阅读说明：本技术 一种黄精甾体皂苷元及其制备方法与应用 (A kind of rhizoma polygonati steroid sapogenin and the preparation method and application thereof ) 是由 吴毅 黄丽萍 袁铭铭 宁火华 殷海霞 邬秋萍 丁银平 潘蕾 于 2019-08-26 设计创作，主要内容包括：本发明公开了黄精甾体皂苷及黄精甾体皂苷元在制备治疗或预防阿尔茨海默症药物中的应用,黄精甾体皂苷及黄精甾体皂苷元结构如下：<Image he="358" wi="700" file="DDA0002179501820000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>其中,R<Sub>1</Sub>代表C1-C6的烷基或烯基,R<Sub>2</Sub>代表H或者单糖或寡聚糖。药理活性研究结果表明,黄精甾体皂苷及黄精甾体皂苷元具有很好的预防、治疗抗老年痴呆的作用。(The invention discloses rhizoma polygonati steroid saponins and rhizoma polygonati steroid sapogenin to treat or prevent the application in Alzheimer disease drug in preparation, and rhizoma polygonati steroid saponin and rhizoma polygonati steroid saponin meta structure are as follows: Wherein, R 1 Represent the alkyl or alkenyl of C1-C6, R 2 Represent H or monosaccharide or oligosaccharide.Pharmacology activity research the result shows that, rhizoma polygonati steroid saponin and rhizoma polygonati steroid sapogenin have the function of preventing, treating anti-senile dementia well.)

1. rhizoma polygonati steroid saponin and rhizoma polygonati steroid sapogenin treat or prevent the application in Alzheimer disease drug in preparation.

2. application as described in claim 1, it is characterised in that the rhizoma polygonati steroid saponin and rhizoma polygonati steroid saponin meta structure are such as Under:

Wherein, R₁Represent the alkyl or alkenyl of C1-C6, R₂Represent H or monosaccharide or oligosaccharide.

3. application as described in claim 1, it is characterised in that the R₁Representative=CH₂Or-CH₃, R₂Represent H；Alternatively, R₁Represent- CH₃, R₂It represents

4. a kind of rhizoma polygonati steroid sapogenin, it is characterised in that have the following structure:

5. the preparation method of rhizoma polygonati steroid sapogenin, the rhizoma polygonati steroid saponin meta structure is as follows:

The R₁Representative=CH₂Or-CH₃, R₂Represent H；

Include the following steps:

(1) after taking the dry rhizome of polygonatum cyrtonema, stew method to process, ethyl alcohol or ethanol water are extracted, are extracted with dichloromethane, dense Contraction obtains medicinal extract；

(2) after dissolving methylene chloride medicinal extract with methanol, silicagel column in dry method takes dry column with methylene chloride-methanol isocratic elution Part is cut into 5 parts from bottom to top, takes the 1st part of upper silicagel column, with the elution of petroleum ether-ethyl acetate linear gradient, successively 22 components carry out chromatographic isolation to the 17th component on ODS column, and with methanol-water solution linear gradient elution, successively obtain 5 components take the 2nd group of lease making high performance liquid preparative chromatography column, with first alcohol-water solution isocratic elution, obtain the rhizoma polygonati steroidal Sapogenin.

6. preparation method as claimed in claim 5, it is characterised in that step (1) is extracted using 70% ethanol solution.

7. preparation method as claimed in claim 5, it is characterised in that dissolved with methanol step (2) methylene chloride medicinal extract part Afterwards, silicagel column in dry method, is eluted with dichloro methane-methanol, and the volume ratio of methylene chloride and methanol is 20:1, elution 2.5 A column volume；Dry post part is taken, is cut into 5 parts from bottom to top, the 1st part of upper silicagel column is taken, with the petroleum of volume ratio 20:1~1:1 Ether-the column volume of ethyl acetate solution gradient elution 3 successively obtains 22 components, carries out chromatography point to the 17th component on ODS column From, and be 20:80~70:30 methanol-water solution linear gradient elution with volume ratio, 5 components are successively obtained, the 2nd component is taken Through high performance liquid preparative chromatography column, is eluted with volume ratio for the first alcohol-water solution of 65:35, obtain rhizoma polygonati steroid sapogenin.

