CN107595838B - A kind of Lignanoids compounds are preparing the application in anti-liver fiber drug - Google Patents
A kind of Lignanoids compounds are preparing the application in anti-liver fiber drug Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/365—Lactones
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
- C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
- C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
- C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D307/58—One oxygen atom, e.g. butenolide
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- C—CHEMISTRY; METALLURGY
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- C07D—HETEROCYCLIC COMPOUNDS
- C07D407/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00
- C07D407/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings
- C07D407/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
Abstract
The present invention provides a kind of Lignanoids compounds and is preparing the application in anti-liver fiber drug.Lignanoids compounds of the present invention are after external, internal, molecular level experiment, prove that it has the function of the effect of stronger anti-hepatic stellate cell proliferation, anti-hepatic fibrosis, and there is lower IC50 value for MMP-2 (MMP2), Matrix Metalloproteinase-9 (MMP9) and transforming growth factor-beta 1 (TGF-β 1), have the function of good combination and inhibits its bioactivity, so can be used as anti-liver fiber drug.
Description
Technical field
The present invention relates to natural product chemistry drug fields, are preparing specifically, being related to a kind of Lignanoids compounds
Application in anti-liver fiber drug.
Background technique
Liver fibrosis is pathological change caused by chronic liver damage caused by many reasons, shows as liver extracellular matrix
Ingredient excessively deposits singularly, and influences the function of liver, is that chronic liver disease develops to the cirrhosis only stage which must be passed by.Research shows that liver
Fibrosis still has reverse to normal possibility, if to cirrhosis without reverse may, therefore find treatment liver fibrosis
Drug is the hot spot of numerous scholar's researchs.Though there are some progress in terms of diagnosis and treatment liver fibrosis at present, but still lacks and determine effectively
Drug.And the monomer of medicinal material and lignanoid rich in lignanoid has preferable drug activity in terms of anti-hepatic fibrosis.
Thinking can be provided for the treatment of liver fibrosis.
Lignanoid is a kind of natural phenolic compound, has the structure and extensive bioactivity of multiplicity.Its structure is two
The dimer of a Phenylpropanoid Glycosides unit (C6-C3).It common are aryl naphthalenes, dibenzylbutyrolactone class, tetrahydrofuran derivatives, dibenzyl
Butanes, the types such as cyclohexyl biphenyl octene class and neolignan.Lignanoid mainly extracts from natural materials and obtains, and is present in and is more than
In 70 kinds of plant, place position is different.Therefore there is presently no the sides for forming system, specification for the extraction and separation of lignanoid
Method is especially a lack of the method for being suitable for batch or prepare with scale.Plant rich in Lignanoids compounds has civil
Quite long medical history, with the extensive use of modern separation means, Structural Identification method and High Throughput Screening Assay, wood
The antitumor, anti-inflammatory of rouge element, pest-resistant, anti-oxidant, antirheumatic, liver protection, inhibits the bioactivity such as plant growth to be reported in succession at antibacterial
Road.
Summary of the invention
The purpose of the present invention is to provide a kind of Lignanoids compounds to prepare the application in anti-liver fiber drug.
The purpose of the present invention is what is be achieved through the following technical solutions:
The present invention provides a kind of Lignanoids compounds and is preparing the application in anti-liver fiber drug.
Lignanoids compounds of the present invention refer to the compound that C6-C3 unit is formed by connecting.
Wherein, preferably compound 1a, 1b, 1c, 1d, 1e, 2a；Most preferably compound 1d, 1e.
Lignanoids compounds of the present invention are to be extracted by natural radix bupleuri and obtained.
