CN105503988A - Natural anti-epilepsy activity compound and uses of the natural anti-epilepsy activity compound in pharmaceutical preparations - Google Patents

Abstract

The present invention relates to a natural anti-epilepsy activity compound and uses of the natural anti-epilepsy activity compound in pharmaceutical preparations. The present invention discloses different monomer compounds having diametrically opposed effects such as epilepsy promoting and epilepsy resistance in Cynanchum otophyllum. According to the present invention, particularly the single component is respectively extracted from Cynanchum, C.auriculatum Royle exWight and C.wilfordii Hemsl, and experiment results prove that each compound has anti-epilepsy activity.

Description

Natural antiepileptic activity compound and the purposes in pharmaceutical preparation thereof

Technical field

The invention belongs to biomedicine field, specifically, the present invention relates to natural antiepileptic activity compound and the purposes in pharmaceutical preparation thereof.

Background technology

Epilepsy (Epilepsy), or claim brain epilepsy, sheep epilepsy, be chronic recurrent of short duration brain function imbalance syndrome.According to statistics, about the ten thousand/year of sickness rate about 23/10 of China's epilepsy, morbidity is 3.5 ‰-4.8 ‰.Epileptic seizures can cause series of symptoms, and spasm is one of typical epilepsy syndromes, comprises limbs repeating motion, irregularities and the whole body tic etc. with the loss of consciousness, even causes body injury or death.The treatment of epilepsy comprises the multiple methods such as pharmacological agent, operative treatment, Physiotherapy and psychotherapeutics, and its drug treatment is the most frequently used, most important means at present.Carbamzepine (Carbamazepine), Sodium Valproate (Depakene), phenytoin Sodium (Dilantin) etc. are comprised clinically at present for the medicine of epilepsy, but these medicines have obviously side effect usually, long-term taking shines into larger impact to health, even may cause suicidal thoughts or behavior in sub-fraction medication patient.Chinese medicine compound prescription, Chinese patent medicine preparation product also occupy many market, but due to Chinese medicine side's complicated component, and plant place of production difference, climate variability, harvesting time, not equal cause influence the content of medicine main component, therefore shine into sizable difficulty to the quality control of Chinese medicine side, which also limits the popularization of Chinese medicine preparation.Therefore, the new antiepileptic medicine finding safety non-toxic effective and quality controllable has extremely important meaning in the prevention and therapy of epilepsy.

Cynanchum (Cynanchum) is a genus of Apocynaceae (Apocynum), about 200 kinds, is distributed in the torrid zone and Temperate Region in China, and about 53 kinds, China, it is all produced in the whole nation.Cynanchum auriculatum Royle ex Wight (C.auriculatum), also known as Root of Bunge Auriculate, be China's endemic plant kind, about 95% is grown on Binhai County, Jiangsu Province, has liver and kidney tonifying, replenishing vital essence and blood, anti-ageing effect of waiting for a long time.Root of Wilford Swallowwort (C.wilfordii) originates in the ground such as Liaoning, Henan, Hubei, conventional with stomach invigorating, disappear glutted, control food etc. of choking.Cynanchum otophvllum (C.otophyllum) originates in the provinces and regions such as Hunan, Guangxi, Guizhou, Yunnan, Sichuan and Tibet, has removing toxic substances, antispastic, the effects such as wind-damp dispelling, clinical in dispelling the wind and dampness pathogens ostalgia, epilepsy and venomous snake bite.

The total glycosides of Cynanchum otophvllum extract Cynanchum otophvllum is the adjuvant drug of clinical treatment epilepsy.But Cynanchum otophvllum sheet complicated component, carrys out certain difficulty to quality control cincture, and action target is failed to understand, the clinical main and antiepileptic drug such as phenytoin Sodium or phenylethyl barbituric acid share treatment.

Summary of the invention

The object of the present invention is to provide natural antiepileptic activity compound and the purposes in pharmaceutical preparation thereof.

A first aspect of the present invention, provides a kind of structure such as formula the compound shown in A or its pharmaceutically acceptable salt, it is characterized in that:

In formula A, R ₁for glycosyl, R ₂for substituted or unsubstituted C1 ~ C6 alkyl or substituted or unsubstituted C1 ~ C6 thiazolinyl, R ₃for substituted or unsubstituted C1 ~ C6 alkyl;

Described replacement refers to that one or more hydrogen atom is selected from the substituting group replacement of lower group: C1 ~ C10 alkyl, C3 ~ C10 cycloalkyl, C1 ~ C10 alkoxyl group, hydroxyl, carboxyl, C1 ~ C10 carbonyl, C1 ~ C10 amide group, C2 ~ C10 ester group, C6 ~ C30 aryl and oxygen (=O).

In another preference, described glycosyl by apocynum cannabinum glycosyl (cymarosyl), Folium seu Cortex Nerii glycosyl (oleandrosyl), diginose base (diginosyl), thevetose base (thevetosyl), digoxigenin glycosyl (digitoxosyl), block in that glycosyl (canarosyl), rhamanopyranosyl (rhamnosyl) and glucosyl group (glucosyl) one or more form.

In another preference, described glycosyl comprises 1 ~ 6 identical or different monosaccharide unit, preferably, comprises 2 ~ 4 identical or different monosaccharide units.

In another preference, R ₁in not containing sugar unit

In another preference, work as R ₂during for hydroxy phenyl, R ₁middle sugar unit quantity is not 3.

In another preference, described sugar unit quantity can be 0,1,2,3 or 4.

In another preference, described R ₃for wherein R ₄for substituted or unsubstituted C1 ~ C6 alkyl; R ₅for substituted or unsubstituted C1 ~ C6 alkyl.

In another preference, described R ₁for n is the positive integer of 1 ~ 4, R ₇for H or methyl or C1 ~ C10 ester group, R ₈for H or C2 ~ C10 ester group.

In another preference, described R1 is selected from lower group:

In another preference, described R ₂be selected from lower group:

In another preference, described R ₃be selected from lower group:

In another preference, described compound structure is such as formula shown in I:

In another preference, described compound structure is such as formula shown in II:

In another preference, described compound structure is such as formula shown in III:

In another preference, described compound structure is such as formula shown in IV:

In another preference, described compound structure is such as formula shown in V:

In another preference, described compound structure is such as formula shown in VI:

In another preference, described compound structure is such as formula shown in VII:

In another preference, described compound structure is such as formula shown in VIII:

In another preference, described compound structure is such as formula shown in Ⅸ:

In another preference, described compound structure is such as formula shown in Ⅹ:

In another preference, described compound structure is such as formula shown in Ⅺ:

In another preference, described compound structure is such as formula shown in Ⅻ:

In another preference, described compound structure is such as formula shown in XIII:

In another preference, described compound structure is such as formula shown in XV:

In another preference, described compound structure is such as formula shown in XVI:

A first aspect of the present invention, provides a kind of pharmaceutical composition, and it comprises compound as described in the first aspect of the invention or its pharmacy acceptable salt, and optional pharmaceutically acceptable carrier or vehicle.

In another preference, also comprise the medicine of group under optional being selected from described pharmaceutical composition: Carbamzepine (Carbamazepine), Sodium Valproate (Depakene) and phenytoin Sodium (Dilantin).

A third aspect of the present invention, provides compound as described in the first aspect of the invention or its pharmacy acceptable salt, wilfosideG or its pharmaceutically acceptable salt or the application of pharmaceutical composition as described in respect of the second aspect of the invention in preparation treatment or prevention epilepsy medicament

In another preference, described wilfosideG structure is such as formula shown in XIV:

A fourth aspect of the present invention, provides the application in preparation treatment or prevention epilepsy medicament of Apocynaceae Cynanchum (Cynanchum) plant Auriculoside A (C.auriculatumRoyleexWight) or Root of Wilford Swallowwort (C.wilfordiiHemsl.) or its extract.

A fifth aspect of the present invention, provide a kind of plant total glycoside or the Chinese medical extract containing described plant total glycoside, contain antiepileptic activity composition in described plant total glycoside or described extract, and the content C1 of antiepileptic activity composition described in described extract meets following formula B:

C1/ (C1+C2) >=50% formula B

In formula B, C1 is the content of antiepileptic activity composition; C2 is the content of short epilepsy activeconstituents.

In another preference, C1/ (C1+C2) >=80%; More preferably, >=90%; Best, >=95%, as 96%, 97%, 98%, 99%, 99.5%.

In another preference, described antiepileptic activity composition be selected from lower group one or more:

Compound shown in formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI.

In another preference, described short epilepsy activeconstituents be selected from lower group one or more:

Compound shown in formula a, b, c and d

In another preference, described plant comprises: Auriculoside A (C.auriculatumRoyleexWight), Root of Wilford Swallowwort (C.wilfordiiHemsl.).

In another preference, the preparation method of described extract is as follows:

Medicinal material is extracted three times with 95% alcohol dipping, concentrating under reduced pressure extracting solution obtains ethanol extract, add water suspendible, with extraction into ethyl acetate, reclaims ethyl acetate, ethyl acetate extract is dissolved in 70% ethanolic soln, filter or after centrifugal segregation insolubles, carry out macroporous resin column chromatography separation, collect 75% ethanol elution flow point, concentrated, to obtain final product.

A sixth aspect of the present invention, provides a kind of pharmaceutical composition, comprises the Chinese medical extract as described in fifth aspect present invention, and optional pharmaceutically acceptable carrier or vehicle.

A seventh aspect of the present invention, provides one and carries out quality controlling means to anti-epileptic product, comprise step:

A () measures the content C2 of short epilepsy activeconstituents in described product or its raw material, and compare with preset value C0;

B () when described content C2 is higher than C0, then judges the off quality of described product; When described content C2 is less than or equal to C0, then judge the up-to-standard of described product.

In another preference, described short epilepsy activeconstituents be selected from lower group one or more: the compound shown in formula a, b, c and d.

In another preference, described anti-epileptic product comprises the anti-epileptic medicine containing Cynanchum otophvllum extract.

In another preference, described C2 is the weight percent of short epilepsy activeconstituents in described product or raw material.

In another preference, described C0≤10% (w/w), preferably, described C0≤5% (w/w), more preferably, described C0≤1%, described C0 can be 0.01%, 0.05%, 0.1%, 0.2%, 0.5%, 1.0%, 5%, 10%.

