CN102924554B - C-3,11,12,20-tetrasubstituted C-21 steroid derivative and its pharmaceutical composition and use in medicine - Google Patents

C-3,11,12,20-tetrasubstituted C-21 steroid derivative and its pharmaceutical composition and use in medicine Download PDF

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CN102924554B
CN102924554B CN201210419579.6A CN201210419579A CN102924554B CN 102924554 B CN102924554 B CN 102924554B CN 201210419579 A CN201210419579 A CN 201210419579A CN 102924554 B CN102924554 B CN 102924554B
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zebra fish
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pyrans glycosyl
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CN102924554A (en
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张颖君
许敏
王东
朱宏涛
杨崇仁
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Kunming Institute of Botany of CAS
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Abstract

The invention provides a C-3,11,12,20-tetrasubstituted C-21 steroid derivative shown in the general formula I and its pharmaceutical salt and glycoside, and also provides a pharmaceutical composition containing the C-3,11,12,20-tetrasubstituted C-21 steroid derivative as an active ingredient and a use of the C-3,11,12,20-tetrasubstituted C-21 steroid derivative in preparation of a medicine for treating epilepsy. In the general formula I, R1, R2 and R3 respectively represent a ketonic group, a hydroxyl group, a substituted acyl ester group, an amino-group or a substituted acylamino group; R4 represents a ketonic group, a hydroxyl group, a substituted acyl ester group, an amino-group, a substituted acylamino group, or a glycosyl group; and R5, R6 and R7 respectively represent hydrogen, a hydroxyl group, a substituted acyl ester group, an amino-group or a substituted acylamino group.

Description

C-3,11,12,20-tetra-replaces C-21 steroid derivatives and pharmaceutical composition and its thereof application in medicine
Technical field:
The invention belongs to field of medicaments, be specifically related to a kind of natural compounds be activeconstituents antiepileptic drug, more specifically, relate to natural physiological active substance C-3,11,12,17-tetra-replaces C-21 steroid derivative and pharmaceutical composition and its thereof application in the medicine of preparation treatment epileptics.
Background technology:
Epilepsy (epilepsy) is commonly called as " insane crazy, the epilepsy of sheep ", is the electric discharge of cerebral neuron paroxysmal abnormality, causes a kind of chronic disease of of short duration cerebral disorder.This disease due to the neurone position difference in the brain of paradoxical discharge, and has diversified symptom.These symptoms comprise relatively common, significant motor symptoms and the visible loss of consciousness in tetanic old contraction outbreak, old the change observed in multi-motion function in addition, the change of such as sensation, sense of smell, vision and higher function, the such as change of mood, memory, language and vision.Wherein, spasm is one of typical epilepsy syndromes, and the normal involuntary movement with stiff property, indirect, also has not with the seizure types of spasm.In addition, may occur unexpected loss of consciousness, memory, faint suddenly, tic of the limbs, spits out white foams, and the symptom such as to shout, epileptic seizures great majority belong to temporary, and generally namely several points recovered as ordinary person to several tens minutes.But it is long that epilepsy has case history, the feature of recurrent exerbation, the sequela caused thus even can cause disturbance of intelligence.Once suffer from epilepsy, be just difficult to radical cure.Slighter can control outbreak with medicine, and anti-frightened epilepsy agent treatment will be lifelong.When medicine can not control epileptic attack, must operate treatment, but expense, risk are high, and still need to take medicine all the life.
According to WHO statistics, current global epileptic 5,000 ten thousand people, wherein 80% in developing country, annual newly-increased 2,000,000 epileptics.The sickness rate of China's epileptic condition is about 7 ‰, wherein 40% has not accepted any treatment, and what the patient of 40% accepted is non-regular treatment, and annual newly-increased patient 400,000 people nearly.
The antiepileptic drug of WHO recommendation in 1980 mainly contains: phenylethyl barbituric acid (Phenobarbital, Luminal), Carbamzepine (Carbamazepine), phenytoin Sodium (Dilantin, Phenytoin, insanely to stop), Sodium Valproate (Depakene), ethosuximide (Ethosuximide, Zarontin), doxenitoin sodium (Primidonum, Mysoline, Hexamidinu, Primidone).At present, market at home, traditional old kind phenylethyl barbituric acid, Carbamzepine, Sodium Valproate, phenytoin Sodium still occupy the absolute market share.In addition, some antiepileptic Chinese patent medicine products are also had, such as Cynanchum otophvllum sheet, the clear sheet of epilepsy, the peaceful sheet of epilepsy, more epilepsy spirit market sale at home.But these traditional Western medicine side effects are large, and long-term taking can cause larger impact to health.Such as, the First Line medication Carbamzepine for the treatment of epilepsy, this medicine can produce the fragrant Johnson Syndrome (Steven-Johnson syndrome) of severe allergy side effect-Shi Supreme Being, and mortality ratio reaches 1 to 4 and becomes.And Chinese patent medicine product exists the features such as curative effect is remarkable, target spot is unclear, there is no the market competitiveness.
In recent years, in world market, there is multiple new antiepileptic medicine, such as: oxcarbazepine (Trileptal), Levetiracetam (Levetiracetam), topiramate (Topiramate), lamotrigine (Lamotrigine) etc.Wherein, Levetiracetam was in 2008, and sales volume just more than 18.64 hundred million dollars, becomes the cookle in antiepileptic drug.The mechanism of action of this medicine is that synaptophysin SV2A is combined in presynaptic nerve teminal, suppresses the paradoxical discharge in epilepsy loop, thus blocks epileptic seizures.Have that anti-outbreak spectrum is wide, good effect, therapeutic index increase, can with the feature such as other AEDs share.Behind Discussion on Chinese Listed, occupy the market share of Chinese antiepileptic drug 10.65% in 2009.
Therefore, study to send out with external antiepileptic drug and enliven, market growth is rapidly compared, the domestic AED operation listing not having novelty.AED market domestic is at present occupied by import medicine, on the high side, and general patient is difficult to bear, the economic problems that long-term taking brings, and is also difficult to solve China epileptics and seeks medical advice the low problem of rate.Meanwhile, existing drug side effect is all comparatively large, and long-term taking affects greatly.
Up to now, there are no C-3 in prior art, 11,12,20-tetra-replaces the report of C-21 steroid derivatives and pharmacologically active thereof, also has no the report that it has antiepileptic activity.
Summary of the invention:
Object of the present invention aims to provide C-3, and 11,12,20-tetra-replaces C-21 steroid derivative, the pharmaceutical composition being activeconstituents with it, and they treat the application in the medicine of epileptics in preparation.
In order to realize above-mentioned purpose of the present invention, the invention provides following technical scheme:
C-3 shown in general formula (I), 11,12,20-tetra-replace C-21 steroid derivatives and pharmacy acceptable salt thereof and glycoside,
Wherein, R 1, R 2, R 3be independently ketone group, hydroxyl and replacement acyl ester group, amino and replacement amido separately; R 4for ketone group, hydroxyl and replacement acyl ester group, amino and replacement acyl ammonia, glycosyl; R 5, R 6, R 7be independently hydrogen, hydroxyl and replacement acyl ester group thereof, amino and replacement amido separately.
For above-mentioned general formula (I) compound, the preferred technical scheme of the present invention is R 1, R 2be respectively isovaleryl ester group, R 3for acetyl ester group, R 4, R 6for hydroxyl, R 5, R 7be respectively hydrogen.
The present invention further preferred technical scheme is R 1for ketone group, R 2para hydroxybenzene formyl ester group, R 3for hydrogen, R 4for 3-O-β-D-glucopyanosyl base-(1 → 4)-β-D-glucopyanosyl base-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-Folium seu Cortex Nerii pyrans glycosyl-(1 → 4)-β-D-digitoxin pyrans glycosyl, R 5, R 6, R 7be respectively hydroxyl.
The present invention further preferred technical scheme is R 1, R 2, R 3be respectively hydroxyl, R 4for O-6-deoxidation-3-O-methyl-β-D-A Luo pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl, R 5, R 7be respectively hydrogen, R 6for hydroxyl.
The present invention further preferred technical scheme is R 1for ketone group, R 2, R 3be respectively acetyl ester group, R 4for O-6-deoxidation-3-O-methyl-β-D-A Luo pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl, R 5, R 7be respectively hydrogen, R 6for hydroxyl.
The present invention further preferred technical scheme is R 1for ketone group, R 2para hydroxybenzene formyl ester group, R 3for hydrogen, R 4for 3-O-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-Folium seu Cortex Nerii pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-digitoxin pyrans glycosyl, R 5, R 6, R 7be respectively hydroxyl.
The present invention further preferred technical scheme is R 1for cis isopentene ester group, R 2for isovaleryl ester group, R 3for acetyl ester group, R 4for O-6-deoxidation-3-O-methyl-β-D-A Luo pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl, R 5, R 6be respectively hydroxyl, R 7for hydrogen.
The present invention further preferred technical scheme is R 1, R 2, R 3be respectively hydroxyl, R 4for O-β-D-glucopyanosyl base-(1 → 4)-6-deoxidation-3-O-methyl-β-D-A Luo pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl, R 5, R 6be respectively hydroxyl, R 7for hydrogen.
The present invention further preferred technical scheme is R 1, R 2, R 3be respectively hydroxyl, R 4for O-β-D-glucopyanosyl base-(1 → 4)-6-deoxidation-3-O-methyl-β-D-A Luo pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl, R 5, R 6be respectively hydroxyl, R 7for hydrogen.
The present invention further preferred technical scheme is R 1for trans isopentene ester group, R 2for isovaleryl ester group, R 3for acetyl ester group, R 4for O-6-deoxidation-3-O-methyl-β-D-A Luo pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl, R 5, R 6be respectively hydroxyl, R 7for hydrogen.
