CN105503988B - Natural antiepileptic activity compound and its purposes in pharmaceutical preparation - Google Patents
Natural antiepileptic activity compound and its purposes in pharmaceutical preparation Download PDFInfo
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Abstract
The present invention relates to natural antiepileptic activity compound and its purposes in pharmaceutical preparation.The invention discloses the different monomers compound in Cynanchum otophyllum Schneid to have the function that the rush epilepsy completely contradicted and anti-epileptic.Specifically, the present inventor extraction one-component from Apocynaceae Cynanchum (Cynanchum) plant cynanchum auriculatum Royle ex Wight (C.auriculatum Royle ex Wight) and cynanchum wilfordii (C.wilfordii Hemsl.), and experiments prove that the antiepileptic activity of each compound.
Description
Technical field
The invention belongs to biomedicine field, specifically, the present invention relates to natural antiepileptic activity compound and its
Purposes in pharmaceutical preparation.
Background technology
Epilepsy (Epilepsy), or brain epilepsy, sheep epilepsy, it is the of short duration brain function imbalance syndrome of chronic recurrent.According to
Statistics, the incidence of disease about or so 23/,100,000/year of China's epilepsy, illness rate are 3.5 ‰ -4.8 ‰.Epileptic attack can trigger a system
Row symptom, spasm are one of typical epilepsy syndromes, including limbs repeating motion, irregularities and with the complete of the loss of consciousness
Body twitch etc., or even cause body injury or death.The treatment of epilepsy includes drug therapy, operative treatment, physical treatment and the heart
A variety of methods such as reason treatment, its drug treatment are current the most frequently used, most important means.Clinically it is used for controlling for epilepsy at present
Treating medicine includes carbamazepine (Carbamazepine), sodium vedproate (Depakene), dilantin sodium (Dilantin) etc., but
These medicines generally have obviously side effect, long-term use of that body is shone into large effect, or even in a small portion
Suicidal thoughts or behavior may be triggered by dividing in medication patient.Chinese medicine compound prescription, Chinese patent medicine preparation product also occupy many markets, but by
In Chinese medicine side's complicated component, and the cause influence such as plant place of production difference, climate variability, harvest time difference medicine mainly into
The content divided, therefore shone to the quality control of Chinese medicine side into sizable difficulty, which also limits the popularization of Chinese medicine preparation.Cause
This, find safe and non-toxic effective and quality controllable new antiepileptic medicine has extremely in the prevention and treatment of epilepsy
Important meaning.
Cynanchum (Cynanchum) is a category of Apocynaceae (Apocynum), about 200 kinds, is distributed in the torrid zone and temperature
It is produced in band area, about 53 kinds of China, the whole nation.Cynanchum auriculatum Royle ex Wight (C.auriculatum), also known as radix cynanchi bungei, it is China's endemic plant
Kind, about 95% is grown on Jiangsu Province Binhai County, has liver and kidney benefiting, replenishing vital essence and blood, anti-aging and other effects.Cynanchum wilfordii
(C.wilfordii) ground such as Liaoning, Henan, Hubei are originated in, it is conventional with stomach invigorating, disappear it is glutted, control choke food etc..Cynanchum otophyllum Schneid
(C.otophyllum) provinces and regions such as Hunan, Guangxi, Guizhou, Yunnan, Sichuan and Tibet are originated in, there is removing toxic substances, antispastic, wind-damp dispelling etc.
Effect, it is clinically used for controlling treating rheumatic ostealgia, epilepsy and venomous snake bite.
The Qingyang total glycosides of conopsea extraction Cynanchum otophyllum Schneid is the adjuvant drug of clinical treatment epilepsy.But Cynanchum otophyllum Schneid piece complicated component,
Certain difficulty is brought to quality control, action target is failed to understand, clinical mainly to be closed with antiepileptics such as dilantin sodium or phenobarbitals
With treatment.
The content of the invention
It is an object of the invention to provide natural antiepileptic activity compound and its purposes in pharmaceutical preparation.
A kind of the first aspect of the present invention, there is provided compound of structure as shown in formula A or it is pharmaceutically acceptable
Salt, it is characterised in that：
In formula A, R1For glycosyl, R2For substituted or unsubstituted C1~C6 alkyl or substituted or unsubstituted C1~C6 alkene
Base, R3For substituted or unsubstituted C1~C6 alkyl；
The substitution refers to that the substituent that one or more hydrogen atoms are selected from the group substitutes：C1~C10 alkyl, C3~
C10 cycloalkyl, C1~C10 alkoxies, hydroxyl, carboxyl, C1~C10 carbonyls, C1~C10 amide groups, C2~C10 ester groups, C6~
C30 aryl and oxygen (=O).
In another preference, the glycosyl is by apocynum cannabinum glycosyl (cymarosyl), oleander glycosyl
(oleandrosyl), diginose base (diginosyl), thevetose base (thevetosyl), digoxigenin glycosyl
(digitoxosyl), block in that glycosyl (canarosyl), rhamnopyranosyl (rhamnosyl) and glucosyl group (glucosyl)
One or more composition.
In another preference, the glycosyl includes 1~6 identical or different monosaccharide unit, it is preferable that including 2~4
Individual identical or different monosaccharide unit.
In another preference, R1In do not contain sugar unit
In another preference, work as R2For hydroxy phenyl when, R1Middle sugar unit quantity is not 3.
In another preference, the sugar unit quantity can be 0,1,2,3 or 4.
In another preference, the R3ForWherein R4For substituted or unsubstituted C1~
C6 alkyl；R5For substituted or unsubstituted C1~C6 alkyl.
In another preference, the R1ForN be 1~4 positive integer, R7For H or
Methyl or C1~C10 ester groups, R8For H or C2~C10 ester groups.
In another preference, the R1 is selected from the group：
In another preference, the R2It is selected from the group：
In another preference, the R3It is selected from the group：
In another preference, the compound structure is as shown in formula I：
In another preference, the compound structure is as shown in formula II：
In another preference, the compound structure is as shown in formula III：
In another preference, the compound structure is as shown in formula IV：
In another preference, the compound structure is as shown in formula V：
In another preference, the compound structure is as shown in formula VI：
In another preference, the compound structure is as shown in formula VII：
In another preference, the compound structure is as shown in formula VIII：
In another preference, the compound structure is as shown in formula Ⅸ：
In another preference, the compound structure is as shown in formula Ⅹ：
In another preference, the compound structure is as shown in formula Ⅺ：
In another preference, the compound structure is as shown in formula Ⅻ：
In another preference, the compound structure is as shown in Formula X III：
In another preference, the compound structure is as shown in Formula X V：
In another preference, the compound structure is as shown in Formula X VI：
The first aspect of the present invention, there is provided a kind of pharmaceutical composition, it includes change as described in the first aspect of the invention
Compound or its pharmaceutically acceptable salt, and optional pharmaceutically acceptable carrier or excipient.
In another preference, the optional medicine being selected from the group also is included in described pharmaceutical composition：Carbamazepine
(Carbamazepine), sodium vedproate (Depakene) and dilantin sodium (Dilantin).
The third aspect of the present invention, there is provided compound as described in the first aspect of the invention or its is pharmaceutically acceptable
Salt, wilfoside G or its pharmaceutically acceptable salt or pharmaceutical composition as described in respect of the second aspect of the invention are being made
The standby application treated or prevented in epilepsy medicament
In another preference, the wilfoside G structures are as shown in Formula X IV：
The fourth aspect of the present invention, there is provided Apocynaceae Cynanchum (Cynanchum) plant cynanchum auriculatum Royle
(C.auriculatum Royle ex Wight) or cynanchum wilfordii (C.wilfordii Hemsl.) or its extract are controlled in preparation
Treat or prevent the application in epilepsy medicament.
The fifth aspect of the present invention, there is provided a kind of plant total glycoside or the Chinese medical extract containing the plant total glycoside, it is described
Contain antiepileptic activity composition, and antiepileptic activity composition described in the extract in plant total glycoside or the extract
Content C1 meets following formula B：
C1/ (C1+C2) >=50% formulas B
In formula B, C1 is the content of antiepileptic activity composition；C2 is the content for promoting epilepsy active component.
In another preference, C1/ (C1+C2) >=80%；More preferably, >=90%；Most preferably, >=95%, such as 96%,
97%th, 98%, 99%, 99.5%.
In another preference, described antiepileptic activity composition be selected from the group in one or more：
Compound shown in Formulas I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI.
In another preference, described rush epilepsy active component be selected from the group in one or more：
Compound shown in formula a, b, c and d
In another preference, the plant includes：Cynanchum auriculatum Royle (C.auriculatum Royle ex Wight),
Cynanchum wilfordii (C.wilfordii Hemsl.).
In another preference, the preparation method of the extract is as follows：
Medicinal material is extracted three times with 95% alcohol dipping, the extract solution that is concentrated under reduced pressure obtains ethanol extract, adds water to be suspended, with second
Acetoacetic ester extracts, and reclaims ethyl acetate, ethyl acetate extract is dissolved in 70% ethanol solution, and filtering or centrifugation remove insoluble matter
Afterwards, macroporous resin column chromatography separation is carried out, 75% ethanol elution flow point is collected, concentration, produces.
The sixth aspect of the present invention, there is provided a kind of pharmaceutical composition, include the Chinese medicine as described in fifth aspect present invention
Extract, and optional pharmaceutically acceptable carrier or excipient.
The seventh aspect of the present invention, there is provided a kind of that method of quality control, including step are carried out to anti-epileptic product：
(a) the content C2 for promoting epilepsy active component in the product or its raw material is determined, and compared with predetermined value C0；
(b) when the content C2 is higher than C0, then the off quality of the product is judged；Be less than as the content C2 or
During equal to C0, then the up-to-standard of the product is judged.
