CN104788525B - Triterpenoid with inhibiting activity of acetylcholinesterase and preparation method thereof - Google Patents

Triterpenoid with inhibiting activity of acetylcholinesterase and preparation method thereof Download PDF

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CN104788525B
CN104788525B CN201510114979.XA CN201510114979A CN104788525B CN 104788525 B CN104788525 B CN 104788525B CN 201510114979 A CN201510114979 A CN 201510114979A CN 104788525 B CN104788525 B CN 104788525B
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dichloromethane
layer
acetylcholinesterase
activity
normal hexane
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CN104788525A (en
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高品
高品一
牟晓锟
刘学贵
蒋福宇
赵鸿伟
王言
张振学
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Shenyang University of Chemical Technology
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07JSTEROIDS
    • C07J63/00Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms
    • C07J63/008Expansion of ring D by one atom, e.g. D homo steroids
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07JSTEROIDS
    • C07J73/00Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms
    • C07J73/001Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms by one hetero atom
    • C07J73/003Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms by one hetero atom by oxygen as hetero atom

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
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Abstract

Triterpenoid with inhibiting activity of acetylcholinesterase and preparation method thereof, relates to technical field of pharmaceuticals, and the present invention uses the technological means of tracking activity isolated and purified two lupinane type triterpenes noval chemical compounds from Chinese medicine Fructus Fructus Rosae Laevigatae skin.

