CN1911924B - Extraction method of Nepal eagle tail isoflavone, its derivative and medicipal composition using said derivative as active component - Google Patents
Extraction method of Nepal eagle tail isoflavone, its derivative and medicipal composition using said derivative as active component Download PDFInfo
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- CN1911924B CN1911924B CN200610068404XA CN200610068404A CN1911924B CN 1911924 B CN1911924 B CN 1911924B CN 200610068404X A CN200610068404X A CN 200610068404XA CN 200610068404 A CN200610068404 A CN 200610068404A CN 1911924 B CN1911924 B CN 1911924B
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- PAMIQIKDUOTOBW-UHFFFAOYSA-N CN1CCCCC1 Chemical compound CN1CCCCC1 PAMIQIKDUOTOBW-UHFFFAOYSA-N 0.000 description 1
- PVOAHINGSUIXLS-UHFFFAOYSA-N CN1CCNCC1 Chemical compound CN1CCNCC1 PVOAHINGSUIXLS-UHFFFAOYSA-N 0.000 description 1
- SJRJJKPEHAURKC-UHFFFAOYSA-N CN1CCOCC1 Chemical compound CN1CCOCC1 SJRJJKPEHAURKC-UHFFFAOYSA-N 0.000 description 1
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Abstract
The present invention provides process of extracting Nepal roof iris rhizome isoflavone, novel Nepal roof iris rhizome isoflavone derivative and its medicinal salt. The present invention also relates to the preparation process of the derivative, medicinal composition with the derivative as the active component, and the application of the derivative and medicinal composition in preparing medicinesfor improving and treating cardiac and cerebral vascular diseases and osteoporosis.
Description
Technical field
The present invention relates to the extracting method of novel Irisolidone, the Irisolidone derivative is the pharmaceutical composition of activeconstituents with this derivative, and their application in the treatment heart, cerebro-vascular diseases, osteoporosis.
Background technology
FI puerariae (Flos Puerariae) has another name called the Pueraria lobota barriness, is the dry flower of pulse family (Leguminosae) plant elegant jessamine (Pueraria Lobata (Willd.) Ohwi).Cool in nature, it is sweet to distinguish the flavor of, and goes into to return Yangming Channel.It has the effect of relieving acute alcoholism and recuperating the spleen record such as Shennong's Herbal, Compendium of Material Medica, cures mainly get sick from drinking too much wine heating polydipsia, anorexia, vomits against diseases such as acid regurgitation, haematemesis, discharging fresh blood stools.
FI puerariae is widely distributed in China, and plant resources is abundant, and wherein effective constituent such as flavones, saponin(e has very strong pharmacologically active.But domestic deep not enough to its chemical ingredients, pharmacological action and Study of Clinical Application, mainly contain FI puerariae in China at present and separate the soup of waking up and be used for clinically, healthcare products such as Pueravia flower tea, FI puerariae dews go on the market.Mainly be confined to relieve the effect of alcohol, hepatoprotective etc.
At publication number is in the patent documentation of CN1723988A, discloses the pharmaceutical composition and the application thereof of Kakkalide.Wherein relevant for Kakkalide (one of main active ingredient kakkalide in the FI puerariae, 5,7-dihydroxy-6,4 '-pharmacological datum of anti-ischemic cardiovascular and cerebral vascular disease of dimethoxyisoflavone-7-O-β-D-xylopyranosyl-6-O-β-D-glucopyranoside), data show, Kakkalide has certain curative effect to the cardiovascular and cerebrovascular ischemic disease, but its solubility problem has limited the development of its preparation, used hydroxyethyl-etc. to have the auxiliary material of dispute for solving this patent of solvability, stayed potential safety hazard for from now on clinical application.
In order to overcome the deficiency of Kakkalide and aglycon thereof (Irisolidone) poorly water-soluble, at publication number is in the patent documentation of CN 1594307A, discloses extraction separation and the sulfonated bodies preparation method and the pharmaceutical use of Irisolidone in the Flos Pueraria omeiensis.It is water-soluble better to utilize sulfonation reaction to make, and the Irisolidone of the certain pharmaceutical use of tool-3`-sodium sulfonate.But, drawing through a large amount of pharmacological evaluation, Irisolidone-3`-sodium sulfonate is unsatisfactory to the result of treatment of the heart, cerebro-vascular diseases.
Summary of the invention
Technical assignment of the present invention is that a kind of extracting method that extracts Irisolidone from FI puerariae is provided.
Another technical assignment of the present invention is according to above-mentioned the deficiencies in the prior art, provides a kind of good pharmaceutical use that has, highly water-soluble Irisolidone derivative.
The further technical assignment of the present invention provides a kind of pharmaceutical composition for the treatment of the heart, cerebro-vascular diseases and osteoporosis.
Another technical assignment of the present invention provides the purposes of said derivative aspect the medicine of the preparation treatment heart, cerebro-vascular diseases and osteoporosis.
The method of the following formula of extraction separation (I) Irisolidone from FI puerariae:
This extracting method may further comprise the steps:
A, dried FI puerariae is extracted 1-3 time with the 80%-95% alcohol heating reflux, each 1-4 hour, filter also collection filtrate;
B, the ethanol that reclaims in the filtrate are not distinguished the flavor of to there being alcohol;
The water of c, 4-8 times of solution amount of adding, placement is spent the night;
D, get supernatant liquor, cross 101 macroporous resins, use the 40-70% ethanol elution;
E, recovery elutriant are placed and are separated out crystallization;
F, crystal filter, after the drying, use acetone recrystallization, and solids is with 60%～80% ethanol or the methanol aqueous solution hydrolysis of 10-50 times of solid substance quantity, contain mass percent and be 3%～6% acid in ethanol or methanol aqueous solution, and acid can be HCl or H
2SO
4
G, cold filtration hydrolysate are washed to neutrality and promptly obtain required product.