A eighth aspect of the present invention, provide the compound being selected from lower group and preparing the purposes in medicament or reagent, described medicament or reagent are for building animal seizure models: the compound shown in formula a, b, c and d.

Should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and can combining mutually between specifically described each technical characteristic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tiredly no longer one by one to state at this.

Accompanying drawing explanation

Figure 1A shows the zebra fish rapid movement track of each concentration group of cynawilfosideA and epilepsy model group; Figure 1B shows the column diagram drawn according to zebra fish rapid movement distance average mean ± SE.

Fig. 2 A shows the zebra fish rapid movement track of each concentration group of wilfosideC1N and epilepsy model group; Fig. 2 B shows the column diagram drawn according to zebra fish rapid movement distance average mean ± SE.

Fig. 3 A shows the zebra fish rapid movement track of each concentration group of wilfosideK1N and epilepsy model group; Fig. 3 B shows the column diagram drawn according to zebra fish rapid movement distance average mean ± SE.

Fig. 4 A shows the zebra fish rapid movement track of each concentration group of taiwanosideC and epilepsy model group; Fig. 4 B shows the column diagram drawn according to zebra fish rapid movement distance average mean ± SE.

Fig. 5 A shows the zebra fish rapid movement track of the own each concentration group of Cynanchum otophvllum glycosides and epilepsy model group; Fig. 5 B shows the column diagram drawn according to zebra fish rapid movement distance average mean ± SE.

Fig. 6 A shows Russell, and he orders the zebra fish rapid movement track of 3-O-β-D-oleandrose pyrans glycosyl-(1 → 4)-β-D-cymarose pyranyl-(1 → 4)-β-D-cymarose [rostratamine3-O-β-D-oleandropyranosyl-(1 → 4)-β-D-cymaropyranosyl-(1 → 4)-β-D-cymaropyranoside] each concentration group and epilepsy model group; Fig. 6 B shows the column diagram drawn according to zebra fish rapid movement distance average mean ± SE.

Fig. 7 A shows the zebra fish rapid movement track of each concentration group of wilfosideC3N and epilepsy model group; Fig. 7 B shows the column diagram drawn according to zebra fish rapid movement distance average mean ± SE.

Fig. 8 A shows the zebra fish rapid movement track of each concentration group of cynauricosideA and epilepsy model group; Fig. 8 B shows the column diagram drawn according to zebra fish rapid movement distance average mean ± SE.

Fig. 9 A shows the zebra fish rapid movement track of each concentration group of Auriculoside A reactive site and epilepsy model group; Fig. 9 B shows the column diagram drawn according to zebra fish rapid movement distance average mean ± SE.

Figure 10 shows Qingyanshengenin (qingyangshengenin), Caudatin (caudatin), Q-A (otophyllosideA), Cynanchum otophvllum glycosides third (otophyllosideC), own (the otophyllosideF of Cynanchum otophvllum glycosides, formula I), Cynanchum otophvllum glycosides M (otophyllosideM, formula b), Caudatin 3-O-β-D-cymarose [caudatin3-O-β-D-cymaropyranoside, formula IX], Qingyanshengenin 3-O-β-D-cymarose pyranyl-(1 → 4)-β-D-digitoxose [qinyangshengenin3-O-β-D-cymaropyranosyl-(1 → 4)-β-D-digitoxopyranoside, formula XVII], Caudatin 3-O-β-D-cymarose pyranyl-(1 → 4)-β-D-cymarose [caudatin3-O-β-D-cymaropyranosyl-(1 → 4)-β-D-cymaropyranoside, formula X], Russell he order 3-O-β-D-oleandrose pyrans glycosyl-(1 → 4)-β-D-cymarose pyranyl-(1 → 4)-β-D-cymarose [rostratamine3-O-β-D-oleandropyranosyl-(1 → 4)-β-D-cymaropyranosyl-(1 → 4)-β-D-cymaropyranoside, formula II], Russell he order 3-O-α-L-cymarose pyrans glycosyl-(1 → 4)-β-D-cymarose pyranyl-(1 → 4)-α-L-diginose pyrans glycosyl-(1 → 4)-β-D-cymarose [rostratamine3-O-α-L-cymaropyranosyl-(1 → 4)-β-D-cymaropyranosyl-(1 → 4)-α-L-diginopyranosyl-β-D-cymaropyranoside, formula XIII], Qingyanshengenin 3-O-β-D-oleandrose pyrans glycosyl-(1 → 4)-β-D-cymarose pyranyl-(1 → 4)-β-D-digitoxose [qinyangshengenin3-O-β-D-oleandropyranosyl-(1 → 4)-β-D-cymaropyranosyl-(1 → 4)-β-D-digitoxopyranoside, formula a], Caudatin 3-O-β-D-Glucose pyrans glycosyl-(1 → 4)-β-D-oleandrose pyranyl-(1 → 4)-β-D-cymarose pyranyl-(1 → 4)-β-D-cymarose [caudatin3-O-β-D-glucopyranoside-(1 → 4)-β-D-oleandropyranosyl-(1 → 4)-β-D-cymaropyranosyl-(1 → 4)-β-D-cymaropyranoside, formula c], Caudatin 3-O-β-D-thevetose pyrans glycosyl-(1 → 4)-β-D-cymarose pyranyl-(1 → 4)-β-D-cymarose [caudatin3-O-β-D-thevetopyranosyl-(1 → 4)-β-D-cymaropyranosyl-(1 → 4)-β-D-cymaropyranoside, formula XII], Qingyanshengenin 3-O-β-D-oleandrose pyrans glycosyl-(1 → 4)-β-D-cymarose [qinyangshengenin3-O-β-D-oleandropyranosyl-(1 → 4)-β-D-cymaropyranoside, formula XI], taiwanosideC (formula III), wilfosideK1N (formula V), cynawilfosideA (formula VI), wilfosideM1N (formula d), cynauricosideE (formula XV), cynauricuosideA (formula XVI), the compounds such as wilfosideG (formula XIV) affect the zebra fish of PTZ induction under maximal non-toxic dosage.

Embodiment

The present inventor, by extensive and deep research, is surprised to find that, the different monomers compound in Cynanchum otophvllum has diametrically opposite short epilepsy and antiepileptic effect.Particularly, the present inventor extracts one-component from Apocynaceae Cynanchum (Cynanchum) plant cynanchum auriculatum Royle ex Wight (C.auriculatumRoyleexWight) and Root of Wilford Swallowwort (C.wilfordiiHemsl.), and confirms its antiepileptic activity by experiment.

Term

As used herein, term " replacement " refers to that the one or more hydrogen atoms on group are selected from the substituting group replacement of lower group: C1 ~ C10 alkyl, C3 ~ C10 cycloalkyl, C1 ~ C10 alkoxyl group, hydroxyl, carboxyl (-COOH), C1 ~ C10 carbonyl, C1 ~ C10 amide group, C2 ~ C20 ester group, C6 ~ C30 aryl, fluorine-based and thioether group.

Term " C1 ~ C10 alkyl " refers to the straight or branched alkyl with 1 ~ 10 carbon atom, such as methyl, ethyl, propyl group, sec.-propyl, butyl, isobutyl-, sec-butyl, the tertiary butyl or similar group.

Term " C3 ~ C10 cycloalkyl " refers to the cycloalkyl with 3 ~ 10 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, suberyl or similar group.

Term " C1 ~ C10 alkoxyl group " refers to the straight or branched alkoxyl group with 1-10 carbon atom, such as methoxyl group, oxyethyl group, propoxy-, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert.-butoxy or similar group.

Term " carbonyl " refers to have group; Such as " C1 ~ 10 carbonyl " refers to the carbonyl with 1 ~ 10 carbon atom.

Term " amide group " refers to have group; Such as " C1 ~ 10 amide group " refers to the amide group with 1 ~ 10 carbon atom.

Term " C2 ~ C20 ester group " refers to have alkyl-COO-structure or have-COO-alkyl structure, and wherein alkyl can be straight or branched.Preferably there is the group of C1 ~ C10 alkyl-COO-structure, such as CH ₃cOO-, C ₂h ₅cOO-, C ₃h ₈cOO-, (CH ₃) ₂cHCOO-, n-C ₄h ₉cOO-, t-C ₄h ₉cOO-, or similar group.Or there is-COO-C1 ~ C19 alkyl structure, such as-COOCH ₃,-COOC ₂h ₅,-COOC ₃h ₈or-COO-(CH ₂) n-CH ₃, n is the integer of 0 ~ 18.

Term " aryl ", preferably there are 6 ~ 30 carbon atoms, refer to the monocycle or Bicycloaromaticity group in ring part with 6 ~ 12 carbon atoms, such as: phenyl, xenyl, naphthyl or similar group, each carbon atom wherein all can be optionally substituted.Preferably have 1 ~ 3 substituting group, described substituting group is selected from: halogen, C1 ~ C10 alkyl, cyano group, OH, nitro, C3 ~ C10 cycloalkyl, C1 ~ C10 alkoxyl group, amino.

Term " thioether group ", refers to have-the group of alkyl-S-alkyl radical structure.

Term " halogenated compound ", refers to that at least one H atom in organic compound is optionally substituted by halogen the compound of rear formation.

Term " halogen " refers to fluorine, chlorine, bromine, iodine.Term " halo " refer to fluoro, chloro, bromo, iodo.

Each group of the present invention can unsubstituted or replace, described replacement refer to that the substituting group being selected from lower group replaced: C1 ~ C6 alkyl of hydroxyl, halogen, C1 ~ C6 alkyl, halo, C1 ~ C6 alkoxyl group, C1 ~ C6 alkoxyl group of halo, cyano group, tertiary fourth carbamyl ,-O-(CH ₂) _n-O-; Wherein, n is the integer between 1 ~ 3.Described substituting group can be substituted on each position of each group.