The present invention further preferred technical scheme is R 1, R 3be respectively hydroxyl, R 2for trans isopentene ester group, R 4for O-β-D-glucopyanosyl base-(1 → 4)-6-deoxidation-3-O-methyl-β-D-A Luo pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl, R 5, R 6be respectively hydroxyl, R 7for hydrogen.
The present invention further preferred technical scheme is R 1, R 3be respectively hydroxyl, R 2for cis isopentene ester group, R 4for O-β-D-glucopyanosyl base-(1 → 4)-6-deoxidation-3-O-methyl-β-D-A Luo pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl, R 5, R 6be respectively hydroxyl, R 7for hydrogen.
The present invention further preferred technical scheme is R 1, R 2be respectively isovaleryl amino, R 3for kharophen, R 4, R 6for hydroxyl, R 5, R 7be respectively hydrogen.
The present invention further preferred technical scheme is R 1for ketone group, R 2for para hydroxybenzene formamido group, R 3for hydrogen, R 4for O-β-D-Folium seu Cortex Nerii pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl, R 5, R 6, R 7be respectively hydroxyl.
The present invention further preferred technical scheme is R 1, R 2, R 3be respectively amino, R 4for O-6-deoxidation-3-O-methyl-β-D-A Luo pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl, R 5, R 7be respectively hydrogen, R 6for hydroxyl.
The present invention further preferred technical scheme is R 1for cis senecioyl is amino, R 2for isovaleryl is amino, R 3for kharophen, R 4for O-6-deoxidation-3-O-methyl-β-D-A Luo pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl, R 5, R 6be respectively hydroxyl, R 7for hydrogen.
The present invention further preferred technical scheme is R 1for trans senecioyl is amino, R 2for isovaleryl is amino, R 3for kharophen, R 4for O-6-deoxidation-3-O-methyl-β-D-A Luo pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl, R 5, R 6be respectively hydroxyl, R 7for hydrogen.
The present invention further preferred technical scheme is R 1, R 3be respectively hydroxyl, R 2for trans senecioyl is amino, R 4for O-β-D-glucopyanosyl base-(1 → 4)-6-deoxidation-3-O-methyl-β-D-A Luo pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl, R 5, R 6be respectively hydroxyl, R 7for hydrogen.
The present invention further preferred technical scheme is R 1, R 3be respectively amino, R 2for cis senecioyl is amino, R 4for O-β-D-glucopyanosyl base-(1 → 4)-6-deoxidation-3-O-methyl-β-D-A Luo pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl, R 5, R 6be respectively hydroxyl, R 7for hydrogen.
The present invention further preferred technical scheme is R 1, R 2, R 3be respectively amino, R 4for O-β-D-glucopyanosyl base-(1 → 4)-6-deoxidation-3-O-methyl-β-D-A Luo pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl, R 5, R 6be respectively hydroxyl, R 7for hydrogen.
The present invention further preferred technical scheme is R 1for ketone group, R 2for para hydroxybenzene formamido group, R 3for hydrogen, R 4for O-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-Folium seu Cortex Nerii pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-digitoxin pyrans glycosyl, R 5, R 6, R 7be respectively hydroxyl.
The present invention further preferred technical scheme is R 1for ketone group, R 2, R 3be respectively kharophen, R 4for O-6-deoxidation-3-O-methyl-β-D-A Luo pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl, R 5, R 7be respectively hydrogen, R 6for hydroxyl.
Pharmaceutical salts described in above-mentioned any one, refer to pharmacy acceptable salt, comprise the salt formed with organic acid or mineral acid, described organic acid includes but not limited to tartrate, citric acid, formic acid, acetic acid, oxalic acid, butyric acid, oxalic acid, toxilic acid, succsinic acid, hexanodioic acid, alginic acid, citric acid, aspartic acid, benzene Phenylsulfonic acid, dextrocamphoric acid, camphorsulfonic acid, didextrose acid, pentamethylene propionic acid, dodecyl sulphate, ethyl sulfonic acid, glucoheptonic acid, Phosphoric acid glycerol esters, hemisulfic acid, enanthic acid, caproic acid, fumaric acid, 2-ethylenehydrinsulfonic acid, lactic acid, toxilic acid, methylsulfonic acid, nicotinic acid, 2-naphthene sulfonic acid, flutter acid, pectinic acid, 3-phenylpropionic acid, picric acid, PIVALIC ACID CRUDE (25), propionic acid, succsinic acid, tartrate, sulfocyanic acid, p-tosylate and undecane hydrochlorate, described mineral acid includes but not limited to hydrochloric acid, Hydrogen bromide, hydroiodic acid HI, sulfuric acid or phosphoric acid.
The present invention provides the pharmaceutical composition of antiepileptic simultaneously, and it comprises the C-3 according to above-mentioned any one, and 11,12,17-tetra-replaces C-21 steroid derivative or its drug salts or glycoside and the pharmaceutically acceptable carrier of at least one.
The present invention still further provides the C-3 described in above-mentioned any one, and 11,12,17-tetra-replaces the application of C-21 steroid derivative in the medicine of preparation treatment epileptics.
Technique scheme of the present invention proposes based on following discovery and principle.
The compound of C-21 steroidal to be a class with pregnane be parent nucleus, its C-11, C-12, C-20 position hydroxyl often forms ester bond, and C-3 position hydroxyl forms glycoside with the sugar chain formed primarily of α-desoxy sugar.This compounds has neural physiologically active, as anticonvulsion, and anti-epileptic, anti-melancholy etc.The neurosteroid that their chemical structure and physiological function and eighties of last century the nineties are risen is closely similar, and infer thus, C-21 steroidal is a kind of class neurosteroid of plant origin.The present invention is based on the basis of document in early stage, a series of new 11-O-ethanoyl-12 is found from asclepiadaceae plant, 20-O-diisoamyl acyl-hardship rope glycoside and Cynanchum otophvllum glycoside derivative, and obtain new 11-O-ethanoyl-12 by hydrolysis, 20-O-diisoamyl acyl-hardship rope aglycon has stronger antiepileptic activity, and do not report this C-21 steroidal compounds and antiepileptic action thereof in document before this, the present invention is this compounds of isolation identification first, and by zebra fish anti-epileptic model validation C-3, 11, 12, it is effective to treatment epilepsy that 20-tetra-replaces C-21 steroid derivative.11-O-ethanoyl-12,20-O-diisoamyl acyl-hardship rope aglycon (11-O-acetyl-12,20-di-O-isovaleryldrevogenin) is C-21 steroid derivative, belongs to a kind of C-21 steroid compound of innovation.Zebra fish anti-epileptic model proves, it has stronger antiepileptic activity (IC 50be 17.2 μMs), be better than positive control drug Levetiracetam (IC far away 50be 170.6 μMs) and Cynanchum otophvllum sheet.And compared with Levetiracetam, Levetiracetam anti-epileptic therapeutic efficiency first increases fast, increase trend afterwards and slow down gradually, and this compound has the pharmacology that anti-epileptic therapeutic efficiency substantially linearly increases along with the increase of concentration.
Found by the model research of zebra fish anti-epileptic, C-3 of the present invention, 11,12,20-tetra-replaces the activity that C-21 steroid derivative and pharmacy acceptable salt and glycoside thereof have treatment epilepsy, significantly reduces attack degree and attack rate, in the concentration range of 1 μM to 100 μMs, anti-epileptic therapeutic efficiency increases along with the increase of concentration, presents concentration dependent.Compound 11-O-ethanoyl-12,20-O-diisoamyl acyl of the present invention-hardship rope aglycon, IC 50be 17.2, be significantly better than positive control Levetiracetam (IC 50be 170.6 μMs) and Cynanchum otophvllum sheet (52.8 μ g/ml).
C-3 of the present invention, 11,12,20-tetra-replaces C-21 steroid derivative and pharmaceutical composition can be any suitable form, such as solid, semi-solid, liquid or aerosol form.Generally, medicine contains compound of the present invention or extract as activeconstituents, with applicable outside, enteron aisle, or the organic or inorganic carrier of administered parenterally or mixed with excipients.Activeconstituents can be compound, such as, can accept carrier and/or vehicle make tablet, piller, capsule, suppository, vaginal suppository, solution, emulsion, suspension and be applicable to other forms of using with conventional non-toxic pharmaceutical.The pharmaceutical acceptable carrier used in the composition comprises, such as, water, glucose, lactose, gum arabic, gelatin, mannitol, starch, Magnesium Trisilicate, talcum, W-Gum, Keratin sulfate, colloidal silica, yam starch, and be adapted at preparing other carriers used in the preparation of solid, semisolid, liquid or aerosol form.Composition can contain stablizer, thickening material in addition, and/or tinting material and spices.
C-3 of the present invention, 11,12,20-tetra-replaces C-21 steroid derivative and pharmacy acceptable salt thereof and glycoside can per os or without mouth administration, dosage is had nothing in common with each other because medicine is different, and concerning adult, every day, 1-100mg was more suitable.
During oral administration administration, first make compound mix as vehicle, disintegrating agent, tamanori, lubricant, antioxidant, Drug coating, tinting material, perfume compound, tensio-active agent etc. with conventional medicinal adjuvant, be made into the form administrations such as granule, capsule, tablet; Can the form administration such as injection liquid, infusion solution or suppository during non-oral administration.When preparing above-mentioned preparation, conventional preparation technique can be used.
Accompanying drawing illustrates:
Fig. 1-1 is the zebra fish movement locus after the process of Compound D V12a each concentration group;
Fig. 1-2 is compound 11-O-ethanoyl-12, zebra fish rapid movement distance after 20-O-diisoamyl acyl-hardship rope aglycon each concentration group process, 11-O-ethanoyl-12,20-O-diisoamyl acyl-hardship restricts each concentration group of aglycon with phenytoin Sodium compared with epilepsy model group, p<0.001;
Fig. 1-3 is the dose-effect curve of compound 11-O-ethanoyl-12,20-O-diisoamyl acyl-hardship rope aglycon antiepileptic action;
Fig. 2-1 is the zebra fish movement locus after the process of Cynanchum otophvllum glycosides H each concentration group;
Fig. 2-2 is the zebra fish rapid movement distance after the process of Cynanchum otophvllum glycosides H each concentration group, each concentration group of Cynanchum otophvllum glycosides H with phenytoin Sodium compared with epilepsy model group, p<0.001;
Fig. 2-3 is the dose-effect curve of Cynanchum otophvllum glycosides H antiepileptic action.