Derivative or its pharmaceutical salts by the Irisolidone of following general formula (II) expression:
Wherein: R
1, R
3Be independently of one another H ,-CH
2CH
2OH;
R
2Be H ,-CH
2NR
4R
5
R
1, R
3, R
2Be not H simultaneously;
And, NR
4R
5For
R
4, R
5Be H, C independently of one another
1-4Alkyl, C
2-4Alkenyl, C
2-4Alkynyl group; R
4During for H, R
5Can not select for use-CH
3R
5During for H, R
4Can not select for use-CH
3
The pharmaceutical salts of the said Irisolidone derivative of the present invention is meant pharmacy acceptable salt, the salt that forms with mineral acids such as hydrochloric acid, sulfuric acid, phosphoric acid, phosphorous acid for example, or the salt that forms with organic acids such as Aspartic Acid, citric acid, succsinic acid, methylsulfonic acid, tosic acid, toxilic acid, fumaric acid, tartrate, or alpha-non-natural amino acid salt etc.
C
1-4Alkyl is meant the alkyl of the straight or branched with 1-4 carbon atom, for example methyl, ethyl, propyl group, sec.-propyl, butyl, isobutyl-, sec-butyl, the tertiary butyl etc.
C
2-4Alkenyl is meant the alkenyl of the straight or branched with 2-4 carbon atom, for example vinyl, 1-propenyl, 2-propenyl, 1-butylene base, crotyl etc.
C
2-4Alkynyl group is meant the alkynyl group of the straight or branched with 2-4 carbon atom, for example ethynyl, 1-proyl, 2-propynyl, ethyl acetylene base, 2-butyne base etc.
Be preferably:
R
1, R
3Be independently of one another H ,-CH
2CH
2OH;
R
2Be H ,-CH
2NR
4R
5
R
1, R
3, R
2Be not H simultaneously;
And, NR
4R
5For
R
4, R
5Be H, C independently of one another
1-4Alkyl, C
2-4Alkenyl, C
2-4Alkynyl group; R
4During for H, R
5Can not select for use-CH
3R
5During for H, R
4Can not select for use-CH
3
The best is:
R
1, R
3Be independently of one another H ,-CH
2CH
2OH;
R
2Be H ,-CH
2NR
4R
5
R
1, R
3, R
2Be not H simultaneously;
And, NR
4R
5For
R
4, R
5Be H, C independently of one another
1-2Alkyl;
R
4During for H, R
5Can not select for use-CH
3R
5During for H, R
4Can not select for use-CH
3
Optimizing compound of the present invention is:
R
1, R
3Be H, R
2For-CH
2NR
4R
5, R
4, R
5For-CH
3, its structure is suc as formula shown in (III):
R
1, R
3Be H, R
2For-CH
2NR
4R
5, NR
4R
5For
Its structure is suc as formula shown in (IV):
R
1, R
3For-CH
2CH
2OH, R
2Be H, its structure is shown in formula V:
In the compound of general formula of the present invention (II) structure, for its concrete group that replaces, difference according to its position of substitution, can be raw material all by the Irisolidone of formula (I) structure, according to preparing the known preparation method and the principle of similar substitution compound, adopt corresponding method to prepare.
For example, the method for preparing the 8-methylene radical-piperidyl-Irisolidone shown in the above-mentioned formula (IV) is: the Irisolidone with formula (I) structure is a raw material, the mixture of one or more in formaldehyde or Paraformaldehyde 96, piperidines and methyl alcohol, ethanol, tetrahydrofuran (THF), the dioxane mixes, be heated to 50-70 ℃ of reaction, the reaction postcooling filters and obtains compound (IV).Reaction equation is:
The method for preparing the hydrochloride of the 8-methylene radical-dimethylamino shown in the above-mentioned formula (III)-Irisolidone correspondence is: the Irisolidone with formula (I) structure is a raw material, the mixture of one or more in the hydrochloride of formaldehyde or Paraformaldehyde 96, dimethylamine and methyl alcohol, ethanol, tetrahydrofuran (THF), the dioxane mixes, 50～70 ℃ of reactions of temperature control, the reaction postcooling, filtration obtains compound (III), and can continue the hydrochloride compound (VI) with HCl prepared in reaction formula (III) structure correspondence.Reaction equation is:
Prepare 5 shown in the above-mentioned formula V, 7-dihydroxy ethyl-6,4 '-synthetic method of dimethoxy Irisolidone is: the Irisolidone with formula (I) structure is a raw material, chloroethanol or oxyethane mix with water, 50～90 ℃ of reactions of temperature control postcooling filters and obtains compound (V) in alkaline environment.Reaction equation is:
But the derivative of Irisolidone of the present invention or its pharmaceutical salts per os or without the mouth administration, dosage is had nothing in common with each other because of medicine is different, and concerning the adult, every day, 100mg-300mg was proper.
During the oral administration administration, this compound and conventional medicinal adjuvant such as vehicle, disintegrating agent, tamanori, lubricant, antioxidant, Drug coating, perfume compound, tensio-active agent etc. are mixed, be made into form administrations such as granule, capsule, tablet; Can injection liquid during non-oral administration, form administration such as infusion solution.When preparing above-mentioned preparation, all can use conventional preparation technique.
Can get through a large amount of pharmacological testings and animal experiment, the derivative of Irisolidone of the present invention or its pharmaceutical salts have well water-soluble, to improving and treating the heart, cerebro-vascular diseases and osteoporosis positive effect are arranged.