Description

Triterpenoid with inhibiting activity of acetylcholinesterase and preparation method thereof
Technical field
The invention belongs to technical field of pharmaceuticals, relate to lupinane type triterpenoid compound, and with it as active component Pharmaceutical composition and preparation method thereof, particularly relates to triterpenoid and the preparation thereof with inhibiting activity of acetylcholinesterase Method.
Background technology
Alzheimer's disease (Alzheimer ' s disease, AD) it is with memory impairment, cognitive dysfunction, behavior barrier Hinder and drop to the chronic neurodegenerative disease of feature with self care ability, be modal disease in senile dementia.Old Dementia endangers the health of patient, and family and society to us bring white elephant.Along with China steps into always In age, therefore, AD is increasingly becoming one of problem the severeest that current geriatrics faces.Use acetyl on therapeutic treatment more Cholinesterase inhibitor (acetylcholinesterase inhibitor, AChEI) suppression AChE activity, delays ACh water The speed solved, improves the level of synaptic space ACh, thus plays the therapeutical effect to AD.The most also there is no medicine at present Thing can protect neuron completely, and the degraded of the internal ACh of inhibitory neuron is the Main Means for the treatment of senile dementia, AChEI It is the unique medicine as treatment AD of U.S. FDA license, is also one for the treatment of maximally effective medicine of AD.Recently from medium-height grass The research screening AChEI in medicine, the metabolite of microorganism and chemosynthesis receives much attention.
Chinese medicine Fructus Rosae Laevigatae Rosa laevigata Michx. is that Rosaceae gul is distributed widely in China. Beginning to be loaded in " another name for Sichuan Province book on Chinese herbal medicine ", its fruit, root and Ye Junke are used as medicine.Fructus Rosae Laevigatae described in Compendium of Material Medica: property is sour, puckery, flat, nontoxic; Cure mainly spleen and rush down dysentery, only normal urination, arresting seminal emission gas.Containing abundant triterpene saponin, flavone and polysaccharide chemical combination in Fructus Rosae Laevigatae fruit Thing, the multiformity of its chemical composition and pharmacologically active becomes the focus of Chinese scholars research in recent years.Through looking into, in prior art The compound that there are no the present invention and the phase of the activity with anti-senile dementia or neurodegenerative diseases value treatment prospect thereof Close report.
Summary of the invention
It is an object of the invention to provide triterpenoid with inhibiting activity of acetylcholinesterase and preparation method thereof, The present invention by Fructus Rosae Laevigatae peel chemical composition is studied, therefrom two lupinane new chemical combination of type triterpenes of isolated Thing, compound has the effect of significant acetylcholine esterase inhibition, is to have anti-senile dementia or neurodegenerative diseases valency The compound of value.
The above-mentioned purpose of the present invention is to be achieved by following technical scheme:
Having the triterpenoid of inhibiting activity of acetylcholinesterase, described compound includes following two, its structural formula For:
Having the triterpenoid preparation method of inhibiting activity of acetylcholinesterase, described method includes following preparation step Rapid:
(1) Fructus Rosae Laevigatae peel 6-10 times amount ethanol: water (3:1) microwave radiation exaraction 2-3 time, merging filtrate, concentrating under reduced pressure Obtain extractum;Extractum is dispersed in water, respectively by equal-volume normal hexane, dichloromethane, ethyl acetate, the repeated multiple times extraction of n-butyl alcohol Take, be concentrated under reduced pressure to give normal hexane layer, dichloromethane layer, ethyl acetate layer, n-butanol layer;
(2) respectively total extract, normal hexane layer, dichloromethane layer, ethyl acetate layer, n-butanol layer are carried out acetylcholine ester Enzyme inhibition activity is tested, and only dichloromethane activity is apparently higher than total extract activity, it is thus determined that it is active site;
(3) dichloromethane extract is carried out polyamide column separation, through normal hexane: dichloromethane: methanol mixes Gradient elution, concentrating under reduced pressure obtains component A-E.5 flow points also pass through inhibiting activity of acetylcholinesterase test, determine that D is alive Property position;
(4) component D is carried out C8 reversed phase chromatography separation, with methanol: water mixed solvent carries out gradient elution mutually for flowing, 10 flow points, wherein the 5th separate to obtain compound 1 and 2 with the 9th flow point through high performance liquid chromatography.
Having the triterpenoid of inhibiting activity of acetylcholinesterase, described compound is used in prepares anti-senile dementia or god In degenerative disease medicine.
The invention have the benefit that
Present invention firstly discloses the structure of two lupinane type tetraterpene derivatives in Chinese medicine Fructus Rosae Laevigatae peel.The present invention By Fructus Rosae Laevigatae peel chemical composition is studied, therefrom two lupinane type triterpenes noval chemical compounds of isolated, warp Crossing the Ellman method improved and carry out the mensuration of inhibiting activity of acetylcholinesterase, result shows that two noval chemical compounds have significantly The effect of acetylcholine esterase inhibition, and it presents obvious dose-effect and time-effect relationship, show this compounds prepare anti-ageing Application prospect in dementia and neurodegenerative diseases medicine.
Accompanying drawing explanation
Fig. 1 is compound 11H NMR spectra;
Fig. 2 is compound 113C NMR spectra;
Fig. 3 is the HSQC collection of illustrative plates of compound 1;
Fig. 4 is the HMBC collection of illustrative plates of compound 1;
Fig. 5 is compound 21H NMR spectra;
Fig. 6 is compound 213C NMR spectra;
Fig. 7 is the HSQC collection of illustrative plates of compound 2;
Fig. 8 is the HMBC collection of illustrative plates of compound 2;
Fig. 9 is sample and the huperzine A suppression curve chart to acetylcholinesterase;
Figure 10 is that lupinane type triterpenes noval chemical compound acetylcholine esterase inhibition presents dose-effect and time-effect relationship figure.
Detailed description of the invention
Further illustrate the essentiality content of the present invention below with embodiments of the invention, but do not limit this with this Invention.The simple modifications that the present invention is carried out by the essence according to the present invention broadly falls into the scope of the present invention.
