CN103393670A - Application of Chukrasone B in preparing anti-hypoxic medicines - Google Patents

Abstract

The invention discloses an application of Chukrasone B in preparing anti-hypoxic medicines. According to the invention, the anti-hypoxic effect of Chukrasone B is observed through integral and cellular hypoxic models. The results show that Chukrasone B can remarkably improve the survival rate and the survival time of mice with asphyxial hypoxia and acute decompressed hypoxia, alleviate myocardial damage of medicine specificity hypoxic mice and further has a remarkable protective effect on hypoxia injury of cardiac myocytes and nerve cells of neonatal rats cultured in vitro. The invention provides a use of Chukrasone B in preparing anti-hypoxic medicines.

Description

The application of Chukrasone B in anti-anoxic medicine

Technical field

The present invention relates to a kind of pharmaceutical preparation, these goods can be used for preventing and treating the hypoxic damage disease.

Technical background

Oxygen is the essential condition that the mankind and many biologies are depended on for existence.Hypoxia (Hypoxia) refers to that the required oxygen of body vital movement can not obtain sufficient supply.Oxygen and hypoxia are the most important key factors of vital movement, are the important topics of life sciences basic theories.The formation of hypoxia can be divided three classes: the first kind is that the external environment oxygen content reduces, and makes the normal physiological activity process can not absorb enough oxygen, as plateau and aviation anoxia; Equations of The Second Kind refers to can not fully arrive in body because disease etc. causes extraneous normal oxygen amount, causes the anoxia of the heart, brain and respiratory system etc.; The 3rd class is the movable requisite oxygen consumption of body, has surpassed the physiology ability of mobilization, causes relative oxygen supply not enough, is common in strenuous exercise and the amount of transfiniting work.Long-term hypoxia is the important hidden danger that is detrimental to health, but the severe patient threat to life.Therefore, hypoxia causes the heart, brain and respiratory system equivalent damage to become one of 21 century medical circle subject matter anxious to be resolved.

the Compound C hukrasone B that the present invention relates to is one and delivered (Liu in 2012, H. B. et al., 2012. Chukrasones A and B:Potential Kv1.2 Potassium Channel Blockers with New Skeletons from Chukrasia tabularis. Organic Letters 14 (17), 4438 – 4441.) New skeleton compound, this compound has brand-new framework types, present purposes only relates to potassium-channel and suppresses active (Liu, H. B. et al., 2012. Chukrasones A and B:Potential Kv1.2 Potassium Channel Blockers with New Skeletons from Chukrasia tabularis. Organic Letters 14 (17), 4438 – 4441.), belong to open first for the purposes of Chukrasone B in preparing anti-anoxic medicine that the present invention relates to, because framework types belongs to brand-new framework types, and its anti-hypoxia is active unexpectedly strong, there do not is the possibility that is provided any enlightenment by other compounds, possesses outstanding substantive distinguishing features, be used for simultaneously anti-hypoxia and obviously have significant progress.

Summary of the invention

The object of the invention is to find that Chukrasone B has significant oxygen lack resistant function, can be used for preventing and treating the anoxia-induced apoptosis disease, thereby increased the application of Chukrasone B.

The application of Chukrasone B of the present invention in preparing anti-anoxic medicine is Chukrasone B prevents and treats application in the hypoxic damage medicine in preparation.

Described Compound C hukrasone B structure is as shown in formula I:

Formula I

It is open first that the purposes of Chukrasone B in preparing anti-anoxic medicine that the present invention relates to belongs to, because framework types belongs to brand-new framework types, and its anti-hypoxia is active unexpectedly strong, there do not is the possibility that is provided any enlightenment by other compounds, possess outstanding substantive distinguishing features, be used for simultaneously anti-hypoxia and obviously have significant progress.

The specific embodiment

The preparation method of Compound C hukrasone B involved in the present invention is referring to document (Liu, H. B. et al., 2012. Chukrasones A and B:Potential Kv1.2 Potassium Channel Blockers with New Skeletons from Chukrasia tabularis. Organic Letters 14 (17), 4438 – 4441.).

The present invention is further detailed explanation by the following examples, but protection scope of the present invention is not subjected to any restriction of specific embodiment, but by claim, be limited.

Embodiment 1: the preparation of Compound C hukrasone B tablet involved in the present invention:

Get 20 and digest compound Chukrasone B, add conventional adjuvant 180 grams that prepare tablet, mix, conventional tablet machine is made 1000.

Embodiment 2: the preparation of Compound C hukrasone B capsule involved in the present invention:

Get 20 and digest compound Chukrasone B, add the conventional adjuvant such as starch 180 grams that prepare capsule, mix, encapsulatedly make 1000.