In embodiments of the present invention, relate to Cynanchum otophvllum glycosides own (otophyllosideF), rostratamine3-O-triglycoside (rostratamine3-O-β-D-oleandropyranosyl-(1 → 4)-β-D-cymaropyra-nosyl-(1 → 4)-β-D-cymaropyranoside), taiwanosideC, wilfosideC1N, wilfosideC3N, wilfosideK1N, cynauricosideA, the monomeric compounds such as cynawilfosideA are in preparation treatment or prevent the application in epilepsy medicament and Apocynaceae Cynanchum (Cynanchum) plant cynanchum auriculatum Royle ex Wight (C.auriculatumRoyleexWight), the application of Root of Wilford Swallowwort (C.wilfordiiHemsl.) extract in preparation or prevention epilepsy medicament.And the antiepileptic efficacy of these monomeric compounds is all better than the middle principal constituent Q-A (otophyllosideA) of Cynanchum otophvllum total glycosides sheet, cynanchogenin (qingyangshengenin) and Caudatin (caudatin).

Relevant Cynanchum otophvllum glycosides own (otophyllosideF), Russell he order 3-O-β-D-oleandrose pyrans glycosyl-(1 → 4)-β-D-cymarose pyranyl-(1 → 4)-β-D-cymarose [rostratamine3-O-β-D-oleandropyranosyl-(1 → 4)-β-D-cymaropyranosyl-(1 → 4)-β-D-cymaropyranoside], taiwanosideC, wilfosideC1N, wilfosideC3N, wilfosideK1N, cynauricosideA, cynauricosideA and cynanchum auriculatum Royle ex Wight, the application of antiepileptic action in related products of Root of Wilford Swallowwort extract has not yet to see bibliographical information, and cynawilfosideA is the compound of isolation identification from natural product first.

In the present invention, monomeric compound antiepileptic action in Cynanchum otophvllum is conducted in-depth research, be surprised to find that the different monomers compound in Cynanchum otophvllum has diametrically opposite short epilepsy and antiepileptic effect.In Cynanchum otophvllum, the Cynanchum otophvllum glycosides of one of principal constituent is own, (otophyllosideF) and Russell he order 3-O-β-D-oleandrose pyrans glycosyl-, (1 → 4)-β-D-cymarose pyranyl-, (1 → 4)-β-D-cymarose [rostratamine3-O-β-D-oleandropyranosyl-, (1 → 4)-β-D-cymaropyranosyl-, (1 → 4)-β-D-cymaropyranoside] there is the significant effect resisting the clonism that Yetrazol causes in zebra fish model, and have as Qingyanshengenin 3-O-β-D-oleandrose pyrans glycosyl-(1 → 4)-β-D-cymarose pyranyl-(1 → 4)-β-D-digitoxose [qinyangshengenin3-O-β-D-oleandropyranosyl-(1 → 4)-β-D-cymaropyranosyl-(1 → 4)-β-D-digitoxopyranoside] of one of the principal constituent of Cynanchum otophvllum and Cynanchum otophvllum glycosides M (otophyllosideM) and promote convulsion effect significantly.

The invention provides the compound having and significantly suppress epilepsy effect, anti-epileptic or the anticonvulsant effect of these compounds all do not have relevant report, cynawilfosideA is separated and the new natural product identified first, because these compounds content in crude drug is larger, extract, preparation is relatively easy, and quality controllable, therefore in exploitation antiepileptic drug, there is larger development prospect.

The present invention passes through animal vivo test, the extract that discovery comes from Apocynaceae Cynanchum (Cynanchum) plant cynanchum auriculatum Royle ex Wight (C.auriculatum) and Root of Wilford Swallowwort (C.wilfordii) has certain antiepileptic activity, and monomeric compound Cynanchum otophvllum glycosides own (otophyllosideF), Russell he order 3-O-β-D-oleandrose pyrans glycosyl-(1 → 4)-β-D-cymarose pyranyl-(1 → 4)-β-D-cymarose [rostratamine3-O-β-D-oleandropyranosyl-(1 → 4)-β-D-cymaropyranosyl-(1 → 4)-β-D-cymaropyranoside], taiwanosideC, wilfosideC1N, wilfosideC3N, wilfosideK1N, cynauricosideA, cynawilfosideA etc. have significant antiepileptic activity, its structure represents by as shown in the formula I ~ formula XVI respectively:

The structure of compound Cynanchum otophvllum glycosides oneself (otophyllosideF, Compound I) is such as formula shown in I:

Compound Russell he order the structure of 3-O-β-D-oleandrose pyrans glycosyl-(1 → 4)-β-D-cymarose pyranyl-(1 → 4)-β-D-cymarose [rostratamine3-O-β-D-oleandropyranosyl-(1 → 4)-β-D-cymaropyranosyl-(1 → 4)-β-D-cymaropyranoside, Compound II per] such as formula shown in II:

The structure of compound taiwanosideC (compound III) is such as formula shown in III:

The structure of compound wilfosideC1N (compound IV) is such as formula shown in IV:

The structure of compound wilfosideK1N (compound V) is such as formula shown in V:

The structure of compound cynawilfosideA (compound VI) is such as formula shown in VI:

The structure of compound wilfosideC3N (compound VI I) is such as formula shown in VII:

The structure of compound cynauricosideA (compound VI II) is such as formula shown in VIII:

Compound Ⅸ:

Compound Ⅹ:

Compound Ⅺ:

Compound Ⅻ:

Compounds X III:

Compounds X V:

Compounds X VI:

And the partial monosomy compound coming from Apocynaceae Cynanchum (Cynanchum) has significant short epilepsy activity.

The structural formula of Qingyanshengenin 3-O-β-D-oleandrose pyrans glycosyl-(1 → 4)-β-D-cymarose pyranyl-(1 → 4)-β-D-digitoxose (compound a) is such as formula shown in a:

The structural formula of Cynanchum otophvllum glycosides M (compound b) is such as formula shown in b:

The structural formula of Caudatin 3-O-β-D-Glucose pyrans glycosyl-(1 → 4)-β-D-oleandrose pyranyl-(1 → 4)-β-D-cymarose pyranyl-(1 → 4)-β-D-cymarose (compound c) is such as formula shown in c:

The structural formula of wilfosideM1N (compound d) is such as formula shown in d:

Pharmaceutical composition and application process

The compounds of this invention has excellent antiepileptic efficacy.The compounds of this invention can be applied to Mammals (as people), can oral, rectum, parenteral (intravenously, intramuscular or subcutaneous), the mode administration such as local.Described compound can be individually dosed, or with other pharmaceutically acceptable compound Combined Preparation.It may be noted that compound of the present invention can mix administration.

Solid dosage for oral administration comprises capsule, tablet, pill, powder and granule.In these solid dosages, active compound mixes with at least one conventional inert excipients (or carrier), as Trisodium Citrate or Si Liaodengji dicalcium phosphate feed grade, or mix with following compositions: (a) filler or expanding material, such as, starch, lactose, sucrose, glucose, N.F,USP MANNITOL and silicic acid; (b) tackiness agent, such as, Walocel MT 20.000PV, alginate, gelatin, Polyvinylpyrolidone (PVP), sucrose and gum arabic; (c) wetting Agent for Printing Inks, such as, glycerine; (d) disintegrating agent, such as, agar, calcium carbonate, yam starch or tapioca (flour), alginic acid, some composition silicate and sodium carbonate; (e) retarding solvent, such as paraffin; F () absorbs accelerator, such as, and quaternary ammonium compound; (g) wetting agent, such as hexadecanol and glyceryl monostearate; (h) sorbent material, such as, kaolin; (i) lubricant, such as, talcum, calcium stearate, Magnesium Stearate, solid polyethylene glycol, sodium lauryl sulphate, or its mixture.In capsule, tablet and pill, formulation also can comprise buffer reagent.

Solid dosage such as tablet, sugar-pill, capsule, pill and granule can adopt dressing and the preparation of shell material, as casing and other material well known in the art.They can comprise opacifying agent, and in this composition, the release of active compound or compound can discharge in certain part in a delayed fashion in digestive tube.The example of adoptable embedding component is polymeric material and Wax.If desired, active compound also can form microencapsulation form with one or more in above-mentioned vehicle.

Liquid dosage form for oral administration comprises pharmaceutically acceptable emulsion, solution, suspension, syrup or tincture.Except active ingredient beyond the region of objective existence, liquid dosage form can comprise the conventional inert diluent adopted in this area, as water or other solvent, solubilizing agent and emulsifying agent, example is known, the mixture etc. of ethanol, Virahol, ethyl-carbonate, ethyl acetate, propylene glycol, 1,3 butylene glycol, dimethyl formamide and oil, particularly Oleum Gossypii semen, peanut oil, maize germ, sweet oil, Viscotrol C and sesame oil or these materials.

Except these inert diluents, composition also can comprise auxiliary agent, as wetting agent, emulsifying agent and suspension agent, sweeting agent and spices.

Except active ingredient beyond the region of objective existence, suspension can comprise suspension agent, such as, and the mixture etc. of ethoxylation isooctadecane alcohol, polyoxyethylene sorbitol and Isosorbide Dinitrate, Microcrystalline Cellulose, aluminum methylate and agar or these materials.

Composition for parenteral injection can comprise physiologically acceptable sterile, aqueous or anhydrous solution, dispersion liquid, suspension or emulsion, and for being again dissolved into aseptic Injectable solution or the sterilized powder of dispersion liquid.Suitable moisture and nonaqueous carrier, thinner, solvent or vehicle comprise water, ethanol, polyvalent alcohol and suitable mixture thereof.

Formulation for the compounds of this invention of topical comprises ointment, powder, patch, propellant and inhalation.Activeconstituents aseptically with physiologically acceptable carrier and any sanitas, buffer reagent, or the propelling agent that may need if desired is mixed together.

The compounds of this invention can be individually dosed, or with other activeconstituentss (as other antiepileptic drug clinically) Combined Preparation.

When making pharmaceutical composition, it is the Mammals (as people) being applicable to the compounds of this invention of safe and effective amount need treatment, when wherein using, dosage is the effective dosage pharmaceutically thought, for the individuality of 60kg body weight, day dosage is generally 1 ~ 1000mg, preferably 20 ~ 500mg.Certainly, concrete dosage also should consider the factor such as route of administration, individual health situation, and these are all within skilled practitioners skill.

