CN106420775A - Application of crenatoside - Google Patents

Application of crenatoside Download PDF

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Publication number
CN106420775A
CN106420775A CN201610803751.6A CN201610803751A CN106420775A CN 106420775 A CN106420775 A CN 106420775A CN 201610803751 A CN201610803751 A CN 201610803751A CN 106420775 A CN106420775 A CN 106420775A
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China
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crenatoside
medicine
application
cerebral ischemia
sugar
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萧伟
王红梅
周建明
顾睿
周军
丁岗
王振中
胡晗绯
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Jiangsu Kanion Pharmaceutical Co Ltd
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Jiangsu Kanion Pharmaceutical Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7032—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a polyol, i.e. compounds having two or more free or esterified hydroxy groups, including the hydroxy group involved in the glycosidic linkage, e.g. monoglucosyldiacylglycerides, lactobionic acid, gangliosides

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  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention relates to the technical field of medicine, in particular to application of crenatoside. The experiment indicates that crenatoside has an obvious improving effect on the nerve cell survival rate and calcium ion content after oxygen-glucose deprivation-oxygen-glucose deprivation and reintroduction injuries. Meanwhile, crenatoside has adjusting functions of different degrees on the activity of total adenosine triphosphatase (ATPase), sodium-potassium-adenosine triphosphatase (Na+-K+ATPase), calcium-magnesium-adenosine triphosphatase (Ca2+-Mg2+-ATPase), superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA) and nitric oxide (NO) in rat hippocampus and cortex after rat acute cerebral ischemia-reperfusion injuries in vivo and has certain dosage dependency. It is indicated that the compound has the function and application value of treating cerebral ischemia diseases.

Description

The purposes of crenatoside
Technical field
The present invention relates to the purposes of pharmaceutical technology field, more particularly, to crenatoside.
Background technology
Cerebrovascular disease is one of modal disease in the world, because of the spy that its sickness rate is high, disability rate is high, mortality rate is high Point, serious threat human life and health.Ischemic cerebrovascular (ischemiccerebrovascular disease, ICVD) confession under directions answer cerebral blood flow major arteries occur narrow or inaccessible, result leads to cerebral tissue (particularly Middle cerebral artery Domain) drastically hypoxic-ischemic, finally cause local brain tissue that ischemia or necrosis occur, corresponding clinical symptoms occur.Ischemic brain Post stroke can recover the hemoperfusion of ischemic region by way of thrombus or machinery lead to again, but some patients after ischemia again So that function of organization is recovered after perfusion, so that the dysfunction caused by ischemia and structural deterioration is increased further, that is, on the contrary Our described cerebral ischemia reperfusion injury (cerebral ischemia-reperfusion (IR) injury).Cerebral ischemia is again Perfusion injury is a complicated pathophysiological process, and research recently finds, cerebral blood flow interrupts and Reperfu- sion makes the cell of brain produce Raw damage is a quick cascade reaction, and this cascade reaction includes many links, such as energy battier, cell acidosis, emerging Put forth energy acidic amino acid release increases, intracellular Ca2+ unstability state, free radical generate, apoptogene activate etc..These link reciprocal causations, Overlap each other, and connect each other, form vicious cycle, ultimately result in apoptosis or necrosis.Although the disease to cerebral ischemia at present Reason Physiological Mechanism makes substantial progress, but therapeutic choice is still limited.At present, common treatment Imaging in Patients with Cerebral Ischemia Disease medicine has Nimodipine, disappear benzene Horizon slow releasing tablet, flunarizine etc., but these medicine side effect are stronger.
In recent years, the research for the treatment of by Chinese herbs Imaging in Patients with Cerebral Ischemia Disease gradually causes the concern of people, and due to a lot of Chinese medicine groups Belong to natural extract medicine, there are Small side effects.Crenatoside is extraction phenethyl alcohol glycosides from Chinese medicinal plant Composition, its structure is shown in formula I：
Pharmacological research shows, this compound has multiple effect such as tonifying YANG, antioxidation, memory reinforcing.But at present not See the report of Crenatoside application in preparation treatment Imaging in Patients with Cerebral Ischemia Disease medicine.