8. the preparation method of rhizoma polygonati steroid saponin, the rhizoma polygonati steroid saponin structure is as follows:

The R₁Representative-CH₃, R₂It represents

Include the following steps:

(1) it after taking the dry rhizome of polygonatum cyrtonema, stew method to process, is extracted using ethyl alcohol or ethanol water, successively uses dichloromethane Butanol extraction liquid is concentrated and medicinal extract is made by alkane, ethyl acetate, extracting n-butyl alcohol water phase；

(2) n-butanol medicinal extract (1900g) is dissolved in water, is eluted through D101 macroporous resin column with ethanol-water solution, is successively obtained 4th component silica gel mixed sample is eluted by silica gel column chromatography with dichloro methane-methanol, obtains 6 by 5 components Component, by the silica gel mixed sample of the 5th component 1:1.5, silicagel column in dry method is eluted with dichloro methane-methanol, successively To 8 components, wherein pressing preparative liquid chromatography in the 5th group lease making, with methanol-water solution gradient elution, 7 components are obtained, The 6th component is taken to obtain 10 components by silica gel column chromatography with dichloro methane-methanol isocratic elution, wherein 8,9 components The rhizoma polygonati steroid saponin is obtained after recrystallizing.

9. preparation method as claimed in claim 8, it is characterised in that step (1) is extracted using 70% ethanol solution.

10. preparation method as claimed in claim 8, it is characterised in that n-butanol medicinal extract is dissolved in water by step (2), through D101 Macroporous resin column respectively elutes 2.5 column volumes with 10%, 30%, 50%, 70%, 95% ethyl alcohol, 70% ethanol eluate is subtracted Pressure is evaporated, and with the silica gel mixed sample of 1:1.5, silicagel column in dry method, is 50:1,30:1,20:1,10:1,5:1,3:2 with volume ratio Dichloro methane-methanol successively respectively elutes 3 column volumes, obtains 6 components, the 5th component is mixed with the silica gel of 1:1.5 Sample, silicagel column in dry method, is the methylene chloride-methanol of 50:1,30:1,20:1,10:1,5:1,3:1,3:2,1:1 with volume ratio Solution successively respectively elutes 3 column volumes, successively obtains 8 components, preparative liquid chromatography will be pressed in the 5th group of lease making, with volume ratio For 3 column volumes of 20:80~90:10 methanol-water solution gradient elution, 7 components are obtained, take the 6th component by silica gel column layer Analysis, is successively that 20:1,15:1,10:1,5:1,7:2,2:1 dichloro methane-methanol respectively elute 3 column volumes with volume ratio, 10 components are obtained, wherein obtaining the rhizoma polygonati steroid saponin after 8,9 groups of lease making recrystallizations.

Technical field

The invention belongs to tcm developments to utilize field, and in particular to a kind of new rhizoma polygonati steroid sapogenin Huangjingsterol B and the preparation method and application thereof.

