CN101234121A - Application of purslane amide alkaloid in preparing antioxidant and neuroprotective agent - Google Patents
Application of purslane amide alkaloid in preparing antioxidant and neuroprotective agent Download PDFInfo
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- CN101234121A CN101234121A CNA2008100144271A CN200810014427A CN101234121A CN 101234121 A CN101234121 A CN 101234121A CN A2008100144271 A CNA2008100144271 A CN A2008100144271A CN 200810014427 A CN200810014427 A CN 200810014427A CN 101234121 A CN101234121 A CN 101234121A
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- herba portulacae
- oleracein
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Abstract
The invention discloses an application of an oleracein alkaloid in preparing antioxidants, neuroprotective agents, ion channel blockers of brain neuronal cells and inhibitor of apoptosis of brain neuronal cells, wherein the oleracein alkaloid refers to the monomer of oleracein A, oleracein B or oleracein E. The application relates to the development of oleracein alkaloid, which supplies a powerful basis for treating neurodegenerative diseases and lays a foundation for further development of the oleracein alkaloid.
Description
Technical field
The present invention relates to the medicinal application of Herba Portulacae amide alkaloid, relate in particular to the application of Herba Portulacae amide alkaloid in preparation antioxidant and Neuroprotective Agents; Belong to medical domain.
Background technology
Neurodegenerative diseases is that a class is chronic, carrying out property sacred disease.Such disease mainly comprises alzheimer's disease, parkinson disease, Huntington chorea, amyotrophic lateral sclerosis and spinal cord muscular atrophy etc.Along with the acceleration of world population aging process, this class neurodegenerative diseases (as senile dementia, parkinson disease) is on the rise to senior health and fitness's harm.Have 85 years old old man of 65 years old old man of 10% and nearly 40% can be subjected to the puzzlement of senile dementia at US and European, its sickness rate and mortality rate are only second to cardiovascular and cerebrovascular disease and cancer.Although it is the research of this class disease has been caused various countries scientist's concern, at present also very limited to the understanding and the treatment means of neurodegenerative diseases.
Though the diseased region and the cause of disease of neurodegenerative diseases have nothing in common with each other, the neurocyte degeneration is their common ground.Discover, cause that the dead mechanism of neurocyte mainly contains oxidative stress mechanism, inflammatory mechanisms and apoptosis mechanism etc., influence each other between them, finally cause maladjusted nervous system and cell death.
Based on the research of nerve retrograde affection mechanism, the someone proposes this notion of neurocyte protection.People attempt to come the neuroprotective cell by following 3 kinds of approach, prevent its degenerative change.Promptly (1) suppresses the startup factor of neurocyte degenerative change (as nitric oxide; free radical; excitatory toxicity and inflammatory cytokine etc.); (2) signal of block nerves cell degenerative change conduction (as apoptosis etc.) process, and (3) activate endogenous Neuroprotective Mechanisms (as neurotrophic factor etc.).
Herba Portulacae (Portulaca oleracea L.) is widely distributed in temperate zone, the whole world and torrid areas, use as medicine food dual purpose plant in China and other many countries, but diuresis, parasite killing, pain relieving, antitussive, anti-hepatitis, treatment gastric ulcer, little tumor, inflammation, urinary tract disorder etc.Modern pharmacological research shows that Herba Portulacae has antibiotic, lax skeletal muscle, excited uterus, blood fat reducing, atherosclerosis, blood sugar lowering, anti-inflammatory and antalgic, promotion wound healing, neuroprotective, antioxidation, the anti-ageing effect of waiting for a long time.
Except chemical compounds such as coumarin, flavone, organic acid, monoterpenes, alkaloid is the important chemical constituent of a class in the Herba Portulacae, comprising neurotransmitter constituents such as DOPA, dopamine, noradrenalins.
Herba Portulacae amide A, B, E are the applicant separated the novel structure that obtains in 2005 from Herba Portulacae amide alkaloid [Xiang L; Xing DM; Wang W; et al.Alkaloids from Portulaca oleracea L..Phytochemistry, 2005,66 (21): 2595-2601]; its structure is as follows; but, up to the present, this type of alkaloidal antioxidation and neuroprotective activity and yet there are no report as the application of medicine.
Herba Portulacae amide E (oleracein E)
Herba Portulacae amide A (oleraceinA) Herba Portulacae amide B (oleracein B)
Summary of the invention
At the deficiencies in the prior art, the problem to be solved in the present invention provides the application of a kind of Herba Portulacae amide alkaloid in preparation antioxidant and Neuroprotective Agents.
