CN103301106A - Application for Nardoaristolones A in preparation for anti-hypoxic medicines - Google Patents

Abstract

The invention discloses an application for Nardoaristolones A in preparation of anti-hypoxic medicines. According to the application disclosed by the invention, the anti-hypoxic effect of Nardoaristolones A is observed via an overall and cellular level hypoxic model, and the result indicates that Nardoaristolones A is capable of remarkably increasing the survival rate and survival time of asphyxiant hypoxic mice and acute decompression hypoxic mice, alleviating the myocardial injury of medicine-specificity hypoxic mice, and remarkably protecting the hypoxic injury of in-vitro cultured neonatal rat cardiac myocytes and neurocytes. An application for Nardoaristolones A in preparation of anti-hypoxic medicines is provided.

Description

The application of Nardoaristolones A in the preparation anti-anoxic medicine

Technical field

The present invention relates to the new purposes of compound N ardoaristolones A, relate in particular to the application of Nardoaristolones A in the preparation anti-anoxic medicine.

Background technology

Oxygen is essential condition human and that 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, such as plateau and aviation anoxia; Equations of The Second Kind refers to can not fully arrive in the 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 the work of strenuous exercise and the amount of transfiniting.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 N ardoaristolones A that the present invention relates to is one and delivered (Ming-Li Liu in 2013; Ying-Hui Duan; Yun-Long Hou; et al.Nardoaristolones A and B; Two Terpenoids with Unusual Skeletons from Nardostachys chinensis Batal.ORGANIC LETTERS; 2013; 15(5): noval chemical compound 1000 – 1003.); this chemical compound has brand-new framework types; the myocardial damage that present purposes is only induced hydrogen peroxide has protective effect (Ming-Li Liu; Ying-Hui Duan; Yun-Long Hou; et al.Nardoaristolones A and B; Two Terpenoids with Unusual Skeletons from Nardostachys chinensis Batal.ORGANIC LETTERS; 2013,15(5): 1000 – 1003.), the purposes of the Nardoaristolones A that the present invention relates in the preparation anti-anoxic medicine belongs to open first.

Summary of the invention

For above-mentioned prior art, the object of the invention is to find that Nardoaristolones A has significant oxygen lack resistant function, can be used for preventing and treating the anoxia-induced apoptosis disease, thereby increased the application of Nardoaristolones A.

Nardoaristolones A of the present invention is in the application of preparation in the anti-anoxic medicine, and being Nardoaristolones A prevents and treats application in the hypoxic damage medicine in preparation.Described compound N ardoaristolones A structure is shown in formula I:

Formula I

The purposes of the Nardoaristolones A that the present invention relates in the preparation anti-anoxic medicine belongs to open first, because framework types belongs to brand-new framework types, and its anti-hypoxia is active unexpectedly strong, there is not the possibility that is provided any enlightenment by other chemical 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 N ardoaristolones A involved in the present invention is referring to document (Ming-Li Liu, Ying-Hui Duan, Yun-Long Hou, et al.Nardoaristolones A and B, Two Terpenoids with Unusual Skeletons from Nardostachys chinensis Batal.ORGANIC LETTERS, 2013,15(5): 1000 – 1003.)

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 limited by claim.

Embodiment 1: the preparation of compound N ardoaristolones A tablet involved in the present invention:

Get 5 and digest compound Nardoaristolones A, add dextrin 195 grams, mixing, conventional tablet machine are made 1000.

Embodiment 2: the preparation of compound N ardoaristolones A capsule involved in the present invention:

Get 5 and digest compound Nardoaristolones A, add starch 195 grams, mixing is encapsulatedly made 1000.

Further specify its pharmaceutically active below by pharmacodynamic experiment.

Experimental example 1: test mice specificity myocardial ischemia experiment

1, method: 75 kunming mices derive from Nanjing University of Traditional Chinese Medicine's Experimental Animal Center, 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 Nardoaristolones A0.015,0.03gKg ^-1, propranolol hydrochloride 0.03gKg ^-1, behind the 50min, except the 1st group, equal lumbar injection isoproterenol (ISO) 15mgKg ^-1, behind the 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 Nardoaristolones A0.015,0.03gKg with the solvent matched group ^-1Can significantly resist the myocardial oxygen consumption increase (P＜0.01) that isoproterenol (ISO) causes, prolong simultaneously the time-to-live (P＜0.01) under the anoxia in mice air-tight state, the results are shown in Table 1.

