosis granulosa both can be on animal body, also can be on human body, according to the clinical onset position of this disease, be commonly referred to as Cystic hydatidosis.
Therefore, the invention also discloses the application of sodium arsenite in preparation treatment Cystic hydatidosis medicine.
As required, can use the sodium arsenite of variable concentrations, in general, in order to effectively kill cause of disease, the concentration of sodium arsenite is not less than 8 μm of ol/L.
For common protoscolex stand density, preferably, the concentration of sodium arsenite is 16-20 μm of ol/L.
Preferably, the continuous use time is not less than 4 days.
Under above-mentioned concentration, to Echinococcus Granulosus Cysts protoscolex, there is more satisfactory suppression and killing effect.
On this basis, the invention also discloses the application of sodium arsenite as Echinococcus Granulosus Cysts protoscolex inhibitor.
It will be appreciated by those skilled in the art that, under kinds of experiments environment, especially in animal isolated experiment situation, in order to prevent due to the growth in vitro of the Echinococcus Granulosus Cysts protoscolex in animal body and cephalomere excessive, need to add necessary inhibitor, such as 20% hypertonic saline etc.
Similar to the above, the concentration of sodium arsenite is not less than 8 μm of ol/L, and preferably, the concentration of sodium arsenite is 16-20 μm of ol/L.
Accompanying drawing explanation
Fig. 1 is the impact of sodium arsenite on Echinococcus Granulosus Cysts protoscolex form, wherein, and A: matched group protoscolex; B:8 μm of ol/LNaAsO
2the protoscolex of effect 4d; C:8 μm of ol/LNaAsO
2the protoscolex of effect 8d; D:16 μm of ol/LNaAsO
2the protoscolex of effect 4d; E:16 μm of ol/LNaAsO
2the protoscolex of effect 8d.
Fig. 2 is that the sodium arsenite of variable concentrations is on the impact of Echinococcus Granulosus Cysts protoscolex vigor.
Fig. 3 is the impact of sodium arsenite on Echinococcus Granulosus Cysts protoscolex Surface Ultrastructure, wherein, and A: matched group protoscolex; B:8 μm of ol/LNaAsO2 effect protoscolex 3d; C:16 μm of ol/LNaAsO2 effect protoscolex 3d.
Fig. 4 is the impact of sodium arsenite on Caspase-3 enzymatic activity in Echinococcus Granulosus Cysts protoscolex.
Detailed description of the invention
In order to sodium arsenite (NaAsO is described
2) effect, applicant carried out experiment in vitro illustrate sodium arsenite to the killing effect of Echinococcus Granulosus Cysts protoscolex.
Experiment 1:NaAsO
2interaction in vitro is in the effect of Echinococcus Granulosus Cysts protoscolex
Get fresh natural infect the sheep liver of Echinococcus Granulosus Cysts and taken back laboratory, rinse liver surface well with water, and with 75% alcohol disinfecting surface, put in sterile culture flask with the capsule liquid that sterile manner extracts containing protoscolex.Supernatant discarded capsule liquid after protoscolex natural sedimentation, aseptically, clean 3-5 time with PBS liquid (PH:7.2), 0.1% eosin stain makees dye liquor and repels experiment, vigor >=98%, observing protoscolex polypide under inverted microscope is interior swaged, is dispersed in densely covered, and polypide structure is full, ovalize, clear in structure is complete, and internal structure is clear, and calcium granule is limpid and obvious.
Collect Echinococcus Granulosus Cysts protoscolex, then protoscolex is divided (penicillin 100U/mL, streptomycin 100U/mL) in the RPMI1640 culture medium be filled to containing 10% calf serum, put 37 DEG C, 5%CO
2cultivate in incubator, every 3d changes a culture fluid, stand-by.
Experiment grouping: RMPI-1640 culture medium blank group, 4 μm of ol/LNaAsO
2group, 8 μm of ol/LNaAsO
2group, 12 μm of ol/LNaAsO
2group, 16 μm of ol/LNaAsO
2group, 20 μm of ol/LNaAsO
2group.Get six orifice plates, often Kong Junshe 5mL system, by above grouping, prepare corresponding culture fluid.
5 days will be cultivated in vitro and the Echinococcus Granulosus Cysts protoscolex PBS (pH=7.2) conformed cleans 3 times, 0.1% eosin stains, observes its survival rate (>=98%) under inverted microscope, often organize in culture fluid and add about 2000 protoscolexs, put 37 DEG C, 5%CO
2cultivate in incubator, under inverted microscope, observe NaAsO
2on the impact of Echinococcus Granulosus Cysts protoscolex form and vigor.