The application of Herba Portulacae amide alkaloid of the present invention in the preparation antioxidant.
Test confirms: Herba Portulacae amide alkaloid of the present invention particularly Herba Portulacae amide A (oleracein A), Herba Portulacae amide B (oleracein B) and Herba Portulacae amide E (oleracein E) has very strong removing 2, the 2-hexichol is for the effect of bitterness hydrazides (DPPH) free radical, its IC
50Be respectively 8.96,5.56,9.87 μ M, its ability of removing free radical is than reference substance vitamin C (IC
50=11.69 μ M) and vitamin E (IC
50=13.13 μ M) activity is strong, wherein antioxidant Butylated hydroxyanisole (the BHA) (IC of Herba Portulacae amide A and Herba Portulacae amide E and synthetic
50=9.07 μ M) ability of removing free radical is suitable; In addition, the Herba Portulacae amide alkaloid can significantly suppress the generation of the inductive Wistar rat cerebral even slurry of hydrogen peroxide lipid peroxide malonaldehyde (MDA), wherein the antioxidation of Herba Portulacae amide E (oleracein E) is the strongest, the generation (p＜0.01) that in concentration 17.84～285.39 μ M scopes, significantly suppresses MDA (Fig. 1), IC
50=73.1 μ M, suitable (IC with the caffeinic activity of reference substance
50=72.1 μ M); Herba Portulacae amide A (oleraceinA) can significantly suppress MDA in the rat cerebral even slurry when concentration 248.51～497.02 μ M generation (p＜0.05) (Fig. 2); Herba Portulacae amide B (oleracein B) can significantly suppress MDA and produce (p＜0.05) (Fig. 3) when concentration 234.52～938.09 μ M.The Herba Portulacae amide alkaloid has tempting DEVELOPMENT PROSPECT as antioxidant.
The application of Herba Portulacae amide alkaloid of the present invention in the preparation Neuroprotective Agents.
Test confirms: Herba Portulacae amide alkaloid of the present invention particularly Herba Portulacae amide A (oleracein A), Herba Portulacae amide B (oleracein B) and Herba Portulacae amide E (oleracein E) has protective effect (Fig. 4) to the corticocerebral neuronal cell injury of mice tire Mus that external excitatory amino acid glutamic acid causes; Herba Portulacae amide E has certain protection effect (Fig. 5) to the corticocerebral neuronal cell injury of mice tire Mus of hydrogen peroxide-induced; Herba Portulacae amide A, the Herba Portulacae amide B of high dose and Herba Portulacae amide E have the certain protection effect to the corticocerebral neuronal damage of mice tire Mus that scarce sugared anoxia causes, can significantly improve the survival rate (Fig. 6) of neuronal cell.
The application of Herba Portulacae amide alkaloid of the present invention in preparation brain neuron cell ion channel antagonist.
Test confirms: scarce sugared anoxia complex sugar reoxygenation again can cause the neuronal cell film Na that mice tire Mus cerebral cortex damages
+, K
+-ATPase and Ca
2+-ATPase is active significantly to be reduced, and Herba Portulacae amide alkaloid of the present invention particularly Herba Portulacae amide E and Herba Portulacae amide B can significantly improve Na
+, K
+-ATPase and Ca
2+-ATPase activity; The Ca and Herba Portulacae amide A significantly raises
2+-ATPase activity is to Na
+, K
+The obviously effect (Fig. 7) of the active nothing of-ATPase.The ion channel antagonist that prompting Herba Portulacae amide alkaloid can be used as in the neuroprotective uses.
The application of Herba Portulacae amide alkaloid of the present invention in preparation brain neuron cell inhibitors of apoptosis.
Test confirms: Herba Portulacae amide alkaloid of the present invention particularly Herba Portulacae amide A, Herba Portulacae amide B and Herba Portulacae amide E all has certain anti-apoptotic effect when concentration is 0.005mg/ml, wherein the anti-apoptotic effect of Herba Portulacae amide E the strongest (seeing Fig. 8, table 1 and Fig. 9).The apoptosis inhibitor that prompting Herba Portulacae amide alkaloid can be used as in the neuroprotective uses.
In the above-mentioned various application: described Herba Portulacae amide alkaloid is meant the monomer of Herba Portulacae amide A (oleracein A), Herba Portulacae amide B (oleracein B) or Herba Portulacae amide E (oleracein E); Or refer to the blended component of any weight ratio of described monomer; Or refer to the monomer of the derivant of described monomeric compound; Or refer to the blended component of any weight ratio of described derivatives monomer.