Content of the invention
In view of this, the technical problem to be solved in the present invention is to provide the purposes of crenatoside, and the present invention tests table Bright, crenatoside all has to Neuronal Survival rate and calcium ion content after scarce sugar anoxia-complex sugar reoxygenation injury and significantly changes Kind effect；Simultaneously in vivo to total adenosinetriphosphataes in rat hippocampus after acute cerebral ischemia/reperfusion in rats damage and cortex (ATP enzyme), sodium-potassium-adenosinetriphosphataes (Na+-K+- ATP enzyme), calcium-magnesium-adenosinetriphosphataes (Ca2+-Mg2+- ATP enzyme), super Superoxide dismutase (SOD), catalase (CAT), malonaldehyde (MDA), the activity of nitric oxide (NO) all have in various degree Adjustment effect, and there is certain dose-dependant, show that this compound has effect and the application valency for the treatment of Imaging in Patients with Cerebral Ischemia Disease Value.
The invention provides Crenatoside metabolic defect in cellular calcium ion after preparation improves and lacks sugar anoxia-complex sugar reoxygenation injury is dense Application in the abnormal medicine raising of degree.
The cell tested in the embodiment of the present invention is neurocyte；It is specifically people's bone marrow neuroblastoma；Preferably , cell strain is SH-SY5Y.
Crenatoside be used for improve lack sugar anoxia-complex sugar reoxygenation injury after metabolic defect in cellular calcium ion concentration abnormality raise dense Spend for 25 μ g/mL～100 μ g/mL；Preferably, concentration is 50 μ g/mL.
Calcium ion is the body items indispensable ion of physiological activity, can be used in maintaining normal nerve conduction work( Energy.Under the conditions of hypoxic-ischemic, the phenomenon that the intracellular calcium ion concentration occurring raises extremely, this is to cause nerve after cerebral ischemia The irreversible damage of unit, the major reason of apoptosis.It is intracellular to SH-SY5Y caused by Anoxia that the present invention measures Crenatoside The impact of the abnormal calcium ion concentration raising, after reoxygenation administration 1h, in the cell of model group, calcium ion concentration significantly raises, Compare with DMEM matched group (DMEM group) that there were significant differences (P ＜ 0.01), each dosage group of Crenatoside is common with reoxygenation complex sugar After effect 1h, intracellular calcium concentration significantly reduces, and compares with model group and has significant difference (P ＜ 0.05, P ＜ 0.01), The high, medium and low dosage group of Crenatoside has preferable adjustment effect to calcium ion change after cell injury.
The invention provides Crenatoside lacks in sugar anoxia-medicine to cell injury for the complex sugar reoxygenation in preparation protection Application.
The cell tested in the embodiment of the present invention is neurocyte；It is specifically people's bone marrow neuroblastoma；Preferably , cell strain is SH-SY5Y.
Crenatoside is used for protecting scarce sugar anoxia-complex sugar reoxygenation to be 25 μ g/mL～100 μ g/ to the concentration of cell injury mL；Preferably, concentration is 50 μ g/mL.
During lacking sugar anoxia-complex sugar reoxygenation, cell can produce a series of reaction, including such as energy battier, cell Acidosis, excitatory amino acid release increase, the generation of intracellular Ca2+ unstability state, free radical, apoptogene activation etc..And then lead to The apoptosis of cell or necrosis.The present invention adopts MTS method to measure Crenatoside to SH-SY5Y caused by scarce sugar anoxia-complex sugar reoxygenation The protective effect of cell injury, after scarce sugar anoxia-complex sugar reoxygenation injury 4h, reoxygenation administration 3h, the cell survival rate of model group is 51.02%, compare with solvent control group (DMSO group) that there were significant differences (P ＜ 0.01)；After positive drug and medicine group effect 3h, carefully Born of the same parents' protective rate substantially increases, and compares with model group and has significant difference (P ＜ 0.05, P ＜ 0.01).Wherein with Crenatoside Compound middle dose group is best to neuroprotective, can be obviously improved cell caused by lacking sugar anoxia-complex sugar reoxygenation injury and damage Wound, has preferable protective effect to it.
The invention provides Crenatoside improves the medicine of cerebral ischemia re-pouring tissues following MCAO in rats oxidation resistance in preparation In application.
In some embodiments, improve cerebral ischemia re-pouring tissues following MCAO in rats oxidation resistance and include：Improve atpase activity, carry High anti-oxidation enzymatic activity, the content of reduction MDA and/or NO.
In some specific embodiments, ATP enzyme is total adenosinetriphosphataes, sodium-potassium-adenosinetriphosphataes, calcium-magnesium-three phosphorus Adenosine monophosphate enzyme；Described antioxidase is superoxide dismutase or catalase.