Major advantage of the present invention is:

(1) first the antiepileptic activity of the single-component extract of Apocynaceae Cynanchum (Cynanchum) plant cynanchum auriculatum Royle ex Wight (C.auriculatum) and Root of Wilford Swallowwort (C.wilfordii) is evaluated, found that, extracting section thing has anti-epileptic function, and extracting section thing has short epilepsy effect;

(2) antiepileptic activity with the monomeric compound of antiepileptic activity involved by is all better than the middle principal constituent Q-A of clinical application Cynanchum otophvllum sheet, Qingyanshengenin and Caudatin;

(3) content in plant of the compound with antiepileptic activity involved by is large, and source is wide, has great researching value and DEVELOPMENT PROSPECT;

(4) compound cynawilfosideA is separated and the new compound identified first, and its antiepileptic activity is better than positive drug phenytoin Sodium;

(5) step obtaining the efficient part being rich in wilfosideC1N, wilfosideC3N, wilfosideK1N, cynauricosideA isoreactivity compound from Chinese medicine Auriculoside A is simple, is convenient to production application.

Below in conjunction with specific embodiment, state the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.Unless otherwise indicated, otherwise per-cent and number calculate by weight.

Embodiment 1

The evaluation of antiepileptic action in compound cynawilfosideA (formula VI) body:

The preparation method of compound cynawilfosideA: fresh Root of Wilford Swallowwort block root 10kg, peeling chip drying obtains 2.7kg dry product, pulverize and extract three times with 95% ethanol percolation, each 2 ~ 3 days, united extraction liquid, concentrating under reduced pressure obtains the alcohol medicinal extract of Root of Wilford Swallowwort, by extract with water suspendible, extraction into ethyl acetate, obtain acetic acid ethyl ester extract, with 100 order silica gel mixed samples, be separated with silica gel column chromatography (200-300 order silica gel), with chloroform-methanol (10:1 → 5:1) gradient elution, obtain component Fr1-Fr9.Fr6 further with sherwood oil-acetone and preparative HPLC repeatedly purifying obtain pure compound cynawilfosideA (1.4g).

The physicochemical data of compound cynawilfosideA is as follows: white amorphous powder, is soluble in methyl alcohol, ethanol, acetone, the organic solvents such as chloroform, and molecular formula is C ₆₃h ₉₆o ₂₀, ¹hNMR (CDCl ₃, 500MHz): δ _h1.49 (s, 3H, H-18), 1.20,1.24,1.26,1.26 (d, each3H, J=6.3Hz, H-6ofsugarmoiety), 1.15 (s, 3H, 19-CH ₃), 1.16 (d, 3H, J=6.5Hz, H-21), 3.39,3.40,3.41,3.45 (s, each3H, 3-OCH ₃ofsugarmoieties), 4.76 (d, 1H, J=9.8, anomericH), 4.77 (brs, 1H, anomericH), 4.84 (1H, d, J=9.6Hz, anomericH), 4.98 (brs, anomericH), 5.37 (brs, 1H, H-6), 6.31 (d, 1H, J=16.0Hz, H _cin-2), 7.37 (m, 3H, H _cin-6,7,8), 7.51 (m, 2H, H _cin-5,9), 7.63 (d, 1H, J=16.0Hz, H _cin-3), 2.33 (m, 1H, H _{2 '-methyl-butyryl}-2), 1.44 (m, 1H, H _{2 '-methyl-butyryl}-3), 0.95 (t, 1H, J=6.5Hz, H _{2 '-methyl-butyryl}-4), 1.17 (d, 1H, J=6.5Hz, H _{2 '-methyl-butyryl}-5), ¹³cNMR (CDCl ₃, 125MHz): δ _c38.9 (t, C-1), 29.1 (t, C-2), 78.0 (d, C-3), 38.8 (t, C-4), 139.3 (s, C-5), 118.6 (d, C-6), 34.6 (t, C-7), 74.2 (s, C-8), 43.5 (d, C-9), 37.1 (s, C-10), 25.2 (t, C-11), 73.9 (d, C-12), 56.3 (s, C-13), 87.8 (s, C-14), 34.4 (t, C-15), 34.6 (t, C-16), 88.1 (s, C-17), 10.4 (q, C-18), 18.4 (q, C-19), 73.6 (d, C-20), 15.1 (q, C-21), 166.9 (s, C _cin-1), 119.1 (d, C _cin-2), 144.7 (d, C _cin-3), 134.7 (s, C _cin-4), 128.2 (d, C _cin-5,9), 130.3 (d, C _cin-6,8), 128.9 (d, C _cin-7), 174.5 (s, C _{2 '-methyl-butyryl}-1), 41.5 (d, C _{2 '-methyl-butyryl}-2), 26.4 (t, C _{2 '-methyl-butyryl}-3), 11.9 (q, C _{2 '-methyl-butyryl}-4), 16.7 (q, C _{2 '-methyl-butyryl}-5), 95.8 (d, C _cym1-1), 34.2 (t, C _cym1-2), 77.2 (d, C _cym1-3), 81.9 (d, C _cym1-4), 68.7 (d, C _cym1-5), 18.3 (q, C _cym1-6), 57.6 (q, C _cym1-OMe), 101.0 (d, C _digin-1), 31.7 (t, C _digin-2), 73.9 (d, C _digin-3), 74.5 (d, C _digin-4), 66.9 (d, C _digin-5), 18.2 (q, C _digin-6), 55.7 (q, C _digin-OMe), 99.5 (d, C _cym2-1), 34.4 (t, C _cym2-2), 77.3 (d, C _cym2-3), 81.9 (d, C _cym2-4), 69.2 (d, C _cym2-5), 18.4 (q, C _cym2-6), 57.1 (q, C _cym2-OMe), 98.8 (d, C _cym3-1), 31.2 (t, C _cym3-2), 74.9 (d, C _cym3-3), 72.1 (d, C _cym3-4), 65.7 (d, C _cym3-5), 17.6 (q, C _cym3-6), 56.5 (q, C _cym3-OMe).

Compound cynawilfosideA induces the provide protection of zebra fish epileptic seizures model to Yetrazol (Pentylenetetrazole):

The breeding of zebrafish embryo is carried out in the mode of natural paired cross, each mating prepares the Adult Zebrafish that 4 ~ 5 pairs of average energies produce 200 ~ 300 embryos, within 6 hours and 24 hours, dead embryo is removed at after fertilization, and select suitable embryo according to the etap of embryo, hatch 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 with fish culture water 28 DEG C time; PH is 6.9 ~ 7.2; Hardness is 53.7 ~ 71.6mg/LCaCO ₃).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.

Epilepsy model group: the zebra fish that Yetrazol (Pentylenetetrazole) processes; Positive controls: phenytoin Sodium (PhenytoinSodium) 300 μMs; Blank group: blank group (1%DMSO), all the other process same model group.After testing compound is joined microwell plate, utilize the motion conditions of zebra fish in motion/behavioural analysis instrument record 60min, quantitative analysis is carried out to the rapid movement (V>20mm/sec) distance (D) of zebra fish, calculate the anti-epileptic therapeutic efficiency of medicine, according to zebra fish movement locus figure, qualitative evaluation is carried out to the anti-epileptic result for the treatment of of medicine.From each concentration group of Figure 1A, cynawilfosideA compared with epilepsy model group, zebra fish rapid movement track obviously reduces.Figure 1B is the column diagram drawn according to zebra fish rapid movement distance average mean ± SE, from Figure 1B, compound cynawilfosideA all has antiepileptic action under 30,100,300 μMs of concentration, therapeutic efficiency is respectively 34%, 44.6%, 61.6% (P<0.05, P<0.01, P<0.01), and dose-effect relationship is clear and definite.Meanwhile, the antiepileptic action of positive control drug phenytoin Sodium under 300 μMs of concentration is only 57.2% (P<0.01).

Embodiment 2

The evaluation of antiepileptic action in compound wilfosideC1N (formula IV) body:

The auricle cynanchum auriculatum Royle ex Wight block root 10kg that the preparation method of WilfosideC1N: a. is fresh, chip drying obtains 3Kg dry product, pulverize and carry three times by 95% ethanol heat, extracting solution merges, and concentrating under reduced pressure obtains ethanol extraction, silica gel mixed sample, cross 200-300 order silica gel, with sherwood oil-acetone (3:1 → 2:1) gradient elution, then use chloroform-methanol (10:1 → 5:1) gradient elution, obtain component Fr1-Fr6.Fr3 uses sherwood oil-acetone silica gel column chromatography purifying repeatedly further, obtains pure compound wilfosideC1N (1.5g); B. fresh Root of Wilford Swallowwort block root 10kg, peeling chip drying obtains 2.7kg dry product, pulverizes and extracts three times with 95% ethanol percolation, each 2 ~ 3 days, united extraction liquid, concentrating under reduced pressure obtains the alcohol medicinal extract of Root of Wilford Swallowwort, by extract with water suspendible, extraction into ethyl acetate, obtain acetic acid ethyl ester extract, with 100 order silica gel mixed samples, be separated with silica gel column chromatography (200-300 order silica gel), with chloroform-methanol (10:1 → 5:1) gradient elution, obtain component Fr1-Fr9.Fr6 further with sherwood oil-acetone and preparative HPLC repeatedly purifying obtain pure compound wilfosideC1N (2.5g).