Embodiment
The following examples, example of formulations can illustrate in greater detail the present invention, but do not limit the present invention in any form.
(embodiment 1): 5,7-dihydroxyl-6,4 '-extraction separation of dimethoxy isoflavones (Irisolidone).
A, the dried FI puerariae of 10kg is pulverized,, each 3 hours, filter also collection filtrate with 90% aqueous ethanolic solution heating and refluxing extraction 2 times;
B, decompression and solvent recovery are to there not being the alcohol flavor;
C, add 6 times of about 30L of water gaging of solution, placement is spent the night;
D, get supernatant liquor, cross 101 macroporous resins, with 60% aqueous ethanolic solution wash-out;
E, elutriant reclaim, and place and separate out crystallization in 10 hours;
After f, crystal filtration, the drying, use acetone recrystallization, solids 70% ethanol hydrolysis of 30 times of amounts of solids, (containing 5% HCl in the ethanol);
G, cold filtration hydrolysate, being washed to neutrality, promptly to obtain required product be faint yellow needle crystal.189～190 ℃ of fusing points (m.p.), extraction yield 1%.UVλmax(MeOH)：266，335nm；λmax(MeOH+AlCl3)：275，315，375nm；λmax(MeOH+AlCl3+HCl)：277，312，374nm；λmax(MeOH+NaOAc)：271，335nm。IR(KBr)cm-1：3450(OH)，3067，2960，1658(C＝O)，1660，1624，1576，1515，1460，1371，1294，1231。Add NaOAc red shift 5nm in the ultraviolet determination, be shown with 7-OH.The AlCl3/HCl spectrum is consistent with the AlCl3 spectrum, shows no adjacent two phenolic hydroxyl structures.And change than the MeOH spectrum, be shown with 3-and/or 5-OH.13CNMR(DMSO-d6，150MHz，δ，ppm)：180.6(C-4)，159.3(C-4′)，157.7(C-7)，154.5(C-2)，153.4(C-5)，152.9(C-9)，131.6(C-6)，130.3(C-2′，C-6′)，123.1(C-3)，121.6(C-1′)，113.9(C-3′，C-5′)，105.0(C-10)，94.1(C-8)，60.1(6-OCH3)，55.3(4′OCH3)。1HNMR(DMSO-d6，600MHz，δ，ppm)：13.05(1H，s，5-OH)，7.90(1H，s，2-H)，7.45(2H，d，J＝8.8Hz，?2′-H，6′-H)，7.05(2H，d，J＝8.8Hz，3′-H，5′-H)，6.60(1H，s，7-OH)，6.55(1H，s，8-H)，3.91(3H，s，6-OCH3)，3.78(3H，s，4′-OCH3)。Spectroscopic data is consistent with the irisolidon bibliographical information.
The derivative of the Irisolidone of table 1. embodiment of the invention and pharmaceutical salts thereof
Annotate: a of group is
The b of group is
The C of group is
(embodiment 2): 8-methylene radical-dimethylamino-Irisolidone (compound 1, structural formula II I)
Stir down embodiment 1 is obtained 5,7-dihydroxyl-6,4 '-dimethoxy isoflavones (1g), formaldehyde 1ml, dimethylamine 2ml be dissolved in ethanol, 70 ℃ of following temperature controls reactions 4 hours, cooling was filtered and is obtained 1.3g compound 1.m.p.185～193℃。
(embodiment 3) can obtain 8-methylene radical-dimethylamino-Irisolidone hydrochloride (compound 2) with the dimethylamine among the hydrochloride replacement embodiment 2 of dimethylamine.m.p.206～212℃。
(embodiment 4) can obtain 8-methylene radical-piperidyl-Irisolidone (compound 3, structural formula IV) with the dimethylamine among the piperidines replacement embodiment 2.m.p.190～193℃。
(embodiment 5) compound 3 and Aspartic Acid acid-base reaction in water obtain 8-methylene radical-piperidyl-Irisolidone aspartate (compound 4) through drying under reduced pressure again.m.p.200～206℃。
(embodiment 6) 5,7-dihydroxy ethyl Irisolidone (compound 5, structural formula V)
With embodiment 1 obtain 5,7-dihydroxyl-6,4 '-dimethoxy isoflavones (1g), oxyethane 50ml, in the water, under the alkaline environment, 70 ℃ of following temperature controls reactions 8 hours, cooling was filtered and is obtained 1.21g compound 5.m.p.172～176℃。
(embodiment 7) 8-methylene radical-diethylin-Irisolidone hydrochloride (compound 6)
Dimethylamine with among the diethylamine hydrochloride replacement embodiment 2 can obtain 8-methylene radical-diethylin-Irisolidone hydrochloride (compound 6).m.p.216～219℃。
(embodiment 8) 7-dimethylin formyl radical-Irisolidone (compound 7)
Stir down embodiment 1 is obtained 5,7-dihydroxyl-6,4 '-dimethoxy isoflavones (1g), acetone 30ml, dimethylin formyl chloride 1ml, salt of wormwood 2g, 70 ℃ of following temperature control reactions 24 hours, cooling was filtered, reclaim solvent, obtain the yellowish solid of 1.4g, compound 7.