The preparation method comprising steps of
1) it takes dry HERBA BUPLEURI pulverizing medicinal materials at coarse powder, measures 75% ethyl alcohol heating and refluxing extraction 2-4 times with 8-10 times,
Each 1-2h, filtering, merging filtrate are concentrated under reduced pressure and remove ethyl alcohol, and stirring is diluted with water, successively with petroleum ether, ethyl acetate, just
The extraction of fourth alcohol and water；Extract liquor is collected, rotary evaporation obtains each extraction part；
2) ethyl acetate extraction part is dissolved in ethyl acetate makes its concentration reach 20-50mg/ml, crosses 200-300 mesh
Silica gel column chromatography, use petroleum ether-ethyl acetate system with 4:1 (v/v), 1:1 (v/v), 1:8 (v/v), 1:20 (v/v) is pure
Methanol system gradient elution；Eluent is collected, rotary evaporation obtains the elution fraction of 1:20；
3) elution fraction of 1:20 is dissolved in ethyl acetate makes its concentration reach 30-80mg/ml, crosses 200-300 purpose
Silica gel column chromatography uses petroleum ether-ethyl acetate system with 2:1 (v/v) isocratic elution；Each fraction collection 50ml, note first
A fraction is 1#, and number consecutively is merged after rotary evaporation by polarity, obtains eluting fraction 94-109# and 110- to be separated
128#；
4) eluting fraction 94-109# to be separated is dissolved in acetonitrile makes its concentration reach 30-80mg/ml, using reverse phase
Isocratic elution of the C18 column through 60% acetonitrile of row, the Detection wavelength of UV detector are 230-280nm, collect 41-43min and occur
Compound absorption peak；Fraction is named as compound 4b；
5) eluting fraction 110-128# to be separated is dissolved in acetonitrile makes its concentration reach 30-80mg/ml, using reverse phase
Isocratic elution of the C18 column through 50% acetonitrile of row, the Detection wavelength of UV detector are 230-280nm, collect 12-14min and occur
Compound absorption peak；Fraction is named as compound 1a, collects the compound absorption peak that 22-25min occurs；Being named as of fraction
Close object 4a；
6) n-butanol portion by 3 times amount acetone soln heating and refluxing extraction 3 times, each 30min.Extracting solution is collected,
Rotary evaporation obtains the insoluble part of acetone of the acetone soluble fraction and extracting n-butyl alcohol of extracting n-butyl alcohol；
7) the acetone soluble fraction of extracting n-butyl alcohol is dissolved in acetone makes its concentration reach 20-50mg/ml, crosses 200-
The silica gel column chromatography of 300 mesh uses acetate-methanol system with 100:1 (v/v), 75:1 (v/v), 25:1 (v/v), pure first
Alcohol system gradient elution；Eluent is collected, rotary evaporation obtains the elution fraction of 75:1；
8) elution fraction of 75:1 is dissolved in acetone makes its concentration reach 30-80mg/ml, crosses the silica gel of 200-300 mesh
Column chromatography uses cyclohexane-acetone system with 4:1 (v/v), 3:2 (v/v), 1:2 (v/v) gradient elution.Each fraction collection
50ml remembers that first fraction is 1#, and number consecutively is merged after rotary evaporation by polarity, obtains eluting fraction 3-5# to be separated
And 6-10#；
9) eluting fraction 3-5# to be separated is dissolved in acetonitrile makes its concentration reach 30-80mg/ml, using reverse phase C18
Isocratic elution of the column through 55% acetonitrile of row, the Detection wavelength of UV detector are 230-280nm, collect the change that 45-47min occurs
Object absorption peak is closed, fraction is named as compound 3a；The compound absorption peak that 48-49min occurs is collected, fraction is named as compound
5a；The compound absorption peak that 61-63min occurs is collected, fraction is named as compound 2a；
9) eluting fraction 6-10# to be separated is dissolved in acetonitrile makes its concentration reach 30-80mg/ml, using reverse phase C18
Isocratic elution of the column through 45% acetonitrile of row, the Detection wavelength of UV detector are 230-280nm, collect the change that 30-32min occurs
Object absorption peak is closed, fraction is named as compound 2b；The compound absorption peak that 35-37min occurs is collected, fraction is named as compound
5b；The compound absorption peak that 45-49min occurs is collected, fraction is named as compound 3b；