The physicochemical data of compound wilfosideC1N is as follows: white amorphous powder, is soluble in methyl alcohol, ethanol, acetone, the organic solvents such as chloroform, and molecular formula is C ₅₆h ₉₀o ₁₉, ESIMSm/z1089 [M+Na] ⁺, ¹hNMR (pyridine-d ₅, 300MHz): δ _h0.95,0.97 (d, each3H, J=6.5Hz, H _ikem-5,6), 1.32 (s, 3H, H-19), 1.36,1.37,1.40,1.48 (d, each3H, J=6.0Hz, H-6ofsugarmoieties), 1.96 (s, 3H, H-18), 2.29 (s, 3H, H _ikem-7), 2.49 (s, 3H, H-21), 3.45,3.48,3.52,3.53 (s, each3H, 3-OCH ₃ofsugarmoieties), 5.02 (brd, 1H, J=3.0Hz, anomericH), 5.09 (d, 1H, J=9.3Hz, anomericH), 5.15 (brd, 1H, J=3.0Hz, anomericH), 5.22 (d, 1H, J=9.0Hz, anomericH), 5.30 (brs, 1H, H-6), 5.84 (brs, 1H, H _ikem-2), ¹³cNMR (Pyridine-d ₅, 100MHz): δ _c39.2 (t, C-1), 29.9 (t, C-2), 77.6 (d, C-3), 38.9 (t, C-4), 139.3 (s, C-5), 119.2 (d, C-6), 33.8 (t, C-7), 74.2 (s, C-8), 44.5 (d, C-9), 37.4 (s, C-10), 25.0 (t, C-11), 72.5 (d, C-12), 57.9 (s, C-13), 89.4 (s, C-14), 34.8 (t, C-15), 32.9 (t, C-16), 92.4 (s, C-17), 10.7 (q, C-18), 18.4 (q, C-19), 209.4 (s, C-20), 27.5 (q, C-21), 166.0 (s, C _ikem-1), 114.1 (d, C _ikem-2), 165.5 (s, C _ikem-3), 38.1 (s, C _ikem-4), 20.8 (q, C _ikem-5), 20.9 (q, C _ikem-6), 16.4 (q, C _ikem-7), 96.1 (d, C _cym1-1), 35.3 (t, C _cym1-2), 77.6 (d, C _cym1-3), 82.4 (d, C _cym1-4), 69.2 (d, C _cym1-5), 18.7 (q, C _cym1-6), 57.3 (q, C _cym1-OMe), 100.9 (d, C _digin-1), 32.5 (t, C _digin-2), 73.8 (d, C _digin-3), 74.6 (d, C _digin-4), 67.4 (d, C _digin-5), 17.9 (q, C _digin-6), 55.3 (q, C _digin-OMe), 99.4 (d, C _cym2-1), 36.3 (t, C _cym2-2), 77.8 (d, C _cym2-3), 82.3 (d, C _cym2-4), 69.4 (d, C _cym2-5), 18.6 (q, C _cym2-6), 58.2 (q, C _cym2-OMe), 99.0 (d, C _cym3-1), 32.1 (t, C _cym3-2), 76.4 (d, C _cym3-3), 73.3 (d, C _cym3-4), 66.4 (d, C _cym3-5), 18.1 (q, C _cym3-6), 56.6 (q, C _cym3-OMe).

Compound wilfosideC1N induces the provide protection of zebra fish epileptic seizures model to Yetrazol (Pentylenetetrazole):

Qualitative evaluation is carried out according to the anti-epileptic result for the treatment of of the method in embodiment 1 to compound.From Fig. 2 A, when the dosage 300 μMs of wilfosideC1N compared with epilepsy model group, zebra fish rapid movement track obviously reduces.Fig. 2 B is the column diagram drawn according to zebra fish rapid movement distance average mean ± SE, and from Fig. 2 B, compound wilfosideC1N, under 30,100 μMs of concentration, has epilepsy therapy trend, but no difference of science of statistics; Under 300 μMs of concentration, epilepsy therapy efficiency is 46.4%, and (P<0.01), dose-effect relationship is clear and definite.

Embodiment 3

The evaluation of antiepileptic action in compound wilfosideK1N (formula V) body:

The cynanchum auriculatum Royle ex Wight block root 10kg that the preparation method of WilfosideK1N: a. is fresh, chip drying obtains 3kg dry product, pulverize and carry three times by 95% ethanol heat, extracting solution merges, and concentrating under reduced pressure obtains ethanol extraction, silica gel mixed sample, cross 200-300 order silica gel, with sherwood oil-acetone (3:1 → 2:1) gradient elution, then use chloroform-methanol (10:1 → 5:1) gradient elution, obtain component Fr1-Fr6.Fr3 uses sherwood oil-acetone silica gel column chromatography purifying repeatedly further, obtains pure compound wilfosideK1N (2.5g); B. fresh Root of Wilford Swallowwort block root 10kg, peeling chip drying obtains 2.7kg dry product, pulverizes and extracts three times with 95% ethanol percolation, each 2 ~ 3 days, united extraction liquid, concentrating under reduced pressure obtains the alcohol medicinal extract of Root of Wilford Swallowwort, by extract with water suspendible, extraction into ethyl acetate, obtain acetic acid ethyl ester extract, with 100 order silica gel mixed samples, be separated with silica gel column chromatography (200-300 order silica gel), with chloroform-methanol (10:1 → 5:1) gradient elution, obtain component Fr1-Fr9.Fr7 further with sherwood oil-acetone and preparative HPLC repeatedly purifying obtain pure compound wilfosideK1N (1.7g).

The physicochemical data of compound wilfosideK1N is as follows: white amorphous powder, is soluble in methyl alcohol, ethanol, acetone, the organic solvents such as chloroform, and molecular formula is C ₅₈h ₈₆o ₁₉, ESIMSm/z1109 [M+Na] ⁺, ¹hNMR (pyridine-d ₅, 300MHz): 1.36 (s, 3H, H-19), 1.37,1.38,1.42,1.49 (d, each3H, J=6.0Hz, H-6ofsugarmoieties), 2.02 (s, 3H, H-18), 2.54 (s, 3H, H-21), 3.42,3.44,3.53,3.54 (s, each3H, 3-OCH ₃ofsugarmoieties), 4.98 (brd, 1H, J=3.0Hz, anomericH), 5.09 (d, 1H, J=8.0Hz, anomericH), 5.15 (brd, 1H, J=3.0Hz, anomericH), 5.23 (d, 1H, J=9.0Hz, anomericH), 5.32 (brs, 1H, H-6), 6.81 (d, 1H, J=16.0Hz, H _cin-2) 7.38 (m, 3H, H _cin-6,7,8), 7.65 (m, 2H, H _cin-5,9), 7.99 (d, 1H, J=16.0Hz, H _cin-3), ¹³cNMR (Pyridine-d ₅, 100MHz): δ _c39.2 (t, C-1), 29.9 (t, C-2), 77.6 (d, C-3), 38.9 (t, C-4), 139.3 (s, C-5), 119.2 (d, C-6), 33.8 (t, C-7), 74.5 (s, C-8), 44.5 (d, C-9), 37.4 (s, C-10), 25.0 (t, C-11), 73.6 (d, C-12), 58.1 (s, C-13), 89.5 (s, C-14), 34.7 (t, C-15), 33.0 (t, C-16), 92.4 (s, C-17), 10.7 (q, C-18), 18.6 (q, C-19), 209.9 (s, C-20), 27.5 (q, C-21), 165.8 (s, C _cin-1), 119.2 (d, C _cin-2), 145.0 (d, C _cin-3), 135.0 (s, C _cin-4), 128.6 (d, C _cin-5,9), 129.3 (d, C _cin-6,8), 130.6 (d, C _cin-7), 96.1 (d, C _cym1-1), 35.3 (t, C _cym1-2), 77.5 (d, C _cym1-3), 82.5 (d, C _cym1-4), 69.2 (d, C _cym1-5), 18.7 (q, C _cym1-6), 57.3 (q, C _cym1-OMe), 100.9 (d, C _digin-1), 32.5 (t, C _digin-2), 73.8 (d, C _digin-3), 74.6 (d, C _digin-4), 67.4 (d, C _digin-5), 17.9 (q, C _digin-6), 55.3 (q, C _digin-OMe), 99.4 (d, C _cym2-1), 36.3 (t, C _cym2-2), 77.8 (d, C _cym2-3), 82.3 (d, C _cym2-4), 69.4 (d, C _cym2-5), 18.7 (q, C _cym2-6), 58.3 (q, C _cym2-OMe), 99.0 (d, C _cym3-1), 32.1 (t, C _cym3-2), 76.4 (d, C _cym3-3), 73.2 (d, C _cym3-4), 66.4 (d, C _cym3-5), 18.2 (q, C _cym3-6), 56.6 (q, C _cym3-OMe).

Compound wilfosideK1N induces the provide protection of zebra fish epileptic seizures model to Yetrazol (Pentylenetetrazole):

Qualitative evaluation is carried out according to the anti-epileptic result for the treatment of of the method in embodiment 1 to compound.From Fig. 3 A, when the dosage 100 μMs of wilfosideK1N compared with epilepsy model group, zebra fish rapid movement track obviously reduces.Fig. 3 B is the column diagram drawn according to zebra fish rapid movement distance average mean ± SE, and from Fig. 3 B, compound wilfosideK1N, under 10,30 μMs of concentration, has epilepsy therapy trend, but no difference of science of statistics; Under 100 μMs of concentration, have antiepileptic action, epilepsy therapy efficiency is 49% (P<0.01), and dose-effect relationship is clear and definite.

Embodiment 4

The evaluation of antiepileptic action in compound taiwanosideC (formula III) body:

The cynanchum auriculatum Royle ex Wight block root 10kg that the preparation method of TaiwanosideC: a. is fresh, chip drying obtains 3kg dry product, pulverize and carry three times by 95% ethanol heat, extracting solution merges, and concentrating under reduced pressure obtains ethanol extraction, silica gel mixed sample, cross 200-300 order silica gel, with sherwood oil-acetone (3:1 → 2:1) gradient elution, then use chloroform-methanol (10:1 → 5:1) gradient elution, obtain component Fr1-Fr6.Fr4 uses chloroform-methanol silica gel column chromatography purifying repeatedly further, obtains pure compound taiwanosideC (80.4mg); B. fresh Root of Wilford Swallowwort block root 10kg, peeling chip drying obtains 2.7kg dry product, pulverizes and extracts three times with 95% ethanol percolation, each 2 ~ 3 days, united extraction liquid, concentrating under reduced pressure obtains the alcohol medicinal extract of Root of Wilford Swallowwort, by extract with water suspendible, extraction into ethyl acetate, obtain acetic acid ethyl ester extract, with 100 order silica gel mixed samples, be separated with silica gel column chromatography (200-300 order silica gel), with chloroform-methanol (10:1 → 5:1) gradient elution, obtain component Fr1-Fr9.Fr6 further with sherwood oil-acetone and preparative HPLC repeatedly purifying obtain pure compound taiwanosideC (640mg).