(embodiment 9) 8-methylene radical-ethene amido-5,7-dihydroxy ethyl Irisolidone (compound 8)
The compound 5 that obtains with embodiment 6 is dissolved in ethanol with formaldehyde 1ml, vinyl-amine 0.8ml, and 70 ℃ of following temperature control reactions 4 hours, cooling was filtered and obtained 1.1g compound 8.m.p.174～179℃。
(embodiment 10) 8-methylene radical-anilino-5,7-dihydroxy ethyl Irisolidone (compound 9)
The compound 5 that obtains with embodiment 6 is dissolved in ethanol with formaldehyde 1ml, aniline 1ml, and 70 ℃ of following temperature control reactions 4 hours, cooling was filtered and obtained 1.5g compound 9.m.p.184～189℃。
(embodiment 11) 8-methylene radical-diethylin-5,7-dipropyl-Irisolidone (compound 10)
Embodiment 1 obtain 5,7-dihydroxyl-6,4 '-dimethoxy isoflavones (1g), propyl alcohol 50ml, vitriol oil 3ml, 80 ℃ of following temperature controls reactions 4 hours, cooling was filtered and is obtained 1.1g compound 10.m.p.157～159℃。
(embodiment 12) 8-methylene radical-dimethylamino-5-benzyl-Irisolidone (compound 11)
Embodiment 1 obtain 5,7-dihydroxyl-6,4 '-dimethoxy isoflavones (1g), acetone 50ml, salt of wormwood 1.5g, benzyl chloride 2g was 60 ℃ of following temperature control reactions 4 hours, and cooling, filtration are steamed to desolventize and obtained the 1.9g compound, formaldehyde 1ml, dimethylamine 2ml are dissolved in methyl alcohol, 70 ℃ of following temperature control reactions 4 hours, cooling was filtered and is obtained 1.9g compound 11.m.p.219～221℃。
(embodiment 13) 8-methylene radical-dimethylamino-5,7-phenylbenzene-Irisolidone (compound 12)
Embodiment 1 obtain 5,7-dihydroxyl-6,4 '-dimethoxy isoflavones (1g), acetone 50ml, salt of wormwood 5g, chlorinated benzene 2g was 60 ℃ of following temperature control reactions 4 hours, and cooling, filtration are steamed to desolventize and obtained the 1.8g compound, formaldehyde 1ml, dimethylamine 2ml are dissolved in methyl alcohol, 70 ℃ of following temperature control reactions 4 hours, cooling was filtered and is obtained 1.9g compound 12.m.p.211～214℃。
(embodiment 14): 8-methylene radical-hexenyl-Irisolidone (compound 13)
Stir down embodiment 1 is obtained 5,7-dihydroxyl-6,4 '-dimethoxy isoflavones (1g), formaldehyde 1ml, hexamethyleneamine 1.9ml be dissolved in ethanol, 70 ℃ of following temperature controls reactions 4 hours, cooling was filtered and is obtained 1.6g compound 13.m.p.201～203℃。
(embodiment 15) 8-methylene radical-piperazine-Irisolidone (compound 14)
Dimethylamine with among the piperazine replacement embodiment 2 can obtain 8-methylene radical-piperazine-Irisolidone (compound 14).m.p.192～194℃。
Illustrate that by following test compound of the present invention is at the positive effect that has that improves and treat the heart, cerebro-vascular diseases and osteoporosis.
(experimental example 1) intravenous drip administration is to the influence of anesthetized dog acute myocardial ischemia
Test sample: the compound in the table 16 is dissolved in the distilled water, and concentration is 80.0mg/kg (test
Group).
Control sample: puerarin injection, 100mg/kg (positive controls);
Irisolidone-3 '-sodium sulfonate (being called for short the sodium sulfonate contrast) is dissolved in distilled water
In, concentration is 80.0mg/kg.
Physiological saline (myocardial infarction and ischemia model group).
Animal: 20 of hybrid dogs, body weight 12.0～16.5kg, male and female have concurrently, available from the suburb, Jinan.Raised and train for 1 week before the experiment, select that it is normal, healthy, female no pregnant person is for experiment.
Instrument: BioPAC leads the physiological signal acquisition analysis system more, U.S. BioPAC company.KNOEPRO type automatic clinical chemistry analyzer, Finland Kang Yi instrument company.XD-2 type epicardial lead, Xiyuan hospital of Beijing academy of traditional Chinese medicine.Q811 type planimeter, the Xinanjiang River, Zhejiang scientific instrument factory.
Method: 20 of normal health dogs, body weight 12.0～16.5kg, male and female have concurrently, are divided into 4 groups at random, i.e. test group 80.0mg/kg, sodium sulfonate control group 80.0mg/kg, positive control drug puerarin injection group 100mg/kg and myocardial infarction and ischemia model group.Animal via 2.5% vetanarcol (25mg/kg) intravenous anesthesia separates tracheae and intubate, meets electric respirator pedestrian worker fully and breathes.Separate femoral vein standby (using) for getting blood.The dog right arm reclining, chest is opened in the 4th intercostal space in the left side, makees the pericardium bed, separates nearly 1/2 place of left anterior descending coronary artery, and lead-in wire is equipped with ligation and uses.Epicardial lead is sewn on the visceral pericardium, leads the physiological signal acquisition analysis system record epicardial electrogram that links to each other more through waver and BioPAC.The slow constant speed intravenous drip of postoperative physiological saline is to replenish body fluid.30min record epicardial electrogram behind the ligation coronary artery, (∑-ST) and the displacement of ST section surpass leading of 2mv and count (N-ST) as being worth before the medicine to calculate the total value of 30 ST sections displacements of leading.40/min of speed is dripped in the intravenous drip administration.The administration volume is 10.0ml/kg, and the myocardial infarction and ischemia model group gives isopyknic physiological saline.Write down the epicardial electrogram that administration begins back 15min, 30min, 60min, 90min, 120min, 180min and 240min respectively, calculate ∑-ST, N-ST and velocity of variation thereof.Simultaneously get blood examination by the dog femoral vein and survey CK-MB, CK, AST, LDH respectively at 2h, 4h before the administration and after the administration.4h after the administration injects burnt black ink 1.0ml/kg in the room left through the left auricle of heart root, and 20-30 has annotated in second, takes off heart rapidly, removes fat, atrium and right ventricle's flesh, and freezing 30-40min under-20 ℃ weighs.Parallel coronary sulcus is cut into left ventricle 5 of uniform thickness under coronary artery ligation point, weigh respectively, measure burnt black ink dyeing district, every myocardium two sides (non-ischemic region) and district's (ischemic region) area that is unstained with planimeter, calculate the percentage that ischemic region accounts for left compartment muscle weight.Then 5 cardiac muscles are placed 37 ℃ of N-BT dye liquors, jolting dyeing 15min takes out, as above measure infarct (light red) and non-infarct (garnet) area, calculate infarct and account for the percentage of left compartment muscle weight, and calculate the percentage that infarct accounts for ischemic region cardiac muscle weight.Experimental result compares with the myocardial infarction and ischemia model group respectively, carries out statistical analysis and handles.