Background technique

Rhizoma polygonati is that defend the newest promulgation of planning commission is both food and one of Chinese medicine of drug to country in 2016, feeding with tonifying Qi Yin, invigorating the spleen, moistening lung, kidney-nourishing effect are used for deficiency of spleen-QI and stomach-QI, fatigue and asthenia, deficiency of stomach-Yin, dry deficiency of food, deficiency syndrome of the lung cough caused by dryness, overstrain cough cough Blood, asthenia of essence and blood, soreness and weakness of waist and knees, poliosis, the illnesss such as Heat Diabetes.If traditionally rhizoma polygonati is not processed or is processed and do not conform to Reason, it is likely that certain side effects will be caused, raw sealwort is clinically should not use and apply RHIZOMA POLYGONATI PREPARATA.By processing, on the one hand Its toxic side effect can be made, numb taste is eliminated, mitigates the stimulation to throat, the clinical efficacy of rhizoma polygonati on the other hand can be improved, enhance medicine Object tonifying spleen moistening lung, the effect of kidney-nourishing.Stew rhizoma polygonati is that one of most representational medicine materical crude slice is helped in Jiangxi Jianchang, and there are history to surpass for stew method Spend 400 years.It is the RHIZOMA POLYGONATI ZINGIBERIFORME medicinal material for selecting East China that traditional Jianchang side, which prepares stew rhizoma polygonati, using traditional stew method Preparation is the prepared slices of Chinese crude drugs with distinct Jiangxi characteristic.Rhizoma polygonati contains there are many chemical component, mainly include polysaccharide, steroid saponin, Triterpene, alkaloid, lignanoid, flavones, plant steroid sapogenin and volatile oil etc., wherein polysaccharide and steroid saponin are its main medicine Ingredient is imitated, has the effects that hypoglycemic, reducing blood lipid, anti-inflammatory, antitumor, adjusting is immune.Modern clinic be usually used in treat diabetes, The diseases such as artery sclerosis.67 kinds of steroid saponin compounds are isolated to obtain from polygonatum kingianurn, polygonatum cyrtonema and rhizoma polygonati, according to aglycon Framework types it is different, can be divided into: spirostan alcohol type (I, II) C-25 is S configuration, and methyl is in axial bond；Different spirostan alcohol type (III) C-25 is R type, and methyl is located at equatorial bond, and chemical property is more stable, and there are more.The pure and mild different spirostan alcohol type of spirostan Aglycon is the precursor of other aglycons, i.e., other aglycons are derived by this 2 seed type.Spirostan alcohol type saponin(e is mostly single Sugar chain saponin(e, glycosyl are connected in the position C-3 of aglycon mostly, and minority is connected with glycosyl on the position C-23, C-24 or C-27 of F ring, are formed Bisdesmoside, C-3 sugar chains are made of a variety of glycosyls with different connection types.Common glycosyl has glucose, gala Sugar, rhamnose, arabinose, xylose and fucose.

Summary of the invention

The present invention passes through the methods of preparative liquid chromatography isolated 4 kinds of steroid saponins and aglycon: (25S) from stew rhizoma polygonati spirostan-5-en-12-one-3-O-D-glucopyranosyl-(1→2)-O-[β-D-Xylopyranosyl(1→ 3)]-O- β-D-glucopyranosyl (1 → 4)-β-D-galactopyranoside (huangjinoside A 1, compound 3), (25S)spirostan-5-en-12-one-3-O-β-D-glucopyranosyl-(1→2)-O-[β-D- Glucopyranosyl- (1 → 3)]-O- β-D-glucopyranosyl- (1 → 4)-β-D-galactopyranoside (rhizoma polygonati Saponin A 2, compound 4), (25R) -3- β-hydroxyspirost-5-en-12-one (huangjingsterol A, compound 1), 3- β-hydroxyspirost-5,25-diene-12-one (huangjingsterol B, compound 2), wherein rhizoma polygonati steroid Body sapogenin B (huangjingsterol B) is the new construction found for the first time, and the present invention further also discloses above-mentioned steroidal soap The preparation method of glycosides and aglycon prevents, treats the application in anti-alzheimer's drug with and preparation.

Specific technical solution of the present invention is as follows:

The application of rhizoma polygonati steroid saponin and rhizoma polygonati steroid sapogenin in preparation treatment or prevention Alzheimer disease drug. Rhizoma polygonati steroid saponin and rhizoma polygonati steroid sapogenin of the present invention are the chemical combination with rhizoma polygonati steroid sapogenin parent nucleus general formula structure Object, including different spirostan alcohol type aglycone-type and derivative obtained compound.

Preferably, the rhizoma polygonati steroid saponin and rhizoma polygonati steroid saponin meta structure are as follows:

Wherein, R₁Represent the alkyl or alkenyl of C1-C6, R₂Represent H or monosaccharide or oligosaccharide.