Further illustrate its pharmaceutically active below by pharmacodynamic experiment.

The description of test of following Chukrasone B oxygen lack resistant function:

Test a mice specificity myocardial ischemia experiment

1, method:

75 kunming mices, body weight (20 ± 2) g.Be divided at random 5 groups, gastric infusion.Front 2 groups give 0.3% sodium carboxymethyl cellulose (CMC-Na) solution, and rear 3 groups give respectively Chukrasone B 0.015,0.03gKg ^-1, propranolol hydrochloride 0.03gKg ^-1, after 50min, except the 1st group, equal lumbar injection isoproterenol (ISO) 15mgKg ^-1, after 15min, mice is put into the normobaric hypoxia device, record mouse diing time and oxygen consumption.

2, result:

Isoproterenol can pass through excited heart beta receptor, and myocardial oxygen consumption is increased.This experiment shows, compares Chukrasone B 0.015,0.03gKg with the solvent matched group ^-1Can significantly resist the myocardial oxygen consumption increase (P＜0.01) that isoproterenol (ISO) causes, extend simultaneously the time-to-live (P＜0.01) under the anoxia in mice air-tight state, the results are shown in Table 1.

Table 1 Chukrasone B causes the impact (x ± s, n=15) of specificity hypoxia mice on isoproterenol

Annotate: ¹⁾P＜0.01, compare with matched group, ²⁾P＜0.01, compare with the isoproterenol group.

Test two mice normal pressure asphyxiating anoxia experiments

1, method:

60 kunming mices, body weight (20 ± 2) g.Be divided at random 4 groups, gastric infusion.The 1st group gives 0.3% sodium carboxymethyl cellulose (CMC-Na) solution, and the 2nd group gives propranolol hydrochloride 0.03gKg ^-1, the pro group.3rd, 4 groups of CMC-Na solution that contain respectively Chukrasone B, concentration is respectively 0.015,0.03 gKg ^-1After administration 50min, be placed in wide mouthed bottle and cover tightly bottle stopper (placing the 5g sodica calx in bottle)., take respiratory arrest as sign, record the mouse survival time.

2, result:

Compare Chukrasone B 0.015,0.03 gKg with the solvent matched group ^-1Make the time-to-live of mice under the atmospheric closed condition extend respectively 29.84% and 41.56%, difference has significance (P＜0.01, P＜0.05).

Test three mice hypobaric hypoxia experiments

1, method:

40 kunming mices, body weight (20 ± 2) g.Be divided at random 4 groups, gastric infusion.The administration group gives Chukrasone B, and concentration is respectively 0.015,0.03 gKg ^-1, matched group gives 0.3% CMC-Na solution, and the gavage volume is 2mlKg ^-1After 50min, administration group and matched group are respectively got 5, put into decompressor, at 26.7Kpa(, be equivalent to approximately 10000m of height above sea level) time stop the decompression, keep this pressure constant, when animal dead 50%, stop immediately decompression, slowly put into air, take out animal, each group death of record and survival number, repetitive operation to experiment is completed.

2, result:

Chukrasone B 0.015,0.030gKg ^-1Make the survival rate of mice under the hypobaric hypoxia condition be increased to 70%, 80% by 30%, 20% of matched group, difference has significance (P＜0.05).

Test the protective effect of four pairs of Myocytes Anoxia damages

1, method:

(1) Neonatal Rat Primary Cardiomyocytes is cultivated: the SD neonatal rat of newborn 1-3d is got ventricular muscles and is cut into approximately 1mm ³The size piece of tissue, add 0.25% trypsin-0.02%EDTA, with 37 ℃ under digestion.Except digesting first supernatant discards, collect each time supernatant and end digestion, centrifugal collection myocardial cell precipitation, add the DMEM/F-12 culture medium and the Brdu(final concentration 0.1mmol/L that contain 10% hyclone), piping and druming mixes gently, make cell suspension, be inoculated in culture bottle, in 37 ℃, 5%CO ₂Hatch 70min in incubator, make most non-myocardial cell adherent.

Draw bottle inner cell suspension counting, adjusting cell concentration is 1 * 10 ⁵Individual/ml, be inoculated into 24 orifice plates, every hole 1ml; 96 orifice plates, every hole 200ul.After the 48h cell attachment, change the DMEM/F-12 culture medium (not containing Brdu) that contains 10% hyclone, changed liquid 1 time in later every 2 days.