In another preference, described rush epilepsy active component be selected from the group in one or more：Formula a, b, c and d
Shown compound.
In another preference, the anti-epileptic product includes the anti-epileptic medicine of the conopsea extraction containing Qingyang.
In another preference, the C2 is the percentage by weight for promoting epilepsy active component in the product or raw material.
In another preference, C0≤10% (w/w), it is preferable that C0≤5% (w/w), it is highly preferred that institute
C0≤1% is stated, the C0 can be 0.01%, 0.05%, 0.1%, 0.2%, 0.5%, 1.0%, 5%, 10%.
The eighth aspect of the present invention, there is provided purposes of the compound being selected from the group in medicament or reagent is prepared, it is described
Medicament or reagent are used to build animal seizure models：Compound shown in formula a, b, c and d.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment)
It can be combined with each other between each technical characteristic of body description, so as to form new or preferable technical scheme.As space is limited, exist
This no longer tires out one by one states.
Brief description of the drawings
Figure 1A shows the quick movement locus of zebra fish of each concentration groups of cynawilfoside A and epilepsy model group；Figure
1B shows the column diagram drawn according to the quick move distance average value mean ± SE of zebra fish.
Fig. 2A shows the quick movement locus of zebra fish of each concentration groups of wilfoside C1N and epilepsy model group；Fig. 2 B
Show the column diagram drawn according to the quick move distance average value mean ± SE of zebra fish.
Fig. 3 A show the quick movement locus of zebra fish of each concentration groups of wilfoside K1N and epilepsy model group；Fig. 3 B
Show the column diagram drawn according to the quick move distance average value mean ± SE of zebra fish.
Fig. 4 A show the quick movement locus of zebra fish of each concentration groups of taiwanoside C and epilepsy model group；Fig. 4 B
Show the column diagram drawn according to the quick move distance average value mean ± SE of zebra fish.
Fig. 5 A show the quick movement locus of zebra fish of oneself each concentration group and epilepsy model group of Cynanchum otophyllum Schneid glycosides；Fig. 5 B are shown
The column diagram drawn according to the quick move distance average value mean ± SE of zebra fish.
Fig. 6 A show Russell, and he orders 3-O- β-D- oleandroses pyranose-(1 → 4)-β-D- cymarose pyrans
Base-(1 → 4)-β-D- cymaroses [rostratamine 3-O- β-D-oleandropyranosyl- (1 → 4)-β-D-
Cymaropyranosyl- (1 → 4)-β-D-cymaropyranoside] each concentration group and epilepsy model group zebra fish it is quick
Movement locus；Fig. 6 B show the column diagram drawn according to the quick move distance average value mean ± SE of zebra fish.
Fig. 7 A show the quick movement locus of zebra fish of each concentration groups of wilfoside C3N and epilepsy model group；Fig. 7 B
Show the column diagram drawn according to the quick move distance average value mean ± SE of zebra fish.
Fig. 8 A show the quick movement locus of zebra fish of each concentration groups of cynauricoside A and epilepsy model group；Figure
8B shows the column diagram drawn according to the quick move distance average value mean ± SE of zebra fish.
Fig. 9 A show the quick movement locus of zebra fish of each concentration group of cynanchum auriculatum Royle active site and epilepsy model group；
Fig. 9 B show the column diagram drawn according to the quick move distance average value mean ± SE of zebra fish.
Figure 10 shows Qingyanshengenin (qingyangshengenin), caudatin (caudatin), Qingyangshenglycoside A
(otophylloside A), Cynanchum otophyllum Schneid glycosides third (otophylloside C), Cynanchum otophyllum Schneid glycosides oneself (otophylloside F, formula
I), Cynanchum otophyllum Schneid glycosides M (otophylloside M, formula b), caudatin 3-O- β-D- cymaroses [caudatin3-O- β-D-
Cymaropyranoside, Formula IX], Qingyanshengenin 3-O- β-D- cymaroses pyranose-(1 → 4)-β-D- digoxigenins
Sugared [qinyangshengenin 3-O- β-D-cymaropyranosyl- (1 → 4)-β-D-digitoxopyranoside, formula
XVII], caudatin 3-O- β-D- cymaroses pyranose-(1 → 4)-β-D- cymaroses [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- cymaroses pyranose-(1 → 4)-β-D- cymaroses [rostratamine3-O- β-D-
Oleandropyranosyl- (1 → 4)-β-D-cymaropyranosyl- (1 → 4)-β-D-cymaropyranoside, formula
II], Russell he order 3-O- α-L- cymaroses pyranose-(1 → 4)-β-D- cymaroses pyranose-(1 → 4)-α-
L- diginoses pyranose-(1 → 4)-β-D- cymaroses [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- oleandroses pyranose-(1 → 4)-β-D- cymaroses pyranose-(1 → 4)-
β-D- digitoxoses [qinyangshengenin3-O- β-D-oleandropyranosyl- (1 → 4)-β-D-
Cymaropyranosyl- (1 → 4)-β-D-digitoxopyranoside, formula a], caudatin 3-O- β-D-Glucose pyranose
Base-(1 → 4)-β-D- oleandroses pyranose-(1 → 4)-β-D- cymaroses pyranose-(1 → 4)-β-D- apocynum cannabinums
Sugar [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 pyranoses
Base-(1 → 4)-β-D- cymaroses pyranose-(1 → 4)-β-D- cymaroses [caudatin 3-O- β-D-
Thevetopyranosyl- (1 → 4)-β-D-cymaropyranosyl- (1 → 4)-β-D-cymaropyranoside, formula
XII], Qingyanshengenin 3-O- β-D- oleandroses pyranose-(1 → 4)-β-D- cymaroses
[qinyangshengenin3-O- β-D-oleandropyranosyl- (1 → 4)-β-D-cymaropyranoside, Formula X I],
Taiwanoside C (formula III), wilfoside K1N (Formula V), cynawilfoside A (Formula IV), wilfoside M1N
(the chemical combination such as formula d), cynauricoside E (Formula X V), cynauricuoside A (Formula X VI), wilfoside G (Formula X IV)
Thing influences under maximal non-toxic dosage on the zebra fish of PTZ inductions.
Embodiment
The present inventor is by extensive and in-depth study, it has unexpectedly been found that, the different monomers compound in Cynanchum otophyllum Schneid has
There is the rush epilepsy completely contradicted and anti-epileptic.Specifically, the present inventor is from Apocynaceae Cynanchum
(Cynanchum) plant cynanchum auriculatum Royle ex Wight (C.auriculatum Royle ex Wight) and cynanchum wilfordii (C.wilfordii
Hemsl. extraction one-component in), and experiments prove that its antiepileptic activity.
Term
As used herein, term " substitution " refers to the substituent that one or more hydrogen atoms on group are selected from the group and taken
Generation：C1~C10 alkyl, C3~C10 cycloalkyl, C1~C10 alkoxies, hydroxyl, carboxyl (- COOH), C1~C10 carbonyls, C1~
C10 amide groups, C2~C20 ester groups, 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, third
Base, isopropyl, butyl, isobutyl group, sec-butyl, the tert-butyl group or similar group.
Term " C3~C10 cycloalkyl " refers to the cycloalkyl with 3~10 carbon atoms, such as cyclopropyl, cyclobutyl, ring penta
Base, suberyl or similar group.
Term " C1~C10 alkoxies " refers to the straight or branched alkoxyl with 1-10 carbon atom, such as methoxyl group, second
Epoxide, propoxyl group, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy or similar group.
Term " carbonyl ", which refers to, to be hadGroup；Such as " C1~10 carbonyl " refers to 1~10 carbon atom
Carbonyl.
Term " amide groups ", which refers to, to be hadGroup；Such as " C1~10 amide groups " refers to there is 1~10
The amide groups of carbon atom.
Term " C2~C20 ester groups " refers to alkyl-COO- structures or has-COO- alkyl structures, and wherein alkyl can
Think straight or branched.Preferably there is the group of C1~C10 alkyl-COO- structures, such as CH3COO-、C2H5COO-、
C3H8COO-、(CH3)2CHCOO-、n-C4H9COO-、t-C4H9COO-, or similar group.Or there is-COO-C1~C19 alkyl
Structure, such as-COOCH3、-COOC2H5、-COOC3H8Or-COO- (CH2)n-CH3, n is 0~18 integer.
Term " aryl ", it is however preferred to have 6~30 carbon atoms, refer to that there is 6~12 carbon atoms in ring part
Monocyclic or Bicycloaromaticity group, such as：Phenyl, xenyl, naphthyl or similar group, each carbon atom therein can be with
Arbitrarily substituted.It is preferred that having 1~3 substituent, the substituent is selected from：Halogen, C1~C10 alkyl, cyano group, OH, nitro,
C3~C10 cycloalkyl, C1~C10 alkoxies, amino.
Term " thioether group ", refer to the group with-alkyl-S- alkyl radical structures.
Term " halogenated compound ", refer to the chemical combination formed after at least one H atom in organic compound is optionally substituted by halogen
Thing.
Term " halogen " refers to fluorine, chlorine, bromine, iodine.Term " halo " refer to fluoro, chloro, bromo, iodo
's.
Each group of the present invention can be with unsubstituted or substituted, the substituted substituent for referring to be selected from the group
Substituted：Hydroxyl, halogen, C1~C6 alkyl, C1~C6 alkyl of halo, C1~C6 alkoxies, halo C1~C6 alkoxies,
Cyano group, tertiary fourth carbamyl ,-O- (CH2)n-O-；Wherein, n is the integer between 1~3.The substituent may replace in each group
Each position on.