Embodiment:
Below in conjunction with accompanying drawing, further illustrate essentiality content of the present invention with embodiments of the invention, these examples are only the explanations to preferred version of the present invention, and also limit the scope of the invention never in any form.
Embodiment 1:
The evaluation of antiepileptic action in 11-O-acetyl ester group-12,20-O-diisoamyl acyl ester group-hardship rope aglycon body:
11-O-ethanoyl-12; the preparation of 20-O-diisoamyl acyl-hardship rope aglycon: dry South Mountain rattan (5.5kg) is pulverized, and extracts three times, each four hours with methanol eddy; cooled and filtered; united extraction liquid, is concentrated into about 1.5L, after petroleum ether extraction removing fat-soluble component; reclaim methyl alcohol to medicinal extract shape; enriched material is suspended in water, with chloroform extraction four times, obtains chloroform extract 168g.Get 150g to dissolve, sample mixed by 300g silica gel (80-100 order), is separated, CHCl with silica gel column chromatography (200-300 order, 3270g) 3-MeOH-H 2o (86:17:1) wash-out, is mainly divided into three part Frs.A-C of opposed polarity.Fr.C (21g) is little polar portion, separating difficulty is relatively little, main utilization silica gel column chromatography is separated (chloroform/methanol or sherwood oil/acetone are eluent), Rp-8 reverse phase silica gel and MCI-gel CHP-20P column chromatography purification (methanol/water is eluent), obtain new compound root of Twisting Dregea glycosides I (97mg).Root of Twisting Dregea glycosides I is dissolved in MeOH (20mL), adds 5%HCl (10mL), after 50 ° of C water-bath 15min, add H 2o (20mL), reclaim under reduced pressure MeOH to 30mL, then after 60 ° of C water-bath 15min, cooling, extracts three times by equal amounts of chloroform, obtains glucoside unit part.Glucoside unit part, through silica gel column chromatography separating purification, with sherwood oil/acetone mixture wash-out, obtains 11-O-ethanoyl-12,20-O-diisoamyl acyl-hardship rope aglycon.
11-O-acetyl ester group-12,20-O-diisoamyl acyl ester group-hardship rope aglycon
11-O-acetyl-12,20-di-O-isovaleryldrevogenin
The physicochemical data of compound 11-O-acetyl-12,20-di-O-isovaleryldrevogenin is as follows: white amorphous powder, C 33h 52o 8; Mp 97-99 ° C, (c=0.27, MeOH), IR (KBr) ν max3441,2961,2934,2873,1742,1631,1467,1415,1370,1294,1254,1236,1196,1168,1120,1094,1061,1029,1000,963,940,901,811,771cm -1.ESI-MS (positive ion) m/z 599 [M+Na] +, HRESI-MS (positive ion) m/z 599.3568 [M (C 33h 52o 8)+Na] +(calcd.599.3559). 1H-NMR(C 5D 5N,400MHz):δ1.46(1H,m,H-1a),2.08(1H,m,H-1b),1.82(1H,m,H-2a),2.10(1H,m,H-2b),3.85(1H,m,H-3),2.41(1H,m,H-4a),2.61(1H,m,H-4b),5.51(1H,d,J=4.9Hz,H-6),2.01(1H,m,H-7a),2.61(1H,m,H-7b),2.22(1H,m,H-8),1.86(1H,m,H-9),5.77(1H,t,J=9.8Hz,H-11),5.28(1H,d,J=9.8Hz,H-12),1.89(1H,m,H-15a),1.98(1H,m,H-15b),1.81(1H,m,H-16a),1.99(1H,m,H-16b),2.77(1H,m,H-17),1.46(3H,s,Me-18),1.31(3H,s,Me-19),5.47(1H,m,H-20),1.20(3H,d,J=5.9Hz,Me-21),2.12(3H,s,Me-2′),2.38(1H,m,H-2″a),2.51(1H,m,H-2″b),2.35(1H,m,H-3″),1.04(3H,d,J=6.4Hz,Me-4″),1.04(3H,d,J=6.4Hz,Me-5″),2.27(2H,m,H-2″′),2.20(1H,m,H-3″′),0.95(3H,d,J=6.4Hz,Me-4″′),0.95(3H,d,J=6.4Hz,Me-5″′)13C-NMR(C5D5N，100MHz)：δ39.0(t,C-1),32.8(t,C-2),70.9(d,C-3),43.9(t,C-4),140.8(s,C-5),121.9(d,C-6),28.2(t,C-7),38.0(d,C-8),48.2(d,C-9),39.6(s,C-10),72.2(d,C-11),78.8(d,C-12),53.0(s,C-13),84.2(s,C-14),33.0(t,C-15),25.0(t,C-16),50.2(d,C-17),11.3(q,C-18),19.5(q,C-19),73.2(d,C-20),19.5(q,C-21),170.2(s,C-1'),21.8(q,C-2'),173.2(s,C-1″),43.9(t,C-2″),25.7(d,C-3″),22.7(q,C-4″),22.8(q,C-5″),172.3(s,C-1″′),44.1(t,C-2″′),25.9(d,C-3″′),22.5(q,C-4″′),22.5(q,C-5″′)。
The breeding of zebrafish embryo is carried out in the mode of natural paired cross.Each mating prepares 4 ~ 5 pairs of Adult Zebrafishs, and on average often pair can be produced 200 ~ 300 embryos.At after fertilization 6 hours (i.e. 6hpf) and 24hpf, embryo is cleared up (removing dead embryo), and select suitable embryo according to the etap of embryo.Embryo's (fish culture water water quality: add 200mg Instant Ocean in every 1L reverse osmosis water, specific conductivity is 480 ~ 510 μ S/cm is hatched with fish culture water under 28 DEG C of conditions; PH is 6.9 ~ 7.2; Hardness is 53.7 ~ 71.6mg/LCaCO 3).Because embryo can obtain nutritive substance from the yolk sac of self, so (9dpf) does not need feeding in after fertilization 9 days.After having tested, with tricaine methylsulfonic acid, over-exposure process is carried out to the zebra fish of each etap, thus zebra fish anesthesia is put to death.The operation steps that anesthesia is put to death meets the code requirement that American Veterinary association (AVMA) puts to death Animal Anesthesia.
The zebra fish of Yetrazol process is epilepsy model group; The zebra fish of phenytoin Sodium and Yetrazol co-treatment is positive controls; The zebra fish of the DMSO process of 1.1% is negative control group (solvent control group); Untreated zebra fish is blank group.11-O-acetyl ester group-12,20-O-diisoamyl acyl ester group-hardship rope aglycon and positive control (Levetiracetam and Cynanchum otophvllum sheet) are dissolved in DMSO respectively, are mixed with the solution to be measured that concentration is 1,3,10,30,60 and 100 μM.Above-mentioned solution to be measured respectively with Yetrazol co-treatment zebra fish for some time after, utilize the movement locus of zebra fish in behavioural analysis instrument record 60min, then to the rapid movement (seizure of zebra fish, V>20mm/sec) distance carries out quantitative analysis, by calculating following index: 1. zebra fish rapid movement distance (distance ofrapid movement-seizure); 2. the anti-epileptic therapeutic efficiency (therapeutic efficacy) of compound; 3. compound time changing curve (compound effect on the time-course of zebrafish seizure) that zebra fish rapid movement is affected.Quantitative analysis is carried out, the anti-epileptic therapeutic efficiency of computerized compound with distance (D) to the movement velocity of zebra fish, and draws dose-effect curve according to compound epilepsy therapy efficiency, the IC of computerized compound antiepileptic action 50; Carry out statistical analysis with variance analysis and T-inspection, p<0.05 is significant difference.In experimentation all there are not the phenomena of mortality in each group zebra fish.Fig. 1-1 is each experimental group zebra fish movement locus in 60min, and restrict each concentration group of aglycon compared with epilepsy model group from Fig. 1-1,11-O-acetyl ester group-12,20-O-diisoamyl acyl ester group-hardship, zebra fish rapid movement track obviously reduces.Fig. 1-2 is the column diagram drawn according to zebra fish rapid movement distance average mean ± SE, and from Fig. 1-2, each concentration group all has statistical significance (p<0.001) compared with solvent control group; Fig. 1-3 is 11-O-acetyl ester group-12; the dose-effect curve of 20-O-diisoamyl acyl ester group-hardship rope aglycon antiepileptic action; from Fig. 1-3; 11-O-ethanoyl-12; 20-O-diisoamyl acyl-hardship rope aglycon is in the concentration range of 1 μM to 100 μMs; anti-epileptic therapeutic efficiency increases along with the increase of concentration, presents concentration dependent, IC 50it is 17.2 μMs.
Table 1.11-O-acetyl ester group-12,20-O-diisoamyl acyl ester group-hardship rope aglycon each concentration group zebra fish rapid movement distance raw data
Embodiment 2:
The evaluation of antiepileptic action in Cynanchum otophvllum glycosides H [Qingyanshengenin-3-O-β-D-glucopyanosyl base-(1 → 4)-β-D-glucopyanosyl base-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-Folium seu Cortex Nerii pyrans glycosyl-(1 → 4)-β-D-digitoxin pyranoside, Otophylloside H] body.