(2) the Myocytes Anoxia model is set up: get the myocardial cell of cultivating 4d, after changing serum-free DMEM/F-12 culture medium continuous culture 12h, with sugar-free D-Hanks liquid, (be filled with in advance 95%N ₂-5%CO ₂The saturated 30min of gaseous mixture) substitute normal culture medium, then rapidly culture plate is moved into and is connected with 95%N ₂-5%CO ₂In the hypoxia device of gaseous mixture, with oxygen analyser, detect air vent oxygen concentration (＜1%), 37 ℃ of anoxias are cultivated 6h.

(3) experiment grouping: experiment is established blank group, anoxia model group, anoxia+Chukrasone B A group (concentration 0.024 mg/ml), B group (concentration 0.012 mg/ml), C and is organized (concentration 0.006 mg/ml), totally 5 groups, every group of 6 holes.Except the blank group, other respectively organize equal anoxic treatment 6h, and Normal group is in 37 ℃, 5%CO ₂Synchronously hatch 6h in incubator.

(4) anoxia-induced apoptosis myocardial cell MTT experiment: take out and respectively organize sample, every hole adds 20ul MTT(5g/L), in 37 ℃, 5%CO ₂Continue to hatch 4h in incubator, stop cultivating a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices.Add 150ul DMSO jolting 15min, crystallization is fully dissolved,, in measuring wavelength 570 nm, reference wavelength 630nm place, measure each hole absorbance (OD) value.

MTT metabolic rate (%)=experimental group OD value/Normal group OD value * 100%

(5) biochemical indicator detects: get be incubated at 24 orifice plates respectively organize cell culture supernatant, active with colorimetric method for determining LDH, CK, measure in cell SOD with xanthine oxidase active; With MDA content in thiobarbituricacidα-colorimetric method for determining cell, concrete testing process and operational approach are undertaken by the test kit description.

2, result:

When myocardial cell sustained damage because of anoxia, mitochondrial function was abnormal, and oxidation-respiration chain is impaired, electronics transmission blocking-up, and ATP produces minimizing.Succinate dehydrogenase is one of compound enzyme important in respiratory chain of succinic acid oxidation, but the dehydrogenation of catalysis succinic acid generates Fumaric acid, thereby make FAD accept two hydrogen atoms, generates FADH ₂, and then hydrogen is passed to C _OQ, generate C _OQH ₂, the electron transfer process of continuation respiratory chain.So, the activity of succinate dehydrogenase, can the reflecting myocardium cell hypoxia degree of damage.The MTT experimental principle, utilize succinate dehydrogenase can catalysis Thiazolyl blue (MTT) to generate the characteristic of the insoluble coloured product of aubergine exactly, reflects the cytoactive of each group by the mensuration of absorbance.This experimental result shows: concentration is respectively 0.024 mg/ml, 0.012 mg/ml, 0.006 mg/ml Chukrasone B, MTT records the OD value and is significantly higher than model group, show and all can significantly increase cytoactive (P＜0.01), illustrate that Chukrasone B can, to the infringement of anti-hypoxia to the myocardial cell succinic dehydrogenase activity, keep the respiratory function of Hypoxic cell; When anoxia causes cell membrane damage, in cell, LDH, CK will leak outside, cause the active rising of LDH in culture medium, CK, 3 dosage groups of this test demonstration are compared LDH in culture medium, CK activity and are all obviously reduced with model group, show that Chukrasone B can be under Conditions of Acute Hypoxia in Human Body, keep the integrity of myocardial cell membrane, the above results is in Table 2.

SOD has reflected the ability of cell clearance free radical, MDA has reflected Cell membrane lipids Peroxidative damage degree, 3 dosage groups of this test raise with SOD is active in model group is compared cell, the MDA level produces and descends,, in Table 3, show the cell membrane oxidative damage that Chukrasone B can cause anti-hypoxia by the ability that strengthens myocardial cell removing free radical.

The impact (x ± s, n=6) of table 2 Chukrasone B on the outer leakage quantity of anoxic myocardial LDH, CK

* P＜0.05, ^*P＜0.01, compare with model group

The impact (x ± s, n=8) of table 3 Chukrasone B on anoxic myocardial vigor, SOD activity and MDA content

* P＜0.05, ^*P＜0.01, compare with model group

Conclusion: Chukrasone B can significantly improve survival rate and the time-to-live of asphyxiating anoxia and acute decompression hypoxia mice; alleviate the myocardial damage of drug specificity hypoxia mice; neonatal rat myocardial cell and neurocyte anoxia-induced apoptosis to In vitro culture also have significant protective effect, and the purposes of Chukrasone B in preparing anti-anoxic medicine is provided.

Claims (1)

1.Chukrasone the application of B in anti-anoxic medicine, described Compound C hukrasone B structure as Formula IShown in:

Formula I.