Wherein: described Herba Portulacae amide alkaloid preferably is meant the monomer of Herba Portulacae amide A (oleracein A), Herba Portulacae amide B (oleracein B) or Herba Portulacae amide E (oleracein E).
The present invention has set forth the biological activity of Herba Portulacae amide alkaloid and as the application of medicine, the medicine that is developed as the treatment neurodegenerative diseases for the Herba Portulacae amide alkaloid provides strong foundation, for the further development and use of Herba Portulacae amide alkaloid are laid a good foundation.
Description of drawings
Fig. 1. Herba Portulacae amide E (oleracein E) to the influence (n=4) of the inductive rat cerebral even slurry lipid peroxidation of hydrogen peroxide (compare with model group numerical value,
*P＜0.05,
*P＜0.01).
Fig. 2. Herba Portulacae amide A (oleracein A) to the influence (n=4) of the inductive rat cerebral even slurry lipid peroxidation of hydrogen peroxide (compare with model group numerical value,
*P＜0.05,
*P＜0.01).
Fig. 3. Herba Portulacae amide B (oleracein B) to the influence (n=4) of the inductive rat cerebral even slurry lipid peroxidation of hydrogen peroxide (compare with model group numerical value,
*P＜0.05,
*P＜0.01).
Fig. 4. Herba Portulacae amide alkaloid (concentration unit mg/ml) is to the protective effect (n=4) of 0.5mM glutamic acid injured neuron cell
(wherein: M is a model group, and C is the blank group, and MA is Herba Portulacae amide A, and MB is Herba Portulacae amide B, and ME is Herba Portulacae amide E, down together.With model group neuronal cell survival rate is 100%, and all the other each groups are compared with the model group respective value,
*P＜0.05,
*P＜0.01).
Fig. 5. Herba Portulacae amide E (concentration unit mg/ml) is to the protective effect (n=4) of hydrogen peroxide injured neuron cell
(wherein: M is a model group, and C is the blank group, and ME is Herba Portulacae amide E.With model group neuronal cell survival rate is 100%, and all the other each groups are compared with the model group respective value,
*P＜0.05,
*P＜0.01).
Fig. 6. the Herba Portulacae amide alkaloid lacks sugared hypoxia protection effect (n=3) to neuronal cell
(wherein: M is a model group, and C is the blank group, and MA is Herba Portulacae amide A, and MB is Herba Portulacae amide B, and ME is Herba Portulacae amide E.With model group neuronal cell survival rate is 100%, and all the other each groups are compared with the model group respective value,
*P＜0.05,
*P＜0.01).
Fig. 7. the Herba Portulacae amide alkaloid is to Na in the neuronal cell that lacks complex sugar reoxygenation after the sugared anoxia
+-K
+ATPase and Ca
2+The active influence of-ATPase (n=3)
(wherein: model is a model group, and control is the blank group, and MA is Herba Portulacae amide A, and MB is Herba Portulacae amide B, and ME is Herba Portulacae amide E.Compare with the model group respective value,
*P＜0.05,
*P＜0.01).
Fig. 8. the Herba Portulacae amide alkaloid is to the influence of the neuronal cell apoptosis rate of scarce sugared anoxic treatment
(A: Herba Portulacae amide A (0.005mg/ml) wherein; B: Herba Portulacae amide E (0.005mg/ml); C: Herba Portulacae amide B (0.005mg/ml); D: model group; E: the normal control group).
Fig. 9. the Herba Portulacae amide alkaloid lacks the influence (n=3) of bax and bcl-2mRNA expression after the sugared anoxia-induced apoptosis to neuronal cell
(the gained result is 100% with the blank group, all the other each groups and blank group ratio (%) expression.Compare with the model group respective value,
*P＜0.05,
*P＜0.01).
The specific embodiment
Embodiment 1: the Herba Portulacae amide alkaloid uses as novel antioxidant
1. experiment material and instrument
The monomer of Herba Portulacae amide A, Herba Portulacae amide B, Herba Portulacae amide E obtains from the separation of Herba Portulacae medicinal material extract; 2, the 2-hexichol for bitterness hydrazides free radical (2,2-diphenyl-2-picryl-hydrazyl, DPPH), ((butylated hydroxyl anisol is BHA) available from Sigma company for α-Tocopherol) and Butylated hydroxyanisole for vitamin E; 1,1,3,3-tetraethoxypropane, thiobarbituricacid (2-thiobarbituric acid, TBA) and sodium lauryl sulphate (sodium dodecyl sulfate is SDS) available from Shanghai Chemical Reagent Co., Ltd., Sinopharm Group; BSA and Coomassie brilliant blue G-250 are available from the magnificent transduction in Beijing Science and Technology Ltd., and other reagent is analytical pure.The TU-1800 ultraviolet spectrophotometer is that the Beijing Puxi General Instrument Co., Ltd produces; Germany produces ULTRA-TURRAX high-shearing dispersion emulsifying machine (refiner).Male Wistar rat is provided by pharmaceutical college of Shandong University Experimental Animal Center.