The present invention has the triterpenoid compound 1 and 2 of following structural formula,
The method of the compound 1 and 2 described in preparation, after dry Fructus Fructus Rosae Laevigatae corium farinosum essence, adds the body of 6-10 times amount 400 watts of heating and refluxing extraction of ethanol water microwave that long-pending ratio is 3:1 2-3 time, merging filtrate, concentrating under reduced pressure obtains extractum;Extractum is divided It is dispersed in water, respectively with equal-volume normal hexane, dichloromethane, ethyl acetate, the repeated multiple times extraction of n-butyl alcohol, is concentrated under reduced pressure to give Normal hexane layer, dichloromethane layer, ethyl acetate layer, n-butanol layer;Respectively to total extract, normal hexane layer, dichloromethane layer, acetic acid Methacrylate layer, n-butanol layer carry out inhibiting activity of acetylcholinesterase test, and only dichloromethane activity is apparently higher than total extract activity, It is thus determined that it is active site;Dichloromethane extract is carried out polyamide column separation, according to normal hexane: dichloromethane: Methanol is 75:25:0,60:40:0,45:65:0,0:100:0,0:95:5,0:80:20,0:60:40,0: 0:100 Carrying out gradient elution, concentrating under reduced pressure obtains component A, B, C, D, E;5 flow points also pass through inhibiting activity of acetylcholinesterase test, Determine that D is active site;Component D is carried out C8 reversed phase chromatography separation gradient elution, by methanol: water is 5:5,6:4,7:3, 8:2,9:1,10:0 carry out gradient elution, and each gradient elution 1L, every 400mL collect a flow point, obtain 10 flow points, Qi Zhong 5 separate to obtain compound 1 and 2 with the 9th flow point through high performance liquid chromatography.
Embodiment 1:
Compound 1 and 2 isolated and purified:
Taking Fructus Rosae Laevigatae medical material 10kg, its crude drug source is Rosaceae gul Fructus Rosae Laevigatae Rosa laevigata Michx. dry peel is after smashing, with 10 times amount 400 watts of heating and refluxing extraction of ethanol water (3:1) microwave 3 times, each 2 little Time, merging filtrate, concentrating under reduced pressure obtains extractum;Extractum is dispersed in 5L water, extracts 3 times with equal-volume dichloromethane, reduce pressure dense Contract to obtain the extract 100g of dichloromethane fractions;Dichloromethane extract is carried out polyamide column separation, according to normal hexane: two Chloromethanes: methanol is 75:25:0,60:40:0,45:65:0,0:100:0,0:95:5,0:80:20,0:60:40, 0:0:100 carries out gradient elution, and concentrating under reduced pressure obtains five flow points of component A-E;By component D(9g) carry out C18 reversed phase chromatography separation Gradient elution, by methanol: water is 5:5,6:4,7:3,8:2,9:1,10:0 carry out gradient elution, each gradient elution 1L, every 400mL collect a flow point, obtain 10 flow points, and wherein the 5th flow point is through high performance liquid chromatography isocratic (methanol-water 80%) point Compound 1 is separated to obtain through high performance liquid chromatography isocratic (methanol-water 73%) from obtaining compound 2 (11.0 mg) same 9th flow point (33.5 mg).
The structural identification of compound 1 and 2:
The structural formula of compound 1 and 2 is as follows:
Compound 1: colorless needle crystals, is soluble in the organic solvents such as dichloromethane, methanol, high resolution mass spectrum HRESIMS m/z: [M+Na]+ 495.3478 (calcd For C30H48O4Na+, 4495.3445), 1H NMR, 13C NMR data is shown in Following table;
Compound 2: colorless needle crystals, is soluble in the organic solvents such as dichloromethane, methanol, high resolution mass spectrum HRESIMS m/z: [M+Na]+ 599.3719 [M + Na]+ (calcd C37H52O5Na+,599.3707), 1H NMR, 13C NMR data See table.
The Structural Identification data of compound 1 and 2:
Embodiment 2:
The compounds of this invention and with the anti-senile dementia of pharmaceutical composition of pharmaceutic adjuvant composition or nervus retrogression disease Sick pharmacological action.
The inhibiting activity of acetylcholinesterase experiment of compound 1 and 2.
Experimental principle: under certain condition, iodo acetylthiocholine resolves into iodo under the effect of acetylcholinesterase Thiocholine, iodo thiocholine acts on rapidly with developer DTNB, and the 5-sulfydryl-2-nitrobenzoic acid of generation is at 405nm Place has the yellow substance of UV Absorption.
Acetylcholinesterase and the effect of iodo acetylthiocholine
Iodo thiocholine and the coloration of DTNB
The preparation of compound 1 and 2 sample solution: accurately weigh sample 2mg, is placed in 15ml EP pipe, adds 10ml and contains The dehydrated alcohol of 0.1%DMSO, is diluted to 0.2mg/ml stock sample solution, stand-by.It is diluted to 2 μ g/ml, 10 μ g/ml, 30 μ respectively g/ml、60μg/ml、100μg/ml、150μg/ml、200μg/ml。
Preparation PBS: PBS powder is settled in 200ml volumetric flask, prepares to obtain the PBS of 0.1mol/L, pH7.4 Buffer.
The preparation of P-D solution: take 10 μ L DMSO and add in 15mlEP pipe, add the PBS of 9990 μ l.
The dilution of acetylcholinesterase: take the acetylcholinesterase of certain mass be diluted to respectively 0.2U/ml, 0.4U/ml, 0.6U/ml、0.8U/ml、1U/ml。
The preparation of DTNB: take 40mg and add 10ml dehydrated alcohol 10mmol/L, occur that yellow is i.e. abandoned, be placed in 4 DEG C of guarantors Deposit for;
The preparation of substrate A TCI: take 43mg and add 10ml dehydrated alcohol, prepare to obtain 15mmol/LATCI.
Laboratory operating procedures
Sample sets: be sequentially added into the PBS of 140 μ L 0.1mol/L pH=7.4,20 μ in 96 hole ELISA Plate L sample solution, 15 μ L 0.4 U/mL AChE, react 20min under the conditions of 4 DEG C.Add 10 μ l 15mmol/L substrate A TCI, Add 20 μ l 10mmol/L DTNB, under the conditions of 37 DEG C, react 30min, at 405nm wavelength, measure it by microplate reader and inhale Shading value, each sample gradient does three groups of parallel laboratory tests.
Group at the bottom of sample copy: replacing 15 μ L AChE solution with 15 μ L PBS buffer, other condition is constant.
Blank group: replacing 20 μ L testing sample solutions with 20 μ L PBS buffer, other condition is constant.
Completely inhibit control wells: with 20 μ L PBS buffer huperzine A solution (0.1mg/mL).
Suppression ratio (%)=[(blank group-completely inhibit group)-(group at the bottom of sample sets-sample copy)]/(blank group-completely inhibit Group) × 100%
The sample according to the different quality Concentraton gradient suppression ratio to acetylcholinesterase, draws sample and huperzine A pair The suppression curve of acetylcholinesterase, such as Figure 10, and obtains the IC of compound 1 and 2 by SPSS18 computed in software50It is worth as follows Table.Result shows that compound 1 and 2 can carry out subsequent derivation as the lead compound of anti-acetylcholinesterase activity and melt Send out.