The preparation of Cynanchum otophvllum glycosides H [Qingyanshengenin-3-O-β-D-glucopyanosyl base-(1 → 4)-β-D-glucopyanosyl base-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-Folium seu Cortex Nerii pyrans glycosyl-(1 → 4)-β-D-digitoxin pyranoside, Otophylloside H]:
Dry Cynanchum otophvllum root (10.0kg) is pulverized, and extracts three times (72,72,48 hours), each consumption 15L by 90% ethanol soaking at room temperature, filters, united extraction liquid, and recycling design, to without alcohol taste, obtains fluid extract 368g.Be suspended in by medicinal extract in 2L water, with chloroform extraction three times, each consumption 1.5L, combining extraction liquid, is concentrated into dry, obtains chloroform extract 155g.Get 150g to be separated through silica gel column chromatography, chloroform/methanol (10:1.5,7.6L) wash-out, obtain five part Frs.1-5.Fr.3 is through silica gel column chromatography, with chloroform/methanol and ethyl acetate/ethanol/water for eluent, and Rp-8 column chromatography (taking methanol/water as eluent) separation and purification repeatedly, obtain Cynanchum otophvllum glycosides H [Qingyanshengenin-3-O-β-D-glucopyanosyl base-(1 → 4)-β-D-glucopyanosyl base-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-Folium seu Cortex Nerii pyrans glycosyl-(1 → 4)-β-D-digitoxin pyranoside, Otophylloside H].
Cynanchum otophvllum glucoside H, Otophylloside H
The physicochemical data of Cynanchum otophvllum glycosides H is as follows: white amorphous powder, C 60h 90o 27; Mp 213-216 ° C, (c=0.83, MeOH), IR (KBr) ν max3442,2934,1709,1610,1591,1504,1452,1384,1309,1276,1163,1072,911,854,773cm -1.FAB-MS (negative ion) m/z 1242 [M] -, 1105 [M-137] -, 943 [M-137-162] -, HRFAB-MS (negative ion) m/z1241.5628 [M (C 60h 90o 27)-H] -(calcd.1241.5591). 1H-NMR(C 5D 5N,500MHz):δ3.54(1H,m,H-3),5.38(1H,d,J=4.7Hz,H-6),4.72(1H,dd,J=11.6Hz,J=4.1Hz,H-12),1.63(3H,s,Me-18),1.14(3H,s,Me-19),2.08(3H,s,Me-21),7.73(1H,d,J=8.8Hz,H-3'),6.71(1H,d,J=8.8Hz,H-4'),6.71(1H,d,J=8.8Hz,H-6'),7.73(1H,d,J=8.8Hz,H-7'),4.94(1H,dd,J=9.7Hz,J=1.7Hz,H-1″),1.67(1H,m,H-2″a),1.93(1H,m,H-2″b),4.21(1H,m,H-3″),3.27(1H,m,H-4″),3.80(1H,m,H-5″),1.21(3H,d,J=5.8Hz,H-6″),4.60(dd,J=9.6Hz,J=1.6Hz,H-1″′),1.41(1H,m,H-2a″′),2.34(1H,m,H-2b″′),3.41(1H,m,H-3″′),3.18(1H,m,H-4″′),3.40(1H,m,H-5″′),1.38(3H,d,J=6.2Hz,H-6″′),3.48(3H,s,Me-3″′),4.82(1H,br d,J=9.7Hz,H-1″″),1.73(1H,m,H-2a″″),2.45(1H,m,H-2b″″),3.55(1H,m,H-3″″),3.51(1H,m,H-4″″),3.52(1H,m,H-5″″),1.43(3H,d,J=6.4Hz,H-6″″),3.51(3H,s,Me-3″″),4.48(1H,d,J=7.9Hz,H-1″″′),3.23(1H,m,H-2″″′),3.29(1H,m,H-3″″′),3.52(1H,m,H-4″″′),3.51(1H,m,H-5″″′),3.82(1H,m,H-6a″″′),3.92(1H,dd,J=12.0Hz,J=2.2Hz,H-6b″″′),4.38(1H,d,J=7.8Hz,H-1″″″),3.21(1H,m,H-2″″″),3.34(1H,m,H-3″″″),3.28(1H,m,H-4″″″),3.32(1H,m,H-5″″″),3.64(1H,dd,J=12.0Hz,J=5.8Hz,H-6a″″″),3.87(1H,dd,J=12.0Hz,J=2.2Hz,H-6b″″″)。 13C-NMR(C 5D 5N,100MHz):δ39.8(t,C-1),30.1(t,C-2),79.3(d,C-3),39.8(t,C-4),140.3(s,C-5),119.4(d,C-6),35.2(t,C-7),75.0(s,C-8),45.1(d,C-9),38.1(s,C-10),25.5(t,C-11),73.7(d,C-12),59.1(s,C-13),90.0(s,C-14),34.3(t,C-15),33.4(t,C-16),93.1(s,C-17),10.6(q,C-18),18.6(q,C-19),211.9(s,C-20),27.7(q,Me-21),167.3(s,C-1'),119.6(s,C-2'),132.8(d,C-3'),117.6(d,C-4'),168.3(s,C-5'),117.6(d,C-6'),132.8(d,C-7'),97.0(d,C-1″),38.8(t,C-2″),68.3(d,C-3″),83.7(d,C-4″),69.5(d,C-5″),18.5(q,C-6″),102.6(d,C-1″′),37.7(t,C-2″′),80.1(d,C-3″′),83.6(d,C-4″′),72.6(d,C-5″′),18.8(q,C-6″′),57.9(q,Me-3″′),100.6(d,C-1″″),36.3(t,C-2″″),78.4(d,C-3″″),83.6(d,C-4″″),70.0(d,C-5″″),18.6(q,C-6″″),58.6(q,Me-3″″),104.0(d,C-1″″′),75.3(d,C-2″″′),76.7(d,C-3″″′),81.0(d,C-4″″′),76.4(d,C-5″″′),62.1(t,C-6″″′),104.6(d,C-1″″″),74.9(d,C-2″″″),78.1(d,C-3″″″),71.4(d,C-4″″″),77.8(d,C-5″″″),62.4(t,C-6″″″)。
The breeding of zebrafish embryo is carried out in the mode of natural paired cross.Each mating prepares 4 ~ 5 pairs of Adult Zebrafishs, and on average often pair can be produced 200 ~ 300 embryos.At after fertilization 6 hours (i.e. 6hpf) and 24hpf, embryo is cleared up (removing dead embryo), and select suitable embryo according to the etap of embryo.Embryo's (fish culture water water quality: add 200mg Instant Ocean in every 1L reverse osmosis water, specific conductivity is 480 ~ 510 μ S/cm is hatched with fish culture water under 28 DEG C of conditions; PH is 6.9 ~ 7.2; Hardness is 53.7 ~ 71.6mg/LCaCO3).Because embryo can obtain nutritive substance from the yolk sac of self, so (9dpf) does not need feeding in after fertilization 9 days.After having tested, with tricaine methylsulfonic acid, over-exposure process is carried out to the zebra fish of each etap, thus zebra fish anesthesia is put to death.The operation steps that anesthesia is put to death meets the code requirement that American Veterinary association (AVMA) puts to death Animal Anesthesia.
The zebra fish of Yetrazol process is epilepsy model group; The zebra fish of phenytoin Sodium and Yetrazol co-treatment is positive controls; The zebra fish of the DMSO process of 1.1% is negative control group (solvent control group); Untreated zebra fish is blank group.Cynanchum otophvllum glycosides H and positive control (Levetiracetam and Cynanchum otophvllum sheet) are dissolved in DMSO respectively, are mixed with the solution to be measured that concentration is 1,3,10,30,60,100,150 and 300 μM.Above-mentioned solution to be measured respectively with Yetrazol co-treatment zebra fish certain hour, utilize the movement locus of zebra fish in behavioural analysis instrument record 60min, then to the rapid movement (seizure of zebra fish, V>20mm/sec) distance carries out quantitative analysis, by calculating following index: 1. zebra fish rapid movement distance (distance of rapid movement-seizure); 2. the anti-epileptic therapeutic efficiency (therapeuticefficacy) of compound; 3. compound time changing curve (compound effect onthe time-course of zebrafish seizure) that zebra fish rapid movement is affected.Quantitative analysis is carried out, the anti-epileptic therapeutic efficiency of computerized compound with distance (D) to the movement velocity of zebra fish, and draws dose-effect curve according to compound epilepsy therapy efficiency, the IC of computerized compound antiepileptic action 50; Carry out statistical analysis with variance analysis and T-inspection, p<0.05 is significant difference.In experimentation all there are not the phenomena of mortality in each group zebra fish.Fig. 2-1 is each experimental group zebra fish movement locus in 60min, and from Fig. 2-1, each concentration group of compound Cynanchum otophvllum glycosides H is compared with epilepsy model group, and zebra fish rapid movement track obviously reduces.Fig. 2-2 is the column diagram drawn according to zebra fish rapid movement distance average mean ± SE, and from Fig. 2-2, each concentration group all has statistical significance (p<0.001) compared with solvent control group; Fig. 2-3 is the dose-effect curve of compound Cynanchum otophvllum glycosides H antiepileptic action, from Fig. 2-3, compound Cynanchum otophvllum glycosides H is in the concentration range of 1 μM to 300 μMs, anti-epileptic therapeutic efficiency increases along with the increase of concentration, present concentration dependent, when concentration is 30 μMs, anti-epileptic therapeutic efficiency is 45% ± 2.13%.
Table 2. Cynanchum otophvllum glycosides H each concentration group zebra fish rapid movement distance raw data
Embodiment 3:
Root of Twisting Dregea glycosides Da 1(Dregeoside Da 1) evaluation of antiepileptic action in body:
Root of Twisting Dregea glycosides Da 1the preparation of (Dregeoside Da1): dry South Mountain rattan (5.5kg) is pulverized, three times are extracted with methanol eddy, each four hours, cooled and filtered, united extraction liquid, be concentrated into about 1.5L, after petroleum ether extraction removing fat-soluble component, reclaim methyl alcohol to medicinal extract shape, enriched material is suspended in water, with chloroform extraction four times, obtain chloroform extract 168g.Get 150g to dissolve, sample mixed by 300g silica gel (80-100 order), is separated, CHCl with silica gel column chromatography (200-300 order, 3270g) 3-MeOH-H 2o (86:17:1) wash-out, is mainly divided into three part Frs.A-C of opposed polarity.Fr.C (21g) is little polar portion, separating difficulty is relatively little, main utilization silica gel column chromatography is separated (chloroform/methanol or sherwood oil/acetone are eluent), Rp-8 reverse phase silica gel and MCI-gel CHP-20P column chromatography purification (methanol/water is eluent), obtain compound root of Twisting Dregea glycosides Da 1(46mg).