In embodiments of the present invention, it is related to Cynanchum otophyllum Schneid glycosides oneself (otophylloside F), rostratamine3-O-
triglycoside(rostratamine 3-O-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyra-
nosyl-(1→4)-β-D-cymaropyranoside)、taiwanoside C、wilfoside C1N、wilfoside C3N、
The monomeric compounds such as wilfoside K1N, cynauricoside A, cynawilfoside A are preparing treatment or prevention epilepsy
Application and Apocynaceae Cynanchum (Cynanchum) plant cynanchum auriculatum Royle ex Wight (C.auriculatum Royle in medicine
Ex Wight), cynanchum wilfordii (C.wilfordii Hemsl.) extract prepare or prevent epilepsy medicament in application.And
The antiepileptic efficacy of these monomeric compounds is superior to the middle principal component Qingyangshenglycoside A of the total glycosides piece of Cynanchum otophyllum Schneid
(otophylloside A), cynanchogenin (qingyangshengenin) and caudatin (caudatin).
Relevant Cynanchum otophyllum Schneid glycosides oneself (otophylloside F), Russell he order 3-O- β-D- oleandroses pyranose-(1 →
4)-β-D- cymaroses pyranose-(1 → 4)-β-D- cymaroses [rostratamine3-O- β-D-
oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranoside]、
taiwanoside C、wilfoside C1N、wilfoside C3N、wilfoside K1N、cynauricoside A、
The application of cynauricoside A and cynanchum auriculatum Royle ex Wight, the antiepileptic action of cynanchum wilfordii extract in Related product is current not yet
See document report, and cynawilfoside A are the compound for separating identification from natural products first.
Monomeric compound antiepileptic action in Cynanchum otophyllum Schneid is had made intensive studies in the present invention, it has unexpectedly been found that Cynanchum otophyllum Schneid
In different monomers compound have the function that with the rush epilepsy completely contradicted and anti-epileptic.One of principal component in Cynanchum otophyllum Schneid
Cynanchum otophyllum Schneid glycosides oneself (otophylloside F) and Russell he order 3-O- β-D- oleandroses pyranose-(1 → 4)-β-D- and add
Put on airs sesame candy pyranose-(1 → 4)-β-D- cymaroses [rostratamine3-O- β-D-oleandropyranosyl- (1
→ 4)-β-D-cymaropyranosyl- (1 → 4)-β-D-cymaropyranoside] have significantly in zebra fish model
Resist the effect of clonic convulsion caused by pentylenetetrazole；And the Qingyanshengenin 3-O- β-D- of one of principal component as Cynanchum otophyllum Schneid
Oleandrose pyranose-(1 → 4)-β-D- cymaroses pyranose-(1 → 4)-β-D- digitoxoses
[qinyangshengenin3-O-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-
β-D-digitoxopyranoside] and Cynanchum otophyllum Schneid glycosides M (otophylloside M) but have and significant promote convulsion effect.
The invention provides with the compound for significantly inhibiting epilepsy effect, the anti-epileptic of these compounds or anticonvulsant
Effect does not have relevant report, and cynawilfoside A are the new natural products for separating and identifying first, due to these chemical combination
Thing content in crude drug is larger, extraction, preparation it is relatively easy and quality controllable, therefore in antiepileptic develop with compared with
Big development prospect.
The present invention comes from Apocynaceae Cynanchum (Cynanchum) plant cynanchum auriculatum Royle ex Wight by animal vivo test, discovery
(C.auriculatum) and the extract of cynanchum wilfordii (C.wilfordii) has certain antiepileptic activity, and monomer chemical combination
Thing Cynanchum otophyllum Schneid glycosides oneself (otophylloside F), Russell he order 3-O- β-D- oleandroses pyranose-(1 → 4)-β-D- and add
Put on airs sesame candy pyranose-(1 → 4)-β-D- cymaroses [rostratamine3-O- β-D-oleandropyranosyl- (1
→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranoside]、taiwanoside C、wilfoside
C1N, wilfoside C3N, wilfoside K1N, cynauricoside A, cynawilfoside A etc. have significant anti-
Epilepsy activity, its structure are represented by such as I~Formula X of following formula VI respectively：
The structure of compound Cynanchum otophyllum Schneid glycosides oneself (otophylloside F, compound I) is as shown in formula I：
Compound Russell he order 3-O- β-D- oleandroses pyranose-(1 → 4)-β-D- cymaroses pyranose-(1
→ 4)-β-D- cymaroses [rostratamine 3-O- β-D-oleandropyranosyl- (1 → 4)-β-D-
Cymaropyranosyl- (1 → 4)-β-D-cymaropyranoside, compound II] structure as shown in formula II：
Compound taiwanoside C (compound III) structure is as shown in formula III：
Compound wilfoside C1N (compound IV) structure is as shown in formula IV：
Compound wilfoside K1N (compound V) structure is as shown in formula V：
Compound cynawilfoside A (compound VI) structure is as shown in formula VI：
Compound wilfoside C3N (compound VII) structure is as shown in formula VII：
Compound cynauricoside A (compound VIII) structure is as shown in formula VIII：
Compound Ⅸ：
Compound Ⅹ：
Compound Ⅺ：
Compound Ⅻ：
Compound XIII：
Compound XV：
Compound XVI：
And there is the partial monosomy compound for coming from Apocynaceae Cynanchum (Cynanchum) the significant epilepsy that promotees to live
Property.
Qingyanshengenin 3-O- β-D- oleandroses pyranose-(1 → 4)-β-D- cymaroses pyranose-(1 →
4) structural formula of-β-D- digitoxoses (compound a) is as shown in formula a：
(compound b) structural formula is as shown in formula b by Cynanchum otophyllum Schneid glycosides M：
Caudatin 3-O- β-D-Glucose pyranose-(1 → 4)-β-D- oleandroses pyranose-(1 → 4)-β-D- add
Put on airs sesame candy pyranose-(compound c) structural formula is as shown in formula c for (1 → 4)-β-D- cymaroses：
(compound d) structural formula is as shown in formula d by wilfoside M1N：
Pharmaceutical composition and application process
The compounds of this invention has excellent antiepileptic efficacy.The compounds of this invention can be applied to mammal (such as people),
Can orally, rectum, parenteral (intravenous, intramuscular or subcutaneous), the mode such as local be administered.The compound can individually give
Medicine, or with other pharmaceutically acceptable compound administering drug combinations.It may be noted that the present invention compound can mix to
Medicine.
Solid dosage forms for oral administration includes capsule, tablet, pill, powder and granule.In these solid formulations
In type, reactive compound mixes with least one conventional inert excipients (or carrier), such as sodium citrate or Dicalcium Phosphate, or with
Following compositions mix：(a) filler or bulking agent, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid；(b) bond
Agent, for example, hydroxymethyl cellulose, alginates, gelatin, PVP, sucrose and Arabic gum；(c) NMF, example
Such as, glycerine；(d) disintegrant, for example, agar, calcium carbonate, farina or tapioca, alginic acid, some composition silicates,
And sodium carbonate；(e) retarding solvent, such as paraffin；(f) absorbsion accelerator, for example, quaternary ammonium compound；(g) wetting agent, such as spermaceti
Alcohol and glycerin monostearate；(h) adsorbent, for example, kaolin；Lubricant, for example, talcum, calcium stearate, tristearin (i)
Sour magnesium, solid polyethylene glycol, lauryl sodium sulfate, or its mixture.In capsule, tablet and pill, formulation can also include
Buffer.
Solid dosage forms such as tablet, sugar-pill, capsule, pill and granule can use coating and shell material to prepare, such as casing and
Other materials well known in the art.They can include opacifying agent, also, reactive compound or compound in this composition
Release can be discharged in certain part in alimentary canal in a delayed fashion.The example of adoptable embedding component is polymeric material
And Wax.If necessary, reactive compound also can be with one or more formation microencapsulation forms in above-mentioned excipient.
Liquid formulation for oral administration includes pharmaceutically acceptable emulsion, solution, suspension, syrup or tincture.
Except active ingredient beyond the region of objective existence, liquid dosage form can include the inert diluent routinely used in this area, such as water or other solvents, increase
Solvent and emulsifying agent, example know, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propane diols, 1,3-BDO, dimethyl formyl
The mixture of amine and oil, particularly cottonseed oil, peanut oil, maize germ, olive oil, castor oil and sesame oil or these materials
Deng.
In addition to these inert diluents, composition can also include auxiliary agent, such as wetting agent, emulsifying agent and suspending agent, sweet taste
Agent and spices.
Except active ingredient beyond the region of objective existence, suspension can include suspending agent, for example, ethoxylation isooctadecane alcohol, polyoxyethylene
Sorbierite and the mixture of Isosorbide Dinitrate, microcrystalline cellulose, aluminium methoxide and agar or these materials etc..
Composition for parenteral injection can include physiologically acceptable sterile, aqueous or anhydrous solution, dispersion liquid,
Suspension or emulsion, and for being dissolved into the aseptic powdery of sterile Injectable solution or dispersion liquid again.It is suitable aqueous and
Nonaqueous carrier, diluent, solvent or excipient include water, ethanol, polyalcohol and its suitable mixture.
The formulation of the compounds of this invention for being locally administered includes ointment, powder, patch, propellant and inhalant.
Active component aseptically with physiologically acceptable carrier and any preservative, buffer, or if necessary may need
Propellant be mixed together.
The compounds of this invention can be administered alone, or with other active components (such as other antiepileptics clinically)
Administering drug combinations.