The result
(1) test group is to (the influence of ∑-ST) of anesthetized dog acute myocardial ischemia degree
240min can obviously alleviate anesthetized dog acute myocardial ischemia degree (∑-ST), with the myocardial infarction and ischemia model group notable difference (P＜0.05) is arranged more all after the test group administration.240min obviously alleviates anesthetized dog acute myocardial ischemia degree (P＜0.05) after the puerarin injection administration.Sodium sulfonate control group no significant difference.The result sees table 2 for details.
(2) test group is to the influence of anesthetized dog acute myocardial ischemia scope (N-ST)
240min obviously dwindles anesthetized dog acute myocardial ischemia scope (N-ST) after the test group administration, with the myocardial infarction and ischemia model group notable difference (P＜0.05) is arranged relatively.240min obviously dwindles anesthetized dog acute myocardial ischemia scope (P＜0.05) after the puerarin injection administration, and 180min has reduction trend.Sodium sulfonate control group no significant difference.The result sees table 3 for details.
(3) test group is to the influence of anesthetized dog acute myocardial ischemia myocardial infarction area
The myocardial infarction area that shows with N-BT dyeing is roughly consistent with the result that epicardial electrogram is measured.The heavy dose of group of test group has the damaging effect that obviously alleviates myocardial ischemia, and anesthetized dog Acute Myocardial Infarction area is obviously dwindled, and with the myocardial infarction and ischemia model group notable difference (P＜0.05) is arranged more all.Puerarin injection is suitable with the test group effect.Sodium sulfonate control group no significant difference.The result sees table 4 for details.
(4) test group is to the influence of anesthetized dog acute myocardial ischemia myocardial enzymes
Test group and puerarin injection respectively at administration after 4h obviously reduce serum CK-MB, with the myocardial infarction and ischemia model group notable difference (P＜0.05) is arranged more all, but CK, AST, LDH is not had obvious influence.Sodium sulfonate control group no significant difference.The result sees table 5, table 6, table 7, table 8 for details.
(experimental example 2) is to the provide protection of rat brain damage
Test sample: the compound in the table 15 is dissolved in the distilled water, and concentration is 100mg/kg (heavy dose of group), 50mg/kg (middle dosage group) and 25mg/kg (small dose group).
Control sample: Ligustrazine Hydrochloride Injection, 7.2mg/kg (positive controls);
Physiological saline (sham operated rats, cerebral ischemic model group).
Animal: the Wistar rat, body weight 160-180g, anti-medical Group Co.,Ltd provides credit number by the Shandong, Shandong: Shandong kinoplaszm word 200001001.
Test method:
(1) test group is to the provide protection of acute cerebral ischemia in rats
90 of Wistar rats, male and female dual-purpose, body weight 160-180g.Be divided into 6 groups at random, be respectively sham operated rats, cerebral ischemic model group, the heavy dose of group of test group 100mg/kg, middle dosage group 50mg/kg, small dose group 25mg/kg, positive control drug Ligustrazine Hydrochloride Injection group 7.2mg/kg.Every group 15.The tail vein injection administration.The administration volume is 1.0ml/200g.Administration time is the preceding 20min of blocking-up cerebral blood flow.Sham operated rats and cerebral ischemic model group all give isopyknic physiological saline.Rat is through 25% urethane (1g/kg) intraperitoneal anesthesia, and neck median incision is separated bilateral carotid, and dual ligation (sham operated rats is only worn two-wire but not ligation) causes acute experiment imperfection cerebral ischemia.The quick broken end of 3h is got brain after the ligation, and the weighing bottle of packing into claims the brain weight in wet base, calculates cerebral index, places 110 ℃ of baking boxs to dry to constant weight then, claims brain stem heavy, the calculating brain water content.Wherein each group all has 5 rat experiments to finish, and takes out cerebral tissue rapidly, through 10% formalin fixed, and conventional section, dyeing changes to detect cerebral morphology.