Root of Twisting Dregea glycosides Da 1, Dregeoside Da 1
The breeding of zebrafish embryo is carried out in the mode of natural paired cross.Each mating prepares 4 ~ 5 pairs of Adult Zebrafishs, and on average often pair can be produced 200 ~ 300 embryos.At after fertilization 6 hours (i.e. 6hpf) and 24hpf, embryo is cleared up (removing dead embryo), and select suitable embryo according to the etap of embryo.Embryo's (fish culture water water quality: add 200mg Instant Ocean in every 1L reverse osmosis water, specific conductivity is 480 ~ 510 μ S/cm is hatched with fish culture water under 28 DEG C of conditions; PH is 6.9 ~ 7.2; Hardness is 53.7 ~ 71.6mg/LCaCO 3).Because embryo can obtain nutritive substance from the yolk sac of self, so (9dpf) does not need feeding in after fertilization 9 days.After having tested, with tricaine methylsulfonic acid, over-exposure process is carried out to the zebra fish of each etap, thus zebra fish anesthesia is put to death.The operation steps that anesthesia is put to death meets the code requirement that American Veterinary association (AVMA) puts to death Animal Anesthesia.
The zebra fish of Yetrazol process is epilepsy model group; The zebra fish of phenytoin Sodium and Yetrazol co-treatment is positive controls; The zebra fish of the DMSO process of 1.1% is negative control group (solvent control group); Untreated zebra fish is blank group.Root of Twisting Dregea glycosides Da1 is dissolved in DMSO, is mixed with the solution to be measured that concentration is 30 μMs.Above-mentioned solution to be measured respectively with Yetrazol co-treatment zebra fish certain hour, utilize the movement locus of zebra fish in behavioural analysis instrument record 60min, then to the rapid movement (seizure of zebra fish, V>20mm/sec) distance carries out quantitative analysis, by calculating following index: 1. zebra fish rapid movement distance (distance of rapid movement-seizure); 2. the anti-epileptic therapeutic efficiency (therapeutic efficacy) of compound; 3. compound time changing curve (compound effect on the time-course of zebrafish seizure) that zebra fish rapid movement is affected.Root of Twisting Dregea glycosides Da1 and phenytoin Sodium are compared with epilepsy model group, and zebra fish rapid movement track obviously reduces, and preliminary deterministic compound root of Twisting Dregea glycosides Da1 has antiepileptic action, and therapeutic efficiency is 40 ± 3.13%.
Embodiment 4:
Root of Twisting Dregea glycosides D [11,12-di-O-acetyldrevogenin P-3-O-6-deoxidation-3-O-methyl-β-D-A Luo pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyranoside, Drevoluoside D] evaluation of antiepileptic action in body
Root of Twisting Dregea glycosides D [11,12-di-O-acetyldrevogenin P-3-O-6-deoxidation-3-O-methyl-β-D-A Luo pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyranoside, Drevoluoside D] preparation:
Dry South Mountain rattan (5.5kg) is pulverized, three times are extracted with methanol eddy, each four hours, cooled and filtered, united extraction liquid, be concentrated into about 1.5L, after petroleum ether extraction removing fat-soluble component, reclaim methyl alcohol to medicinal extract shape, enriched material is suspended in water, with chloroform extraction four times, obtain chloroform extract 168g.Get 150g to dissolve, sample mixed by 300g silica gel (80-100 order), is separated, CHCl with silica gel column chromatography (200-300 order, 3270g) 3-MeOH-H 2o (86:17:1) wash-out, is mainly divided into three part Frs.A-C of opposed polarity.Fr.C (21g) is little polar portion, separating difficulty is relatively little, main utilization silica gel column chromatography is separated (chloroform/methanol or sherwood oil/acetone are eluent), Rp-8 reverse phase silica gel and MCI-gel CHP-20P column chromatography purification (methanol/water is eluent), obtain root of Twisting Dregea glycosides D (8mg).
Root of Twisting Dregea glycosides D, Drevoluoside D
The physicochemical data of root of Twisting Dregea glycosides D is as follows: white amorphous powder, C 46h 72o 17; Mp 116-120 ° C, (c=0.20, MeOH), IR (KBr) ν max3442,2971,2932,2856,1748,1700,1612,1450,1369,1318,1247,1226,1195,1166,1127,1084,1030,1004,964,915,866,816,785,721cm -1.FAB-MS (negative ion) m/z 895 [M-1] -, HRESI-MS (negative ion) m/z 931.4434 [M (C 46h 72o 17)+Cl] -(calcd.931.4458). 1H-NMR(C 5D 5N,500MHz):δ1.33(1H,m,H-1a),1.98(1H,m,H-1b),1.74(1H,m,H-2a),2.10(1H,m,H-2b),3.77(1H,m,H-3),2.30(1H,m,H-4a),2.50(1H,m,H-4b),5.43(1H,d,J=5.4Hz,H-6),1.94(1H,m,H-7a),2.58(1H,m,H-7b),2.03(1H,m,H-8),3.08(1H,m,H-9),5.65(1H,t,J=10.3Hz,H-11),5.12(1H,d,J=10.3Hz,H-12),1.95(1H,m,H-15a),2.05(1H,m,H-15b),1.96(1H,m,H-16a),2.29(1H,m,H-16b),3.08(1H,m,H-17),1.30(3H,s,Me-18),1.19(3H,s,Me-19),2.20(3H,s,Me-21),2.02(3H,s,Me-2'),2.18(3H,s,Me-2″),5.23(1H,br d,J=9.3Hz,cym-1),1.85(1H,m,cym-2a),2.27(1H,m,cym-2b),4.03(1H,m,cym-3),3.44(1H,dd,J=9.8Hz,J=2.4Hz,cym-4),4.19(1H,m,cym-5),1.33(3H,d,J=6.4Hz,cym-6),3.57(3H,s,Me-cym-3),5.09(1H,br d,J=9.3Hz,cym-1'),1.82(1H,m,cym-2a'),2.33(1H,m,cym-2b'),4.08(1H,m,cym-3'),3.53(1H,dd,J=9.8Hz,J=2.4Hz,cym-4'),4.18(1H,m,cym-5'),1.49(3H,d,J=6.4Hz,cym-6'),3.58(3H,s,Me-cym-3'),5.12(1H,d,J=7.8Hz,alm-1),3.88(1H,m,alm-2),4.07(1H,m,alm-3),3.60(1H,br d,J=9.8Hz,alm-4),4.15(1H,m,alm-5),1.53(3H,d,J=6.4Hz,cym-6'),3.84(3H,s,Me-alm-3')。 13C-NMR(C 5D 5N,125MHz)：δ38.8(t,C-1),30.5(t,C-2),77.2(d,C-3),39.8(t,C-4),139.7(s,C-5),122.6(d,C-6),28.3(t,C-7),37.4(d,C-8),47.9(d,C-9),39.5(s,C-10),71.9(d,C-11),78.0(d,C-12),54.6(s,C-13),84.0(s,C-14),34.5(t,C-15),24.0(t,C-16),58.3(d,C-17),11.5(q,C-18),19.3(q,C-19),213.6(s,C-20),31.8(q,Me-21),170.2(s,C-1'),21.4(q,C-2'),171.1(s,C-1″),20.8(q,C-2″),96.5(d,cym-1),37.3(t,cym-2),78.0(d,cym-3),83.3(d,cym-4),69.1(d,cym-5),18.6(q,cym-6),58.8(q,Me-cym-3),100.4(d,cym-1'),36.9(t,cym-2'),78.2(d,cym-3'),83.3(d,cym-4'),69.4(d,cym-5'),18.6(q,cym-6'),58.8(q,Me-cym-3'),104.2(d,alm-1),73.1(d,alm-2),84.0(d,alm-3),74.5(d,alm-4),70.8(d,alm-5),18.6(d,alm-6),62.2(q,Me-alm-3')。
The breeding of zebrafish embryo is carried out in the mode of natural paired cross.Each mating prepares 4 ~ 5 pairs of Adult Zebrafishs, and on average often pair can be produced 200 ~ 300 embryos.At after fertilization 6 hours (i.e. 6hpf) and 24hpf, embryo is cleared up (removing dead embryo), and select suitable embryo according to the etap of embryo.Embryo's (fish culture water water quality: add 200mg Instant Ocean in every 1L reverse osmosis water, specific conductivity is 480 ~ 510 μ S/cm is hatched with fish culture water under 28 DEG C of conditions; PH is 6.9 ~ 7.2; Hardness is 53.7 ~ 71.6mg/LCaCO 3).Because embryo can obtain nutritive substance from the yolk sac of self, so (9dpf) does not need feeding in after fertilization 9 days.After having tested, with tricaine methylsulfonic acid, over-exposure process is carried out to the zebra fish of each etap, thus zebra fish anesthesia is put to death.The operation steps that anesthesia is put to death meets the code requirement that American Veterinary association (AVMA) puts to death Animal Anesthesia.