10) its concentration is made to reach 20-50mg/ml insoluble be partially soluble in acetone of the acetone of extracting n-butyl alcohol, mistake
The silica gel column chromatography of 100-200 mesh uses acetate-methanol system with 100:1 (v/v), and 4:1 (v/v), 1:1 (v/v) are pure
Methanol system gradient elution；Eluent is collected, rotary evaporation obtains the elution fraction of 4:1；
11) elution fraction of 4:1 is dissolved in acetone makes its concentration reach 30-80mg/ml, crosses the silica gel of 200-300 mesh
Column chromatography uses acetate-methanol system with 3:4 (v/v) isocratic elution；Each fraction collection 50ml remembers first fraction
For 1#, number consecutively is merged after rotary evaporation by polarity, obtains eluting fraction 1-3#, 4-8# and 9-12# to be separated；
12) eluting fraction 1-3# to be separated is dissolved in acetonitrile makes its concentration reach 30-80mg/ml, using reverse phase C18
Isocratic elution of the column through 35% acetonitrile of row, the Detection wavelength of UV detector are 230-280nm, collect the change that 18-20min occurs
Object absorption peak is closed, fraction is named as compound 1e；The compound absorption peak that 26-29min occurs is collected, fraction is named as compound
1d；
13) eluting fraction 4-8# to be separated is dissolved in acetonitrile makes its concentration reach 30-80mg/ml, using reverse phase C18
Isocratic elution of the column through 35% acetonitrile of row, the Detection wavelength of UV detector are 230-280nm, collect the change that 31-33min occurs
Object absorption peak is closed, fraction is named as compound 6a；The compound absorption peak that 36-37min occurs is collected, fraction is named as compound
1b；
14) eluting fraction 4-8# to be separated is dissolved in acetonitrile makes its concentration reach 30-80mg/ml, using reverse phase C18
Isocratic elution of the column through 30% acetonitrile of row, the Detection wavelength of UV detector are 230-280nm, collect the change that 25-28min occurs
Object absorption peak is closed, fraction is named as compound 1c；The compound absorption peak that 48-50min occurs is collected, fraction is named as compound
7a。
The compound that the present invention extracts, after external, internal, molecular level experiment, it was demonstrated that it is with stronger
The effect of anti-hepatic stellate cell proliferation, the effect of anti-hepatic fibrosis, and for MMP-2 (MMP2), matrix gold
Proteases -9 (MMP9) and transforming growth factor-beta 1 (TGF-β 1) have lower IC50 value, have good combination and press down
The effect of its bioactivity is made, so can be used as anti-liver fiber drug.
When the compounds of this invention is used for drug, the medicine of other useful clinically different dosage forms is can be used alone or is made
Object, dosage form include powder, injection, capsule, pill, microcapsules, tablet, film, soft capsule, paste, suppository, aerosol
Agent, tincture, oral solution, granule.It can add medically acceptable pharmaceutic adjuvant according to pharmaceutics, including filler,
Adhesive, wetting agent, disintegrating agent, pH adjusting agent or lubricant etc..
Specific embodiment
The present invention is described further below with reference to embodiment.
Unless stated otherwise, all percentages are percent by volume in the present invention.
The preparation method of 1 compound of embodiment
Lignanoids compounds described in the present embodiment are to be extracted by natural radix bupleuri and obtained, and detailed process is as follows:
1) it takes dry HERBA BUPLEURI medicinal material (originating from Chinese yunnan) 15kg to be ground into coarse powder, is added with 9 times of 75% ethyl alcohol of amount
Circumfluence distillation 3 times, each 1h, filtering, merging filtrate is concentrated under reduced pressure and removes ethyl alcohol, and stirring is diluted with water to 40L, successively uses
Petroleum ether, ethyl acetate, n-butanol and water extraction.Extract liquor is collected, rotary evaporation obtains each extraction part.
2) ethyl acetate extraction part is dissolved in ethyl acetate makes its concentration reach 20-50mg/ml, crosses 200-300 mesh
Silica gel column chromatography, use petroleum ether-ethyl acetate system with 4:1 (v/v), 1:1 (v/v), 1:8 (v/v), 1:20 (v/v) is pure
Methanol system gradient elution.Eluent is collected, rotary evaporation obtains the elution fraction of 1:20.