(2) test group is to the influence of rat cerebral tissue's capillary permeability
60 of Wistar rats, body weight 160-180g, male and female half and half.Be divided into 6 groups at random, i.e. sham operated rats, cerebral ischemic model group, the heavy dose of group of test group 100mg/kg, middle dosage group 50mg/kg, small dose group 25mg/kg, positive control drug Ligustrazine Hydrochloride Injection group 7.2mg/kg.The tail vein injection administration.The administration volume is 1.0ml/200g.Administration time is the preceding 20min of blocking-up cerebral blood flow.Sham operated rats and cerebral ischemic model group give isopyknic physiological saline.Animal is with 25% urethane (1g/kg) intraperitoneal anesthesia.Neck median incision is separated bilateral carotid, and threading is standby.Ischemia model group and each drug study group then before the ligation bilateral carotid 5min by tail vein injection Evans Blue 50mg/kg.Sham operated rats is identical with time, the dosage of model group and each experimental group injection Evans Blue, but not ligation bilateral carotid.Behind the ligation 3h, broken end is got brain and is weighed, and is soaked in formamide soln respectively in (4ml/ brain), incubation 72h in 45 ℃ of thermostat containers, treat that the Evans Blue pigment all leaches in the cerebral tissue, get and contain the Evans Blue pigment solution and carry out colorimetric, measure the OD value with 722 type grating spectrophotometer 620nm places.
The result:
(1) to the influence of cerebral index and brain water content
The result shows that the cerebral index of cerebral ischemic model group and brain water content are apparently higher than sham operated rats, and learning by statistics to handle has significant difference (P＜0.05), illustrates that cerebral ischemic model is successful.The cerebral index that big or middle two the dosage groups of test group all obviously reduce acute imperfection rats with cerebral ischemia raises and the brain water content increase, shows that test group can obviously alleviate the caused cerebral edema of rat acute imperfection cerebral ischemia.The result sees table 9 for details.
Table 9. test group to the influence of acute imperfection rats with cerebral ischemia cerebral index, brain water content (X ± s, n=10)
Annotate: compare with sham operated rats,
*P＜0.05,
*P＜0.01; Compare with the cerebral ischemic model group,
ΔP＜0.05,
The Δ ΔP＜0.01
(2) cerebral tissue pathomorphism check result:
Sham operated rats: weave constructions such as pallium, cerebellum, hippocampus are normal, do not have changes such as hyperemia, oedema and encephalomalacia kitchen range.Neurocyte, spongiocyte are not seen morphological changes such as sex change, necrosis.
Cerebral ischemic model group: cerebral tissue hyperemia, oedema, little vasodilation.Partial nerve unit mild swelling, the obvious enlargement of hippocampus partial nerve cell, partial necrosis.
The heavy dose of group of test group: cerebral tissue mild hyperaemia, little blood vessel are slightly expanded.Partial nerve unit mild swelling, the enlargement of hippocampus partial nerve cell, a small amount of downright bad.The ischemic change obviously is lighter than model group.
Dosage group in the test group: cerebral tissue mild hyperaemia, little blood vessel are slightly expanded.Partial nerve unit mild swelling, the enlargement of hippocampus partial nerve cell, a small amount of downright bad.The ischemic change is lighter than model group.
The test group small dose group: cerebral tissue mild hyperaemia, little blood vessel are slightly expanded.Partial nerve unit mild swelling, the enlargement of hippocampus partial nerve cell, a small amount of downright bad.The ischemic change is lighter than model group.
The Ligustrazine Hydrochloride Injection group: cerebral tissue mild hyperaemia, little blood vessel are slightly expanded.Partial nerve unit mild swelling, the enlargement of hippocampus partial nerve cell, a small amount of downright bad.The ischemic change is lighter than model group.
(3) to the influence of cerebral ischemic model rat brain capillary permeability
The result shows that cerebral ischemic model group brain capillary permeability is apparently higher than sham operated rats.The unit's of showing as cerebral tissue Evans Blue content significantly raises; The big or middle dosage group of Ligustrazine Hydrochloride Injection group and test group Evans Blue content all obviously reduces, and it is normal or approaching normal that unit organization Evans Blue content is returned to, and with model group significant difference arranged relatively.The results are shown in Table 10.
Table 10. test group to the influence of rat cerebral tissue's capillary permeability (X ± s, n=10)
Annotate: compare with sham operated rats
*P＜0.05,
*P＜0.01; Compare with model group
ΔP＜0.05,
The Δ ΔP＜0.01
The result shows that cerebral ischemic model group brain capillary permeability is apparently higher than sham operated rats.The heavy dose of group of test group, middle dosage group can obviously reduce the capillary permeability that cerebral ischemia causes.
This experimental example shows, when ligation rat bilateral common carotid arteries forms acute imperfection cerebral ischemia, cerebral index, brain water content and capillary permeability all obviously increase, and test group is by reducing cerebral index, alleviate capillary permeability, alleviating cerebral edema, blood-brain barrier permeability changes, and the brain regional blood flow increases.Can improve the brain microcirculation blood perfusion, improve the ischemia condition of cerebral tissue, alleviate the ischemia injury of cerebral tissue, thereby the rat ischemia brain injury is had good provide protection, be expected to become the good medicine of treatment ischemic cerebrovascular disease.
The function of resisting osteoporosis of (experimental example 3) test group
Test sample: the compound in the table 14 is dissolved in the distilled water, and concentration is 100mg/kg (heavy dose of group), 50mg/kg (middle dosage group) and 25mg/kg (small dose group).
Control sample: nilestriol, 150mg/kg (positive controls);
Physiological saline (sham operated rats, model group).
Animal:
The Wistar rat, Shandong University's Experimental Animal Center provides, credit number: Shandong kinoplaszm word 200001001.