It is the mammal that the compounds of this invention of safe and effective amount is applicable to treatment during using pharmaceutical composition
(such as people), wherein dosage is the effective dosage pharmaceutically thought when applying, for the individual of 60 kg body weight, day is given
Pharmaceutical quantities are usually 1~1000mg, preferably 20~500mg.Certainly, specific dosage is also contemplated that method of administration, individual health shape
The factors such as condition, within the scope of these are all skilled practitioners technical ability.
Main advantages of the present invention are：
(1) first to Apocynaceae Cynanchum (Cynanchum) plant cynanchum auriculatum Royle ex Wight (C.auriculatum) and cynanchum wilfordii
(C.wilfordii) antiepileptic activity of one-component extract is evaluated, and is as a result found, extracting section thing has anti-insane
Epilepsy function, and extracting section thing has rush epilepsy effect；
(2) antiepileptic activity of the monomeric compound with antiepileptic activity involved by is superior to clinical application Cynanchum otophyllum Schneid
Middle principal component Qingyangshenglycoside A, Qingyanshengenin and the caudatin of piece；
(3) compound with antiepileptic activity involved by the content in plant is big, and source is wide, has greatly research
Value and DEVELOPMENT PROSPECT；
(4) compound cynawilfoside A are the noval chemical compound for separating and identifying first, and its antiepileptic activity is excellent
In positive drug dilantin sodium；
(5) obtained from Chinese medicine cynanchum auriculatum Royle rich in wilfoside C1N, wilfoside C3N, wilfoside
K1N, cynauricoside A isoreactivity compounds active component the step of it is simple, be easy to production application.
With reference to specific embodiment, further statement is of the invention.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.Unless otherwise indicated, otherwise percentage and number are calculated by weight.
Embodiment 1
The evaluation of the internal antiepileptic actions of compound cynawilfoside A (Formula IV)：
Compound cynawilfoside A preparation method：Fresh cynanchum wilfordii root tuber 10kg, peeling chip drying obtain
2.7kg dry products, crush with the extraction of 95% ethanol percolation three times, 2~3 days every time, merge extract solution, be concentrated under reduced pressure to give cynanchum wilfordii
Alcohol medicinal extract, extract is suspended with water, ethyl acetate extraction, obtain acetic acid ethyl ester extract, with 100 mesh silica gel mixed samples, use silicon
Plastic column chromatography (200-300 mesh silica gel) separates, with chloroform-methanol (10:1→5:1) gradient elution, component Fr1-Fr9 is obtained.
Fr6 further purifies to obtain pure compound cynawilfoside A (1.4g) repeatedly with petroleum ether-acetone and preparation HPLC.
Compound cynawilfoside A physicochemical data is as follows：White amorphous powder, is soluble in methanol, ethanol, and third
The organic solvents such as ketone, chloroform, molecular formula C63H96O20,1H NMR(CDCl3,500 MHz):δH 1.49(s,3H,H-18),
1.20,1.24,1.26,1.26 (d, each 3H, J=6.3 Hz, H-6 of sugar moiety), 1.15 (s, 3H, 19-
CH3), 1.16 (d, 3H, J=6.5 Hz, H-21), 3.39,3.40,3.41,3.45 (s, each 3H, 3-OCH3 of sugar
), moieties 4.76 (d, 1H, J=9.8, anomeric H), 4.77 (brs, 1H, anomeric H), 4.84 (1H, d, J=
9.6 Hz, anomeric H), 4.98 (brs, anomeric H), 5.37 (brs, 1H, H-6), 6.31 (d, 1H, J=16.0
Hz,HCin-2),7.37(m,3H,HCin-6,7,8),7.51(m,2H,HCin-5,9), 7.63 (d, 1H, J=16.0 Hz, HCin-3),2.33
(m,1H,H2′-methyl-butyryl-2),1.44(m,1H,H2′-methyl-butyryl- 3), 0.95 (t, 1H, J=6.5Hz,
H2′-methyl-butyryl- 4), 1.17 (d, 1H, J=6.5 Hz, H2′-methyl-butyryl-5)；13C NMR(CDCl3,125MHz):δC
38.9(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,CCin-1),119.1(d,CCin-2),144.7
(d,CCin-3),134.7(s,CCin-4),128.2(d,CCin-5,9),130.3(d,CCin-6,8),128.9(d,CCin-7),174.5(s,
C2′-methyl-butyryl-1),41.5(d,C2′-methyl-butyryl-2),26.4(t,C2′-methyl-butyryl-3),11.9(q,
C2′-methyl-butyryl-4),16.7(q,C2′-methyl-butyryl-5),95.8(d,Ccym1-1),34.2(t,Ccym1-2),77.2(d,
Ccym1-3),81.9(d,Ccym1-4),68.7(d,Ccym1-5),18.3(q,Ccym1-6),57.6(q,Ccym1-OMe),101.0(d,
Cdigin-1),31.7(t,Cdigin-2),73.9(d,Cdigin-3),74.5(d,Cdigin-4),66.9(d,Cdigin-5),18.2(q,
Cdigin-6),55.7(q,Cdigin-OMe),99.5(d,Ccym2-1),34.4(t,Ccym2-2),77.3(d,Ccym2-3),81.9(d,
Ccym2-4),69.2(d,Ccym2-5),18.4(q,Ccym2-6),57.1(q,Ccym2-OMe),98.8(d,Ccym3-1),31.2(t,
Ccym3-2),74.9(d,Ccym3-3),72.1(d,Ccym3-4),65.7(d,Ccym3-5),17.6(q,Ccym3-6),56.5(q,
Ccym3-OMe)。
Compound cynawilfoside A induce zebra fish epileptic attack mould to pentylenetetrazole (Pentylenetetrazole)
The protective effect of type：
The breeding of zebrafish embryo is carried out in a manner of natural paired cross, and mating every time prepares 4~5 pairs of average energy productions
The Adult Zebrafish of 200~300 embryos, dead embryo is removed within 6 hours and 24 hours in after fertilization, and according to the hair of embryo
The stage of educating selects suitable embryo, and embryo's (fish culture water water quality is incubated with fish culture water at 28 DEG C：Add in per 1L reverse osmosis waters
Enter 200mg Instant Oceans, electrical conductivity is 480~510 μ S/cm；PH is 6.9~7.2；Hardness is 53.7~71.6mg/L
CaCO3).After the completion of experiment, over-exposure processing is carried out to the zebra fish of each stage of development with tricaine methanesulfonic acid, so that will
Zebra fish anesthesia is put to death, and the operating procedure for anaesthetizing execution meets the specification that American Veterinary association (AVMA) puts to death to Animal Anesthesia and wanted
Ask.
Epilepsy model group：The zebra fish of pentylenetetrazole (Pentylenetetrazole) processing；Positive controls：Dilantin sodium
(Phenytoin Sodium)300μM；Blank group：Blank group (1%DMSO), remaining handles same model group.By testing compound
After being added to microwell plate, using the motion conditions of zebra fish in motion/behavioural analysis instrument record 60min, to the quick of zebra fish
Move (V>20mm/sec) distance (D) carries out quantitative analysis, the anti-epileptic therapeutic efficiency of medicine is calculated, according to zebra fish movement rail
Mark figure, qualitative evaluation is carried out to the anti-epileptic therapeutic effect of medicine.From Figure 1A, each concentration groups of cynawilfoside A with
Epilepsy model group is compared, and the quick movement locus of zebra fish significantly reduces.Figure 1B is according to the quick move distance average value of zebra fish
The column diagram that mean ± SE is drawn, from Figure 1B, compound cynawilfoside A have under 30,100,300 μM of concentration
Antiepileptic action, therapeutic efficiency are respectively 34%, 44.6%, 61.6% (P<0.05、P<0.01、P<0.01), dose-effect relationship is bright
Really.At the same time, antiepileptic action of the positive control drug dilantin sodium under 300 μM of concentration is only 57.2% (P<0.01).
Embodiment 2
The evaluation of the internal antiepileptic actions of compound wilfoside C1N (formula IV)：
Wilfoside C1N preparation method：A. fresh auricle cynanchum auriculatum Royle ex Wight root tuber 10kg, chip drying obtain 3Kg and done
Product, crushing are carried three times with 95% ethanol heat, and extract solution merges, and are concentrated under reduced pressure to give ethanol extract, silica gel mixed sample, are crossed 200-
300 mesh silica gel, with petroleum ether-acetone (3：1→2：1) gradient elution, then with chloroform-methanol (10:1→5:1) gradient elution,
Obtain component Fr1-Fr6.Fr3 is further purified repeatedly with petroleum ether-acetone silica gel column chromatography, obtains pure compound
wilfoside C1N(1.5g)；B. fresh cynanchum wilfordii root tuber 10kg, peeling chip drying obtain 2.7kg dry products, crushed with 95%
Ethanol percolation extracts three times, 2~3 days every time, merges extract solution, is concentrated under reduced pressure to give the alcohol medicinal extract of cynanchum wilfordii, by extract with
Water is suspended, and ethyl acetate extraction, acetic acid ethyl ester extract is obtained, with 100 mesh silica gel mixed samples, with silica gel column chromatography (200-300 mesh silicon
Glue) separation, with chloroform-methanol (10:1→5:1) gradient elution, component Fr1-Fr9 is obtained.Fr6 further uses petroleum ether-acetone
Purify to obtain pure compound wilfoside C1N (2.5g) repeatedly with HPLC is prepared.