The zebra fish of Yetrazol process is epilepsy model group; The zebra fish of phenytoin Sodium and Yetrazol co-treatment is positive controls; The zebra fish of the DMSO process of 1.1% is negative control group (solvent control group); Untreated zebra fish is blank group.Root of Twisting Dregea glycosides D is dissolved in DMSO, is mixed with the solution to be measured that concentration is 30 μMs.Above-mentioned solution to be measured respectively with Yetrazol co-treatment zebra fish certain hour, utilize the movement locus of zebra fish in behavioural analysis instrument record 60min, then to the rapid movement (seizure of zebra fish, V>20mm/sec) distance carries out quantitative analysis, by calculating following index: 1. zebra fish rapid movement distance (distance of rapid movement-seizure); 2. the anti-epileptic therapeutic efficiency (therapeutic efficacy) of compound; 3. compound time changing curve (compound effect on the time-course of zebrafish seizure) that zebra fish rapid movement is affected.Root of Twisting Dregea glycosides D and phenytoin Sodium are compared with epilepsy model group, and zebra fish rapid movement track obviously reduces, and preliminary deterministic compound root of Twisting Dregea glycosides D has antiepileptic action, and therapeutic efficiency is 38 ± 4.43%.
Embodiment 5:
The evaluation of antiepileptic action in Cynanchum otophvllum glycosides N [Qingyanshengenin-3-O-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-Folium seu Cortex Nerii pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-digitoxin pyranoside, Otophylloside N] body
The preparation of Cynanchum otophvllum glycosides N [Qingyanshengenin-3-O-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-Folium seu Cortex Nerii pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-digitoxin pyranoside, Otophylloside N]:
Dry Cynanchum otophvllum root (10.0kg) is pulverized, and extracts three times (72,72,48 hours), each consumption 15L by 90% ethanol soaking at room temperature, filters, united extraction liquid, and recycling design, to without alcohol taste, obtains fluid extract 368g.Be suspended in by medicinal extract in 2L water, with chloroform extraction three times, each consumption 1.5L, combining extraction liquid, is concentrated into dry, obtains chloroform extract 155g.Get 150g to be separated through silica gel column chromatography, chloroform/methanol (10:1.5,7.6L) wash-out, obtain five part Frs.1-5.Fr.1 is separated repeatedly through silica gel column chromatography, with chloroform/methanol and sherwood oil/acetone for eluent, obtains Cynanchum otophvllum glycosides N (10mg).
Cynanchum otophvllum glycosides N, Otophylloside N
The physicochemical data of Cynanchum otophvllum glucoside N (Otophylloside N): white amorphous powder, C 55h 82o 20; Mp163-165 ° of C, (c=0.20, MeOH), IR (KBr) ν max3446,2971,2932,1713,1610,1594,1516,1452,1382,1369,1308,1275,1164,1093,1004,912,866,852,772cm -1.FAB-MS (negative ion) m/z 1061 [M-1] -, HRESI-MS (negative ion) m/z 1061.5284 [M (C 55h 82o 20)-H] -(calcd.1061.5321). 1H-NMR(C 5D 5N,500MHz):δ3.87(1H,m,H-3),5.26(1H,br s,H-6),5.33(1H,dd,J=11.5Hz,J=4.0Hz,H-12),2.09(3H,s,Me-18),1.30(3H,s,Me-19),2.41(3H,s,Me-21),8.30(1H,d,J=8.6Hz,H-3'),7.23(1H,d,J=8.6Hz,H-4'),7.23(1H,d,J=8.6Hz,H-6'),8.30(1H,d,J=8.6Hz,H-7'),5.48(1H,br d,J=9.2Hz,H-1″),2.07(1H,m,H-2″a),2.40(1H,m,H-2″b),4.64(1H,m,H-3″),3.52(1H,m,H-4″),4.30(1H,m,H-5″),1.43(3H,d,J=6.4Hz,H-6″),5.17(1H,br d,J=9.8Hz,H-1″′),1.78(1H,m,H-2a″′),2.29(1H,m,H-2b″′),4.01(1H,m,H-3″′),3.41(1H,m,H-4″′),4.18(1H,m,H-5″′),1.32(3H,d,J=6.1Hz,H-6″′),3.56(3H,s,Me-3″′),4.68(1H,br d,J=9.7Hz,H-1″″),1.73(1H,m,H-2a″″),2.45(1H,m,H-2b″″),3.55(1H,m,H-3″″),3.51(1H,m,H-4″″),3.52(1H,m,H-5″″),1.43(3H,d,J=6.4Hz,H-6″″),3.51(3H,s,Me-3″″),5.25(1H,br d,J=9.3Hz,H-1″″′),1.77(1H,m,H-2a″″′),2.38(1H,m,H-2b″″′),3.77(1H,m,H-3″″′),3.54(1H,m,H-4″″′),4.14(1H,m,H-5″″′),1.54(3H,d,J=6.3Hz,H-6″″′),3.46(3H,s,Me-3″″′)。 13C-NMR(C 5D 5N,100MHz)：δ39.3(t,C-1),29.9(t,C-2),77.6(d,C-3),38.9(t,C-4),139.4(s,C-5),119.2(d,C-6),34.8(t,C-7),74.3(s,C-8),44.5(d,C-9),37.4(s,C-10),25.2(t,C-11),73.4(d,C-12),58.4(s,C-13),89.6(s,C-14),33.9(t,C-15),33.2(t,C-16),92.5(s,C-17),10.9(q,C-18),18.2(q,C-19),209.9(s,C-20),27.9(q,Me-21),165.4(s,C-1'),122.0(s,C-2'),132.5(d,C-3′),116.2(d,C-4'),163.7(s,C-5'),116.2(d,C-6'),132.5(d,C-7'),96.4(d,C-1″),39.1(t,C-2″),67.6(d,C-3″),83.4(d,C-4″),68.6(d,C-5″),18.7(q,C-6″),99.8(d,C-1″′),36.7(t,C-2″′),77.7(d,C-3″′),83.2(d,C-4″′),69.1(d,C-5″′),18.5(q,C-6″′),58.9(q,Me-3″′),102.0(d,C-1″″),37.7(t,C-2″″),78.9(d,C-3″″),82.7(d,C-4″″),71.8(d,C-5″″),18.7(q,C-6″″),57.4(q,Me-3″″),98.6(d,C-1″″′),35.9(t,C-2″″′),79.0(d,C-3″″′),74.2(d,C-4″″′),71.3(d,C-5″″′),19.1(q,C-6″″′),58.1(q,Me-3″″′)。
The breeding of zebrafish embryo is carried out in the mode of natural paired cross.Each mating prepares 4 ~ 5 pairs of Adult Zebrafishs, and on average often pair can be produced 200 ~ 300 embryos.At after fertilization 6 hours (i.e. 6hpf) and 24hpf, embryo is cleared up (removing dead embryo), and select suitable embryo according to the etap of embryo.Embryo's (fish culture water water quality: add 200mg Instant Ocean in every 1L reverse osmosis water, specific conductivity is 480 ~ 510 μ S/cm is hatched with fish culture water under 28 DEG C of conditions; PH is 6.9 ~ 7.2; Hardness is 53.7 ~ 71.6mg/LCaCO 3).Because embryo can obtain nutritive substance from the yolk sac of self, so (9dpf) does not need feeding in after fertilization 9 days.After having tested, with tricaine methylsulfonic acid, over-exposure process is carried out to the zebra fish of each etap, thus zebra fish anesthesia is put to death.The operation steps that anesthesia is put to death meets the code requirement that American Veterinary association (AVMA) puts to death Animal Anesthesia.
The zebra fish of Yetrazol process is epilepsy model group; The zebra fish of phenytoin Sodium and Yetrazol co-treatment is positive controls; The zebra fish of the DMSO process of 1.1% is negative control group (solvent control group); Untreated zebra fish is blank group.Cynanchum otophvllum glycosides N is dissolved in DMSO, is mixed with the solution to be measured that concentration is 30 μMs.Above-mentioned solution to be measured respectively with Yetrazol co-treatment zebra fish certain hour, utilize the movement locus of zebra fish in behavioural analysis instrument record 60min, then to the rapid movement (seizure of zebra fish, V>20mm/sec) distance carries out quantitative analysis, by calculating following index: 1. zebra fish rapid movement distance (distance of rapid movement-seizure); 2. the anti-epileptic therapeutic efficiency (therapeutic efficacy) of compound; 3. compound time changing curve (compound effect on the time-course of zebrafish seizure) that zebra fish rapid movement is affected.Cynanchum otophvllum glycosides N and phenytoin Sodium are compared with epilepsy model group, and zebra fish rapid movement track obviously reduces, and preliminary deterministic compound Cynanchum otophvllum glycosides N has antiepileptic action, and therapeutic efficiency is 32 ± 4.50%.
Embodiment 6:
The evaluation of antiepileptic action in 11-O-acetyl-12-O-angeloyl-17 β-marsdenin-3-O-6-deoxidation-3-O-methyl-β-D-A Luo pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyranoside body
The preparation of 11-O-acetyl-12-O-angeloyl-17 β-marsdenin-3-O-6-deoxidation-3-O-methyl-β-D-A Luo pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyranoside:
Dry South Mountain rattan (5.5kg) is pulverized, three times are extracted with methanol eddy, each four hours, cooled and filtered, united extraction liquid, be concentrated into about 1.5L, after petroleum ether extraction removing fat-soluble component, reclaim methyl alcohol to medicinal extract shape, enriched material is suspended in water, with chloroform extraction four times, obtain chloroform extract 168g.Get 150g to dissolve, sample mixed by 300g silica gel (80-100 order), and be separated with silica gel column chromatography (200-300 order, 3270g), CHCl3-MeOH-H2O (86:17:1) wash-out, is mainly divided into three part Frs.A-C of opposed polarity.Fr.C (21g) is little polar portion, separating difficulty is relatively little, main utilization silica gel column chromatography is separated (chloroform/methanol or sherwood oil/acetone are eluent), Rp-8 reverse phase silica gel and MCI-gel CHP-20P column chromatography purification (methanol/water is eluent), obtain 11-O-acetyl-12-O-angeloyl-17 β-marsdenin-3-O-6-deoxidation-3-O-methyl-β-D-A Luo pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyranoside (3mg).
