CN1768739A - Application of soybean isoflavone as medicine for preventing cerebral ischemia - Google Patents

Abstract

The invention discloses the novel uses of soybean isoflavone in pharmacy, the medicament prepared by using soybean isoflavone as the effective composition can be used for the prevention of cerebral ischemia injury, and can be made into the dosage forms of tablets, capsules and powder preparations.

Description

Soybean isoflavone is as the application of medicine for preventing cerebral ischemia

Technical field

The present invention relates to the medicinal usage of soybean isoflavone, particularly it is as the purposes of medicine for preventing cerebral ischemia.

Semen sojae atricolor is as the grain consumption history of existing nearly one thousand years, along with people's going deep into to the research of the Nutrition of Semen sojae atricolor and goods thereof, find still not outstanding protein of Semen sojae atricolor and greasy important source, also contain many beneficial biological activities materials, as soybean oligo saccharide, soybean isoflavone, soybean phospholipid etc.Studies show that especially in recent years, soybean isoflavone have antioxidation, anticancer, angiocardiopathy preventing, prevention of osteoporosis, the physiological functions such as Woman climacteric obstacle of improving.Particularly along with human living standard's raising day by day, people more and more pay attention to the health care of self, and soybean isoflavone is to the useful adjusting of the physiological metabolism of human body and the soybean resource of China's abundant, for bright prospects have been showed in the exploitation of soybean isoflavone.

Summary of the invention

The object of the present invention is to provide the new purposes of soybean isoflavone, specifically is exactly the application of soybean isoflavone as medicine for preventing cerebral ischemia.

Soybean isoflavone in the Semen sojae atricolor (Soybean Isoflavone) existence form mainly is to exist with malonyl glycocide form.Soybean isoflavone is a kind of mixture, for having the compound group of 2-phenylchromone skeleton.The soybean isoflavone that native state exists has 12 kinds.Free isoflavone and glucosides isoflavone structure are as follows respectively:

With

Find out that from the molecular structure of soybean isoflavone the phenolic hydroxyl group in the soybean isoflavone is easily oxidized, can be used as antioxidant and produce a series of biological activitys in vivo.Free radical is the intermediate product of biochemical reaction in human life activity's process.Under normal circumstances, the generation of intravital free radical and its removing are among the dynamic equilibrium.But, if it is slow that interior free yl produces too much or removed, then free radical can cause damage to body on molecular level, cellular level and organ level, can cause function and metabolism disorder, accelerate the aging course of body, even bring out many diseases such as cancer, cardiovascular disease.The soybean isoflavone specific molecular structure has determined soybean isoflavone can react with superoxide anion in vivo, stops free radical to cause, and with metal ion-chelant, stops hydroxyl radical free radical to generate, and with the lipid peroxidation radical reaction, stops the lipid peroxidation process.In addition, Epidemiological study confirms, often eating Semen sojae atricolor, that health taken place, promotes for prophylaxis of cancer and cardiovascular disease is useful.Soybean isoflavone (SIF) as the class microcomponent in the Semen sojae atricolor owing to possess multiple biological activity, as estrogenic activity, antioxidation, anticancer and suppress vascular smooth muscle contraction etc. and be subjected to people's attention day by day.Because soybean isoflavone can effectively be removed too much active oxygen and free radical thereof in the brain; cerebral ischemia is played positive protective effect; therefore, be that the prevention of brain ischemic injuries class medicine of active ingredient provides further theoretical foundation with the soybean isoflavone for exploitation.

The present invention is by following test, promptly by setting up and observe rat cerebral ischemia damage re-perfusion model, inquired into the relation of cerebral ischemia and iron metabolism, the confirmation soybean isoflavone may be finished by reducing the inductive peroxidization of ferrum the preventive effect of cerebral ischemia.

Zoopery

One, materials and methods

Laboratory animal and main agents:

200-220g Wistar male rat effluent north medical university zoopery center provides: soybean isoflavone is presented by Beijing University of Chemical Technology.