Compound wilfoside C1N physicochemical data is as follows：White amorphous powder, is soluble in methanol, ethanol, and third
The organic solvents such as ketone, chloroform, molecular formula C56H90O19, ESIMS m/z 1089 [M+Na]+；1H NMR(pyridine-d5,300
MHz):δH0.95,0.97 (d, each 3H, J=6.5Hz, HIkem-5,6),1.32(s,3H,H-19),1.36,1.37,1.40,
1.48 (d, each 3H, J=6.0Hz, H-6 of sugar moieties), 1.96 (s, 3H, H-18), 2.29 (s, 3H,
HIkem-7),2.49(s,3H,H-21),3.45,3.48,3.52,3.53(s,each 3H,3-OCH3 of sugar
), moieties 5.02 (brd, 1H, J=3.0 Hz, anomeric H), 5.09 (d, 1H, J=9.3 Hz, anomeric H),
5.15 (brd, 1H, J=3.0 Hz, anomeric H), 5.22 (d, 1H, J=9.0 Hz, anomeric H), 5.30 (brs,
1H,H-6),5.84(br s,1H,HIkem-2)；13C NMR(Pyridine-d5,100 MHz):δC 39.2(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,CIkem-1),114.1(d,CIkem-2),165.5(s,CIkem-3),38.1(s,
CIkem-4),20.8(q,CIkem-5),20.9(q,CIkem-6),16.4(q,CIkem-7),96.1(d,Ccym1-1),35.3(t,Ccym1-2),
77.6(d,Ccym1-3),82.4(d,Ccym1-4),69.2(d,Ccym1-5),18.7(q,Ccym1-6),57.3(q,Ccym1-OMe),100.9
(d,Cdigin-1),32.5(t,Cdigin-2),73.8(d,Cdigin-3),74.6(d,Cdigin-4),67.4(d,Cdigin-5),17.9(q,
Cdigin-6),55.3(q,Cdigin-OMe),99.4(d,Ccym2-1),36.3(t,Ccym2-2),77.8(d,Ccym2-3),82.3(d,
Ccym2-4),69.4(d,Ccym2-5),18.6(q,Ccym2-6),58.2(q,Ccym2-OMe),99.0(d,Ccym3-1),32.1(t,
Ccym3-2),76.4(d,Ccym3-3),73.3(d,Ccym3-4),66.4(d,Ccym3-5),18.1(q,Ccym3-6),56.6(q,
Ccym3-OMe)。
Compound wilfoside C1N induce zebra fish epileptic attack model to pentylenetetrazole (Pentylenetetrazole)
Protective effect：
Qualitative evaluation is carried out to the anti-epileptic therapeutic effect of compound according to the method in embodiment 1.From Fig. 2A,
During 300 μM of wilfoside C1N dosage compared with epilepsy model group, the quick movement locus of zebra fish significantly reduces.Figure
2B is the column diagram drawn according to the quick move distance average value mean ± SE of zebra fish, from Fig. 2 B, compound
Wilfoside C1N have epilepsy therapy trend, but no difference of science of statistics under 30,100 μM of concentration；It is insane under 300 μM of concentration
Epilepsy therapeutic efficiency is 46.4%, (P<0.01), dose-effect relationship is clear and definite.
Embodiment 3
The evaluation of the internal antiepileptic actions of compound wilfoside K1N (Formula V)：
Wilfoside K1N preparation method：A. fresh cynanchum auriculatum Royle ex Wight root tuber 10kg, chip drying obtain 3kg dry products, powder
Broken to be carried three times with 95% ethanol heat, extract solution merges, and is concentrated under reduced pressure to give ethanol extract, silica gel mixed sample, crosses 200-300 mesh silicon
Glue, with petroleum ether-acetone (3：1→2：1) gradient elution, then with chloroform-methanol (10:1→5:1) gradient elution, group is obtained
Divide Fr1-Fr6.Fr3 is further purified repeatedly with petroleum ether-acetone silica gel column chromatography, obtains pure compound wilfoside K1N
(2.5g)；B. fresh cynanchum wilfordii root tuber 10kg, peeling chip drying obtain 2.7kg dry products, crush and extracted with 95% ethanol percolation
Three times, every time 2~3 days, merge extract solution, be concentrated under reduced pressure to give the alcohol medicinal extract of cynanchum wilfordii, extract is suspended with water, acetic acid second
Ester extracts, and obtains acetic acid ethyl ester extract, with 100 mesh silica gel mixed samples, is separated with silica gel column chromatography (200-300 mesh silica gel), with chlorine
Imitation-carbinol (10:1→5:1) gradient elution, component Fr1-Fr9 is obtained.Fr7 is further anti-with petroleum ether-acetone and preparation HPLC
Multiple purifying obtains pure compound wilfoside K1N (1.7g).
Compound wilfoside K1N physicochemical data is as follows：White amorphous powder, is soluble in methanol, ethanol, and third
The organic solvents such as ketone, chloroform, molecular formula C58H86O19, ESIMS m/z 1109 [M+Na]+；1H NMR(pyridine-d5,300
MHz):1.36 (s, 3H, H-19), 1.37,1.38,1.42,1.49 (d, each 3H, J=6.0Hz, H-6 of sugar
moieties),2.02(s,3H,H-18),2.54(s,3H,H-21),3.42,3.44,3.53,3.54(s,each 3H,3-
OCH3Of sugar moieties), 4.98 (brd, 1H, J=3.0 Hz, anomeric H), 5.09 (d, 1H, J=8.0
Hz, anomeric H), 5.15 (brd, 1H, J=3.0 Hz, anomeric H), 5.23 (d, 1H, J=9.0 Hz, anomeric
), H 5.32 (brs, 1H, H-6), 6.81 (d, 1H, J=16.0 Hz, HCin-2)7.38(m,3H,HCin-6,7,8),7.65(m,2H,
HCin-5,9), 7.99 (d, 1H, J=16.0 Hz, HCin-3)；13C NMR(Pyridine-d5,100 MHz):δC 39.2(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,CCin-1),119.2(d,CCin-2),145.0(d,CCin-3),
135.0(s,CCin-4),128.6(d,CCin-5,9),129.3(d,CCin-6,8),130.6(d,CCin-7),96.1(d,Ccym1-1),
35.3(t,Ccym1-2),77.5(d,Ccym1-3),82.5(d,Ccym1-4),69.2(d,Ccym1-5),18.7(q,Ccym1-6),57.3(q,
Ccym1-OMe),100.9(d,Cdigin-1),32.5(t,Cdigin-2),73.8(d,Cdigin-3),74.6(d,Cdigin-4),67.4(d,
Cdigin-5),17.9(q,Cdigin-6),55.3(q,Cdigin-OMe),99.4(d,Ccym2-1),36.3(t,Ccym2-2),77.8(d,
Ccym2-3),82.3(d,Ccym2-4),69.4(d,Ccym2-5),18.7(q,Ccym2-6),58.3(q,Ccym2-OMe),99.0(d,
Ccym3-1),32.1(t,Ccym3-2),76.4(d,Ccym3-3),73.2(d,Ccym3-4),66.4(d,Ccym3-5),18.2(q,Ccym3-6),
56.6(q,Ccym3-OMe)。
Compound wilfoside K1N induce zebra fish epileptic attack model to pentylenetetrazole (Pentylenetetrazole)
Protective effect：
Qualitative evaluation is carried out to the anti-epileptic therapeutic effect of compound according to the method in embodiment 1.From Fig. 3 A,
During 100 μM of wilfoside K1N dosage compared with epilepsy model group, the quick movement locus of zebra fish significantly reduces.Figure
3B is the column diagram drawn according to the quick move distance average value mean ± SE of zebra fish, from Fig. 3 B, compound
Wilfoside K1N have epilepsy therapy trend, but no difference of science of statistics under 10,30 μM of concentration；Have under 100 μM of concentration anti-
Epilepsy acts on, and epilepsy therapy efficiency is 49% (P<0.01), dose-effect relationship is clear and definite.
Embodiment 4
The evaluation of the internal antiepileptic actions of compound taiwanoside C (formula III)：
Taiwanoside C preparation method：A. fresh cynanchum auriculatum Royle ex Wight root tuber 10kg, chip drying obtain 3kg dry products, powder
Broken to be carried three times with 95% ethanol heat, extract solution merges, and is concentrated under reduced pressure to give ethanol extract, silica gel mixed sample, crosses 200-300 mesh silicon
Glue, with petroleum ether-acetone (3：1→2：1) gradient elution, then with chloroform-methanol (10:1→5:1) gradient elution, group is obtained
Divide Fr1-Fr6.Fr4 is further purified repeatedly with chloroform-methanol silica gel column chromatography, obtains pure compound taiwanoside C
(80.4mg)；B. fresh cynanchum wilfordii root tuber 10kg, peeling chip drying obtain 2.7kg dry products, and crushing is carried with 95% ethanol percolation
Take three times, 2~3 days every time, merge extract solution, be concentrated under reduced pressure to give the alcohol medicinal extract of cynanchum wilfordii, extract is suspended with water, acetic acid
Ethyl ester extracts, and obtains acetic acid ethyl ester extract, with 100 mesh silica gel mixed samples, is separated with silica gel column chromatography (200-300 mesh silica gel), with
Chloroform-methanol (10:1→5:1) gradient elution, component Fr1-Fr9 is obtained.Fr6 further with petroleum ether-acetone and prepares HPLC
Purifying obtains pure compound taiwanoside C (640mg) repeatedly.