In some embodiments, cerebral tissue is Hippocampus and/or cortex.
In some specific embodiments, cerebral tissue is rat cerebral tissue.
The dosage that Crenatoside is used for improving cerebral ischemia re-pouring tissues following MCAO in rats oxidation resistance is 6.5mg/kg d-1 ～26mg/kg rat body weight d-1.Preferably, dosage is 26mg/kg rat body weight d-1.Preferably, administering mode is vein Injection.
ATP enzyme is also called adenosinetriphosphataes, be a class can by adenosine triphosphate (ATP) catalyzing hydrolysis be adenosine diphosphate (ADP) (ADP) and phosphate anion enzyme, experiment shows, in ischemia-reperfusion rat hippocampus and cortex, atpase activity reduces, tool Body, total adenosinetriphosphataes (ATP enzyme), sodium-potassium-adenosinetriphosphataes (Na+-K+- ATP enzyme), calcium-magnesium-adenosinetriphosphataes (Ca2+-Mg2+- ATP enzyme) activity reduction.
Antioxidase refers to the scavenging capacity oxygen-derived free radicals existing in vivo, prevents the enzyme of oxidative stress status illeffectss, Experiment shows, in ischemia-reperfusion rat hippocampus and cortex, activities of antioxidant enzymes reduces.Specifically, superoxide dismutase (SOD), the activity of catalase (CAT) reduces.
Malonaldehyde (MDA) mainly results in Lipid peroxidation metabolism, damage biological membrane structure, changes the permeability of film, and then affects A series of biochemical reactions be normally carried out.Nitric oxide (NO) passes through oxidative stress, interfering energy metabolism, directly damages DNA, the apoptosis activating many Poly ADP-ribose polymerases or making the many aspects inducing cells such as cytosol Ca2+ regulation disorder.Experiment table Bright, in ischemia-reperfusion rat hippocampus and cortex, the concentration abnormality of malonaldehyde (MDA) and nitric oxide (NO) raises.The present invention is real Test and show, Crenatoside can improve total ATP enzyme, Na in ischemia-reperfusion rat hippocampus and cortex+-K+- ATP enzyme, Ca2+- Mg2+- ATP enzyme, the activity of SOD, CAT and MDA, NO content, can pass through free radical resisting peroxidation, protection membrane structure Integrity, and then, on protection cell membrane, the activity of multiple ATP enzyme, plays the protective effect to ischemical reperfusion injury rat.This Invention measures, in intravenous administration mode, the protective effect that Crenatoside damages to acute cerebral ischemia/reperfusion in rats, knot Fruit shows, Crenatoside extremely has different degrees of to the cell viability level calcium ion caused by scarce sugar anoxia complex sugar reoxygenation Improve it is possible to improve total ATP enzyme, Na in ischemia-reperfusion rat hippocampus and cortex+-K+- ATP enzyme, Ca2+-Mg2+- ATP enzyme, The activity of SOD, CAT, MDA and NO, can pass through free radical resisting peroxidation, protect the integrity of membrane structure, and then, protect On shield cell membrane, the activity of multiple ATP enzyme, plays the protective effect to ischemical reperfusion injury rat.
Based on above experimental result, present invention also offers Crenatoside is in preparation treatment and/or prevention cerebrovascular disease Application in the medicine of disease；Described cerebrovascular disease includes ischemic cerebrovascular and/or cerebral ischemia reperfusion injury.
In some embodiments, the dosage of Crenatoside treatment treatment cerebrovascular disease is 6.5mg/kg d-1～26mg/ kg·d-1.
Present invention also offers the medicine of a kind for the treatment of and/or prevention cerebrovascular disease, including Crenatoside.
Pharmaceutically acceptable adjuvant is also included in the medicine of the treatment of present invention offer and/or prevention cerebrovascular disease.
The medicine of the treatment of present invention offer and/or prevention cerebrovascular disease is specifically for preventing and treating ischemic cerebrovascular And/or cerebral ischemia reperfusion injury.
The treatment that the present invention provides and/or the dosage form of the medicine of prevention cerebrovascular disease are oral administered dosage form, are administered to Pharmaceutically dosage form, topical administration formulations.
Described oral administered dosage form is tablet, capsule, granule, pill, oral solutionses, soft extract, suspending agent, dispersion Agent, syrup.
Described injecting medicine-feeding form is injection liquor or powder ampoule agent for injection.
Described topical administration formulations are suppository, patch or gel.