2. experimental procedure
(1) the Herba Portulacae amide alkaloid is removed the research of DPPH free radical effect
0.1ml add 3.9ml25 μ g/ml DPPH methanol solution in the drug solution of variable concentrations; Concuss shakes up the back room temperature and keeps away dark placement 30 minutes, measures absorbance, replication three times at 515nm.(annotate:, then do blank zeroing in methanol with methanol if sample dissolves fully; Otherwise with the DMSO dissolving, the mixed liquor that adds 3.9ml methanol with 0.1mlDMSO is done blank zeroing).Calculate DPPH concentration in the solution according to DPPH concentration and absorbance standard curve.The initial concentration of DPPH free radical is expressed as [DPPH
]
T=0, medicine and DPPH reaction after 30 minutes remaining DPPH number of free radical be [DPPH
]
T=30, the percentage rate of remaining DPPH free radical (%)=[DPPH
]
T=30/ [DPPH
]
T=0Medicine is removed concentration (IC to the removing ability of free radical with half
50) expression, be meant and remove the required drug level of 50%DPPH free radical.
(2) research of Herba Portulacae amide alkaloid lipoid peroxidization resistant
Male Wistar rat (225-275g) is used etherization, peels off cerebral tissue after the sacrificed by decapitation rapidly, with the normal saline flush away blood stains of pre-cooling, removes normal saline with the filter paper suction.With cerebral tissue by weight volume ratio 1: 10 add the 20mMTris-HCl buffer (pH 7.4) of pre-cooling, use refiner in rotating speed 18000rpm homogenate three times rapidly, each 10s, 30s at interval, be prepared into brain homogenate (1: 10, w/v).Experiment is divided into three groups: autoxidation matched group, model of oxidative group and medicine group.After getting 150 μ l brain homogenates and 25 μ l Herba Portulacae amide A, B, E solution mixing in the medicine group, hatched 10 minutes, add the H of 25 μ l 400mM afterwards at 37 ℃
2O
2, hatched 60 minutes in 37 ℃ behind the mixing, then ten minutes cessation reactions of ice bath.Autoxidation matched group (control) replaces medicine and does not add hydrogen peroxide with distilled water, and model of oxidative group (model) replaces medicine and adds hydrogen peroxide with distilled water, and all the other conditions are identical with the medicine group.Brain homogenate after the cessation reaction under rotating speed 3000rpm centrifugal 10 minutes, get 100 μ l supernatant and place teat glass, add 200 μ l, 8.1% sodium lauryl sulphate, 1.5ml 0.2M acetic acid-sodium-acetate buffer (pH 3.5), 1.5ml 0.8%TBA aqueous solution and 700 μ l deionized waters.Behind the aforesaid liquid mixing, test tube is sealed and stays an aperture with plastic foil, in 95 ℃ of heating in water bath 60 minutes, ice bath cessation reaction then.With 1,1,3, the 3-tetraethoxypropane is measured malonaldehyde (MDA) content as standard control according to the absorbance of 532nm.With the protein content in Coomassie brilliant blue method (Bradford) the method mensuration brain homogenate.
3. experimental result
(1) the Herba Portulacae amide alkaloid has very strong scavenging action to the DPPH free radical
DPPH (2, the 2-hexichol is for the bitterness hydrazides) free radical is widely used in the resistance to oxidation evaluation of material.DPPH is a kind of stable free radical in organic solvent, and its methanol solution (showing darkviolet) has maximum absorption band at the 515nm place.When doing the time spent with the material with free radical scavenging activity, its lone pair electrons are matched, and cause at 515nm place absorbance to disappear or weaken, and absorb the degree that weakens by measuring, and can estimate the activity of free radical scavenger.The Herba Portulacae amide alkaloid is removed the ability of free radical with IC
50Represent IC
50Be the concentration that DPPH free radical number reduces by 50% o'clock required sample.The result: Herba Portulacae amide A, B, E have the effect of very strong removing DPPH free radical, its IC
50Be respectively 8.96,5.56,9.87 μ M, its ability of removing free radical is than reference substance vitamin C (IC
50=11.69 μ M) and vitamin E (IC
50=13.13 μ M) activity is strong, wherein antioxidant Butylated hydroxyanisole (the BHA) (IC of Herba Portulacae amide A and Herba Portulacae amide E and synthetic
50=9.07 μ M) ability of removing free radical is suitable.