Two, Mus Focal Cerebral Ischemia Reperfusion model preparation:

1. the preparation of plug wire: every section 5cm of sub-thread nylon wire, with match the nylon line end is scalded into round end, then be immersed in the 75% ethanol liquid standby.2. MCAO modelling: all rats are all selected MCA district, left side, 3% pentobarbital sodium 1ml/100g anesthesia, and the rat dorsal position is fixed, and the positive split shed of neck exposes left carotid (CCA) and external carotid artery (ECA), sends about 0.8cm place's ligation at ECA.Ligation CCA proximal part, do a wedge-formed incision thereon, nylon wire is inserted CCA gently from incision, in neck, the external carotid artery furcation enters internal carotid artery (ICA), continue to push ahead, flow into the blood flow of middle cerebral artery (MCA) up to the blocking-up of ICA intracranial furcation.The about 17mm of this external distance CCA furcation, fixing nylon wire, stump stays 1cm to be longer than outside the skin.Behind the ischemia 0.5h, nylon wire extracted out gently can make tremulous pulse recover again logical, realize perfusion again.The sign of model success is after waiting to regain consciousness, observe phenomena: the right fore paralysis, be crossed as the shears shape with left fore, and to turn-take to the right during walking, left eye diminishes.Broken end behind the rat perfusion is got brain, and there is white macula left brain part.

Three, animal grouping

Rat is divided into four groups: prevention group, non-prevention group, matched group, normal group, three every group.The continuous irrigation stomach is one month before wherein prevention group and the matched group modeling.Prevention group rat oral gavage soybean isoflavone, dosage is 2.5mg/100g, matched group is with the volume normal saline.Non-prevention group is not irritate the rat of stomach before the modeling.

Four, Nissl's staining and ABC immunohistochemical staining

Nissl's staining:

6min → 70% ethanol 5s → 80% ethanol 5min → 90% ethanol 5min → 95% ethanol 5min → 100% ethanol I 5min → transparent the 5min4 of the 100% ethanol II 5min → dimethylbenzene I → neutral gum mounting that dyes in the Nissl liquid is observed.

The ABC immunohistochemical staining:

0.01M phosphate buffer PBS (PH 7.4) cleans section 3 times, 5min/ time.Hatch 30min under the 0.3% methanol hydrogen peroxide at room temperature.Microwave is repaired 5min, low fire, natural cooling.0.01M PBS cleans 3 times, 5min/ time.Normal serum is hatched 60min at 37 ℃ of incubators in the wet box.After with filter paper serum being blotted, add one anti-1: 500 (the Mus HP of the rabbit Chinese People's Anti-Japanese Military and Political College), spend the night in 4 ℃ of wet boxes of refrigerators.Take out wet box, rewarming under the room temperature (about 0.5-1h) PBS cleans 3 times, 5min/ time.After with filter paper PBS being blotted, add goat-anti rabbit biotinylation two anti-working solutions and in 37 ℃ of incubators, wet box, hatch 60min.PBS cleans 3 times, 5min/ time.After with filter paper PBS being blotted, add VECTASTAIN Elite ABC reagent, in 37 ℃ of incubators, wet box, hatch 60min.Take out wet box, PBS cleans section 3 times, 5min/ time.The DAB colour developing, tap water flowing water fully washes.Gradient alcohol dehydration, transparent, the neutral gum mounting.

Five, histopathology is observed

Rat cerebral ischemia 0.5h pours into 24h again, carries out quick whole body perfusion fixation with 4 ℃ 4% paraformaldehyde fixatives, and getting brain, to put into 4% formaldehyde fixing.Take out fixedly brain, conventional dehydration, transparent, get the dorsal part Hippocampus (bregma-3～-3.8mm) conventional 12 μ m continuous coronal are cut into slices.HE dyeing and immunohistochemical staining, light microscopic are observed the strong side in Hippocampus CA1 district down and are hindered the side cellular morphology and change.

Six, experimental result

Nissl's staining result (200 *):

By section is observed under 20 times of mirrors, one side cerebral tissue of prevention group damaged obviously reduces than the degree of matched group damaged, Hippocampus CA1 district neuronal cell group has evident difference at aspects such as porousness and quantity, shows that soybean isoflavone has certain preventive effect to focal cerebral ischemia injury.Prevention is organized big brain injury side Hippocampus CA1 district neuronal cell group and obviously and is comparatively disperseed not as strong side layering, horse CA1 district, matched group brain both sides neuronal cell group does not then have obvious difference, the prevention group is hindered side Hippocampus CA1 district neuronal cell and is compared with non-prevention group, and cell is arranged comparatively neat.

Immunohistochemical staining result (200 *):

The result confirms that the Hp positive cell of prevention group cerebral lesion side is obviously expressed significantly than matched group damage side and normal group damage side after the operation, and by comparing with offside, finds the difference minimum of prevention group.