In some embodiments, the tablet for the treatment of and/or prevention cerebrovascular disease includes：Crenatoside, starch, carboxylic Methyl starch sodium, Pulvis Talci, dextrin, magnesium stearate and starch slurry.
Specifically, the mass ratio of Crenatoside, starch, carboxymethyl starch sodium, Pulvis Talci, dextrin and magnesium stearate is 350∶5:7.5:0.8:50:0.8.
In some embodiments, the content of the capsule for the treatment of and/or prevention cerebrovascular disease includes： Crenatoside, starch, low-substituted hydroxypropyl cellulose, micropowder silica gel, magnesium stearate and starch slurry.
Specifically, the mass ratio of Crenatoside, starch, low-substituted hydroxypropyl cellulose, micropowder silica gel and magnesium stearate For 350:32:6:4.5:1.5.
In some embodiments, the granule for the treatment of and/or prevention cerebrovascular disease includes：Crenatoside, sucrose and Dextrin.
Specifically, the mass ratio of Crenatoside, sucrose and dextrin is 350:1000:500.
In some embodiments, the pill for the treatment of and/or prevention cerebrovascular disease includes：Crenatoside, poly- second two Alcohol -6000 and Tween-80.
Specifically, the mass ratio of Crenatoside, PEG-4000 and Tween-80 is 350:12:80.5.
In some embodiments, the injection liquor for the treatment of and/or prevention cerebrovascular disease includes：Crenatoside, Semen sojae atricolor Phospholipid, G ＆ W.
Specifically, the concentration of Crenatoside is 200g/L；The concentration of soybean phospholipid is 15g/L；The concentration of glycerol is 25g/L.
During injection, after the injection dilution agent for the treatment of and/or prevention cerebrovascular disease, intravenous drip；Described dilution is adopted Use 5% Glucose Injection；The ratio of described dilution is 1:250.
A kind of method that the present invention additionally provides treatment and/or prevention cerebrovascular disease simultaneously, it is to give the present invention to carry For treatment and/or prevention cerebrovascular disease medicine.Described give as oral or inject.
Treatment that the present invention provides and/or the daily injection dosage of medicine of prevention cerebrovascular disease are with Crenatoside Quality be calculated as 6.5mg/kg rat body weight～26mg/kg rat body weight.It is preferably 26mg/kg rat body weight.
The invention provides the purposes of crenatoside, experiments indicate that, crenatoside is to scarce sugar anoxia-multiple After sugared reoxygenation injury, Neuronal Survival rate and calcium ion content all improve significantly；Simultaneously in vivo to rat acute Total adenosinetriphosphataes (ATP enzyme), sodium-potassium-adenosinetriphosphataes (Na in rat hippocampus and cortex after cerebral ischemia reperfusion injury+-K+- ATP enzyme), calcium-magnesium-adenosinetriphosphataes (Ca2+-Mg2+- ATP enzyme), superoxide dismutase (SOD), catalase (CAT), malonaldehyde (MDA), the activity of nitric oxide (NO) all have different degrees of adjustment effect, and have certain dosage according to Rely, show that this compound has effect and the using value for the treatment of Imaging in Patients with Cerebral Ischemia Disease.
Specific embodiment
The invention provides the purposes of crenatoside, those skilled in the art can use for reference present disclosure, be suitably modified Technological parameter is realized.Specifically, all similar replacements and change be for a person skilled in the art aobvious and It is clear to, they are considered as including in the present invention.The method of the present invention and application are retouched by preferred embodiment State, related personnel substantially in without departing from present invention, spirit and scope, methods herein and application can be modified or Suitably change and combine, to realize and to apply the technology of the present invention.
Medicine, biomaterial or instrument that the present invention adopts are all common commercially available product, all can buy in market.
With reference to embodiment, the present invention is expanded on further：
Embodiment 1：The protective effect to cell injury caused by scarce sugar anoxia/complex sugar reoxygenation injury for the Crenatoside
1st, experiment material
SH-SY5Y cell line, Nanjing KaiJi Biology Science Development Co., Ltd.