(2) the Herba Portulacae amide alkaloid significantly suppresses the generation of rat cerebral even slurry MDA
Level of lipid in intact animal's brain homogenate is lower.Concentration (the hydrogen peroxide instability of free radical in the scalable normal cell of the hydrogen peroxide of external source high concentration, very easily produce hydroxy radical OH) and the degree of accelerated oxidation damage, OH can excite lipid peroxidation in conjunction with the H on the immobilized artificial membrane, produces a series of lipid peroxidation products.Wherein malonaldehyde (MDA) is exactly the typical product of lipid peroxidation.Thiobarbituricacid (TBA) can react under low pH, heating condition with MDA, generates a kind of pink chromophoric material of being with: 3,5, and 5-trimethyl oxazole 2,4-diketone.Measure the height of MDA content by the absorbance of measuring this kind material, but the size of assess sample oxidation resistance.Result of the test of the present invention: compare with model group, the Herba Portulacae amide alkaloid can significantly suppress the generation of the inductive Wistar rat cerebral even slurry of hydrogen peroxide lipid peroxide malonaldehyde (MDA), wherein the antioxidation of Herba Portulacae amide E (oleracein E) is the strongest, the generation (p＜0.01) that in concentration 17.84～285.39 μ M scopes, significantly suppresses MDA (Fig. 1), IC
50=73.1 μ M, suitable (IC with the caffeinic activity of reference substance
50=72.1 μ M); Herba Portulacae amide A (oleracein A) can significantly suppress MDA in the rat cerebral even slurry when concentration 248.51～497.02 μ M generation (p＜0.05) (Fig. 2); Herba Portulacae amide B (oleracein B) can significantly suppress MDA and produce (p＜0.05) (Fig. 3) when concentration 234.52～938.09 μ M.
Embodiment 2: the Herba Portulacae amide alkaloid uses as novel Neuroprotective Agents
1. experiment material and instrument
MEM culture medium and F12 culture medium (Gibco), calf serum and hyclone are available from the Hangzhou Ilex purpurea Hassk.[I.chinensis Sims; Poly-D-lysine, MTT are the Sigma product; The D-glucose is a Sigma import packing product; The LDH test kit is available from giving birth to biological high-technology company in Beijing, all the other reagent are homemade analytical pure.Tissue Culture Plate and culture dish are the Costar product.SANYO (MCO-15AC) CO2 gas incubator, Olympus inverted microscope and fluorescence microscope; The SORVALL high-speed refrigerated centrifuge, T52 type ultraviolet-uisible spectrophotometer (OHAUS), Bio-RAD microplate reader.
2. experimental procedure
(1) neuronal cell is cultivated
After getting newborn mice (0～3 day) death by suffocation, 75% alcohol disinfecting 5 minutes, place the interior ice bath of super-clean bench to separate brain, after carefully peeling off pia mater encephali, after rinsing out blood with the ice-cold PBS buffer that contains 0.1% glucose, the clip cerebral cortex, place test tube, add in the PBS buffer of 0.1% ice-cold glucose of 3ml, for several times cortex is disperseed slightly with suction pipe (elbow) piping and druming, add 300 μ l, 0.5% tryptic digestive juices (1: 10), 37 ℃ of vibrations added MEM and the F12 mixed culture medium 3ml of 10%BS after 30 minutes, suction pipe piping and druming is for several times until almost there not being macroscopic large crumb, Digestive system is crossed 200 order stainless steel filtering nets, collects filtrate, centrifugal 5 minutes of 800rpm, abandoning supernatant, wash cell 2 times with the PBS buffer that contains 0.1% glucose, add the MEM and the F12 mixed culture medium of 2ml 10%BS and 10% horse serum after the last supernatant discarded, counting also is diluted to 2～5 * 10 with the same mixed culture medium that contains calf serum and horse serum
6Individual cell/ml solution is inoculated in advance in the culture plate with poly-D-lysine bag quilt, cultivates in 37 ℃, 5% CO2 gas incubator.Change after 24 hours and keep culture fluid: the mixed culture medium of 5% horse serum, changed liquid every 3 days.(final concentration is 10 to cultivate the 3rd day adding cytosine arabinoside
-5M) incubated altogether 48 hours with cell, to suppress the propagation of non-neuronal cell.
