CN104997792A - Application of lithocholic acid serving as active ingredient in preparation of echinococcosis granulosis cyst treatment medicine - Google Patents
Application of lithocholic acid serving as active ingredient in preparation of echinococcosis granulosis cyst treatment medicine Download PDFInfo
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- CN104997792A CN104997792A CN201510457052.6A CN201510457052A CN104997792A CN 104997792 A CN104997792 A CN 104997792A CN 201510457052 A CN201510457052 A CN 201510457052A CN 104997792 A CN104997792 A CN 104997792A
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Abstract
The invention discloses application of lithocholic acid serving as an active ingredient in preparation of echinococcosis granulosis cyst treatment medicine. The echinococcosis granulosis cyst treatment medicine can kill pathogeny-echinococcosis granulosis cyst tapeworms of echinococcosis granulosis cyst through lithocholic acid, has the remarkable restraining and killing effects, and can be used for treating echinococcosis granulosis cyst.
Description
Technical field
The present invention relates to the pharmaceutical applications of lithocholic acid, belong to medicinal chemistry art.
Background technology
Echinococcosis granulosa (echinococcosis) is also known as cystic echinococcosis (cystic echinococcosis, CE), by a kind of serious harm people ' s health of middle silk ribbon phase of Echinococcus granulosus (Echinococcus granulosus, Eg) caused by larva and the parasitic zoonoses of animal husbandry development.This disease is global distribution, at China's Major Epidemic in Xinjiang, Qinghai, the Inner Mongol, the animal husbandry prosperity such as Tibet provinces and regions.Cystic echinococcosis major effect liver, also can develop in lung.At present, the treatment mainly operating method of this disease is directed to, i.e. external capsule Complete excision in Bile fistula adventitia.Although the complete enucleation of external capsule can reduce the relapse rate of echinococcosis greatly in Bile fistula adventitia adopted in recent years, 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 the complete enucleation of external capsule in open adventitia.This kind of art formula may have leaking outside of cephalomere and remain, and prevents Echinococcus hydatid cyst recurrence in art with the postoperative medicine auxiliary treatment that needs.Therefore all in all, in art, dealing with improperly of the excessive and cephalomere of cephalomere is the defect being difficult to overcome that operative treatment cystic echinococcosis exists.
For the problems referred to above, this area attempts adopting high-concentration acidic wastewater (HCl) and alkaline solution (KOH) in vitro, and clinical conventional local chemotherapeutic drug has 20% hypertonic saline, but has certain destruction to surrounding normal hepatic tissue; This area also once studies have found that bile can suppress the growth of hydatidoma, but bile component is complicated, effect target is many, therapeutic effect is changeable, such as the delay in liver and blood of bile, bile acid can produce multiple toxic action, comprise cytotoxicity and acute and chronic toxic action, cause hepatic injury and whole body multisystem, multiple organ injury, finally cause liver cirrhosis, liver failure and death; Also once studies have found that bile acid formed damage etc. to pancreatic acinar cell.
Therefore, obtain a kind ofly act on definite, clear curative effect, the medicine that effectively can prevent and treat echinococcosis granulosa remains this area problem urgently to be resolved hurrily.
Summary of the invention
Applicant finds that in chronic study procedure lithocholic acid (LCA) can effectively suppress and kill the growth of protoscolex, thus can be used for the clinical treatment of echinococcosis granulosa.
In the present invention, lithocholic acid (LCA), its chemical name is 3a-hydroxyl-5 β-cholanic acid, is the material produced by bacterial metabolism at enteral by chenodeoxy cholic acid.
First, the invention discloses lithocholic acid as the application of active component in preparation treatment Echinococcus Granulosus Cysts medicine.
Above-mentioned medicine, both can be simple active raw materials, can also be the various preparations that lithocholic acid 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 lithocholic acid as the application of active component in preparation treatment Cystic hydatidosis medicine.
As required, can use the lithocholic acid of variable concentrations, in general, in order to effectively kill cause of disease, the concentration of lithocholic acid is not less than 500 μm of ol/L.
For common protoscolex stand density, preferably, the concentration of lithocholic acid is 2000-3000 μm of ol/L.
Preferably, the continuous use time is not less than 2 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 lithocholic acid 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 lithocholic acid is not less than 500 μm of ol/L, and preferably, the concentration of lithocholic acid is 2000-3000 μm of ol/L.
Accompanying drawing explanation
Fig. 1 lithocholic acid on the impact of Echinococcus Granulosus Cysts protoscolex form, wherein, A: matched group protoscolex; The protoscolex of B:500 μm of ol/L LCA effect 3d; The protoscolex of C:1000 μm of ol/L LCA effect 3d; The protoscolex of D:2000 μm of ol/L LCA effect 3d; The protoscolex of E:3000 μm of ol/L LCA effect 3d.
Fig. 2 is that the lithocholic acid of variable concentrations is on the impact of Echinococcus Granulosus Cysts protoscolex vigor.
Fig. 3 is the impact of lithocholic acid on Echinococcus Granulosus Cysts protoscolex Surface Ultrastructure, wherein, and A: matched group protoscolex; B, C:2000 μm of ol/L lithocholic acid effect 1d protoscolex.