2nd, experimental technique
2.1 cell culture and administration
Passage is inoculated in culture bottle, in 37 DEG C, 5%CO2, saturated humidity incubator in cultivate, work as culture bottle In cell substantially cover with after (cell is maintained at exponential phase), discard culture fluid, add appropriate PBS solution gently to swing and wash, Discard, be subsequently adding 0.25% trypsin solution 0.5ml digestion 2-3 minute, add complete medium 2ml to terminate the work of pancreatin With, cell is proceeded in glass centrifuge tube, 1000r, it is centrifuged 4min, supernatant discarded, adds complete medium, carefully blow and beat into single Cell.Auto-counting of Cells instrument meter number, according to 1.5 × 105Individual/mL, every hole 100 μ L is inoculated in 96 porocyte culture plates, side Marginal pore adds 100 μ L sterilized water, continues at 37 DEG C, 5%CO2Carry out modeling, medicine-feeding test after culture 18-20 hour in incubator.
2.2 packets and administration
Experiment sets DMSO group, model group, positive controls and sample sets.Every group sets 5 multiple holes.The every hole of DMSO group adds 100 μ L contain the blank cultures of 1/1000DMSO, in 37 DEG C, 5%CO2Normally cultivate in incubator, model group, positive controls Every hole adds 100 μ L BSS buffer (NaCl122mmol/L, KCl 5.4mmol/L, MgSO with sample sets4·7H2O 0.8mmol/L、NaH2PO42.6mmol/L、NaHCO326.2mmol/L、CaCl21.8mmol/L、HEPES 20.1mmol/ L) as anoxia liquid, 37 DEG C, 5%CO are put into2, 95%N2The little interior of anoxia carry out anoxia, anoxia start before ventilation 20min make Anoxia cell saturation, anoxia will access 95%N at once after 4 hours2Interface disconnect.After modeling, the every hole of model group adds 100 μ L Blank cultures containing 1/1000DMSO, the every hole of positive controls adds 100 μ L to contain the blank cultures of 20 μM of berberine, DMSO 1/1000, the every hole of sample sets adds the blank cultures of 100 μ L various dose Crenatoside to content, and DMSO content is 1/ 1000, reoxygenation is administered 3h.
2.3 cell viabilities measure
After scarce sugar anoxia/complex sugar reoxygenation injury 4h, reoxygenation administration 3h, discard all liq in plate, PBS washes plate once, respectively Hole adds cell proliferation detection solution (final concentration 190 μ g/ml) of 100 μ L, after putting into culture 4h in cell culture incubator, microplate reader Light absorption value is surveyed, by formula at 490nm：
Survival rate=(different dosing group OD490/DMSO group OD490) * 100% calculates each group cell survival rate.
3rd, experimental result
Table 1Crenatoside scarce sugar anoxia/complex sugar reoxygenation injury is caused the protective effect of SH-SY5Y cell injury (N=9)
Group Concentration (μ g/ml) OD±SD Survival rate (%)
DMSO group —— 0.49±0.11 100
Model group 10μM 0.25±0.03△△ 51.02
Positive controls 20μM 0.38±0.02** 77.55
Crenatoside low dosage 25 0.40±0.04** 81.63
Crenatoside middle dosage 50 0.56±0.03** 114.29
Crenatoside high dose 100 0.48±0.03** 97.96
Note：△△P ＜ 0.01, is compared with DMSO group；*P ＜ 0.05,**P ＜ 0.01, is compared with model group
Result is as shown in table 1, after scarce sugar anoxia/complex sugar reoxygenation injury 4h, reoxygenation administration 3h, the cell survival rate of model group For 51.02%, compare with solvent control group (DMSO group) that there were significant differences (P ＜ 0.01)；After positive drug and medicine group effect 3h, Cytoprotective rate substantially increases, and compares with model group and has significant difference (P ＜ 0.05, P ＜ 0.01).Wherein with Crenatoside compound middle dose group is best to neuroprotective, can be obviously improved and lack sugar anoxia/complex sugar reoxygenation injury Caused cell injury, has preferable protective effect to it.
Embodiment 2 Crenatoside is to the abnormal reduction effect raising calcium ion concentration intracellular caused by Anoxia
1st, experiment material
SH-SY5Y cell line, Nanjing KaiJi Biology Science Development Co., Ltd；Fluo-3AM (Sigma, 73881-1MG)； BSA(BIO RAD,E588).
2nd, experimental technique
2.1 cell culture
SH-SY5Y cell is using the DMEM culture fluid containing 10% hyclone, in 37 DEG C, 5%CO2The culture of saturated humidity Secondary Culture in case.