(2) protective effect of Herba Portulacae amide alkaloid neuronal damage that glutamic acid is caused
The former mouse brain cortical neurogenic cell of supporting 5 days of being commissioned to train is with after keeping culture medium and washing 1 time, adding glutamic acid (final concentration is 0.5mM) damaging cells, and the blank group is with keeping culture medium.Sucking-off culture medium behind the 15min, cell adds the medicine of variable concentrations respectively with after keeping culture medium and washing 2 times, each concentration four multiple hole.Model group and blank group cell substitute with the culture medium of keeping that contains 0.1%DMSO.Cell places incubator to hatch 24 hours.Take out supernatant, quantitatively add 200 μ l/ hole normal saline, place-20 ℃ of refrigerator multigelations.Measure LDH vigor in supernatant and the freeze thawing liquid respectively according to method shown in the lactic dehydrogenase enzyme reagent kit, the result represents with LDH (lactic acid dehydrogenase) the escape rate of cell.LDH escape rate (%)=LDH
Supernatant/ (LDH
Supernatant+ LDH
Freeze thawing liquid).
Other gets former generation mouse brain cortical neuron cell of cultivating 5 days, the Herba Portulacae amides compound of glutamic acid damage back adding variable concentrations is cultivated sucking-off culture medium after 24 hours according to the method described above, clean cell once after, cell is hatched sucking-off supernatant after 4 hours with the MTT of 0.5mg/ml, add DMSO concussion dissolution precipitation, microplate reader is measured trap with the dual wavelength colorimetry at 490nm, and reference wavelength is 655nm.The result represents with the cell survival percentage rate, is 100% with model group cell absorbance, and all the other each groups are compared with model group and obtained respective value.
(3) protective effect of Herba Portulacae amide alkaloid neuronal damage that hydrogen peroxide is caused
The former mouse brain cortical neurogenic cell of being commissioned to train and supporting 5 days is the hydrogen peroxide replacement glutamic acid damaging cells 15min of 0.5mM, the same experimental procedure of operating procedure (2) afterwards with final concentration.
(4) the Herba Portulacae amide alkaloid is to the protective effect of the neuronal damage that lacks sugared anoxia and cause
The former mouse brain cortical neuron cell of being commissioned to train and supporting 5 days discards original fluid, and cell (contains 122mM NaCl, 5.4mM KCl, 1.8mM CaCl with sugar-free Earle liquid
2, 0.8mM MgSO
4, 1.0mM NaH
2PO
4, 26.2mMNaHCO
3) wash 2 times after, cellular control unit (contains 116.4mM NaCl, 5.4mM KCl, 1.8mM CaCl with Earle liquid
2, 0.8mM MgSO
4, 1.0mM NaH
2PO
4, 26.2mM NaHCO
3With the 5.6mM glucose) the same processing.Sugar-free group cell is placed hermetic container, feed 95%N
2And 5%CO
2Drove oxygen in the container in 10 minutes away, closed container is placed in 37 ℃ of incubators behind the 2hr, and Tissue Culture Plate is taken out from container.Cellular control unit directly places cell culture incubator same long-time.After sugar-free group cell discards culture fluid, add the medicine (medicine is with the dissolving of sugar-free Earle liquid) of variable concentrations respectively, each concentration three multiple hole places incubator to hatch 24hr.After cellular control unit discarded culture fluid, the Earle liquid that more renews continued to cultivate 24hr.All cells detects cell LDH escape rate and mtt assay and detects cell survival rate according to the method in the experimental procedure (2).
3. experimental result:
Herba Portulacae amide A, Herba Portulacae amide B and Herba Portulacae amide E have protective effect to the corticocerebral neuronal cell injury of mice tire Mus that external excitatory amino acid glutamic acid causes, the survival rate of medicine group neuronal cell is compared be significantly increased (Fig. 4) with model group; Herba Portulacae amide E has certain protection effect (Fig. 5) to the corticocerebral neuronal cell injury of mice tire Mus of hydrogen peroxide-induced; The Herba Portulacae A of high dose, Herba Portulacae amide B and Herba Portulacae amide E have the certain protection effect to the corticocerebral neuronal damage of mice tire Mus that scarce sugared anoxia causes, significantly improve the survival rate (Fig. 6) of neuronal cell.