Data statistical approach: after dosing, 24h starts, often organizes and evenly extracts 150 μ L culture fluid (average containing procephalon saving 80-100) every day, with 0.1% eosin stains 15min, observe the vigor of protoscolex under inverted microscope.The protoscolex of living refuses dye, not painted, and has activeness; Dead or that vigor is poor protoscolex is red stained with color, with structural deterioration.Calculate and often organize average mortality every day, Continuous Observation 9d.Change liquid once (when often organizing system and concentration dosing of same first time) every 3d, the independent repetition of experiment is averaged for 3 times, and draws Dynamic Curve figure.
Experiment 2:NaAsO
2the change of protoscolex Surface Ultrastructure after effect
While survey vigor, get corresponding protoscolex at random and do scanning electron microscope, observe the Ultrastructural change of protoscolex.PBS washs 3 times, each 5min; Put 4% glutaraldehyde and fix 24h.Protoscolex is put into the ethanol serial dehydration step by step that concentration is 50%, 70%, 80%, 90%, 100%, dewatering time is respectively 5min, 10min, 30min, 1h to spending the night, critical point drying, vacuum metal spraying LOOA, observes the change of protoscolex Surface Ultrastructure under then putting LE01430VP scanning electron microscope.
Experiment 3:NaAsO
2on the impact of Caspase-3 enzymatic activity in protoscolex
Get NaAsO
2each group of Echinococcus Granulosus Cysts protoscolex after effect 24h, PBS adds liquid nitrogen grinding after cleaning 3 times, illustrate by test kit, in every 3mg cephalomere, add 100 μ l lysates, then pulverize with cell Ultrasonic Pulverization instrument, 10min is placed, 4 DEG C, after the centrifugal 15min of 1600g, Aspirate supernatant in ice bath.Protein concentration is measured, by the concentration trim of each group of institute's leach protein by Bradford determination of protein concentration method.By specification reaction system adds ELISA Plate, and establishes blank group, and 37 DEG C of constant-temperature incubation casees are hatched, and detect absorbance (A by microplate reader
405value).Yellow paranitroanilinum pNA is produced with tetrapeptides compound Ac-DEVD-pNA colourless in the caspase-3 catalytic reagent activated in sample, according to the A405 value of standard curve and sample, the pNA concentration generated in calculation sample, infers the activity of caspase-3 thus.Independent repetition 3 times will be tested.
Experimental result:
With reference to figure 1, be NaAsO
2the metamorphosis of In vitro culture Echinococcus Granulosus Cysts protoscolex, 0.1% eosin stains, observes under inverted microscope, NaAsO
2protoscolex after effect is many in the type that turns up, the little hook arrangement disorder on protoscolex rostellum, partial exfoliation, sucker projection, distortion, hypoergy or death.Along with NaAsO
2the prolongation of action time, more obvious to the lethal effect of Echinococcus Granulosus Cysts protoscolex, as seen by the protoscolex increasing number of red dye under light microscopic, the contracting of polypide group, smaller volume, edge irregularity, calcium granule obviously reduces.And the protoscolex of blank group to refuse dye not painted, rostellum protrudes or recessed, activeness good (Fig. 1).
With reference to figure 2, be NaAsO
2the external lethal effect to Echinococcus Granulosus Cysts protoscolex.Variable concentrations NaAsO
2the Echinococcus Granulosus Cysts protoscolex vitality test of (4 μm of ol/L, 8 μm of ol/L, 12 μm of ol/L, 16 μm of ol/L, 20 μm of ol/L) In vitro culture 9d the results are shown in Figure 2, display variable concentrations NaAsO
2all inhibitory action is had to external Echinococcus Granulosus Cysts protoscolex.Wherein 20 μm of ol/LNaAsO
2effect 6d protoscolex mortality rate reaches 100%.16 μm of ol/L effect 6d protoscolex mortality rates are 16.76%.As can be seen from Dynamic Curve figure, along with the increase of drug level and the prolongation of administration time, the suppression ratio of protoscolex increases, and this effect has dose dependent and time dependence.Each experimental group protoscolex mortality rate difference compared with matched group all has statistical significance (P < 0.05).
Meanwhile, applicant observes NaAsO under scanning electron microscope (SEM)
2protoscolex Surface Ultrastructure change after effect, observe display blank group protoscolex smooth surface, many in interior swaged, cephalomere is spherical in shape or oval, and rostellum is complete, and microtriche is high-visible; With 8 μm of ol/LNaAsO
2after effect Echinococcus Granulosus Cysts protoscolex 3d, observe discovery under a scanning electron microscope, protoscolex is in the type that turns up, and body slightly subsides, rostellum slight deformation, and microtriche arrangement is more regular, has no and obviously comes off.16 μm of ol/LNaAsO
2after effect Echinococcus Granulosus Cysts protoscolex 3d, there is digitation in surface, sucker distortion, depression, and head hook defect, rostellum destroys, microtriche arrangement disorder or disappearance (Fig. 3).