2.2 packets and administration
Trophophase cell of taking the logarithm is used for testing, 0.25% collected by trypsinisation cell, counts and adjusts cell with DMEM Concentration is 2 × 105Cells/mL, every hole 200 μ l are inoculated in culture in black 96 orifice plate.Be divided into normal group, model group, Crenatoside low dose group, Crenatoside middle dose group, Crenatoside high dose group, model group and DMEM are normal Matched group, every group of 3 multiple holes.37 DEG C of culture 24h, clean once with sugar-free DMEM, each hole adds 100 μ l sugar-free DMEM, puts and contains 5%CO2And 95%N2Three gas incubators cultures, take out 96 orifice plates after anoxia 1h, each hole adds 0.5 μ l various dose Crenatoside medicine, to normal incubator reoxygenation 1h using Fluo-3AM probe in detecting each hole calcium ion concentration
2.3 calcium ion concentrations measure
Each hole adds 25 μ l to contain the Fluo-3am probe of 20uM and the HBSS of 0.25% F-127, places 30min for 37 DEG C, 105 μ l supernatants are abandoned in suction, add 80 μ l HBSS, gently mix, and inhale and abandon 80 μ l supernatants, are repeated 3 times altogether, add 80 μ l HBSS- BSA, detects each hole F=485/525nm fluorescence intensity by 37 DEG C × 20min；Each hole adds 25 μ l to contain 1%Triton X-100's BSS, lucifuge room temperature shakes 5min, ibid detects Fmax；Each hole adds the EGTA of 25 μ l 500mM, ibid detects Fmin, passes through Each hole calcium ion concentration of formula calcium ion=Kd* (F-Fmin)/(Fmax-F).
3rd, experimental result
The tune that table 2 Crenatoside causes SH-SY5Y metabolic defect in cellular calcium ion extremely to raise scarce sugar anoxia/complex sugar reoxygenation injury Section effect (N=5)
Note：△ △ P ＜ 0.01, model is compared with DMEM group；* P ＜ 0.05, * * P ＜ 0.01, is compared with model group
As shown in table 2, after reoxygenation administration 1h, in the cell of model group, calcium ion concentration significantly raises result, with DMEM matched group (DMEM group) compares that there were significant differences (P ＜ 0.01)；The each dosage group of Crenatoside and reoxygenation complex sugar are made jointly After 1h, intracellular calcium concentration significantly reduces, and compares with model group and has significant difference (P ＜ 0.05, P ＜ 0.01), The high, medium and low dosage group of Crenatoside has preferable adjustment effect to calcium ion change after cell injury.
The protective effect that embodiment 3 Crenatoside damages to acute cerebral ischemia/reperfusion in rats.
1st, experiment material
Male Wistar rat, SPF level, body weight 240～280g, provided by Yangzhou University's comparative medicine center；Buddhist nun is not Flat injection；Catalase (CAT), superoxide dismutase (SOD), malonaldehyde (MDA), nitric oxide (NO), total ATPase、Na+-K+-ATPase、Ca2+-Mg2+- ATPase detection kit；Nipride ampoule.
2nd, experimental technique
The preparation of 2.1 acute cerebral ischemia in rats models
Take healthy male Wistar rat, preoperative 12h fasting, normal water.Bilateral carotid arteries are closed using folder and merges blood pressure lowering Method prepares cerebral ischemia/reperfusion injury of rats model.3.5% chloral hydrate (350mg/kg) intraperitoneal injection of anesthesia, back of the body position is fixed on On operating-table, cervical region unhairing, routine disinfection.Neck median incision, blunt separation bilateral common carotid arteries simultaneously gently peel off vagus nerve, Threading is standby.Before folder closes bilateral common carotid arteries, lumbar injection sodium nitroprusside (2.5mg/kg), presss from both sides immediately and closes bilateral common carotid arteries After 10min, Reperfu- sion 10min, then press from both sides again and close 10min, unclamp bulldog clamp, more logical rear wound localized pulverization penicillin 400,000 u, To prevent to infect.Sew up a wound, 5% iodophor disinfection wound, after its revival, normal raising.Rats in sham-operated group does not inject nitre General sodium, does not carry out bilateral common carotid arteries folder and closes Reperfu- sion, remaining operating procedure is ibid.
2.2 animal packets and administration
After male Wistar rat revival, it is randomly divided into model group by body weight, nimodipine group (0.36mg kg-1), RC-10 low dose group, RC-10 middle dose group, RC-10 high dose group, separately set sham operated rats, every group of 10 animals.After Reperfu- sion 3h intravenously administrable first, is administered for second after 24h, is administered once a day later, be administered 7 times altogether.