Further, the R₁Representative=CH₂Or-CH₃, R₂Represent H；Alternatively, R₁Representative-CH₃, R₂It represents

Compound 1: rhizoma polygonati steroid sapogenin B, compound 2: rhizoma polygonati steroid sapogenin A, compound 3: rhizoma polygonati steroid saponin A1 and compound 4: rhizoma polygonati trilling 2, structure is as follows:

Another object of the present invention is to provide a kind of new rhizoma polygonati steroid sapogenin B, have the following structure:

Another object of the present invention is to provide the preparation method of above-mentioned rhizoma polygonati steroid sapogenin, include the following steps:

(1) after taking the dry rhizome of polygonatum cyrtonema, stew method to process, ethyl alcohol or ethanol water are extracted, and are extracted with methylene chloride It takes, extract liquor, which is concentrated under reduced pressure, is made medicinal extract；

(2) after methylene chloride medicinal extract being dissolved with methanol, silicagel column in dry method, using methylene chloride-methanol as eluant, eluent etc. Degree elution, successively meets lower 5 parts of 2000ml eluent F1~F5 and 5 parts is cut to obtain dry column component F6~F10., with petroleum ether-acetic acid second Ester solution linear gradient elution successively obtains 22 components, carries out chromatographic isolation to the 17th component on ODS column, and use methanol-water Linear gradient elution, successively obtains 5 components, the 2nd group of lease making high performance liquid preparative chromatography column is taken, with first alcohol-water solution Isocratic elution obtains the rhizoma polygonati steroid sapogenin.

Preferably, step (1) is extracted using 70% ethanol solution.

Preferably, after step (2) methylene chloride medicinal extract part is dissolved with methanol, silicagel column in dry method, with methylene chloride-first Alcohol (20:1) is eluant, eluent isocratic elution, elutes 2.5 column volumes, successively meets lower 5 parts of 2000ml eluents F1~F5；Take dry column Part is cut into 5 parts from bottom to top, takes the 1st part of upper silicagel column, is washed with the petroleum ether-ethyl acetate solution of volume ratio 20:1~1:1 3 column volumes are taken off, 22 components are successively obtained, chromatographic isolation are carried out to the 17th component on ODS column, and be 20:80 with volume ratio ~70:30 methanol-water solution linear gradient elution successively obtains 5 components, takes the 2nd group of lease making high performance liquid preparative chromatography column, It is eluted with volume ratio for the first alcohol-water solution of 65:35, obtains rhizoma polygonati steroid sapogenin.

Specific operating procedure is as follows:

(1) after taking the dry rhizome of polygonatum cyrtonema, stew method to process, ethyl alcohol or ethanol water are extracted, and are extracted with methylene chloride It takes, extract liquor, which is concentrated, is made medicinal extract；

Specific operation are as follows: the dry rhizome for taking polygonatum cyrtonema cuts sheet, helps traditional concocting method stew method to carry out through Jianchang It processes, 50 DEG C are dried, crushed into coarse powder, and 70% ethyl alcohol soaked overnight, heating and refluxing extraction 2 times, each 2h, filtration, filtrate is closed And it is concentrated into small size, it is extracted with dichloromethane, extract liquor is concentrated into medicinal extract；

(2) after dissolving methylene chloride medicinal extract part with methanol, silicagel column is in dry method with methylene chloride-methanol (20:1) Eluant, eluent isocratic elution elutes 2.5 column volumes, successively meets lower 5 parts of 2000ml eluents F1~F5；Dry post part is taken, under It is cut into 5 parts on and, the 1st part of upper silicagel column is taken, with petroleum ether-ethyl acetate (preferably with volume ratio 20:1~1:1 linear elution) 3 column volumes are eluted, 22 component F6-a~F6-v are obtained, under TLC monitoring, component F6-q (1.46g) is carried out on ODS column Chromatography, and with methanol-water (20:80~70:30) gradient elution, obtain 5 subfraction F6-q-1~F6-q-5.Pass through height Effect liquid phantom preparing chromatogram method (methanol-water, 65:35) purifies subfraction F6-q-2, obtains 1 rhizoma polygonati steroid sapogenin of compound Huangjingsterol B (12.6mg) (noval chemical compound) and 2 rhizoma polygonati steroid sapogenin huangjingsterol A (30.2mg).It is preferred that reversed phase high efficiency preparative liquid chromatography condition is chromatographic column: YMC-Pack ODS-A (250 × 20mm, S-5 μm, 12nm) mobile phase: methanol-water=65:35 (V/V), flow velocity 7mL/min.