Embodiment 3: the Herba Portulacae amide alkaloid uses as the ion channel antagonist in the neuroprotective
1. experiment material and instrument
MEM culture medium and F12 culture medium (Gibco), calf serum and hyclone are available from the Hangzhou Ilex purpurea Hassk.[I.chinensis Sims; PI, EB, poly-D-lysine, Coomassie brilliant blue G250 are the Sigma product; The D-glucose is a sigm import packing product; ATPase test kit (bio-engineering research institute is built up in Nanjing), all the other reagent are homemade analytical pure.Tissue Culture Plate and culture dish are the Costar product.SANYO (MCO-15AC) CO2 gas incubator, the inverted microscope of Olympus and fluorescence microscope; The SORVALL high-speed refrigerated centrifuge, ZS semi-automatic biochemical analyzer, T52 type ultraviolet-uisible spectrophotometer (OHAUS), Bio-RAD microplate reader.
2. experimental procedure
Medicine is to lacking the active influence of ATPase on the sugared anoxia-induced apoptosis neuronal cell film: neuronal cell is cultivated with 24 orifice plates, and lack sugared anoxia operation (method is seen before and stated), after medicine is incubated 24hr with it altogether, abandoning supernatant, after PBS cleans cell 2 times, trypsinization and collecting cell precipitation, every duplicate samples adds behind the 500 μ lPBS smudge cells in the ultrasonic ice bath, the centrifugal 10min of 1000rpm measures ATPase activity in the supernatant, measures albumen quality in the supernatant with the Coomassie brilliant blue method simultaneously.Na
+-K
+ATPase and Ca
2+The active mensuration of-ATPase adopts ATPase test kit (bio-engineering research institute is built up in Nanjing), and operation is measured to specifications.Each drug level repeats three multiple holes, and the result is the unit of activity of enzyme with the per hour newly-generated Pi amount (μ mol) of every mg protein in the supernatant.
3. experimental result
Na in the cerebral tissue
+, K
+-ATPase and Ca
2+-ATPase activity plays crucial effect to normal configuration and the function of keeping intracellular ion concentration, keeping cell.Na during ischemia-reperfusion
+, K
+-ATPase and Ca
2+-ATPase is active significantly to descend.Result of the test of the present invention: Herba Portulacae amide alkaloid particularly Herba Portulacae amide E and Herba Portulacae amide B can significantly improve Na
+, K
+-ATPase and Ca
2+-ATPase activity; The Ca and Herba Portulacae amide A significantly raises
2+-ATPase activity is to Na
+, K
+The obviously effect (Fig. 7) of the active nothing of-ATPase.
Embodiment 4: the Herba Portulacae amide alkaloid uses as the neural apoptosis inhibitor in the neuroprotective
1. experiment material and instrument
MEM culture medium and F12 culture medium (Gibco), calf serum and hyclone are available from the Hangzhou Ilex purpurea Hassk.[I.chinensis Sims; PI, EB, poly-D-lysine are the Sigma product; The D-glucose is a sigma import packing product; DNA MarkerII and Trizol are the sky and are Time Inc.'s product.The AMV reverse transcriptase (Aifaifamosaievirus, AMV) one-step method RT-PCR test kit is that chemical product is given birth in Shanghai; All the other reagent are homemade analytical pure.Tissue Culture Plate and culture dish are the Costar product.It is synthetic that primer is the living worker in Shanghai.SANYO (MCO-15AC) CO2 gas incubator, the inverted microscope of Olympus and fluorescence microscope; The SORVALL high-speed refrigerated centrifuge, Chemicon flow cytometer, the PCR instrument of DYY-10 type electrophresis apparatus (Beijing Liu Yichang), BIOER and gel imaging analysis system (multiple day of Shanghai).
2. experimental procedure
(1) flow cytometer is observed medicine to lacking the protective effect of sugared anoxia-induced apoptosis neuronal cell apoptosis
6 orifice plates are cultivated mouse brain cortical neuron cell, lack sugared anoxia-induced apoptosis, after medicine is incubated 24 hours with it altogether, trypsinization obtains cell suspension, the collecting cell suspension is in the ep pipe, and abandoning supernatant behind the centrifugal 3min of 800rpm, precipitation use the PBS of pre-cooling to clean 3 times.After for the last time centrifugal, add fixedly 1hr of 75% ethanol in the precipitation.Supernatant discarded behind the centrifugal 3min of 800rpm, cell is washed once the back with 100 μ g/ml RNase A room temperature treatment 30min with cold PBS, subsequently with 50 μ g/ml PI dyeing 30min, flow cytometer detection apoptosis rate.