Compound taiwanoside C physicochemical data is as follows：White amorphous powder, is soluble in methanol, ethanol, and third
The organic solvents such as ketone, chloroform, molecular formula C49H80O18, ESIMS m/z 979 [M+Na]+；1H NMR(CDCl3,300 MHz):
δH1.14 (s, 3H, H-18), 1.20,1.21,1.22,1.24 (d, each 3H, J=6.3 Hz, H-6 of sugar
Moieties), 1.27 (s, 3H, H-19), 2.34 (s, 3H, H-21), 3.25,3.30 (dd, each 1H, J=9.6,2.7 Hz,
Hcym-4),3.37,3.40,3.41,3.45(s,each 3H,3-OCH3 of sugar moieties),3.70,3.77(q,
Each 1H, J=3.0 Hz, Hcym-3), 3.83,3.86,4.04 (dq, each 1H, J=8.9,6.4 Hz, Hcym-5),3.85
(brs,1H,Hdigin-4), 3.96 (brq, 1H, J=6.3 Hz, Hdigin-5), 4.75 (d, 1H, J=9.8, anomeric H),
4.77 (brs, 1H, anomeric H), 4.81 (1H, d, J=9.6 Hz, anomeric H), 4.97 (brd, J=3.0Hz,
anomeric H),5.36(brs,1H,H-6)。13C NMR(Pyridine-d5,125 MHz):δC 39.4(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,CAc-1),21.2(q,CAc-2),96.6(d,Ccym1-1),35.7(t,Ccym1-2),
78.0(d,Ccym1-3),82.8(d,Ccym1-4),69.7(d,Ccym1-5),19.2(q,Ccym1-6),57.7(q,Ccym1-OMe),101.4
(d,Cdigin-1),32.9(t,Cdigin-2),74.3(d,Cdigin-3),75.0(d,Cdigin-4),67.9(d,Cdigin-5),18.3(q,
Cdigin-6),55.8(q,Cdigin-OMe),99.8(d,Ccym2-1),36.8(t,Ccym2-2),78.1(d,Ccym2-3),82.8(d,
Ccym2-4),69.9(d,Ccym2-5),18.9(q,Ccym2-6),58.4(q,Ccym2-OMe),99.5(d,Ccym3-1),32.6(t,
Ccym3-2),76.9(d,Ccym3-3),73.7(d,Ccym3-4),66.9(d,Ccym3-5),19.1(q,Ccym3-6),57.0(q,
Ccym3-OMe)。
Compound taiwanoside C induce zebra fish epileptic attack model to pentylenetetrazole (Pentylenetetrazole)
Protective effect：
Qualitative evaluation is carried out to the anti-epileptic therapeutic effect of compound according to the method in embodiment 1.From Fig. 4 A,
Under each dosages of taiwanoside C compared with epilepsy model group, the quick movement locus of zebra fish significantly reduces.Fig. 4 B are root
The column diagram drawn according to the quick move distance average value mean ± SE of zebra fish, from Fig. 4 B, compound taiwanoside C
Under 100 μM of concentration, epilepsy therapy efficiency is 28.1%, no difference of science of statistics；There is anti-epileptic under 300,1000 μM of concentration
Effect, therapeutic efficiency is respectively 48.5%, 84.8% (P<0.01、P<0.01), dose-effect relationship is clear and definite.
Embodiment 5
The evaluation of compound Cynanchum otophyllum Schneid glycosides oneself (otophylloside F) (Formulas I) internal antiepileptic action：
The preparation method of Cynanchum otophyllum Schneid glycosides oneself (otophylloside F)：Dry Cynanchum otophyllum Schneid root tuber 5kg, crush with 95%
Ethanol percolation extracts three times, 2~3 days every time, merges extract solution, is concentrated under reduced pressure to give the alcohol medicinal extract of Cynanchum otophyllum Schneid, by extract with
Water is suspended, and ethyl acetate extraction, obtains acetic acid ethyl ester extract, C-18 reversed-phase column chromatographies with the aqueous solution of methanol (10%~
100%) gradient elution, component Fr1-Fr5, Fr4 is obtained and further purifies to obtain pure compound Cynanchum otophyllum Schneid repeatedly with preparation HPLC
Glycosides oneself (otophylloside F, 1.1g).
Oneself physicochemical data of compound Cynanchum otophyllum Schneid glycosides is as follows：White amorphous powder, it is soluble in methanol, ethanol, acetone, chlorine
It is imitative to wait organic solvent, molecular formula C63H96O20,1H NMR(CDCl3,400 MHz):δH1.04,1.06 (d, each 3H, J=
6.5Hz,HIkem-5,6), 1.11 (s, 3H, H-19), 1.24,1.26,1.33 (d, each 3H, J=6.0Hz, H-6 of sugar
moieties),1.40(s,3H,H-18),2.11(s,3H,HIkem-7),2.16(s,3H,H-21),3.38,3.45(s,each
3H,3-OCH3Of sugar moieties), 4.49 (dd, 1H, J=2.0,9.6 Hz, anomeric H), 4.81 (dd, 1H,
J=2.2,9.3 Hz, anomeric H), 4.92 (dd, 1H, J=2.2,9.6 Hz, anomeric H), 5.35 (brs, 1H, H-
6),5.51(br s,1H,HIkem-2)；13C NMR(CDCl3,125 MHz):δC 38.9(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,CIkem-1),113.1(d,CIkem-2),166.1(s,CIkem-3),38.3(s,CIkem-4),20.9(q,
CIkem-5),21.0(q,CIkem-6),16.7(q,CIkem-7),95.9(d,Cdigit-1),37.2(t,Cdigit-2),75.5(d,
Cdigit-3),82.6(d,Cdigit-4),68.2(d,Cdigit-5),18.3(q,Cdigit-6),98.5(d,Ccym-1),35.7(t,
Ccym-2),77.0(d,Ccym-3),82.5(d,Ccym-4),68.8(d,Ccym-5),18.1(q,Ccym-6),58.5(q,Ccym-OMe),
101.6(d,Cole-1),35.4(t,Cole-2),80.7(d,Cole-3),75.5(d,Cole-4),71.6(d,Cole-5),18.1(q,
Cole-6),56.4(q,Cole-OMe)。
Compound Cynanchum otophyllum Schneid glycosides oneself zebra fish epileptic attack model is induced to pentylenetetrazole (Pentylenetetrazole)
Protective effect：
Qualitative evaluation is carried out to the anti-epileptic therapeutic effect of compound according to the method in embodiment 1.From Fig. 5 A,
Under oneself each dosage of Cynanchum otophyllum Schneid glycosides compared with epilepsy model group, the quick movement locus of zebra fish significantly reduces.According to Fig. 5 B
The column diagram that the quick move distance average value mean ± SE of zebra fish is drawn, from Fig. 5 B, compound Cynanchum otophyllum Schneid glycosides oneself 30,
Under 100 μM of concentration, there are epilepsy therapy trend, but no difference of science of statistics；There are antiepileptic action, epilepsy therapy under 300 μM of concentration
Efficiency is 55% (P<0.01), dose-effect relationship is clear and definite.
Embodiment 6
Compound Russell he order 3-O- β-D- oleandroses pyranose-(1 → 4)-β-D- cymaroses pyranose-(1
→ 4)-β-D- cymaroses [rostratamine 3-O- β-D-oleandropyranosyl- (1 → 4)-β-D-
Cymaropyranosyl- (1 → 4)-β-D-cymaropyranoside] and (Formula II) (hereinafter referred to as " compound II ") it is anti-in vivo
The evaluation of epilepsy effect：
Compound II preparation method：Dry Cynanchum otophyllum Schneid root tuber 5kg, crush with the extraction of 95% ethanol percolation three times, often
Secondary 2~3 days, merge extract solution, be concentrated under reduced pressure to give the alcohol medicinal extract of Cynanchum otophyllum Schneid, extract is suspended with water, ethyl acetate extraction,
Acetic acid ethyl ester extract is obtained, C-18 reversed-phase column chromatographies obtain component with the aqueous solution (10%~100%) gradient elution of methanol
Fr1-Fr5, Fr4 further purify to obtain pure compound II 100mg repeatedly with preparation HPLC.
rostratamine3-O-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→
4)-β-D-cymaropyranoside
Compound II physicochemical data is as follows：White, needle-shaped crystals, it is organic molten to be soluble in methanol, ethanol, acetone, chloroform etc.
Agent, molecular formula C63H96O20,1H NMR(CDCl3,400 MHz):δH 1.46(s,3H,H-18),1.20,1.22,1.31(d,
Each 3H, J=6.3 Hz, H-6 of sugar moiety), 1.12 (s, 3H, 19-CH3),2.19(s,3H,H-21),
3.38,3.44,3.44(s,each 3H,3-OCH3Of sugar moieties), 4.48 (dd, 1H, J=1.9,9.8,
Anomeric H), 4.74 (dd, J=1.9,9.8,1H, anomeric H), 4.84 (dd, 1H, J=1.8,9.6 Hz,
Anomeric H), 5.37 (brs, 1H, H-6), 6.30 (d, 1H, J=16.0 Hz, HCin-2),7.38(m,3H,HCin-6,7,8),
7.51(m,2H,HCin-5,9), 7.62 (d, 1H, J=16.0 Hz, HCin-3)；13C NMR(Pyridine-d5,125 MHz):δC
39.9(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,CCin-1),119.1(d,CCin-2),
144.7(d,CCin-3),135.0(s,CCin-4),128.6(d,CCin-5,9),129.3(d,CCin-6,8),130.6(d,CCin-7),
96.3(d,Ccym1-1),37.9(t,Ccym1-2),77.8(d,Ccym1-3),83.4(d,Ccym1-4),68.7(d,Ccym1-5),18.3(q,
Ccym1-6),58.6(q,C),100.5(d,Ccym2-1),37.2(t,Ccym2-2),78.0(d,Ccym2-3),83.2(d,Ccym2-4),
68.9(d,Ccym2-5),18.5(q,Ccym2-6),58.8(q,Ccym2-OMe),102.2(d,Cole-1),37.2(d,Cole-2),81.3
(d,Cole-3),76.2(d,Cole-4),72.8(d,Cole-5),18.6(d,Cole-6),57.1(d,Cole-OMe),
Compound II induces pentylenetetrazole (Pentylenetetrazole) protective effect of zebra fish epileptic attack model：
Qualitative evaluation is carried out to the anti-epileptic therapeutic effect of compound according to the method in embodiment 1.From Fig. 6 A,
Under each dosages of compound II compared with epilepsy model group, the quick movement locus of zebra fish significantly reduces.Fig. 6 B are according to spot
The column diagram that the quick move distance average value mean ± SE of horse fish is drawn, is had from Fig. 6 B, compound II under 30 μM of concentration
Epilepsy therapy trend, but no difference of science of statistics；There is antiepileptic action under 100,200 μM of concentration, therapeutic efficiency is respectively
65%th, 52% (P<0.01、P<0.01).