Method:
Therapeutic action to spay osteoporosis rat model:
Get 60 of female rats, body weight 230-270g, only opening abdomen except that sham operated rats separates and twoly to survey ovaries and do not do the excision, all the other respectively organize rat under aseptic condition, abdominal injection 1% vetanarcol (40mg/kg) anesthesia, the two ovaries of surveying of excision are set up the osteoporosis rat model, postoperative abdominal injection 0.5ml gentamicin preventing infection.The beginning administration of 2 week of postoperative, rat is divided into 5 groups at random, be respectively sham operated rats, model group, tried thing big (100mg/kg), in (50mg/kg), little (25mg/kg) dosage group and positive control nilestriol group, every group 10, administration volume 1ml/200g, administration every day 1 time, continuous 60 days, sham operated rats and model control group gave isopyknic distilled water.Survey serum calcium, phosphorus, alkaline phosphatase and oestrogenic hormon at 50 days extracting vein bloods of administration, rat is put to death in administration 60 days, dissect rat uterus visual inspection developing womb situation and carry out histopathologic examination, get two femurs of surveying, a side is made calcium content of bone and is measured, and a side is done the pathology histological examination.
The result:
(1) to the influence of serum calcium, phosphorus and alkaline phosphatase, the results are shown in Table 11.
The influence of table 11. pair serum calcium, phosphorus and alkaline phosphatase (n=10, X ± SD)
By table 11 as seen, the big or middle dosage group of test group rat blood serum calcium, phosphorus, serum alkaline phosphatase and model group relatively have rising trend, but do not have marked difference.
(2) to the influence of serum oestrogenic hormon, bone calcium, the results are shown in Table 12.
The influence of table 12. pair serum oestrogenic hormon, bone calcium (n=10, X ± SD)
By table 12 as seen, being tried thing big or middle dosage group calcium content of bone and model group relatively has rising trend, and tried the thing heavy dose and make the serum estrogen level that obvious rising trend be arranged, but not statistically significant.
(3) result of uterus histopathologic examination:
The castration model group: the slight atrophy of endometrial epithelium, secrete inactive; Most of body of gland is mild to moderate atrophy, and part body of gland reactivity expands but glandular cell does not have secreting function, no secretory product in the chamber; Blood vessel obviously reduces flesh layer and adventitia attenuation in the mesenchymal cell reactive hyperplasia, lamina propria.
Sham operated rats: endometrial epithelium and body of gland are normal, and epithelial cell and glandular cell secretion are active; No stromal reaction hyperplasia, lamina propria has cell infiltration, and blood vessel is normal, does not have obvious dilatation and congestion, and flesh layer and adventitia are normal.
Sun is to organizing: endometrial epithelium is normal, and body of gland is less-developed, and the glandular cell secretion is inactive; Lamina propria has cell infiltration, compares with the castration group, and a matter is loose, and no mesenchymal cell hyperplasia has a small amount of blood vessel; Flesh is subnormal layer by layer, but blood vessel is less-developed, and adventitia is normal.
The test group small dose group: the slight atrophy of endometrial epithelium, secrete inactive; Mesenchymal cell mild reaction hyperplasia, the prosperity of lamina propria body of gland, the glandular cell secretion is active, and blood vessel is abundant to organizing than castration model group and sun; Flesh layer and adventitia are normal.
Dosage group in the test group: endometrial epithelium is tending towards normally, and secretion is active; Between matter normal, the prosperity of lamina propria body of gland, glandular cell secretion is active, blood vessel is abundanter; Flesh layer and adventitia are normal.
The heavy dose of group of test group: endometrial epithelium is normal, and secretion is active; Between matter normal, the prosperity of lamina propria body of gland, glandular cell secretion is active, with little, middle dosage group no significant difference relatively; Blood vessel is abundant more, full; Flesh layer and adventitia are normal.
(4) result of femur histopathologic examination:
Sham operated rats: the bone trabecula dense arrangement, the interconnection reticulated structure that is, bone trabecula thickness is big, and its spacing is little, and osteocyte is normal.
The castration model group: bone trabecula attenuates after removing ovary, the part fracture, and the bone resorption pouch increases, and pulp cavity enlarges, the cortex bone attenuation.Compare with sham operated rats, the bone trabecular connection point of interruption is many under the same multiple, and bone trabecula obviously reduces, and the pulp cavity spacing increases, and the normal bone cell reduces, and the empty pouch of osteocyte increases.
Sun is to organizing: compare the bone trabecula rule with the castration group, and the number showed increased, thickness increases, and fracture is few, the pulp cavity gap smaller.The normal bone cell is near sham operated rats under the same multiple.
The test group small dose group: compare with the castration group, the bone trabecula number slightly increases, and thickness slightly increases, and the empty pouch of osteocyte reduces, but the normal bone cell is still less.
Dosage group in the test group: the bone trabecula number increases than the test group small dose group, and thickness increases, the pulp cavity gap smaller, and the normal bone cytosis, but still be lower than sun to group.
The heavy dose of group of test group: bone trabecula number showed increased, thickness increases, and fracture is few, the pulp cavity gap smaller, to group, learn the change degree and overweight sham operated rats slightly by osseous tissue near sun for the normal bone cell number under the same multiple.
Conclusion: in this tested used dosage range, test group can promote bone calcium deposition, alleviates spay rat bone cortex attenuation degree, increases bone trabecula quantity, reduces amount of osteoclast; Illustrate that test group has tangible prevention and therapeutic action to osteoporosis, and this effect presents the doses dependency.
The following examples explanation comprises the medicinal preparations by compound provided by the invention.
(example of formulations 1) tablet
Prepare tablet according to methods known in the art, every contains following composition:
Compound 1 60mg, lactose 80mg, Magnesium Stearate 3mg,
Polyvinylpyrrolidone 7mg.
(example of formulations 2) capsule
Prepare capsule according to methods known in the art, contain following composition in each capsule:
Compound 2 60mg, lactose 85mg, W-Gum 20mg
Magnesium Stearate 1mg polyvinylpyrrolidone 4mg.