3) elution fraction of 1:20 is dissolved in ethyl acetate makes its concentration reach 30-80mg/ml, crosses 200-300 purpose
Silica gel column chromatography uses petroleum ether-ethyl acetate system with 2:1 (v/v) isocratic elution.Each fraction collection 50ml, note first
A fraction is 1#, and number consecutively is merged after rotary evaporation by polarity, obtains eluting fraction 94-109# and 110- to be separated
128#。
4) eluting fraction 94-109# to be separated is dissolved in acetonitrile makes its concentration reach 30-80mg/ml, using reverse phase
Isocratic elution of the C18 column through 60% acetonitrile of row, the Detection wavelength of UV detector are 230-280nm, collect 41-43min and occur
Compound absorption peak.Fraction is named as compound 4b.
5) eluting fraction 110-128# to be separated is dissolved in acetonitrile makes its concentration reach 30-80mg/ml, using reverse phase
Isocratic elution of the C18 column through 50% acetonitrile of row, the Detection wavelength of UV detector are 230-280nm, collect 12-14min and occur
Compound absorption peak.Fraction is named as compound 1a, collects the compound absorption peak that 22-25min occurs.Being named as of fraction
Close object 4a.
6) n-butanol portion by 3 times amount acetone soln heating and refluxing extraction 3 times, each 30min.Extracting solution is collected,
Rotary evaporation obtains the insoluble part of acetone of the acetone soluble fraction and extracting n-butyl alcohol of extracting n-butyl alcohol.
7) the acetone soluble fraction of extracting n-butyl alcohol is dissolved in acetone makes its concentration reach 20-50mg/ml, crosses 200-
The silica gel column chromatography of 300 mesh uses acetate-methanol system with 100:1 (v/v), 75:1 (v/v), 25:1 (v/v), pure first
Alcohol system gradient elution.Eluent is collected, rotary evaporation obtains the elution fraction of 75:1.
8) elution fraction of 75:1 is dissolved in acetone makes its concentration reach 30-80mg/ml, crosses the silica gel of 200-300 mesh
Column chromatography uses cyclohexane-acetone system with 4:1 (v/v), 3:2 (v/v), 1:2 (v/v) gradient elution.Each fraction collection
50ml remembers that first fraction is 1#, and number consecutively is merged after rotary evaporation by polarity, obtains eluting fraction 3-5# to be separated
And 6-10#.
9) eluting fraction 3-5# to be separated is dissolved in acetonitrile makes its concentration reach 30-80mg/ml, using reverse phase C18
Isocratic elution of the column through 55% acetonitrile of row, the Detection wavelength of UV detector are 230-280nm, collect the change that 45-47min occurs
Object absorption peak is closed, fraction is named as compound 3a；The compound absorption peak that 48-49min occurs is collected, fraction is named as compound
5a；The compound absorption peak that 61-63min occurs is collected, fraction is named as compound 2a.
9) eluting fraction 6-10# to be separated is dissolved in acetonitrile makes its concentration reach 30-80mg/ml, using reverse phase C18
Isocratic elution of the column through 45% acetonitrile of row, the Detection wavelength of UV detector are 230-280nm, collect the change that 30-32min occurs
Object absorption peak is closed, fraction is named as compound 2b；The compound absorption peak that 35-37min occurs is collected, fraction is named as compound
5b；The compound absorption peak that 45-49min occurs is collected, fraction is named as compound 3b.
10) its concentration is made to reach 20-50mg/ml insoluble be partially soluble in acetone of the acetone of extracting n-butyl alcohol, mistake
The silica gel column chromatography of 100-200 mesh uses acetate-methanol system with 100:1 (v/v), and 4:1 (v/v), 1:1 (v/v) are pure
Methanol system gradient elution.Eluent is collected, rotary evaporation obtains the elution fraction of 4:1.