The physicochemical data of compound taiwanosideC is as follows: white amorphous powder, is soluble in methyl alcohol, ethanol, acetone, the organic solvents such as chloroform, and molecular formula is C ₄₉h ₈₀o ₁₈, ESIMSm/z979 [M+Na] ⁺; ¹hNMR (CDCl ₃, 300MHz): δ _h1.14 (s, 3H, H-18), 1.20,1.21,1.22,1.24 (d, each3H, J=6.3Hz, H-6ofsugarmoieties), 1.27 (s, 3H, H-19), 2.34 (s, 3H, H-21), 3.25,3.30 (dd, each1H, J=9.6,2.7Hz, H _cym-4), 3.37,3.40,3.41,3.45 (s, each3H, 3-OCH ₃ofsugarmoieties), 3.70,3.77 (q, each1H, J=3.0Hz, H _cym-3), 3.83,3.86,4.04 (dq, each1H, J=8.9,6.4Hz, H _cym-5), 3.85 (brs, 1H, H _digin-4), 3.96 (brq, 1H, J=6.3Hz, H _digin-5), 4.75 (d, 1H, J=9.8, anomericH), 4.77 (brs, 1H, anomericH), 4.81 (1H, d, J=9.6Hz, anomericH), 4.97 (brd, J=3.0Hz, anomericH), 5.36 (brs, 1H, H-6). ¹³CNMR(Pyridine-d ₅,125MHz):δ _C39.4(t,C-1),30.3(t,C-2),78.3(d,C-3),39.4(t,C-4),139.7(s,C-5),119.7(d,C-6),34.2(t,C-7),74.7(s,C-8),44.9(d,C-9),37.8(s,C-10),25.3(t,C-11),74.0(d,C-12),58.7(s,C-13),89.9(s,C-14),35.2(t,C-15),33.3(t,C-16),92.8(s,C-17),10.9(q,C-18),18.6(q,C-19),210.7(s,C-20),28.0(q,C-21),170.4(s,C _Ac-1),21.2(q,C _Ac-2),96.6(d,C _cym1-1),35.7(t,C _cym1-2),78.0(d,C _cym1-3),82.8(d,C _cym1-4),69.7(d,C _cym1-5),19.2(q,C _cym1-6),57.7(q,C _cym1-OMe),101.4(d,C _digin-1),32.9(t,C _digin-2),74.3(d,C _digin-3),75.0(d,C _digin-4),67.9(d,C _digin-5),18.3(q,C _digin-6),55.8(q,C _digin-OMe),99.8(d,C _cym2-1),36.8(t,C _cym2-2),78.1(d,C _cym2-3),82.8(d,C _cym2-4),69.9(d,C _cym2-5),18.9(q,C _cym2-6),58.4(q,C _cym2-OMe),99.5(d,C _cym3-1),32.6(t,C _cym3-2),76.9(d,C _cym3-3),73.7(d,C _cym3-4),66.9(d,C _cym3-5),19.1(q,C _cym3-6),57.0(q,C _cym3-OMe)。

Compound taiwanosideC induces the provide protection of zebra fish epileptic seizures model to Yetrazol (Pentylenetetrazole):

Qualitative evaluation is carried out according to the anti-epileptic result for the treatment of of the method in embodiment 1 to compound.From Fig. 4 A, under each dosage of taiwanosideC compared with epilepsy model group, zebra fish rapid movement track obviously reduces.Fig. 4 B is the column diagram drawn according to zebra fish rapid movement distance average mean ± SE, and from Fig. 4 B, compound taiwanosideC is under 100 μMs of concentration, and epilepsy therapy efficiency is 28.1%, no difference of science of statistics; Under 300,1000 μMs of concentration, all have antiepileptic action, therapeutic efficiency is respectively 48.5%, 84.8% (P<0.01, P<0.01), and dose-effect relationship is clear and definite.

Embodiment 5

The evaluation of antiepileptic action in oneself (otophyllosideF) (formula I) body of compound Cynanchum otophvllum glycosides:

The preparation method of Cynanchum otophvllum glycosides oneself (otophyllosideF): dry Cynanchum otophvllum block root 5kg, pulverize and extract three times with 95% ethanol percolation, each 2 ~ 3 days, united extraction liquid, concentrating under reduced pressure obtains the alcohol medicinal extract of Cynanchum otophvllum, by extract with water suspendible, extraction into ethyl acetate, obtain acetic acid ethyl ester extract, C-18 reversed-phase column chromatography is with the aqueous solution of methyl alcohol (10% ~ 100%) gradient elution, obtain component Fr1-Fr5, Fr4 further with preparative HPLC repeatedly purifying obtain pure compound Cynanchum otophvllum glycosides oneself (otophyllosideF, 1.1g).

Oneself physicochemical data of compound Cynanchum otophvllum glycosides is as follows: white amorphous powder, is soluble in methyl alcohol, ethanol, acetone, the organic solvents such as chloroform, and molecular formula is C ₆₃h ₉₆o ₂₀, ¹hNMR (CDCl ₃, 400MHz): δ _h1.04,1.06 (d, each3H, J=6.5Hz, H _ikem-5,6), 1.11 (s, 3H, H-19), 1.24,1.26,1.33 (d, each3H, J=6.0Hz, H-6ofsugarmoieties), 1.40 (s, 3H, H-18), 2.11 (s, 3H, H _ikem-7), 2.16 (s, 3H, H-21), 3.38,3.45 (s, each3H, 3-OCH ₃ofsugarmoieties), 4.49 (dd, 1H, J=2.0,9.6Hz, anomericH), 4.81 (dd, 1H, J=2.2,9.3Hz, anomericH), 4.92 (dd, 1H, J=2.2,9.6Hz, anomericH), 5.35 (brs, 1H, H-6), 5.51 (brs, 1H, H _ikem-2), ¹³cNMR (CDCl ₃, 125MHz): δ _c38.9 (t, C-1), 29.0 (t, C-2), 78.0 (d, C-3), 38.9 (t, C-4), 140.8 (s, C-5), 117.7 (d, C-6), 34.3 (t, C-7), 74.4 (s, C-8), 43.8 (d, C-9), 37.3 (s, C-10), 24.4 (t, C-11), 71.7 (d, C-12), 58.0 (s, C-13), 88.1 (s, C-14), 33.2 (t, C-15), 32.0 (t, C-16), 91.6 (s, C-17), 9.5 (q, C-18), 18.7 (q, C-19), 209.0 (s, C-20), 27.3 (q, C-21), 166.0 (s, C _ikem-1), 113.1 (d, C _ikem-2), 166.1 (s, C _ikem-3), 38.3 (s, C _ikem-4), 20.9 (q, C _ikem-5), 21.0 (q, C _ikem-6), 16.7 (q, C _ikem-7), 95.9 (d, C _digit-1), 37.2 (t, C _digit-2), 75.5 (d, C _digit-3), 82.6 (d, C _digit-4), 68.2 (d, C _digit-5), 18.3 (q, C _digit-6), 98.5 (d, C _cym-1), 35.7 (t, C _cym-2), 77.0 (d, C _cym-3), 82.5 (d, C _cym-4), 68.8 (d, C _cym-5), 18.1 (q, C _cym-6), 58.5 (q, C _cym-OMe), 101.6 (d, C _ole-1), 35.4 (t, C _ole-2), 80.7 (d, C _ole-3), 75.5 (d, C _ole-4), 71.6 (d, C _ole-5), 18.1 (q, C _ole-6), 56.4 (q, C _ole-OMe).

The own provide protection of Yetrazol (Pentylenetetrazole) being induced to zebra fish epileptic seizures model of compound Cynanchum otophvllum glycosides:

Qualitative evaluation is carried out according to the anti-epileptic result for the treatment of of the method in embodiment 1 to compound.From Fig. 5 A, under oneself each dosage of Cynanchum otophvllum glycosides compared with epilepsy model group, zebra fish rapid movement track obviously reduces.Fig. 5 B is the column diagram drawn according to zebra fish rapid movement distance average mean ± SE, and from Fig. 5 B, compound Cynanchum otophvllum glycosides is own has epilepsy therapy trend under 30,100 μMs of concentration, but no difference of science of statistics; Under 300 μMs of concentration, have antiepileptic action, epilepsy therapy efficiency is 55% (P<0.01), and dose-effect relationship is clear and definite.

Embodiment 6

Compound Russell he order the evaluation of antiepileptic action in 3-O-β-D-oleandrose pyrans glycosyl-(1 → 4)-β-D-cymarose pyranyl-(1 → 4)-β-D-cymarose [rostratamine3-O-β-D-oleandropyranosyl-(1 → 4)-β-D-cymaropyranosyl-(1 → 4)-β-D-cymaropyranoside] (formula II) (hereinafter referred to as " Compound II per ") body:

The preparation method of Compound II per: dry Cynanchum otophvllum block root 5kg, pulverize and extract three times with 95% ethanol percolation, each 2 ~ 3 days, united extraction liquid, concentrating under reduced pressure obtains the alcohol medicinal extract of Cynanchum otophvllum, by extract with water suspendible, extraction into ethyl acetate, obtains acetic acid ethyl ester extract, and C-18 reversed-phase column chromatography is with the aqueous solution of methyl alcohol (10% ~ 100%) gradient elution, obtain component Fr1-Fr5, Fr4 further with preparative HPLC repeatedly purifying obtain pure compound II100mg.