Embodiment 7
The evaluation of the internal antiepileptic actions of compound wilfoside C3N (Formula VII)：
Wilfoside C3N preparation method：Dry cynanchum auriculatum Royle root tuber 10kg, crushing are carried with 95% ethanol percolation
Take three times, 2~3 days every time, merge extract solution, be concentrated under reduced pressure to give the alcohol medicinal extract of cynanchum auriculatum Royle, extract is suspended with water,
Ethyl acetate extracts, and obtains acetic acid ethyl ester extract, ethyl acetate extract is dissolved in 70% ethanol solution, and filtering or centrifugation remove
After insoluble matter, macroporous resin column chromatography separation is carried out, collects 75% ethanol elution flow point, active site, active portion are obtained after concentration
Position further purifies to obtain pure Wilfoside C3N repeatedly with preparation HPLC.
Wilfoside C3N physicochemical data is as follows：White amorphous powder, it is soluble in methanol, ethanol, acetone, chloroform
Deng organic solvent, molecular formula C49H78O16,1H NMR(Pyridine-d5,400 MHz):δH 0.92,0.94(d,each 3H,
J=6.5Hz, HIkem-5,6), 1.32 (s, 3H, H-19), 1.36,1.34,1.51 (d, each 3H, J=6.0Hz, H-6 of
sugar moieties),1.97(s,3H,H-18),2.26(s,3H,HIkem-7),2.50(s,3H,H-21),3.42,3.44,
3.51(s,each 3H,3-OCH3Of sugar moieties), 5.24 (dd, 1H, J=1.9,9.4, anomeric H),
5.15 (d, J=3.3,1H, anomeric H), 5.10 (dd, 1H, J=1.8,9.8 Hz, anomeric H), 5.86 (br s,
1H,HIkem-2)；13C NMR(Pyridine-d5,125 MHz):δC 39.0(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,CIkem-1),114.1(d,CIkem-2),165.5(s,CIkem-3),38.2(s,CIkem-4),20.9(q,
CIkem-5),20.9(q,CIkem-6),16.5(q,CIkem-7),96.1(d,Ccym1-1),35.3(t,Ccym1-2),77.5(d,Ccym1-3),
82.5(d,Ccym1-4),69.2(d,Ccym1-5),18.8(q,Ccym1-6),57.2(q,C),101.0(d,Cdigin-1),32.5(t,
Cdigin-2),74.5(d,Cdigin-3),73.8(d,Cdigin-4),67.5(d,Cdigin-5),17.9(q,Cdigin-6),55.3(q,
Cdigin-OMe),99.5(d,Ccym2-1),35.3(d,Ccym2-2),79.9(d,Ccym2-3),74.1(d,Ccym2-4),71.0(d,
Ccym2-5),18.7(d,Ccym2-6),58.0(d,Ccym2-OMe).
Wilfoside C3N induce pentylenetetrazole (Pentylenetetrazole) protection of zebra fish epileptic attack model
Effect：
Qualitative evaluation is carried out to the anti-epileptic therapeutic effect of compound according to the method in embodiment 1.From Fig. 7 A,
Under each dosages of wilfoside C3N compared with epilepsy model group, the quick movement locus of zebra fish significantly reduces.Fig. 7 B are root
The column diagram drawn according to the quick move distance average value mean ± SE of zebra fish, from Fig. 7 B, wilfoside C3N are in 100 μ
Under M concentration, there are epilepsy therapy trend, but no difference of science of statistics；There is antiepileptic action under 200 μM of concentration, therapeutic efficiency is
55.2% (P<0.001).
Embodiment 8
The evaluation of the internal antiepileptic actions of compound cynauricoside A (Formula VIII)：
Cynauricoside A preparation method：Dry cynanchum auriculatum Royle root tuber 10kg, crush with 95% ethanol percolation
Extraction three times, 2~3 days every time, merges extract solution, is concentrated under reduced pressure to give the alcohol medicinal extract of Cynanchum otophyllum Schneid, extract is suspended with water, second
Acetoacetic ester extracts, and obtains acetic acid ethyl ester extract, ethyl acetate extract is dissolved in 70% ethanol solution, and filtering or centrifugation remove not
After molten thing, macroporous resin column chromatography separation is carried out, collects 75% ethanol elution flow point, active site, active site are obtained after concentration
Further purify to obtain pure cynauricoside A repeatedly with preparation HPLC.
Cynauricoside A physicochemical data is as follows：White amorphous powder, it is soluble in methanol, ethanol, acetone, chlorine
It is imitative to wait organic solvent, molecular formula C51H74O16,1H NMR(Pyridine-d5,400 MHz):δH 2.03(s,3H,H-18),
1.37,1.45,1.52 (d, each 3H, J=6.3 Hz, H-6 of sugar moiety), 1.35 (s, 3H, 19-CH3),
2.49(s,3H,H-21),3.52,3.42,3.45(s,each 3H,3-OCH3 of sugar moieties),5.24(dd,
1H, J=1.9,9.4, anomeric H), 5.15 (d, J=3.3,1H, anomeric H), 5.10 (dd, 1H, J=1.8,9.8
Hz, anomeric H), 5.30 (brs, 1H, H-6), 6.81 (d, 1H, J=16.0 Hz, HCin-2),7.34(m,3H,
HCin-6,7,8),7.63(m,2H,HCin-5,9), 8.00 (d, 1H, J=16.0 Hz, HCin-3)；13C NMR(Pyridine-d5,125
MHz):δC 38.9(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,CCin-1),119.2(d,CCin-2),
144.9(d,CCin-3),135.0(s,CCin-4),128.6(d,CCin-5,9),129.3(d,CCin-6,8),130.6(d,CCin-7),
96.1(d,Ccym1-1),35.3(t,Ccym1-2),77.5(d,Ccym1-3),82.5(d,Ccym1-4),69.2(d,Ccym1-5),18.8(q,
Ccym1-6),57.2(q,C),101.0(d,Cdigin-1),32.5(t,Cdigin-2),74.5(d,Cdigin-3),73.8(d,Cdigin-4),
67.5(d,Cdigin-5),17.9(q,Cdigin-6),55.3(q,Cdigin-OMe),99.5(d,Ccym2-1),35.3(d,Ccym2-2),79.9
(d,Ccym2-3),74.1(d,Ccym2-4),71.0(d,Ccym2-5),18.7(d,Ccym2-6),58.0(d,Ccym2-OMe).
Cynauricoside A induce pentylenetetrazole (Pentylenetetrazole) guarantor of zebra fish epileptic attack model
Shield acts on：
Qualitative evaluation is carried out to the anti-epileptic therapeutic effect of compound according to the method in embodiment 1.From Fig. 8 A,
Under each dosages of cynauricoside A compared with epilepsy model group, the quick movement locus of zebra fish significantly reduces.Fig. 8 B are
The column diagram drawn according to the quick move distance average value mean ± SE of zebra fish, from Fig. 8 B, cynauricoside A exist
30th, under 100 μM of concentration, there are epilepsy therapy trend, but no difference of science of statistics；There is antiepileptic action under 200 μM of concentration, treat
Efficiency is respectively 57.5% (P<0.01).