Another object of the present invention is to provide the preparation method of above-mentioned rhizoma polygonati steroid saponin, include the following steps:

(1) after taking the dry rhizome of polygonatum cyrtonema, stew method to process, ethyl alcohol or ethanol water are extracted, and successively use dichloromethane Alkane, ethyl acetate, extracting n-butyl alcohol water phase, butanol extraction liquid, which is concentrated, is made medicinal extract；

(2) n-butanol medicinal extract (1900g) is dissolved in water, is eluted through D101 macroporous resin column with ethanol-water solution, successively 5 components are obtained, by the 4th component silica gel mixed sample, by silica gel column chromatography, 3 columns are eluted with dichloro methane-methanol Volume obtains 6 components, by the 5th component silica gel mixed sample, by silica gel column chromatography, with dichloro methane-methanol elution 3 A column volume successively obtains 8 components, F5-a~F5-h.Preparative liquid chromatography wherein is pressed in F5-e warp, with methanol-water solution Gradient elution obtains 7 components, takes the 6th component by silica gel column chromatography, elutes 3 cylinders with dichloro methane-methanol Product, obtains 10 components, wherein obtaining the rhizoma polygonati steroid saponin after 8,9 groups of lease making recrystallizations.

Preferably, step (1) is extracted using 70% ethanol solution.

Preferably, medicinal extract is dissolved in water by step (2), through D101 macroporous resin column, with 10%, 30%, 50%, 70%, 95% column volume of ethanol elution 3,70% ethanol eluate are concentrated to dryness, with the silica gel mixed sample of 1:1.5, silica gel in dry method Column (100-200 mesh) is that eluant, eluent successively elutes 3 with methylene chloride-methanol (50:1,30:1,20:1,10:1,5:1,3:2) Column volume obtains 6 component F1~F6, and the 5th component F5 (10.88g) is evaporated, with the silica gel mixed sample of 1:1.5, silica gel in dry method Column (100-200 mesh), with methylene chloride-methanol (50:1,30:1,20:1,10:1,5:1,3:1,3:2,1:1) be eluant, eluent according to Secondary elution obtains 8 components of component F5-a~F5-h, preparative liquid chromatography will be pressed in the 5th component F5-e (1.9g) warp, with volume Than obtaining 7 components, the 6th component being taken to be evaporated, mixed with the silica gel of 1:1.5 for 20:80~90:10 methanol-water solution gradient elution Sample, silicagel column (100-200 mesh) in dry method, is successively 20:1,15:1,10:1,5:1,7:2,2:1 methylene chloride-with volume ratio Methanol solution respectively elutes 3 column volumes, obtains 10 components, wherein obtaining the rhizoma polygonati steroidal soap after 8,9 groups of lease making recrystallizations Glycosides.

Specific operating procedure is as follows:

(1) after taking the dry rhizome of polygonatum cyrtonema, stew method to process, 70% ethanol solution is extracted, and successively uses methylene chloride, second Acetoacetic ester, extracting n-butyl alcohol water phase, butanol extraction liquid, which is concentrated, is made medicinal extract；

Specific operation are as follows: the dry rhizome for taking polygonatum cyrtonema cuts sheet, helps traditional concocting method stew method to carry out through Jianchang It processes, 50 DEG C are dried, crushed into coarse powder, 70% ethyl alcohol soaked overnight, and heating and refluxing extraction 2h filters, is concentrated into small size, just Butanol, before immunoassay is concentrated into medicinal extract；