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

Compare with matched group, #P＜0.01 is compared with the isoproterenol group in * P＜0.01

Test the experiment of two mice normal pressure asphyxiating anoxias

1, method:

60 kunming mices derive from Nanjing University of Traditional Chinese Medicine's Experimental Animal Center, 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 Nardoaristolones A, concentration are respectively 0.015,0.03gKg _-1Behind the administration 50min, place wide mouthed bottle and cover tightly bottle stopper (placing the 5g sodica calx in the bottle).Take respiratory arrest as sign, the record mouse survival time.

2, result:

Compare Nardoaristolones A0.015,0.03gKg with the solvent matched group ^-1Make the time-to-live of mice under the atmospheric closed condition prolong respectively 31.24% and 42.40%, difference has significance (P＜0.01, P＜0.05).

Test the experiment of three mice hypobaric hypoxias

1, method:

40 kunming mices derive from Nanjing University of Traditional Chinese Medicine's Experimental Animal Center, body weight (20 ± 2) g.Be divided at random 4 groups, gastric infusion.The administration group gives Nardoaristolones A, and concentration is respectively 0.015,0.03gKg ^-1, matched group gives 0.3%CMC-Na solution, and the gavage volume is 2mlKg ^-1Behind the 50min, administration group and matched group are respectively got 5, put into decompressor, be equivalent to approximately 10000m of height above sea level at 26.7Kpa() time stop the decompression, keep this pressure constant, when treating 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 finished.

2, result:

Nardoaristolones A0.015,0.030gKg _-1Make the survival rate of mice under the hypobaric hypoxia condition be increased to 75%, 85% by 35%, 25% 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 adds 0.25% trypsin-0.02%EDTA, with 37 ℃ of lower 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), blow and beat gently mixing, make cell suspension, be inoculated in the culture bottle, in 37 ℃, 5%CO ₂Hatch 70min in the 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.Behind the 48h cell attachment, change the DMEM/F-12 culture medium (not containing Brdu) that contains 10% hyclone, changed liquid 1 time in later per 2 days.

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

(3) experiment grouping: experiment is established blank group, anoxia model group, anoxia+Nardoaristolones A A group (concentration 0.024mg/ml), B group (concentration 0.012mg/ml), C and is organized (concentration 0.006mg/ml), totally 5 groups, every group of 6 holes.Except the blank group, other respectively organize equal anoxic treatment 6h, and Normal group is then in 37 ℃, 5%CO ₂Hatch synchronously 6h in the 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 the 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 570nm, 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 the cell SOD with xanthine oxidase active; With MDA content in the 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 unusual, and oxidation-respiration chain is impaired, electronics transmission blocking-up, and ATP produces minimizing.Succinate dehydrogenase is one of compound enzyme important in the respiratory chain of succinic acid oxidation, but the dehydrogenation of catalysis succinic acid generates Fumaric acid, generates FADH thereby make FAD accept two hydrogen atoms ₂, and then hydrogen passed to C _OQ generates COQH 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 utilizes 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 0.024mg/ml, 0.012mg/ml, 0.006mg/ml Nardoaristolones A, MTT records the OD value and is significantly higher than model group, show and all can significantly increase cytoactive (P＜0.01), illustrate that Nardoaristolones A 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, LDH, CK will leak outside in the cell, cause the active rising of LDH in the culture medium, CK, 3 dosage groups of this test demonstration are compared LDH in the culture medium, CK activity and are all obviously reduced with model group, show that Nardoaristolones A can be under Conditions of Acute Hypoxia in Human Body, keep the myocardial cell membrane integrity, the above results sees 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, see Table 3, show the cell membrane oxidative damage that Nardoaristolones A can cause anti-hypoxia by the ability that strengthens myocardial cell removing free radical.

Table 2Nardoaristolones A is on the impact (x ± s, n=6) of the outer leakage quantity of anoxic myocardial LDH, CK

Compare * * P＜0.01, * P＜0.05 with model group

Table 3Nardoaristolones A is on the impact (x ± s, n=8) of anoxic myocardial vigor, SOD activity and MDA content

Compare * * P＜0.01, * P＜0.05 with model group

Conclusion: Nardoaristolones A 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 Nardoaristolones A in the preparation anti-anoxic medicine is provided.

Claims (1)

1.Nardoaristolones the application of A in the preparation anti-anoxic medicine, described compound N ardoaristolonesA structure is shown in formula I:

Formula I.