Fig. 4 is lithocholic acid on the inner Ultrastructural impact of Echinococcus Granulosus Cysts protoscolex, wherein, and A: matched group protoscolex; B, C:2000 μm of ol/L lithocholic acid effect 1d protoscolex.
Detailed description of the invention
In order to the effect of lithocholic acid (LCA) is described, applicant carried out experiment in vitro and illustrate that lithocholic acid is to the killing effect of Echinococcus Granulosus Cysts protoscolex.
Experiment 1:LCA interaction in vitro is in the effect of Echinococcus Granulosus Cysts protoscolex
Get the sheep liver of fresh natural infection Echinococcus Granulosus Cysts, clean Hepar Caprae seu ovis surface, with 75% alcohol disinfecting surface, extract the capsule liquid containing protoscolex under aseptic condition, move in aseptic bottle.Clean protoscolex 3 extremely clarifications with PBS (PH=7.2), do dye exclusion experiment with 0.1% eosin stain, more than 98% refuses dye.Perusal protoscolex is tenderly white husky sample single-size, and the active good protoscolex rate of settling is fast, and the active protoscolex rate of settling that is bad or death is relatively slow.
Observing protoscolex polypide under inverted microscope is interior swaged, is dispersed in densely covered, and polypide structure is full, ovalize, and clear in structure is complete, and calcium granule is limpid and obvious.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, the interior cultivation of 5%CO2 incubator.
Experiment grouping: DMSO matched group, 500 μm of ol/L LCA groups, 1000 μm of ol/L LCA groups, 2000 μm of ol/LLCA groups, 3000 μm of ol/L LCA.Get six orifice plates, 5mL system is established in every hole, by above grouping, prepares corresponding culture fluid.
Echinococcus Granulosus Cysts protoscolex PBS (pH=7.2) after cultivating 5 days is in vitro cleaned 3 times, often organizes in culture fluid and add about 2000 protoscolexs, put 37 DEG C, 5%CO
2cultivate in incubator, under inverted microscope, observe the impact of LCA on Echinococcus Granulosus Cysts protoscolex form and vigor.
Data statistical approach: after dosing, 24h starts, often organizes and evenly extracts 200 μ L culture fluid (average containing procephalon saving 90-120) every day, with 0.1% eosin stains 15min, observe the vigor of protoscolex under inverted microscope.Cephalomere of living refuses dye, not painted, and has activeness; The protoscolex that dead or vigor weakens is red stained with color, with structural deterioration.Calculate and often organize average mortality every day.Change liquid once (when often organizing system and concentration dosing of same first time) every 4d, Continuous Observation, until the protoscolex of maximum effect concentration is all dead.Experiment in triplicate.
The change of protoscolex surface texture after experiment 2:LCA effect
While survey vigor, get corresponding protoscolex at random and do Electronic Speculum, 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 protoscolex top layer Change of Ultrastructure under LEO1430VP scanning electron microscope.
The change of protoscolex internal structure after experiment 3:LCA effect
While survey vigor, get corresponding protoscolex at random and do Electronic Speculum, observe the Ultrastructural change of protoscolex.PBS washs 3 times, each 5min; Fixing 24h before putting 4% glutaraldehyde.Centrifugal with little centrifuge tube, remove supernatant, use Agarose embedding cephalomere, take out gel and continue to put into 4% glutaraldehyde internal fixtion 24h.The protoscolex PBS (pH7.4) fixed is rinsed 3 times, each 15min, 2%O
safter in O4, fixing 1h, rinses twice again with PBS.Ethanol is serial dehydration step by step, 30% → 50% → 60% each 10min, and 70% spends the night, and puts 80% → 90% each 10min, 100%30min next day again.Acetone dewaters step by step afterwards, epoxy resin (Epon812) embeds, and repaiies type after fixing, and LKB2088V type ultramicrotome is cut into slices, acetic acid uranium and plumbi nitras double staining, be placed in the inner Ultrastructural change of Hitachi H-600 type transmission electron microscope observation protoscolex.
Experimental result:
With reference to figure 1, be the metamorphosis of LCA In vitro culture Echinococcus Granulosus Cysts protoscolex, observe under inverted microscope, protoscolex after LCA 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.After drug effect 3d, along with the increase of LCA concentration, the metamorphosis of protoscolex is obvious.The protoscolex rostellum of DMSO group protrudes or is recessed into, and volume is comparatively large, movable good.
With reference to figure 2, for LCA is to the lethal effect of external Echinococcus Granulosus Cysts protoscolex.The Echinococcus Granulosus Cysts protoscolex vitality test of variable concentrations LCA (500 μm of ol/L, 1000 μm of ol/L, 2000 μm of ol/L, 3000 μm of ol/L) In vitro culture 11d the results are shown in Figure 2, and display variable concentrations LCA all has inhibitory action to external Echinococcus Granulosus Cysts protoscolex.Wherein 3000 μm of ol/L LCA effect 5d protoscolex mortality rates reach 100%.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 and matched group comparing difference all have statistical significance (P < 0.01).