(example of formulations 3) injection liquid
Prepare injection liquid according to methods known in the art, contain following composition in each injection liquid:
Compound 5 100mg, water for injection 10ml.
Adopt the preparation technology of conventional method injection to make, every bottle of 10ml contains compound 5 (100mg).Usage: the glucose injection dilution, intramuscular injection, a twice-daily, each 1-2 props up; Intravenous drip, once-a-day, each 2-3 props up.
(example of formulations 4) freeze-dried preparation, sterilized powder
Prepare freeze-dried preparation, sterilized powder according to methods known in the art.Contain following composition in each freeze-dried preparation, the sterilized powder:
Compound 7 100mg, N.F,USP MANNITOL 120mg.
Adopt the preparation technology of conventional method freeze-dried preparation, sterilized powder to make, every bottle contains compound 7 (100mg).Usage: glucose injection dilution, intramuscular injection, a twice-daily, each 1-2 bottle; Intravenous drip, once-a-day, each 2-3 bottle.
Claims (9)
1. the method for the following formula of extraction separation (I) Irisolidone from FI puerariae:
This extracting method may further comprise the steps:
A, dried FI puerariae is extracted 1-3 time with the 80%-95% alcohol heating reflux, each 1-4 hour, filter also collection filtrate;
B, the ethanol that reclaims in the filtrate are not distinguished the flavor of to there being alcohol;
The water of c, 4-8 times of solution amount of adding, placement is spent the night;
D, get supernatant liquor, cross 101 macroporous resins, use the 40-70% ethanol elution;
E, recovery elutriant are placed and are separated out crystallization;
F, crystal filter, after the drying, use acetone recrystallization, and solids is with 60%～80% ethanol or the methanol aqueous solution hydrolysis of 10-50 times of solid substance quantity, contain mass percent and be 3%～6% acid in ethanol or methanol aqueous solution, and acid is HCl or H
2SO
4
G, cold filtration hydrolysate are washed to neutrality and promptly obtain required product.
2. by derivative or its pharmaceutical salts of the Irisolidone of following general formula (II) expression:
Wherein: R
1, R
3Be independently of one another H ,-CH
2CH
2OH;
R
2Be H ,-CH
2NR
4R
5
R
1, R
3, R
2Be not H simultaneously;
And, NR
4R
5For
R
4, R
5Be H, C independently of one another
1-4Alkyl, C
2-4Alkenyl, C
2-4Alkynyl group;
R
4During for H, R
5Can not select for use-CH
3R
5During for H, R
4Can not select for use-CH
3
3. according to the described compound of claim 2, wherein,
R
1, R
3Be independently of one another H ,-CH
2CH
2OH;
R
2Be H ,-CH
2NR
4R
5
R
1, R
3, R
2Be not H simultaneously;
And, NR
4R
5For
R
4, R
5Be H, C independently of one another
1-4Alkyl, C
2-4Alkenyl, C
2-4Alkynyl group;
R
4During for H, R
5Can not select for use-CH
3R
5During for H, R
4Can not select for use-CH
3
4. according to the described compound of claim 2, wherein,
R
1, R
3Be independently of one another H ,-CH
2CH
2OH;
R
2Be H ,-CH
2NR
4R
5
R
1, R
3, R
2Be not H simultaneously;
And, NR
4R
5For
R
4, R
5Be H, C independently of one another
1-2Alkyl;
R
4During for H, R
5Can not select for use-CH
3R
5During for H, R
4Can not select for use-CH
3
5. according to the described compound of claim 2, wherein, R
1, R
3Be H, R
2For-CH
2NR
4R
5, R
4, R
5For-CH
3, its structure is suc as formula shown in (III):
6. according to the described compound of claim 2, wherein, R
1, R
3Be H, R
2For-CH
2NR
4R
5, NR
4R
5For
Its structure is suc as formula shown in (IV):
7. according to the described compound of claim 2, wherein, R
1, R
3For-CH
2CH
2OH, R
2Be H, its structure is shown in formula V:
8. be used for improving or the treatment heart, cerebro-vascular diseases, the pharmaceutical composition of osteoporosis wherein contains the Irisolidone derivative of claim 2 or its pharmaceutical salts as effective constituent, and contains pharmaceutically acceptable carrier.
9. any one compound is preparing the improvement or the treatment heart, cerebro-vascular diseases, the application in the medicine of osteoporosis among the claim 2-7.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1384110A (en) * | 2002-05-20 | 2002-12-11 | 吉林天药科技股份有限公司 | Prepn of hederagenin |
| CN1394603A (en) * | 2002-08-09 | 2003-02-05 | 陕西镇坪制药厂 | Application of hydroxyethyl puerarin in preparation of new medicine for curing cerebrovascular diseases |
| CN1594307A (en) * | 2004-06-25 | 2005-03-16 | 陕西师范大学 | Extraction separation for Nepal irid isoflavone from kudzu, process for preparing sulfonated compounds thereof , and their pharmaceutical uses |
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2006
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1384110A (en) * | 2002-05-20 | 2002-12-11 | 吉林天药科技股份有限公司 | Prepn of hederagenin |
| CN1394603A (en) * | 2002-08-09 | 2003-02-05 | 陕西镇坪制药厂 | Application of hydroxyethyl puerarin in preparation of new medicine for curing cerebrovascular diseases |
| CN1594307A (en) * | 2004-06-25 | 2005-03-16 | 陕西师范大学 | Extraction separation for Nepal irid isoflavone from kudzu, process for preparing sulfonated compounds thereof , and their pharmaceutical uses |
Non-Patent Citations (3)
| Title |
|---|
| 说明书11页实施例20 |
| 说明书11页实施例21. |
| 说明书第1页第1段 |
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