Comparative example 1
Separation and Extraction is to the larger compound Qingyanshengenin of content in three kinds of plants of Cynanchum
(qingyangshengenin), caudatin (caudatin), Qingyangshenglycoside A (otophylloside A), Cynanchum otophyllum Schneid glycosides third
(otophylloside C), Cynanchum otophyllum Schneid glycosides oneself (otophylloside F, Formulas I), Cynanchum otophyllum Schneid glycosides M (otophylloside M,
Formula b), caudatin 3-O- β-D- cymaroses [caudatin 3-O- β-D-cymaropyranoside, Formula IX], Cynanchum otophyllum Schneid
Aglycon 3-O- β-D- cymaroses pyranose-(1 → 4)-β-D- digitoxoses [qinyangshengenin3-O- β-D-
Cymaropyranosyl- (1 → 4)-β-D-digitoxopyranoside, Formula X VII], caudatin 3-O- β-D- apocynum cannabinums
Sugared pyranose-(1 → 4)-β-D- cymaroses [caudatin 3-O- β-D-cymaropyranosyl- (1 → 4)-β-D-
Cymaropyranoside, Formula X], Russell he order 3-O- β-D- oleandroses pyranose-(1 → 4)-β-D- cymaroses
Pyranose-(1 → 4)-β-D- cymaroses [rostratamine 3-O- β-D-oleandropyranosyl- (1 → 4)-β-
D-cymaropyranosyl- (1 → 4)-β-D-cymaropyranoside, Formula II], Russell he order 3-O- α-L- apocynum cannabinums
Sugared pyranose-(1 → 4)-β-D- cymaroses pyranose-(1 → 4)-α-L- diginoses pyranose-(1 → 4)-β-D-
Cymarose [rostratamine3-O- α-L-cymaropyranosyl- (1 → 4)-β-D-cymaropyranosyl- (1
→ 4)-α-L-diginopyranosyl- β-D-cymaropyranoside, Formula X III], Qingyanshengenin 3-O- β-D- folder bamboo
Peach sugar pyranose-(1 → 4)-β-D- cymaroses pyranose-(1 → 4)-β-D- digitoxoses
[qinyangshengenin3-O-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-
β-D-digitoxopyranoside, formula a], caudatin 3-O- β-D-Glucose pyranose-(1 → 4)-β-D- oleandroses
Pyranose-(1 → 4)-β-D- cymaroses pyranose-(1 → 4)-β-D- cymaroses [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- thevetoses pyranose-(1 → 4)-β-D- apocynum cannabinums
Sugared pyranose-(1 → 4)-β-D- cymaroses [caudatin 3-O- β-D-thevetopyranosyl- (1 → 4)-β-D-
Cymaropyranosyl- (1 → 4)-β-D-cymaropyranoside, Formula X II], Qingyanshengenin 3-O- β-D- oleandroses
Pyranose-(1 → 4)-β-D- cymaroses [qinyangshengenin3-O- β-D-oleandropyranosyl- (1 →
4)-β-D-cymaropyranoside, Formula X I], taiwanoside C (formula III), wilfoside K1N (Formula V),
Cynawilfoside A (Formula IV), wilfoside M1N (formula d), cynauricoside E (Formula X V), cynauricuoside
A (Formula X VI), wilfoside G (Formula X IV) influence under maximal non-toxic dosage on the zebra fish of PTZ inductions：
Qualitative evaluation is carried out to the anti-epileptic therapeutic effect of compound according to the method in embodiment 1.As shown in Figure 10, formula
I, the monomeric compound antiepileptic efficacy such as II, III, V, VI is superior to principal component Qingyanshengenin in Cynanchum otophyllum Schneid
(qingyangshengenin), caudatin (caudatin), Qingyangshenglycoside A (otophylloside A), Cynanchum otophyllum Schneid glycosides third
(otophylloside C), formula b compounds, formula c compounds, Formula IX compound, and Cynanchum otophyllum Schneid principal component formula a compounds, formula
Component formula d compounds have the epileptic attack of significant enhancing pentylenetetrazole induction in b compounds, formula c compounds and cynanchum wilfordii
Effect.
The preparation of the cynanchum auriculatum Royle extract extract of embodiment 9
By cynanchum auriculatum Royle medicinal material 10kg, 95% ethanol 5L is added, three times, the extract solution that is concentrated under reduced pressure obtains second to Soakage extraction
Alcohol medicinal extract, add water to be suspended, extracted with 5 times of volume of ethylacetate, reclaim ethyl acetate, ethyl acetate extract is dissolved in 3L 70%
In ethanol solution, filter off except after insoluble matter, macroporous resin column (resin model ZH801) chromatographic isolation is carried out, collect 75% second
Alcohol eluted fraction, concentration, obtains extract 15g.
After testing, in extract, antiepileptic activity ingredient w ilfoside C1N, wilfoside C3N, wilfoside
K1N, cynauricoside A content are respectively 17.4%, 42.1%, 7.1%, 14.2%, antiepileptic activity composition it is total
Content reaches more than 80%.In terms of promoting epilepsy composition, Cynanchum otophyllum Schneid glycosides M, Qingyanshengenin 3-O- β-D- oleandroses pyranose-
(1 → 4)-β-D- cymaroses pyranose-(1 → 4)-β-D- digitoxoses are not detected by.
Using the antiepileptic activity of the method evaluation cynanchum auriculatum Royle extract in embodiment 1, the results showed that, in 50 μ g/
Under mL concentration, there are epilepsy therapy trend, but no difference of science of statistics；There are antiepileptic action, epilepsy therapy efficiency under 100 μM of concentration
For 61.4% (p<0.001).
The preparation of the Qingyang conopsea extraction of embodiment 10
Cynanchum otophyllum Schneid medicinal material is extracted three times with 95% alcohol dipping, the extract solution that is concentrated under reduced pressure obtains ethanol extract, adds water to mix
It is outstanding, extracted with ethyl acetate, reclaim ethyl acetate, ethyl acetate extract is dissolved in 70% ethanol solution, filtering or centrifugation are gone
After insoluble matter, macroporous resin column chromatography separation is carried out, 75% ethanol elution flow point is collected, concentration, produces.
All it is incorporated as referring in this application in all documents that the present invention refers to, it is independent just as each document
It is incorporated as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read, those skilled in the art can
To be made various changes or modifications to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited
Enclose.
Claims (17)
1. a kind of plant total glycoside or the plant extracts containing the plant total glycoside, it is characterised in that the plant total glycoside is described
Contain antiepileptic activity composition in extract and promote epilepsy active component, and antiepileptic activity composition described in the extract
Content C1 meet following formula B：
C1/ (C1+C2) >=50% formulas B
In formula B, C1 is the content of antiepileptic activity composition；C2 is the content for promoting epilepsy active component；
Wherein, the one or more during described antiepileptic activity composition is selected from the group：
Compound shown in Formulas I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI
Described rush epilepsy active component be selected from the group in one or more：
Compound shown in formula a, b, c and d
2. plant total glycoside as claimed in claim 1 or the plant extracts containing the plant total glycoside, wherein, C1/ (C1+C2) >=
80%.
3. plant total glycoside as claimed in claim 2 or the plant extracts containing the plant total glycoside, wherein, C1/ (C1+C2) >=
90%.
4. plant total glycoside as claimed in claim 2 or the plant extracts containing the plant total glycoside, wherein, C1/ (C1+C2) >=
95%.
5. plant total glycoside as claimed in claim 2 or the plant extracts containing the plant total glycoside, wherein, C1/ (C1+C2) is
96% to 99.5%.
6. plant total glycoside as claimed in claim 2 or the plant extracts containing the plant total glycoside, wherein, C1/ (C1+C2) is
96%th, 97%, 98%, 99% or 99.5%.
7. plant total glycoside as claimed in claim 1 or the plant extracts containing the plant total glycoside, wherein, the plant is ear
Leaf cynanchum auriculatum Royle ex Wight (C.auriculatum Royle ex Wight) or cynanchum wilfordii (C.wilfordii Hemsl.).
8. a kind of pharmaceutical composition, it is characterised in that comprising plant total glycoside as claimed in claim 1 or containing the plant total glycoside
Plant extracts, and optional pharmaceutically acceptable carrier or excipient.
9. a kind of carry out method of quality control to anti-epileptic product, it is characterised in that including step：
(a) the content C2 for promoting epilepsy active component in the product or its raw material is determined, and compared with predetermined value C0；
(b) when the content C2 is higher than C0, then the off quality of the product is judged；When the content C2 is less than or equal to
During C0, then the up-to-standard of the product is judged；
Described rush epilepsy active component be selected from the group in one or more：
Compound shown in formula a, b, c and d
10. method as claimed in claim 9, it is characterised in that the anti-epileptic product includes the anti-of the conopsea extraction containing Qingyang
Epilepsy medicine.
11. method as claimed in claim 9, it is characterised in that the C2 is rush epilepsy active component in the product or original
Percentage by weight in material.
12. method as claimed in claim 9, it is characterised in that C0≤10% (w/w).
13. method as claimed in claim 9, it is characterised in that C0≤5% (w/w).
14. method as claimed in claim 9, it is characterised in that C0≤1% (w/w).
15. method as claimed in claim 9, it is characterised in that the C0 is 0.01% to 10% (w/w).
16. method as claimed in claim 9, it is characterised in that the C0 be 0.01%, 0.05%, 0.1%, 0.2%,
0.5%th, 1.0%, 5% or 10%.
17. purposes of the compound being selected from the group in medicament or reagent is prepared, the medicament or reagent are insane for building animal
Epilepsy model：Compound shown in formula a, b, c and d：
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| CN113069462A (en) * | 2021-04-06 | 2021-07-06 | 昆明理工大学 | New use of cynanchum otophyllum saponin M1 |
| CN113968837B (en) * | 2021-11-12 | 2022-09-06 | 中国科学院兰州化学物理研究所 | Compound with antiepileptic activity and application thereof in preparing antiepileptic medicine |
| CN117304032B (en) * | 2023-11-28 | 2024-03-22 | 江西省药品检验检测研究院 | Steroid sapogenin compound in radix clinopodii, and preparation method and application thereof |
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| CN102924554A (en) * | 2012-10-29 | 2013-02-13 | 中国科学院昆明植物研究所 | C-3,11,12,20-tetrasubstituted C-21 steroid derivative and its pharmaceutical composition and use in medicine |
| CN102946877A (en) * | 2010-03-18 | 2013-02-27 | 生物科技研究有限公司 | Neuro-protective effects of adelostemma gracillimum and its isolated compounds |
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| CN102946877A (en) * | 2010-03-18 | 2013-02-27 | 生物科技研究有限公司 | Neuro-protective effects of adelostemma gracillimum and its isolated compounds |
| CN102924554A (en) * | 2012-10-29 | 2013-02-13 | 中国科学院昆明植物研究所 | C-3,11,12,20-tetrasubstituted C-21 steroid derivative and its pharmaceutical composition and use in medicine |
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