The physicochemical data of 11-O-acetyl-12-O-angeloyl-17 β-marsdenin-3-O-6-deoxidation-3-O-methyl-β-D-A Luo pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyranoside: white amorphous powder, C 49h 76o 18; FAB-MS (negative ion) m/z 951 [M-1] -, 869 [M-83] -. 1H-NMR(C 5D 5N,500MHz):δ2.50(1H,m,H-1a),2.56(1H,m,H-1b),1.92(1H,m,H-2a),2.15(1H,m,H-2b),3.87(1H,m,H-3),1.34(1H,m,H-4a),2.36(1H,m,H-4b),5.33(1H,d,J=5.6Hz,H-6),2.05(1H,m,H-7a),2.22(1H,m,H-7b),2.14(1H,m,H-9),6.27(1H,t,J=10.7Hz,H-11),5.42(1H,d,J=10.7Hz,H-12),2.30(1H,m,H-15a),2.59(1H,m,H-15b),2.03(1H,m,H-16a),2.29(1H,m,H-16b),3.15(1H,m,H-17),1.63(3H,s,Me-18),1.59(3H,s,Me-19),2.18(3H,s,Me-21),1.59(3H,s,Me-2'),7.19(1H,H-3″),1.65(3H,d,J=6.8Hz,Me-4″),1.97(3H,s,Me-5″),5.27(1H,br d,J=9.4Hz,cym-1),1.85(1H,m,cym-2a),2.26(1H,m,cym-2b),4.02(1H,m,cym-3),3.44(1H,dd,J=9.4Hz,J=2.6Hz,cym-4),4.19(1H,m,cym-5),1.34(3H,d,J=6.0Hz,cym-6),3.57(3H,s,Me-cym-3),5.09(1H,br d,J=9.4Hz,cym-1'),1.81(1H,m,cym-2a'),2.33(1H,m,cym-2b'),4.07(1H,m,cym-3'),3.52(1H,dd,J=9.4Hz,J=2.6Hz,cym-4'),4.17(1H,m,cym-5'),1.48(3H,d,J=6.0Hz,cym-6'),3.57(3H,s,Me-cym-3'),5.13(1H,d,J=8.1Hz,alm-1),3.87(1H,m,alm-2),4.07(1H,m,alm-3),3.59(1H,m,alm-4),4.16(1H,m,alm-5),1.53(3H,d,J=6.4Hz,cym-6'),3.85(3H,s,Me-alm-3')。 13C-NMR(C 5D 5N,125MHz)：δ39.8(t,C-1),30.2(t,C-2),77.8(d,C-3),40.5(t,C-4),139.7(s,C-5),118.8(d,C-6),36.8(t,C-7),76.0(s,C-8),49.1(d,C-9),39.3(s,C-10),71.7(d,C-11),78.4(d,C-12),55.7(s,C-13),85.6(s,C-14),35.5(t,C-15),24.4(t,C-16),59.5(d,C-17),13.7(q,C-18),18.1(q,C-19),213.3(s,C-20),31.4(q,Me-21),169.9(s,C-1'),21.5(q,C-2'),168.0(s,C-1″),128.6(t,C-2″),139.1(d,C-3″),14.4(q,C-4″),12.2(q,C-5″),96.5(d,cym-1),37.3(t,cym-2),78.0(d,cym-3),83.4(d,cym-4),69.1(d,cym-5),18.6(q,cym-6),58.8(q,Me-cym-3),100.4(d,cym-1'),36.9(t,cym-2'),78.2(d,cym-3'),83.3(d,cym-4'),69.4(d,cym-5'),18.6(q,cym-6'),58.8(q,Me-cym-3'),104.2(d,alm-1),73.2(d,alm-2),84.0(d,alm-3),74.5(d,alm-4),70.8(d,alm-5),18.6(d,alm-6),62.2(q,Me-alm-3')。
11-O-acetyl-12-O-angeloyl-17 β-marsdenin-3-O-6-deoxidation-3-O-methyl-β-D-A Luo pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyranoside
The breeding of zebrafish embryo is carried out in the mode of natural paired cross.Each mating prepares 4 ~ 5 pairs of Adult Zebrafishs, and on average often pair can be produced 200 ~ 300 embryos.At after fertilization 6 hours (i.e. 6hpf) and 24hpf, embryo is cleared up (removing dead embryo), and select suitable embryo according to the etap of embryo.Embryo's (fish culture water water quality: add 200mg Instant Ocean in every 1L reverse osmosis water, specific conductivity is 480 ~ 510 μ S/cm is hatched with fish culture water under 28 DEG C of conditions; PH is 6.9 ~ 7.2; Hardness is 53.7 ~ 71.6mg/LCaCO 3).Because embryo can obtain nutritive substance from the yolk sac of self, so (9dpf) does not need feeding in after fertilization 9 days.After having tested, with tricaine methylsulfonic acid, over-exposure process is carried out to the zebra fish of each etap, thus zebra fish anesthesia is put to death.The operation steps that anesthesia is put to death meets the code requirement that American Veterinary association (AVMA) puts to death Animal Anesthesia.
The zebra fish of Yetrazol process is epilepsy model group; The zebra fish of phenytoin Sodium and Yetrazol co-treatment is positive controls; The zebra fish of the DMSO process of 1.1% is negative control group (solvent control group); Untreated zebra fish is blank group.Glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyranoside is dissolved in DMSO 11-O-acetyl-12-O-angeloyl-17 β-marsdenin-3-O-6-deoxidation-3-O-methyl-β-D-A Luo pyrans, is mixed with the solution to be measured that concentration is 30 μMs.Above-mentioned solution to be measured respectively with Yetrazol co-treatment zebra fish certain hour, utilize the movement locus of zebra fish in behavioural analysis instrument record 60min, then to the rapid movement (seizure of zebra fish, V>20mm/sec) distance carries out quantitative analysis, by calculating following index: 1. zebra fish rapid movement distance (distance of rapidmovement-seizure); 2. the anti-epileptic therapeutic efficiency (therapeutic efficacy) of compound; 3. compound time changing curve (compound effect on the time-course ofzebrafish seizure) that zebra fish rapid movement is affected.11-O-acetyl-12-O-angeloyl-17 β-marsdenin-3-O-6-deoxidation-3-O-methyl-β-D-A Luo pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyranoside with phenytoin Sodium compared with epilepsy model group, zebra fish rapid movement track obviously reduces, preliminary deterministic compound 11-O-acetyl-12-O-angeloyl-17 β-marsdenin-3-O-6-deoxidation-3-O-methyl-β-D-A Luo pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyranoside has antiepileptic action, therapeutic efficiency is 24 ± 4.32%.
Embodiment 7:
The evaluation of antiepileptic action in root of Twisting Dregea glycosides N [Marsectohexol-3-O-β-D-glucopyanosyl base-(1 → 4)-6-deoxidation-3-O-methyl-β-D-A Luo pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyranoside, Drevoluoside N] body
The preparation of root of Twisting Dregea glycosides N [Marsectohexol-3-O-β-D-glucopyanosyl base-(1 → 4)-6-deoxidation-3-O-methyl-β-D-A Luo pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyrans glycosyl-(1 → 4)-β-D-apocynum cannabinum pyranoside, Drevoluoside N]:
Dry South Mountain rattan (5.5kg) is pulverized, three times are extracted with methanol eddy, each four hours, cooled and filtered, united extraction liquid, be concentrated into about 1.5L, after petroleum ether extraction removing fat-soluble component, reclaim methyl alcohol to medicinal extract shape, enriched material is suspended in water, with chloroform extraction four times, obtain chloroform extract 168g.Get 150g to dissolve, sample mixed by 300g silica gel (80-100 order), is separated, CHCl with silica gel column chromatography (200-300 order, 3270g) 3-MeOH-H 2o (86:17:1) wash-out, is mainly divided into three part Frs.A-C of opposed polarity.Fr.A (27g) is large polar portion, be separated after (methanol/water is eluent) through Rp-8 reverse phase silica gel, recycle silicon glue (chloroform/methanol is eluent) and MCI-gel CHP-20P (methanol/water is eluent) column chromatography purification, obtain compound root of Twisting Dregea glycosides N (6mg).