In addition, Caspase-3 test kit is adopted to detect NaAsO
2fig. 4 is shown in, NaAsO to the impact of Caspase-3 enzymatic activity in external Echinococcus Granulosus Cysts protoscolex
2after effect 24h, in each group of protoscolex, Caspase-3 activity expression all strengthens (P < 0.05), and increase along with activity, Caspase-3 activity expression also strengthens gradually, 20 μMs of group effects the strongest (P < 0.05).
To sum up can see, variable concentrations NaAsO
2external all have inhibitory action to Echinococcus Granulosus Cysts protoscolex, and concentration is that the NaAsO2 of 20 μm of ol/L is the most obvious to Echinococcus Granulosus Cysts protoscolex lethal effect, and along with the prolongation of time, inhibitory action significantly increases (P < 0.05).At confirmation NaAsO
2have on inhibiting basis to protoscolex, explore the NaAsO of variable concentrations
2to the Morphology Effects of Echinococcus Granulosus Cysts protoscolex.Result of study shows, NaAsO
2after effect, the form generation change in various degree of external Echinococcus Granulosus Cysts protoscolex can be caused.Interior swaged and the type protoscolex that turns up are the multi-form of polypide existence.When condition is suitable for, protoscolex rostellum indent, can protect a hook, sucker, microtriche to be without prejudice; When adding NaAsO
2after, the protoscolex of the type that turns up as seen under light microscopic increases, ditch arrangement disorder, and sucker is out of shape.The microtriche of the Echinococcus Granulosus Cysts protoscolex stratum germinativum of normal cultivation physically well develops, quantity is many, adhere to and extend in horny layer, stratum germinativum and cuticular contact area can be increased, be conducive to the absorption of nutrient substance, without when blood supply when echinococcus still can comparatively fast grow and constantly form brood capsule.Variable concentrations NaAsO
2echinococcus Granulosus Cysts protoscolex after effect, observes under scanning electron microscope, and defect in various degree appears in microtriche, and digitation appears in body surface, and rostellum destroys serious, the growth of protoscolex is suppressed and dead.
In a word, NaAsO
2external have lethal effect to Echinococcus Granulosus Cysts protoscolex.Further, variable concentrations NaAsO
2after effect Echinococcus Granulosus Cysts protoscolex, Caspase-3 enzymatic activity in protoscolex can be made to strengthen, and showed cell apoptosis take part in NaAsO
2suppress the process of Echinococcus Granulosus Cysts protoscolex growth.
Claims (7)
1. the application of sodium arsenite in preparation treatment Echinococcus Granulosus Cysts medicine.
2. application according to claim 1, is characterized in that the concentration of sodium arsenite is not less than 8 μm of ol/L.
3. application according to claim 2, is characterized in that the concentration of sodium arsenite is 16-20 μm of ol/L.
4. the application of sodium arsenite in preparation treatment Cystic hydatidosis medicine.
5. sodium arsenite is as the application of Echinococcus Granulosus Cysts protoscolex inhibitor.
6. application according to claim 5, is characterized in that the concentration of sodium arsenite is not less than 8 μm of ol/L.
7. application according to claim 6, is characterized in that the concentration of sodium arsenite is 16-20 μm of ol/L.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104357445A (en) * | 2014-10-23 | 2015-02-18 | 吕海龙 | Interfering RNA for inhibiting and treating echinococcus granulosus disease and application thereof |
| CN104382911A (en) * | 2014-10-23 | 2015-03-04 | 姜玉峰 | Application of tauroursodeoxycholic acid salt used as active ingredient in preparation of drug for treating echinococcosis |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104357445A (en) * | 2014-10-23 | 2015-02-18 | 吕海龙 | Interfering RNA for inhibiting and treating echinococcus granulosus disease and application thereof |
| CN104382911A (en) * | 2014-10-23 | 2015-03-04 | 姜玉峰 | Application of tauroursodeoxycholic acid salt used as active ingredient in preparation of drug for treating echinococcosis |
Non-Patent Citations (3)
| Title |
|---|
| JULIA A.LOOS: "Identification and pharmacological induction of autophagy in the larval stages of Echinococcus:granulosus-an active catabolic process in calcareous corpuscles", 《INTERNATIONAL JOURNAL FOR PARASITOLOGY》 * |
| 彭心宇等: "不同药物对小鼠肝细粒棘球蚴术后感染的抑制作用", 《世界华人消化杂志》 * |
| 祁周约: "砷及砷剂的治疗作用和毒性", 《中国兽医科技》 * |
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Application publication date: 20151223 |
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