2.3 Indexs measure
Draw materials after last dose 24h, quick broken end takes brain, is placed on the surface plate of underlying ice cube, with filter paper by surface blood Blot, peel off bilateral hippocampus and brain cortex tissue, be placed in quick freezing in liquid nitrogen, weigh, frozen standby in -80 DEG C.Carry out During biochemical indicator detection, with weight/volume 1:94 DEG C of normal saline of addition, make 10% brain tissue homogenate, 3000r/min Low-temperature centrifugation 15min, takes supernatant, illustrates by test kit, with total in 722 spectrophotometric determination rat hippocampus and cerebral cortex ATPase、Na+-K+-ATPase、Ca2+-Mg2+- ATPase, SOD, CAT activity and MDA and NO content.
3rd, experimental result
Table 3Crenatoside is to total in acute cerebral ischemic reperfusion injury rat hippocampus ATPase, Na+-K+-ATPase、 Ca2+-Mg2+The impact of-ATPase, SOD and CAT activity
Compare with sham operated rats#P<0.05,##P<0.01,###P<0.001；Compare * P with model group<0.05, * * P<0.01
Result as shown in table 3, compared with sham operated rats, total adenosinetriphosphataes (ATP enzyme) in model group rats Hippocampus, Sodium-potassium-adenosinetriphosphataes (Na+-K+- ATP enzyme), calcium-magnesium-adenosinetriphosphataes (Ca2+-Mg2+- ATP enzyme), superoxides discrimination Change enzyme (SOD), catalase (CAT), malonaldehyde (MDA), nitric oxide (NO) activity all significantly reduce, with sham operated rats ratio Relatively, there is significant difference (p<0.05；p<0.01), compare with model group, each concentration compound group dose dependent raises total Adenosinetriphosphataes, calcium-magnesium-adenosinetriphosphataes, superoxide dismutase and catalase content, have significant difference (p<0.05；p<0.01).
Table 4 Crenatoside is to total in acute cerebral ischemic reperfusion injury rat cerebral cortex ATP enzyme, Na+-K+-ATP Enzyme, Ca2+-Mg2+-ATP enzyme, the impact of SOD and CAT activity
Compare #P with sham operated rats<0.05, ##P<0.01；Compare * P with model group<0.05, * * P<0.01
Result is as shown in table 4, compared with sham operated rats, total ATPase, Na in model group rats cerebral cortex+-K+- ATPase、Ca2+-Mg2+- ATPase, SOD and CAT activity significantly reduce, compared with model group, Crenatoside can dosage according to Bad property ground raises the activity of above-mentioned 5 kinds of enzymes hence it is evident that improving the enzymatic activity reduction that ischemia-reperfusion leads to.
Table 5 Crenatoside is to acute cerebral ischemic reperfusion injury rat hippocampus, brain cortical tissue's MDA, NO content Impact
Compare ##P with sham operated rats<0.01, ###P<0.001；Compare * P with model group<0.05, * * P<0.01, * * P< 0.001
As shown in table 5, compared with sham operated rats, the content of model group rats Hippocampus and cerebral cortex MDA and NO shows result Write and raise, compared with model group, Crenatoside each dosage group MDA, NO content significantly reduces.
Acute cerebral ischemia/reperfusion in rats result shows, in rat hippocampus and cortex after reperfusion after acute cerebral ischemia in total three Adenosine phosphate enzyme (ATP enzyme), sodium-potassium-adenosinetriphosphataes (Na+-K+- ATP enzyme), calcium-magnesium-adenosinetriphosphataes (Ca2+-Mg2 +- ATP enzyme), superoxide dismutase (SOD), the activity of catalase (CAT) substantially reduce, and malonaldehyde (MDA), an oxygen Change nitrogen (NO) then significantly to raise, comparing with Normal group all has significant difference, and each concentration C renatoside can be improved scarce Total ATP enzyme, Na in blood Reperfu- sion rat hippocampus and cortex+-K+- ATP enzyme, Ca2+-Mg2+- ATP enzyme, SOD, CAT, MDA, NO Activity, and there is certain dose-dependant, show that it has cerebral protection to cerebral ischemic rats.
This result of study shows, Crenatoside is different to the cell viability level calcium ion caused by scarce sugar anoxia/complex sugar reoxygenation Often there is different degrees of improvement it is possible to improve total ATP enzyme, Na in ischemia-reperfusion rat hippocampus and cortex+-K+- ATP enzyme, Ca2+-Mg2+- ATP enzyme, the activity of SOD, CAT, MDA and NO, can pass through free radical resisting peroxidation, protect membrane structure Integrity, and then, on protection cell membrane, the activity of multiple ATP enzyme, plays protective effect to ischemical reperfusion injury rat.