(2) take n-butanol portion dry cream 1900g, be dissolved in water to 10L, through D101 macroporous resin column (10%, 30%, 50%, 70%, 95% ethyl alcohol) elution, 70% ethanol eluate is concentrated to dryness, dry with the silica gel mixed sample of 1:1.5 Silicagel column (100-200 mesh) in method, with methylene chloride-methanol (50:1,30:1,20:1,10:1,5:1,3:2) be eluant, eluent according to 3 column volumes of secondary elution obtain 6 component F1~F6, and the 5th component F5 (10.88g) is evaporated, dry with the silica gel mixed sample of 1:1.5 Silicagel column (100-200 mesh) in method be with methylene chloride-methanol (50:1,30:1,20:1,10:1,5:1,3:1,3:2,1:1) Eluant, eluent successively elutes, and obtains 8 components of component F5-a~F5-h.Preparative liquid chromatography wherein is pressed in F5-e (1.9g) warp, with first Alcohol-water (20:80~90:10) gradient elution, monitors through TLC, and merging obtains 7 component F5-e-1~F5-e-7.F5-e-6 (900mg) passes through silica gel column chromatography (100-200 mesh), with methylene chloride-methanol (20:1,15:1,10:1,5:1,7:2,2:1) 3 column volumes are successively respectively eluted for eluant, eluent, obtain 10 component F5-e-6-1~F5-e-6-10 of component, wherein F5-e-6-8 (164mg) and F5-e-6-9 (122mg), which is precipitated, to be crystallized, and ((25S) spirostan-5- of compound 3 is respectively obtained after recrystallizing en-12-one-3-O-D-glucopyranosyl-(1→2)-O-[β-D-xylopyranosyl(1→3)]-O-β-D- Glucopyranosyl (1 → 4)-β-D-galactopyranoside) (45.2mg) (huangjinoside A 1) and compound 4 ((25S)spirostan-5-en-12-one-3-O-β-D-glucopyra nosyl-(1→2)-O-[β-D- glucopyranosyl-(1→3)]-O-β-D-glucopyranosyl-(1→4)-β-D-gal actopyranoside) (38.7mg) (huangjinoside A 2).

Alzheimer's disease (Alzheimer ' s disease, AD, also known as senile dementia) be a kind of person in middle and old age it is common with The gradually central nervous system degenerative disease that row failure of memory, cognition dysfunction, personality change are characterized.It is global old Dementia patient alreadys exceed 40,000,000, and with the aggravation of world population ages, patient will continue to increase, it is contemplated that the year two thousand fifty Patients of senile dementia number will be more than 1.5 hundred million, that is, 85 years old or more old man, just have 1 senile dementia to suffer from every 2 people Thus person will bring many society and economic problems, therefore have become very urgent society to the prevention and treatment of AD and ask with medicine Topic.Therapeutic agent is many kinds of at present, but mostly symptomatic treatment, and uncertain therapeutic efficacy is cut, specificity is not strong, toxic side effect is big, no Easily through blood-brain barrier etc., its clinical application is limited, therefore developing high-efficiency low-toxicity senile dementia protective agents is always state The research hotspot of inside and outside the world of medicine.The present invention isolated 4 kinds of steroid saponin constituents from stew rhizoma polygonati, especially obtain one kind New rhizoma polygonati steroid sapogenin huangjingsterol B, and the pharmacological activity of 4 kinds of steroid saponins is studied, as a result Show rhizoma polygonati steroid sapogenin huangjingsterol B and rhizoma polygonati steroid sapogenin huangjingsterol A and rhizoma polygonati soap Glycosides A1 and A2 have the function of preventing, treating anti-senile dementia well.

Detailed description of the invention

Fig. 1 is rhizoma polygonati steroid sapogenin huangjingsterol B and rhizoma polygonati steroid sapogenin huangjingsterol A Detection wavelength is that the HPLC of 210nm prepares chromatogram (t_{R huangjingsterol B}=182min, t_{R huangjingsterol A}= 212min)。

Specific embodiment

Illustrate specific steps of the invention by the following examples, but is not limited by the example.

Term as used in the present invention generally there are those of ordinary skill in the art usually to manage unless otherwise indicated The meaning of solution.

The present invention is described in further detail below with reference to specific example and referring to data.It should be understood that these embodiments are In order to demonstrate the invention, it rather than limits the scope of the invention in any way.

In the examples below, the various processes and method being not described in detail are conventional methods as known in the art.