Claims (1)

1. there is the triterpenoid of inhibiting activity of acetylcholinesterase, it is characterised in that described compound includes following two, Its structural formula is:
The preparation method of described triterpenoid comprises the following steps:
(1), after Fructus Fructus Rosae Laevigatae corium farinosum is broken, the ethanol water that volume ratio is 3:1 of Fructus Rosae Laevigatae peel 6-10 times amount, microwave-assisted are added Extracting 2-3 time, merging filtrate, concentrating under reduced pressure obtains extractum;Extractum is dispersed in water, respectively with equal-volume normal hexane, dichloromethane The repeated multiple times extraction of alkane, ethyl acetate, n-butyl alcohol, is concentrated under reduced pressure to give normal hexane layer, dichloromethane layer, ethyl acetate layer, just Butanol layer;
(2) respectively extractum total extract, normal hexane layer, dichloromethane layer, ethyl acetate layer, n-butanol layer are carried out acetylcholine ester Enzyme inhibition activity is tested, and only dichloromethane activity is apparently higher than total extract activity, it is thus determined that it is active site;
(3) dichloromethane extract is carried out polyamide column separation, through normal hexane: dichloromethane: methanol carries out mixing ladder Degree eluting, concentrating under reduced pressure obtains component A-E;
5 flow points also pass through inhibiting activity of acetylcholinesterase test, determine that D is active site;
(4) component D is carried out C8 reversed phase chromatography separation, with methanol: water mixed solvent carries out gradient elution mutually for flowing, obtains 10 streams Point, wherein the 5th separate to obtain compound 1 and 2 with the 9th flow point through high performance liquid chromatography.
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