Embodiment 4 capsule
By 350gCrenatoside and 32g starch, 6g low-substituted hydroxypropyl cellulose, 4.5g micropowder silica gel, 1.5g Hard Fat Sour magnesium and appropriate 10% starch slurry mixing, load capsule, obtain capsule preparations 1000.3 times a day, 1 every time.
Embodiment 5 granule
By 350gCrenatoside and 1000g sucrose and the mixing of 500g dextrin, conventionally make 1000 bag granules Agent.3 times a day, 1 every time.
Embodiment 6 tablet
Will be hard to 350gCrenatoside and 50g starch, 7.5g carboxymethyl starch sodium, 0.8g Pulvis Talci, 50g dextrin, 0.8g Fatty acid magnesium and appropriate 10% starch slurry fit mixing, conventionally make 1000, tablet.3 times a day, and 1 tablet once.
Embodiment 7 pill
350gCrenatoside and 12g PEG-4000,80.5g Tween-80, appropriate liquid Paraffin are mixed Close, conventionally make pill 1000.3 times a day, 1 every time.
Embodiment 8 injection
By 200gCrenatoside and 15g injection soybean phospholipid, 25g glycerol for injection, water for injection is settled to 1000mL, conventionally makes injection 1000.One time a day, 1 every time, at least adopts 250mL 5% glucose to note Penetrate intravenous drip after liquid dilution.
The above is only the preferred embodiment of the present invention it is noted that coming for those skilled in the art Say, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should be regarded as Protection scope of the present invention.

Claims (10)

  1. The 1.Crenatoside medicine that metabolic defect in cellular calcium ion concentration abnormality raises after preparation improves and lacks sugar anoxia-complex sugar reoxygenation injury Application in thing.
  2. 2.Crenatoside lacks the application in sugar anoxia-medicine to cell injury for the complex sugar reoxygenation in preparation protection.
  3. 3.Crenatoside improves the application in the medicine of cerebral ischemia re-pouring tissues following MCAO in rats oxidation resistance in preparation.
  4. 4. application according to claim 3 is it is characterised in that described improve cerebral ischemia re-pouring tissues following MCAO in rats antioxidation energy Power includes：Improve atpase activity, the content improving activities of antioxidant enzymes, reducing MDA and/or NO.
  5. 5. application according to claim 4 is it is characterised in that described ATP enzyme is total adenosinetriphosphataes, sodium-potassium-three phosphorus Adenosine monophosphate enzyme, calcium-magnesium-adenosinetriphosphataes；Described antioxidase is superoxide dismutase or catalase.
  6. 6. the application according to any one of claim 3～4 is it is characterised in that described cerebral tissue is Hippocampus and/or cortex.
  7. Application in the medicine of preparation treatment and/or prevention cerebrovascular disease for the 7.Crenatoside；Described cerebrovascular disease bag Include ischemic cerebrovascular and/or cerebral ischemia reperfusion injury.
  8. 8. application according to claim 7 is it is characterised in that cerebrovascular disease is treated in described Crenatoside treatment Dosage is 6.5mg/kg d-1～26mg/kg d-1.
  9. 9. the medicine of a kind for the treatment of and/or prevention cerebrovascular disease is it is characterised in that include Crenatoside.
  10. 10. medicine according to claim 9 it is characterised in that its dosage form be oral administered dosage form, injecting medicine-feeding form, Topical administration formulations.
CN201610803751.6A 2016-09-05 2016-09-05 Application of crenatoside Pending CN106420775A (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1526400A (en) * 2003-03-04 2004-09-08 杭州天力药业有限公司 Tubiflorous desert cistanche prepn containing phenethyl alcohol glycoside and its prepn process and use

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1526400A (en) * 2003-03-04 2004-09-08 杭州天力药业有限公司 Tubiflorous desert cistanche prepn containing phenethyl alcohol glycoside and its prepn process and use

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
TING SHEN等: "Hepatoprotective effect of phenylethanoid glycosides from Incarvillea compacta against CCl4-induced cytotoxicity in HepG2 cells", 《J KOREAN SOC APPL BIOL CHEM》 *
赵新杰等: "肉苁蓉的药理作用研究进展", 《中国药业》 *

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Application publication date: 20170222