(2) the RT-PCR method is observed medicine to lacking the influence of sugared anoxia-induced apoptosis neuron expression apoptosis-related genes Bcl-2 and Bax
It is described to press embodiment 2 experimental procedures (4), and neurocyte lacked sugared anoxia-induced apoptosis after 2 hours, adds different pharmaceutical and cultivates altogether 24 hours.Cell adds Trizol after washing 3 times with PBS, and collecting cell liquid extracts cell total rna according to the Trizol operational approach in the EP pipe of DEPC processing, be dissolved in the water of DEPC processing.All samples A
260nm/ A
280nm=1.8-2.0 illustrates that its purity satisfies the molecular biology experiment requirement.The concentration computing formula of RNA is:
(wherein L represents the thickness of cuvette, is generally 1cm, A
260For RNA at the 260nm absorbance value)
Every part of total RNA sample is got 4 μ g, according to the synthetic cDNA of the operating instruction of the MMLV first chain cDNA synthetic agent box (BBI), carry out pcr amplification according to the explanation of one-step method PCR test kit with different primers then, product agarose gel electrophoresis analysis, offset plate dyeed 25 minutes with the EB solution chamber relaxing the bowels with purgatives of warm nature of 0.5 μ g/ml.With taking pictures and analyzing the gray value of each electrophoretic band, the result represents that to increase or to reduce percentage ratio wherein model group band gray value is 100%.
3. experimental result
The apoptotic result of flow cytometry analysis shows, the corticocerebral neuronal cell of mice tire Mus lacks sugared anoxia complex sugar reoxygenation 24 hours again after 2 hours, and apoptosis rate can significantly increase, and reaches about 8%.Result of the test of the present invention: Herba Portulacae amide A, Herba Portulacae amide B and Herba Portulacae amide E all have certain anti-apoptotic effect when concentration is 0.005mg/ml, wherein the anti-apoptotic effect the strongest (seeing Fig. 8 and table 1) of Herba Portulacae amide E.In addition, the present invention tests and confirms that the Herba Portulacae amide alkaloid mainly is the effect of playing anti-neuronal apoptosis by the mediated apoptosis expression of gene.Short apoptogene bax up-regulated during neuronal apoptosis, while apoptosis suppressor bcl-2 down-regulated expression, the expression of finally inducing caspase 3, trigger cell apoptotic response.Compare with scarce sugared anoxia model group cell, Herba Portulacae amide A and Herba Portulacae amide B can significantly reduce the expression of short apoptogene bax, but the expression of bcl-2 is also demonstrated significant downward modulation effect; Herba Portulacae amide E can significantly reduce the expression of short apoptogene bax, and the expression of simultaneously remarkable upregulation of apoptosis suppressor gene bcl-2 demonstrates powerful anti-apoptosis activity (see figure 9).
Table 1 flow cytometer result shows the influence of Herba Portulacae amide alkaloid to the Neuron Apoptosis rate that lacks sugared anoxia and cause
| Groups | Dose(mg/ml) | G2/G1(％) | Dip S(％) | Apoptosis(％) |
| Herba Portulacae amide A Herba Portulacae amide E Herba Portulacae amide B model group (model) normal control group (control) | 0.005 0.005 0.005 | 1.63 1.58 1.79 1.65 1.73 | 21.78 21.35 29.57 42.87 31.55 | 0.27 0 0.01 8.09 0 |
Claims (6)
1. the application of Herba Portulacae amide alkaloid in the preparation antioxidant.
2. the application of Herba Portulacae amide alkaloid in the preparation Neuroprotective Agents.
3. the application of Herba Portulacae amide alkaloid in preparation brain neuron cell ion channel antagonist.
4. the application of Herba Portulacae amide alkaloid in preparation brain neuron cell inhibitors of apoptosis.
5. as the described application of one of claim 1～4, it is characterized in that: described Herba Portulacae amide alkaloid is meant the monomer of Herba Portulacae amide A (oleracein A), Herba Portulacae amide B (oleracein B) or Herba Portulacae amide E (oleracein E); Or refer to the blended component of any weight ratio of described monomer; Or refer to the monomer of the derivant of described monomeric compound; Or refer to the blended component of any weight ratio of described derivatives monomer.
6. application as claimed in claim 5 is characterized in that: described Herba Portulacae amide alkaloid preferably is meant the monomer of Herba Portulacae amide A (oleracein A), Herba Portulacae amide B (oleracein B) or Herba Portulacae amide E (oleracein E).
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