rostratamine3-O-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranoside

The physicochemical data of Compound II per is as follows: white, needle-shaped crystals, is soluble in methyl alcohol, ethanol, acetone, the organic solvents such as chloroform, and molecular formula is C ₆₃h ₉₆o ₂₀, ¹hNMR (CDCl ₃, 400MHz): δ _h1.46 (s, 3H, H-18), 1.20,1.22,1.31 (d, each3H, J=6.3Hz, H-6ofsugarmoiety), 1.12 (s, 3H, 19-CH ₃), 2.19 (s, 3H, H-21), 3.38,3.44,3.44 (s, each3H, 3-OCH ₃ofsugarmoieties), 4.48 (dd, 1H, J=1.9,9.8, anomericH), 4.74 (dd, J=1.9,9.8,1H, anomericH), 4.84 (dd, 1H, J=1.8,9.6Hz, anomericH), 5.37 (brs, 1H, H-6), 6.30 (d, 1H, J=16.0Hz, H _cin-2), 7.38 (m, 3H, H _cin-6,7,8), 7.51 (m, 2H, H _cin-5,9), 7.62 (d, 1H, J=16.0Hz, H _cin-3), ¹³cNMR (Pyridine-d ₅, 125MHz): δ _c39.9 (t, C-1), 29.8 (t, C-2), 77.6 (d, C-3), 38.8 (t, C-4), 139.3 (s, C-5), 119.2 (d, C-6), 34.6 (t, C-7), 74.2 (s, C-8), 44.5 (d, C-9), 37.1 (s, C-10), 25.2 (t, C-11), 73.9 (d, C-12), 58.3 (s, C-13), 89.5 (s, C-14), 34.7 (t, C-15), 33.6 (t, C-16), 92.1 (s, C-17), 10.7 (q, C-18), 18.1 (q, C-19), 209.9 (s, C-20), 27.7 (q, C-21), 165.9 (s, C _cin-1), 119.1 (d, C _cin-2), 144.7 (d, C _cin-3), 135.0 (s, C _cin-4), 128.6 (d, C _cin-5,9), 129.3 (d, C _cin-6,8), 130.6 (d, C _cin-7), 96.3 (d, C _cym1-1), 37.9 (t, C _cym1-2), 77.8 (d, C _cym1-3), 83.4 (d, C _cym1-4), 68.7 (d, C _cym1-5), 18.3 (q, C _cym1-6), 58.6 (q, C), 100.5 (d, C _cym2-1), 37.2 (t, C _cym2-2), 78.0 (d, C _cym2-3), 83.2 (d, C _cym2-4), 68.9 (d, C _cym2-5), 18.5 (q, C _cym2-6), 58.8 (q, C _cym2-OMe), 102.2 (d, C _ole-1), 37.2 (d, C _ole-2), 81.3 (d, C _ole-3), 76.2 (d, C _ole-4), 72.8 (d, C _ole-5), 18.6 (d, C _ole-6), 57.1 (d, C _ole-OMe),

Compound II per induces the provide protection of zebra fish epileptic seizures model to Yetrazol (Pentylenetetrazole):

Qualitative evaluation is carried out according to the anti-epileptic result for the treatment of of the method in embodiment 1 to compound.From Fig. 6 A, under each dosage of Compound II per compared with epilepsy model group, zebra fish rapid movement track obviously reduces.Fig. 6 B is the column diagram drawn according to zebra fish rapid movement distance average mean ± SE, and from Fig. 6 B, Compound II per, under 30 μMs of concentration, has epilepsy therapy trend, but no difference of science of statistics; Under 100,200 μMs of concentration, all have antiepileptic action, therapeutic efficiency is respectively 65%, 52% (P<0.01, P<0.01).

Embodiment 7

The evaluation of antiepileptic action in compound wilfosideC3N (formula VII) body:

The preparation method of WilfosideC3N: dry Auriculoside A block root 10kg, pulverize and extract three times with 95% ethanol percolation, each 2 ~ 3 days, united extraction liquid, concentrating under reduced pressure obtains the alcohol medicinal extract of Auriculoside A, by extract with water suspendible, extraction into ethyl acetate, obtain acetic acid ethyl ester extract, ethyl acetate extract is dissolved in 70% ethanolic soln, filter or centrifugal segregation insolubles after, carry out macroporous resin column chromatography separation, collect 75% ethanol elution flow point, reactive site is obtained after concentrated, reactive site further with preparative HPLC repeatedly purifying obtain pure WilfosideC3N.

The physicochemical data of WilfosideC3N is as follows: white amorphous powder, is soluble in methyl alcohol, ethanol, acetone, the organic solvents such as chloroform, and molecular formula is C ₄₉h ₇₈o ₁₆, ¹hNMR (Pyridine-d ₅, 400MHz): δ _h0.92,0.94 (d, each3H, J=6.5Hz, H _ikem-5,6), 1.32 (s, 3H, H-19), 1.36,1.34,1.51 (d, each3H, J=6.0Hz, H-6ofsugarmoieties), 1.97 (s, 3H, H-18), 2.26 (s, 3H, H _ikem-7), 2.50 (s, 3H, H-21), 3.42,3.44,3.51 (s, each3H, 3-OCH ₃ofsugarmoieties), 5.24 (dd, 1H, J=1.9,9.4, anomericH), 5.15 (d, J=3.3,1H, anomericH), 5.10 (dd, 1H, J=1.8,9.8Hz, anomericH), 5.86 (brs, 1H, H _ikem-2), ¹³cNMR (Pyridine-d ₅, 125MHz): δ _c39.0 (t, C-1), 29.9 (t, C-2), 77.5 (d, C-3), 39.3 (t, C-4), 139.3 (s, C-5), 119.2 (d, C-6), 34.8 (t, C-7), 74.3 (s, C-8), 44.6 (d, C-9), 37.4 (s, C-10), 25.1 (t, C-11), 72.6 (d, C-12), 58.0 (s, C-13), 89.5 (s, C-14), 33.9 (t, C-15), 33.0 (t, C-16), 92.4 (s, C-17), 10.8 (q, C-18), 18.2 (q, C-19), 209.5 (s, C-20), 27.6 (q, C-21), 166.0 (s, C _ikem-1), 114.1 (d, C _ikem-2), 165.5 (s, C _ikem-3), 38.2 (s, C _ikem-4), 20.9 (q, C _ikem-5), 20.9 (q, C _ikem-6), 16.5 (q, C _ikem-7), 96.1 (d, C _cym1-1), 35.3 (t, C _cym1-2), 77.5 (d, C _cym1-3), 82.5 (d, C _cym1-4), 69.2 (d, C _cym1-5), 18.8 (q, C _cym1-6), 57.2 (q, C), 101.0 (d, C _digin-1), 32.5 (t, C _digin-2), 74.5 (d, C _digin-3), 73.8 (d, C _digin-4), 67.5 (d, C _digin-5), 17.9 (q, C _digin-6), 55.3 (q, C _digin-OMe), 99.5 (d, C _cym2-1), 35.3 (d, C _cym2-2), 79.9 (d, C _cym2-3), 74.1 (d, C _cym2-4), 71.0 (d, C _cym2-5), 18.7 (d, C _cym2-6), 58.0 (d, C _cym2-OMe).

WilfosideC3N induces the provide protection of zebra fish epileptic seizures model to Yetrazol (Pentylenetetrazole):

Qualitative evaluation is carried out according to the anti-epileptic result for the treatment of of the method in embodiment 1 to compound.From Fig. 7 A, under each dosage of wilfosideC3N compared with epilepsy model group, zebra fish rapid movement track obviously reduces.Fig. 7 B is the column diagram drawn according to zebra fish rapid movement distance average mean ± SE, from Fig. 7 B, wilfosideC3N under 100 μMs of concentration, has epilepsy therapy trend, but no difference of science of statistics; Under 200 μMs of concentration, have antiepileptic action, therapeutic efficiency is 55.2% (P<0.001).

Embodiment 8

The evaluation of antiepileptic action in compound cynauricosideA (formula VIII) body:

The preparation method of CynauricosideA: dry Auriculoside A block root 10kg, pulverize and extract three times with 95% ethanol percolation, each 2 ~ 3 days, united extraction liquid, concentrating under reduced pressure obtains the alcohol medicinal extract of Cynanchum otophvllum, by extract with water suspendible, extraction into ethyl acetate, obtain acetic acid ethyl ester extract, ethyl acetate extract is dissolved in 70% ethanolic soln, filter or centrifugal segregation insolubles after, carry out macroporous resin column chromatography separation, collect 75% ethanol elution flow point, reactive site is obtained after concentrated, reactive site further with preparative HPLC repeatedly purifying obtain pure cynauricosideA.

The physicochemical data of CynauricosideA is as follows: white amorphous powder, is soluble in methyl alcohol, ethanol, acetone, the organic solvents such as chloroform, and molecular formula is C ₅₁h ₇₄o ₁₆, ¹hNMR (Pyridine-d ₅, 400MHz): δ _h2.03 (s, 3H, H-18), 1.37,1.45,1.52 (d, each3H, J=6.3Hz, H-6ofsugarmoiety), 1.35 (s, 3H, 19-CH ₃), 2.49 (s, 3H, H-21), 3.52,3.42,3.45 (s, each3H, 3-OCH ₃ofsugarmoieties), 5.24 (dd, 1H, J=1.9,9.4, anomericH), 5.15 (d, J=3.3,1H, anomericH), 5.10 (dd, 1H, J=1.8,9.8Hz, anomericH), 5.30 (brs, 1H, H-6), 6.81 (d, 1H, J=16.0Hz, H _cin-2), 7.34 (m, 3H, H _cin-6,7,8), 7.63 (m, 2H, H _cin-5,9), 8.00 (d, 1H, J=16.0Hz, H _cin-3), ¹³cNMR (Pyridine-d ₅, 125MHz): δ _c38.9 (t, C-1), 29.8 (t, C-2), 77.6 (d, C-3), 39.2 (t, C-4), 139.2 (s, C-5), 119.1 (d, C-6), 34.7 (t, C-7), 74.2 (s, C-8), 44.5 (d, C-9), 37.4 (s, C-10), 25.0 (t, C-11), 73.6 (d, C-12), 58.1 (s, C-13), 89.5 (s, C-14), 33.8 (t, C-15), 33.0 (t, C-16), 92.4 (s, C-17), 10.7 (q, C-18), 18.1 (q, C-19), 209.9 (s, C-20), 27.7 (q, C-21), 165.8 (s, C _cin-1), 119.2 (d, C _cin-2), 144.9 (d, C _cin-3), 135.0 (s, C _cin-4), 128.6 (d, C _cin-5,9), 129.3 (d, C _cin-6,8), 130.6 (d, C _cin-7), 96.1 (d, C _cym1-1), 35.3 (t, C _cym1-2), 77.5 (d, C _cym1-3), 82.5 (d, C _cym1-4), 69.2 (d, C _cym1-5), 18.8 (q, C _cym1-6), 57.2 (q, C), 101.0 (d, C _digin-1), 32.5 (t, C _digin-2), 74.5 (d, C _digin-3), 73.8 (d, C _digin-4), 67.5 (d, C _digin-5), 17.9 (q, C _digin-6), 55.3 (q, C _digin-OMe), 99.5 (d, C _cym2-1), 35.3 (d, C _cym2-2), 79.9 (d, C _cym2-3), 74.1 (d, C _cym2-4), 71.0 (d, C _cym2-5), 18.7 (d, C _cym2-6), 58.0 (d, C _cym2-OMe).