Meanwhile, applicant observes the protoscolex Change of Ultrastructure after LCA effect under a scanning electron microscope, and observe display blank group protoscolex smooth surface, many in interior swaged, cephalomere is spherical in shape or oval; After 3000 μm of ol/L LCA effect Echinococcus Granulosus Cysts protoscolex 1d, observe discovery under a scanning electron microscope, protoscolex is many in the type that turns up, rostellum destruction, the distortion of head hook defect, sucker, there is many lacunas in body surface, cortex is in sample infringement (Fig. 3) of damaging by worms.
, observe under transmission electron microscope, the normal Echinococcus Granulosus Cysts protoscolex syncytium band structure cultivated is finer and close meanwhile, inside have differ in size, vesicle that shape differs, arrange more regular, there is the microtriche of more marshalling in outside; Containing dissimilar cell in protoscolex, the core of parenchyma is large and obviously, kernel is placed in the middle, and in core, chromatin is finer and smoother, and heterochromatin is less.Echinococcus Granulosus Cysts protoscolex syncytium band after LCA effect is thinning, loosely organized, and vesicle is expanded, and cilium reduces, and irregular arrangement; Part parenchyma substrate concentrates, and perinuclear space is slightly broadening, and kernel disappears; There is cavity (Fig. 4) in syncytium band and intracavity.
Comprehensive above-mentioned experimental result can be seen, variable concentrations LCA all has inhibitory action to external Echinococcus Granulosus Cysts protoscolex, concentration is that the LCA of 3000 μm of ol/L is the most obvious to Echinococcus Granulosus Cysts protoscolex lethal effect, along with the prolongation of time, inhibitory action significantly increases (P < 0.01).Have on inhibiting basis at confirmation LCA to protoscolex, explore the LCA of variable concentrations to the Morphology Effects of Echinococcus Granulosus Cysts protoscolex.Result of study shows, and after LCA effect, can cause the form generation change in various degree of external Echinococcus Granulosus Cysts protoscolex.Interior swaged and the type protoscolex that turns up are the different existence forms of polypide.When condition is suitable, protoscolex rostellum indent, can protect a hook, sucker, microtriche to be without prejudice; After adding LCA, the protoscolex of the type that turns up increases.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.Echinococcus Granulosus Cysts protoscolex after variable concentrations LCA effect, observes under transmission electron microscope, and defect in various degree appears in microtriche, and cell in stratum germinativum by cavity and fat drip substitute.It is unfavorable for forming brood capsule, the growth of protoscolex is suppressed and dead.
Generally speaking, LCA has lethal effect to external Echinococcus Granulosus Cysts protoscolex.
Claims (7)
1. lithocholic acid is as the application of active component in preparation treatment Echinococcus Granulosus Cysts medicine.
2. application according to claim 1, is characterized in that the concentration of lithocholic acid is not less than 500 μm of ol/L.
3. application according to claim 2, is characterized in that the concentration of lithocholic acid is 2000-3000 μm of ol/L.
4. lithocholic acid is as the application of active component in preparation treatment Cystic hydatidosis medicine.
5. lithocholic acid is as the application of Echinococcus Granulosus Cysts protoscolex inhibitor.
6. application according to claim 5, is characterized in that the concentration of lithocholic acid is not less than 500 μm of ol/L.
7. application according to claim 6, is characterized in that the concentration of lithocholic acid is 2000-3000 μm of ol/L.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113559013A (en) * | 2021-09-01 | 2021-10-29 | 南充市中心医院 | Application of lithocholic acid compound in preparation of antibacterial product |
| CN115025101A (en) * | 2022-06-29 | 2022-09-09 | 扬州大学 | Lithocholic acid-added medicament for preventing and treating mycotoxin poisoning and preparation method and application thereof |
-
2015
- 2015-07-27 CN CN201510457052.6A patent/CN104997792A/en active Pending
Non-Patent Citations (3)
| Title |
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| J.D.SMYTH: "Lysis of Echinococcus granulosus by surface-active agents in bile and the role of this phenomenon in determining host specificity in helminths", 《PROCEEDINGS OF THE ROYAL SOCIETY B》 * |
| J.D.SMYTH等: "THE BIOCHEMISTRY OF BILE AS A FACTOR IN DETERMINING HOST SPECIFICITY IN INTESTINAL PARASITES, WITH PARTICULAR REFERENCE TO ECHINOCOCCUS GRANULOSUS", 《ANNALS OF NEW YORK ACADEMY OF SCIENCES》 * |
| 孙冯等: "胆汁对体外细粒棘球蚴原头节的作用", 《中国现代医学杂志》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113559013A (en) * | 2021-09-01 | 2021-10-29 | 南充市中心医院 | Application of lithocholic acid compound in preparation of antibacterial product |
| CN113559013B (en) * | 2021-09-01 | 2023-02-28 | 南充市中心医院 | Application of lithocholic acid compound in preparation of antibacterial product |
| CN115025101A (en) * | 2022-06-29 | 2022-09-09 | 扬州大学 | Lithocholic acid-added medicament for preventing and treating mycotoxin poisoning and preparation method and application thereof |
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