Seven, discuss

Hippocampus CA1 district neuron is very responsive to ischemia, have in the time of may be with cerebral ischemia to have neurovirulent excitatory amino acid (EAA) in a large number and be released into extracellular fluid, and the CA1 district has a large amount of glutamate receptors relevant.Though from the perforating branches of posterior cerebral artery, because higher its vascularity that adds of ischemia sensitivity in CA1 district is sparse than other subprovince, so middle cerebral artery (MCA) obstruction can cause its irreversible damage to Hippocampus blood for mainly.Studies show that: behind the cerebral ischemia, can produce a large amount of free radical and active oxygen, they can cause ischemic brain injury by the damage macromolecular substances, also can pass through the signal transduction path injured brain tissue, soybean isoflavone then has certain antioxidation, can eliminate too much free radical and active oxygen in the brain.Passing through in this experiment observed the brain section of Nissl's staining, behind the filling stomach soybean isoflavone continuous 1 month to rat, prevention group rat hippocampus CA1 district's neure damage degree and normal rats difference are less, to compare difference bigger with non-prevention group, shows that soybean isoflavone has certain preventive effect really to ischemic brain injury.Hp albumen is that the important ferrum of finding at present discharges auxilin, participate in the release of cell ferrum, result of study also shows that soybean isoflavone can obviously improve the distribution of this albumen at neurocyte, points out the effect of its anti-oxidative damage to realize by reducing the cell iron level.

Eight, conclusion

Soybean isoflavone is as the class microcomponent in the Semen sojae atricolor, and its antioxidation can be removed too much free radical and active oxygen in the brain, can play certain preventive effect to the generation of cerebral ischemia disease.

It is the tablet of effective ingredient that soybean isoflavone can be prepared into the soybean isoflavone, capsule, and dosage forms such as powder, in order to the prevention of brain ischemic injuries, the amount that its day taken the effective ingredient soybean isoflavone is 10-60mg.

The specific embodiment

Following examples are in order to explanation the present invention.

Embodiment

With the soybean isoflavone is that effective ingredient can be the several formulations form as medicine for preventing cerebral ischemia.

One, the preparation technology of tablet:

The adjuvant title	Soybean isoflavone	Soybean lecithin	Gelling starch	Microcrystalline Cellulose	Magnesium stearate	Protein sugar	Swollen fast king
								Usage ratio	1	1-1.5∶1	5-9∶1	2-4∶1	0.02-0.05：	0.002-0.005∶1	0.6-1∶1

1, in proportion with soybean isoflavone, soybean lecithin, gelling starch, the protein sugar powder mixes is even, and to going in the clean mortar, the alcoholic solution that adds 50-75% grinds the system soft material rapidly, and is agglomerating to hold, and touching slightly promptly looses is advisable;

2, with soft material with defeated 18 order nylon mesh granulate rectangular not have, bulk, fine powder is advisable;

3, the soft material granule is dried in 60 ℃ of calorstats, behind the 3-6min in 16 mesh sieves granulate;

4, the granule behind the granulate is placed beaker, add the microcrystalline Cellulose mix homogeneously;

5, add swollen fast king and magnesium stearate, mix homogeneously successively;

6, filler mix homogeneously, tabletting then.

Two, the preparation technology of capsule:

Press soybean isoflavone: soybean lecithin: starch=0.5～1.2: 0.4～1.3: 4～10 mix homogeneously, the alcoholic solution of 50-75% grinds rapidly, dries in 60 ℃ of calorstats, and granule is ground into fine powder, sieves, and mixing incapsulates, promptly.

Three, the preparation technology of powder:

With soybean isoflavone (a day effective dose is 10-50mg), soybean phospholipid, maltose (or maltose alcohol) and common soybeans powder fully mix, and four ratios are 1-1.5: 0.5-1: 1.5-5: 5-12, grind then, and packing is promptly.

Respectively in order to the prevention of brain ischemic injuries, the amount that day is taken the effective ingredient soybean isoflavone is 10-60mg with above-mentioned preparation, the generation that confirmation can the prevention of brain ischemia diseases.

Claims (1)

1, the medicinal application of soybean isoflavone is characterized in that: be that effective ingredient prepares medicine for preventing cerebral ischemia with the soybean isoflavone.