CynauricosideA induces the provide protection of zebra fish epileptic seizures model to Yetrazol (Pentylenetetrazole):

Qualitative evaluation is carried out according to the anti-epileptic result for the treatment of of the method in embodiment 1 to compound.From Fig. 8 A, under each dosage of cynauricosideA compared with epilepsy model group, zebra fish rapid movement track obviously reduces.Fig. 8 B is the column diagram drawn according to zebra fish rapid movement distance average mean ± SE, from Fig. 8 B, cynauricosideA under 30,100 μMs of concentration, has epilepsy therapy trend, but no difference of science of statistics; Under 200 μMs of concentration, all have antiepileptic action, therapeutic efficiency is respectively 57.5% (P<0.01).

Comparative example 1

Derive from separation and Extraction in Cynanchum three kind of plant to the larger compound Qingyanshengenin (qingyangshengenin) of content, Caudatin (caudatin), Q-A (otophyllosideA), Cynanchum otophvllum glycosides third (otophyllosideC), own (the otophyllosideF of Cynanchum otophvllum glycosides, formula I), Cynanchum otophvllum glycosides M (otophyllosideM, formula b), Caudatin 3-O-β-D-cymarose [caudatin3-O-β-D-cymaropyranoside, formula IX], Qingyanshengenin 3-O-β-D-cymarose pyranyl-(1 → 4)-β-D-digitoxose [qinyangshengenin3-O-β-D-cymaropyranosyl-(1 → 4)-β-D-digitoxopyranoside, formula XVII], Caudatin 3-O-β-D-cymarose pyranyl-(1 → 4)-β-D-cymarose [caudatin3-O-β-D-cymaropyranosyl-(1 → 4)-β-D-cymaropyranoside, formula X], Russell he order 3-O-β-D-oleandrose pyrans glycosyl-(1 → 4)-β-D-cymarose pyranyl-(1 → 4)-β-D-cymarose [rostratamine3-O-β-D-oleandropyranosyl-(1 → 4)-β-D-cymaropyranosyl-(1 → 4)-β-D-cymaropyranoside, formula II], Russell he order 3-O-α-L-cymarose pyrans glycosyl-(1 → 4)-β-D-cymarose pyranyl-(1 → 4)-α-L-diginose pyrans glycosyl-(1 → 4)-β-D-cymarose [rostratamine3-O-α-L-cymaropyranosyl-(1 → 4)-β-D-cymaropyranosyl-(1 → 4)-α-L-diginopyranosyl-β-D-cymaropyranoside, formula XIII], Qingyanshengenin 3-O-β-D-oleandrose pyrans glycosyl-(1 → 4)-β-D-cymarose pyranyl-(1 → 4)-β-D-digitoxose [qinyangshengenin3-O-β-D-oleandropyranosyl-(1 → 4)-β-D-cymaropyranosyl-(1 → 4)-β-D-digitoxopyranoside, formula a], Caudatin 3-O-β-D-Glucose pyrans glycosyl-(1 → 4)-β-D-oleandrose pyranyl-(1 → 4)-β-D-cymarose pyranyl-(1 → 4)-β-D-cymarose [caudatin3-O-β-D-glucopyranoside-(1 → 4)-β-D-oleandropyranosyl-(1 → 4)-β-D-cymaropyranosyl-(1 → 4)-β-D-cymaropyranoside, formula c], Caudatin 3-O-β-D-thevetose pyrans glycosyl-(1 → 4)-β-D-cymarose pyranyl-(1 → 4)-β-D-cymarose [caudatin3-O-β-D-thevetopyranosyl-(1 → 4)-β-D-cymaropyranosyl-(1 → 4)-β-D-cymaropyranoside, formula XII], Qingyanshengenin 3-O-β-D-oleandrose pyrans glycosyl-(1 → 4)-β-D-cymarose [qinyangshengenin3-O-β-D-oleandropyranosyl-(1 → 4)-β-D-cymaropyranoside, formula XI], taiwanosideC (formula III), wilfosideK1N (formula V), cynawilfosideA (formula VI), wilfosideM1N (formula d), cynauricosideE (formula XV), cynauricuosideA (formula XVI), wilfosideG (formula XIV) affects the zebra fish of PTZ induction under maximal non-toxic dosage:

Qualitative evaluation is carried out according to the anti-epileptic result for the treatment of of the method in embodiment 1 to compound.As shown in Figure 10, the monomeric compound such as formula I, II, III, V, VI antiepileptic efficacy is all better than principal constituent Qingyanshengenin (qingyangshengenin) in Cynanchum otophvllum, Caudatin (caudatin), Q-A (otophyllosideA), Cynanchum otophvllum glycosides third (otophyllosideC), formula b compound, formula c compound, formula IX compound, and in Cynanchum otophvllum principal constituent formula a compound, formula b compound, formula c compound and Root of Wilford Swallowwort, component formula d compound has the significant effect strengthening the epileptic seizures of Yetrazol induction.

The preparation of embodiment 9 Auriculoside A extract extract

By Auriculoside A medicinal material 10kg, add 95% ethanol 5L, Soakage extraction three times, concentrating under reduced pressure extracting solution obtains ethanol extract, add water suspendible, with 5 times of volume of ethylacetate extractions, reclaims ethyl acetate, ethyl acetate extract is dissolved in 3L70% ethanolic soln, filter after removing insolubles, carry out macroporous resin column (resin model ZH801) chromatographic separation, collect 75% ethanol elution flow point, concentrated, obtain extract 15g.

After testing, in extract, the content of antiepileptic activity ingredient w ilfosideC1N, wilfosideC3N, wilfosideK1N, cynauricosideA is respectively 17.4%, 42.1%, 7.1%, 14.2%, and the total content of antiepileptic activity composition reaches more than 80%.Short epilepsy composition aspect, Cynanchum otophvllum glycosides M, Qingyanshengenin 3-O-β-D-oleandrose pyrans glycosyl-(1 → 4)-β-D-cymarose pyranyl-(1 → 4)-β-D-digitoxose do not detect.

Adopt the antiepileptic activity of the method evaluation Auriculoside A extract in embodiment 1, result shows, under 50 μ g/mL concentration, has epilepsy therapy trend, but no difference of science of statistics; Under 100 μMs of concentration, have antiepileptic action, epilepsy therapy efficiency is 61.4% (p<0.001).

The preparation of embodiment 10 Cynanchum otophvllum extract

Cynanchum otophvllum medicinal material is extracted three times with 95% alcohol dipping, concentrating under reduced pressure extracting solution obtains ethanol extract, add water suspendible, with extraction into ethyl acetate, reclaims ethyl acetate, ethyl acetate extract is dissolved in 70% ethanolic soln, filter or after centrifugal segregation insolubles, carry out macroporous resin column chromatography separation, collect 75% ethanol elution flow point, concentrated, to obtain final product.

The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.

Claims (10)

1. structure is such as formula the compound shown in A or its a pharmaceutically acceptable salt, it is characterized in that:

In formula A, R ₁for glycosyl, R ₂for substituted or unsubstituted C1 ~ C6 alkyl or substituted or unsubstituted C1 ~ C6 thiazolinyl, R ₃for substituted or unsubstituted C1 ~ C6 alkyl;

Described replacement refers to that one or more hydrogen atom is selected from the substituting group replacement of lower group: C1 ~ C10 alkyl, C3 ~ C10 cycloalkyl, C1 ~ C10 alkoxyl group, hydroxyl, carboxyl, C1 ~ C10 carbonyl, C1 ~ C10 amide group, C2 ~ C10 ester group, C6 ~ C30 aryl and oxygen (=O).

2. compound as claimed in claim 1, is characterized in that, described R ₃for or wherein R ₄for substituted or unsubstituted C1 ~ C6 alkyl; R ₅for substituted or unsubstituted C1 ~ C6 alkyl; And/or

Described R ₁for n is the positive integer of 1 ~ 4, R ₇for H, methyl or C1 ~ C10 ester group, R ₈for H or C2 ~ C10 ester group; And/or

Described R ₂be selected from lower group:

3. compound as claimed in claim 1, it is characterized in that, described compound is selected from lower group:

4. a pharmaceutical composition, is characterized in that, comprises as the compound as described in arbitrary in claim 1-3 or its pharmacy acceptable salt, and optional pharmaceutically acceptable carrier or vehicle.

5. the compound as described in as arbitrary in claim 1-3 or its pharmacy acceptable salt, wilfosideG or its pharmaceutically acceptable salt or the application of pharmaceutical composition in preparation treatment or prevention epilepsy medicament as claimed in claim 2.

6. Apocynaceae Cynanchum (Cynanchum) plant Auriculoside A (C.auriculatumRoyleexWight) and Root of Wilford Swallowwort (C.wilfordiiHemsl.) or the application of its extract in preparation treatment or prevention epilepsy medicament.

7. plant total glycoside or the plant milk extract containing described plant total glycoside, is characterized in that, contain antiepileptic activity composition, and the content C1 of antiepileptic activity composition described in described extract meets following formula B in described plant total glycoside or described extract:

C1/ (C1+C2) >=50% formula B

In formula B, C1 is the content of antiepileptic activity composition; C2 is the content of short epilepsy activeconstituents;

Preferably, described antiepileptic activity composition be selected from lower group one or more:

Compound shown in formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI; And/or

Described short epilepsy activeconstituents be selected from lower group one or more:

Compound shown in formula a, b, c and d

8. a pharmaceutical composition, is characterized in that, comprises Chinese medical extract as claimed in claim 7, and optional pharmaceutically acceptable carrier or vehicle.

9. a quality controlling means is carried out to anti-epileptic product, it is characterized in that, comprise step:

A () measures the content C2 of short epilepsy activeconstituents in described product or its raw material, and compare with preset value C0;

B () when described content C2 is higher than C0, then judges the off quality of described product; When described content C2 is less than or equal to C0, then judge the up-to-standard of described product.

10. the compound being selected from lower group is preparing the purposes in medicament or reagent, and described medicament or reagent are for building animal seizure models: the compound shown in formula a, b, c and d.