Root of Twisting Dregea glycosides N, Drevoluoside N
The physicochemical data of root of Twisting Dregea glucoside N (Drevoluoside N): white amorphous powder, C 48h 80o 21; Mp 177-179 ° C, (c=0.22, MeOH), IR (KBr) ν max3442,3433,2970,2933,1637,1452,1402,1378,1371,1319,1262,1235,1196,1162,1080,1004,953,909,864,816,780,720cm -1.FAB-MS (negative ion) m/z 991 [M-1] -, HRESI-MS (negative ion) m/z 1027.4870 [M (C 48h 80o 21)+Cl] -(calcd.1027.4880). 1H-NMR(C 5D 5N,500MHz):δ2.59(2H,m,H-1),2.02(1H,m,H-2a),2.13(1H,m,H-2b),3.92(1H,m,H-3),1.38(1H,m,H-4a),3.33(1H,m,H-4b),5.35(1H,d,J=4.9Hz,H-6),1.89(2H,m,H-7),1.83(1H,m,H-9),4.66(1H,t,J=10.3Hz,H-11),3.67(1H,d,J=10.3Hz,H-12),2.35(1H,m,H-15a),2.54(1H,m,H-15b),1.89(2H,m,H-16),2.50(1H,m,H-17),1.88(3H,s,Me-18),1.80(3H,s,Me-19),4.09(1H,m,H-20),1.41(3H,d,J=6.4Hz,Me-21),5.28(1H,br d,J=9.3Hz,cym-1),1.86(1H,m,cym-2a),2.27(1H,m,cym-2b),4.00(1H,m,cym-3),3.43(1H,br d,J=8.8Hz,cym-4),4.19(1H,m,cym-5),1.33(3H,d,J=6.4Hz,cym-6),3.55(3H,s,Me-cym-3),5.08(1H,br d,J=9.3Hz,cym-1'),1.80(1H,m,cym-2a'),2.30(1H,m,cym-2b'),4.01(1H,m,cym-3'),3.45(1H,dd,J=8.8Hz,J=2.6Hz,cym-4'),4.15(1H,m,cym-5'),1.43(3H,d,J=6.4Hz,cym-6'),3.58(3H,s,Me-cym-3′),5.07(1H,d,J=8.3Hz,alm-1),3.80(1H,m,alm-2),4.44(1H,m,alm-3),3.71(1H,br d,J=9.8Hz,alm-4),4.24(1H,m,alm-5),1.61(3H,d,J=6.4Hz,cym-6'),3.82(3H,s,Me-alm-3'),4.97(1H,d,J=7.8Hz,glc-1),4.04(1H,m,glc-2),4.23(1H,m,glc-3),4.20(1H,m,glc-4),4.25(1H,m,glc-5),4.37(1H,dd,J=11.7Hz,J=5.4Hz,glc-6)。 13C-NMR(C 5D 5N,125MHz)：δ39.9(t,C-1),30.4(t,C-2),78.3(d,C-3),40.9(t,C-4),141.0(s,C-5),118.6(d,C-6),36.1(t,C-7),76.4(s,C-8),51.1(d,C-9),39.7(s,C-10),70.1(d,C-11),82.2(d,C-12),54.4(s,C-13),85.4(s,C-14),36.0(t,C-15),27.5(t,C-16),56.8(d,C-17),12.2(q，C-18),17.9(q，C-19),69.7(s,C-20),23.3(q,Me-21),96.4(d,cym-1),37.3(t,cym-2),78.1(d,cym-3),83.4(d,cym-4),69.1(d,cym-5),18.7(q,cym-6),58.9(q,Me-cym-3),100.4(d,cym-1'),37.1(t,cym-2'),78.2(d,cym-3'),83.2(d,cym-4'),69.3(d,cym-5'),18.6(q,cym-6'),59.1(q,Me-cym-3'),104.0(d,alm-1),72.5(d,alm-2),83.1(d,alm-3),83.1(d,alm-4),69.3(d,alm-5),18.3(d,alm-6),61.8(q,Me-alm-3'),106.6(d,glc-1),75.5(d,glc-2),78.5(d,glc-3),71.9(d,glc-4),78.4(d,glc-5),63.0(t,glc-6)。
The breeding of zebrafish embryo is carried out in the mode of natural paired cross.Each mating prepares 4 ~ 5 pairs of Adult Zebrafishs, and on average often pair can be produced 200 ~ 300 embryos.At after fertilization 6 hours (i.e. 6hpf) and 24hpf, embryo is cleared up (removing dead embryo), and select suitable embryo according to the etap of embryo.Embryo's (fish culture water water quality: add 200mg Instant Ocean in every 1L reverse osmosis water, specific conductivity is 480 ~ 510 μ S/cm is hatched with fish culture water under 28 DEG C of conditions; PH is 6.9 ~ 7.2; Hardness is 53.7 ~ 71.6mg/LCaCO 3).Because embryo can obtain nutritive substance from the yolk sac of self, so (9dpf) does not need feeding in after fertilization 9 days.After having tested, with tricaine methylsulfonic acid, over-exposure process is carried out to the zebra fish of each etap, thus zebra fish anesthesia is put to death.The operation steps that anesthesia is put to death meets the code requirement that American Veterinary association (AVMA) puts to death Animal Anesthesia.
The zebra fish of Yetrazol process is epilepsy model group; The zebra fish of phenytoin Sodium and Yetrazol co-treatment is positive controls; The zebra fish of the DMSO process of 1.1% is negative control group (solvent control group); Untreated zebra fish is blank group.Root of Twisting Dregea glycosides N is dissolved in DMSO, is mixed with the solution to be measured that concentration is 30 μMs.Above-mentioned solution to be measured respectively with Yetrazol co-treatment zebra fish certain hour, utilize the movement locus of zebra fish in behavioural analysis instrument record 60min, then to the rapid movement (seizure of zebra fish, V>20mm/sec) distance carries out quantitative analysis, by calculating following index: 1. zebra fish rapid movement distance (distance of rapid movement-seizure); 2. the anti-epileptic therapeutic efficiency (therapeutic efficacy) of compound; 3. compound time changing curve (compound effect on the time-course of zebrafish seizure) that zebra fish rapid movement is affected.Root of Twisting Dregea glycosides N and phenytoin Sodium are compared with epilepsy model group, and zebra fish rapid movement track obviously reduces, and tentatively determine that root of Twisting Dregea glycosides N has antiepileptic action, therapeutic efficiency is 19 ± 2.27%.
According to the pathogeny of epilepsy, the zebra fish that the above embodiment of the present invention system selects Yetrazol (Pentylenetetrazole) to process is used as epilepsy model group, testing compound and Yetrazol co-treatment zebra fish certain hour, utilize the motion conditions of zebra fish in motion/behavioural analysis instrument record certain hour, with distance (D), quantitative analysis is carried out to the movement velocity of zebra fish, the anti-epileptic therapeutic efficiency of computerized compound, and draw dose-effect curve according to compound epilepsy therapy efficiency, the IC of computerized compound antiepileptic action 50; Carry out statistical analysis with variance analysis and T-inspection, p<0.05 is significant difference.
Rapid movement refers to that zebra fish movement velocity is greater than the motion of 20mm/sec, is equivalent to mammiferous status epilepticus; Rapid movement track refers to utilize the movement velocity of behavioural analysis instrument record to be greater than the movement locus of 20mm/sec, i.e. the movement locus of zebra fish under status epilepticus, represents with red lines in behavior analytic record result; Rapid movement distance refers to that zebra fish movement velocity is greater than the move distance of 20mm/sec, i.e. the move distance of zebra fish under status epilepticus.
All there are not the zebra fish phenomena of mortality in all experimental group in whole experimentation.
Trial-product selects 11-O-acetyl ester group-12,20-O-diisoamyl acyl ester group-hardship to restrict aglycon and derivative thereof and Cynanchum otophvllum glycoside derivates.
Positive reference substance selects Levetiracetam and Cynanchum otophvllum sheet.
In zebra fish anti-epileptic model, trial-product 11-11-O-acetyl ester group-12,20-O-diisoamyl acyl ester group-hardship restricts aglycon and derivative, Cynanchum otophvllum glycoside derivates and positive control Levetiracetam and Cynanchum otophvllum sheet respectively with DMSO dissolving, and final concentration is 1 μM.Observe each experimental group zebra fish movement locus in 60min, the IC of calculation sample 50.
Example of formulations 1:
By the method first obtained above-claimed cpd of the present invention of embodiment 1-7, and utilize organic acid (tartrate, citric acid, formic acid, oxalic acid etc.) or mineral acid (hydrochloric acid, sulfuric acid, phosphoric acid etc.) salt made, inject with water routinely, essence filter, injection liquid is made in embedding sterilizing.
Example of formulations 2:
By the method first obtained above-claimed cpd of the present invention of embodiment 1-7, and utilize organic acid (tartrate, citric acid, formic acid, oxalic acid etc.) or the salt made of mineral acid (hydrochloric acid, sulfuric acid, phosphoric acid etc.), be dissolved in sterile water for injection, stirring makes molten, filters with aseptic suction funnel, more aseptic essence filter, be sub-packed in 2 ampoules, after frozen drying, aseptic sealing by fusing obtains powder injection.
Example of formulations 3:
By the method first obtained above-claimed cpd of the present invention of embodiment 1-7, and utilize organic acid (tartrate, citric acid, formic acid, oxalic acid etc.) or the salt made of mineral acid (hydrochloric acid, sulfuric acid, phosphoric acid etc.), add vehicle with excipient weight than the ratio for 9:1, make pulvis.
Example of formulations 4:
By the method first obtained above-claimed cpd of the present invention of embodiment 1-7, and utilize organic acid (tartrate, citric acid, formic acid, oxalic acid etc.) or the salt made of mineral acid (hydrochloric acid, sulfuric acid, phosphoric acid etc.), vehicle is added, pelletizing press sheet than the ratio for 1:5-1:10 in itself and excipient weight.
Example of formulations 5:
By the method first obtained above-claimed cpd of the present invention of embodiment 1-7, and utilize organic acid (tartrate, citric acid, formic acid, oxalic acid etc.) or mineral acid (hydrochloric acid, sulfuric acid, phosphoric acid etc.) salt made, oral liquid method for making makes oral liquid routinely.
Example of formulations 6:
By the method first obtained above-claimed cpd of the present invention of embodiment 1-7, and utilize organic acid (tartrate, citric acid, formic acid, oxalic acid etc.) or the salt made of mineral acid (hydrochloric acid, sulfuric acid, phosphoric acid etc.), add vehicle in itself and excipient weight than the ratio for 5:1, make capsule or granule or electuary.
Example of formulations 7:
By the method first obtained above-claimed cpd of the present invention of embodiment 1-7, and utilize organic acid (tartrate, citric acid, formic acid, oxalic acid etc.) or the salt made of mineral acid (hydrochloric acid, sulfuric acid, phosphoric acid etc.), add vehicle in itself and excipient weight than the ratio for 3:1, make capsule or granule or electuary.

Claims (3)

1. the C shown in structural formula (I) ?3,11,12,20 ?four replace C ?21 steroid derivatives and pharmaceutical salts thereof and glycoside,
2. the pharmaceutical composition of antiepileptic, wherein containing C according to claim 1 ?3,11,12,17 ?four replace C ?21 steroid derivative and pharmaceutical salts thereof and glycoside and the pharmaceutically acceptable carrier of at least one.
3. C according to claim 1 ?3,11,12,17 ?four replace C ?21 steroid derivative and pharmaceutical salts thereof and the application of glycoside in the medicine of preparation treatment epileptics.
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