11) elution fraction of 4:1 is dissolved in acetone makes its concentration reach 30-80mg/ml, crosses the silica gel of 200-300 mesh
Column chromatography uses acetate-methanol system with 3:4 (v/v) isocratic elution.Each fraction collection 50ml remembers first fraction
For 1#, number consecutively is merged after rotary evaporation by polarity, obtains eluting fraction 1-3#, 4-8# and 9-12# to be separated.
12) eluting fraction 1-3# to be separated is dissolved in acetonitrile makes its concentration reach 30-80mg/ml, using reverse phase C18
Isocratic elution of the column through 35% acetonitrile of row, the Detection wavelength of UV detector are 230-280nm, collect the change that 18-20min occurs
Object absorption peak is closed, fraction is named as compound 1e；The compound absorption peak that 26-29min occurs is collected, fraction is named as compound
1d。
13) eluting fraction 4-8# to be separated is dissolved in acetonitrile makes its concentration reach 30-80mg/ml, using reverse phase C18
Isocratic elution of the column through 35% acetonitrile of row, the Detection wavelength of UV detector are 230-280nm, collect the change that 31-33min occurs
Object absorption peak is closed, fraction is named as compound 6a；The compound absorption peak that 36-37min occurs is collected, fraction is named as compound
1b。
14) eluting fraction 4-8# to be separated is dissolved in acetonitrile makes its concentration reach 30-80mg/ml, using reverse phase C18
Isocratic elution of the column through 30% acetonitrile of row, the Detection wavelength of UV detector are 230-280nm, collect the change that 25-28min occurs
Object absorption peak is closed, fraction is named as compound 1c；The compound absorption peak that 48-50min occurs is collected, fraction is named as compound
7a。
15 compounds13C NMR data is as shown in table 1.
2 mtt assay of embodiment presses down the hepatic stellate cells experiment of activation in vitro
The present embodiment carries out the hepatic stellate cells reality that mtt assay presses down activation in vitro for Lignanoids compounds of the present invention
It tests.
Experimental material: human liver microsome proteins strain LX-2, cultured rat hepatic stellate cells strain T-6
Test medicine: it is 100 μM, 20 μM, 4 μM, 0.8 μM and 0.16 μM that 15 compounds, which prepare final concentration gradient,.
Positive control: it is 100 μM, 20 μM, 4 μM, 0.8 μM and 0.16 μM that saikoside d, which prepares final concentration gradient,.
By taking human liver microsome proteins strain LX-2 as an example, 15 compounds are measured to hepatic stellate cells after activation using mtt assay
Inhibiting effect.
(1) strain of LX-2 human liver microsome proteins counts, and cell is made 5 × 104The cell suspension of a/mL.
(2) cell suspension is taken, is inoculated in 96 porocyte culture plates, every 200 μ L of hole.37 DEG C, 5%CO2Saturated humidity
It is cultivated in incubator for 24 hours, it is adherent to cell.
(3) every hole sequentially add TGF-β 1 to final concentration of 10ng/mL be incubated for for 24 hours.
(3) thereafter, test medicine is added in 96 orifice plates, every group is all provided with 5 multiple holes.Solvent control group and bavin are set up simultaneously
Hu saponin(e d positive controls.37 DEG C, 5%CO248h is cultivated in the incubator of saturated humidity.
(4) 10 μ L MTT solution (5mg/mL is dissolved in PBS, 0.22 μm of film filtering), culture is added in every culture hole thereafter
Stationary culture 4h again in case.
(5) supernatant is sucked out, 150 μ L dimethyl sulfoxides (DMSO) are added in every hole, and micro oscillator vibrates 5min.
(6) finally with microplate reader at 490nm wavelength, each hole light absorption value is measured.Statistical data simultaneously carries out data analysis.
T-6 cultured rat hepatic stellate cells are also adopted to be tested through row with the aforedescribed process.Compound is starlike to the people liver after activation thin
The IC of born of the same parents' strain LX-2 and mouse hepatic stellate cells T-650Value see the table below 2.
Table 2
By table 2, statistics indicate that, the compound in the present invention has the significant proliferation function for inhibiting activation hepatic stellate cells,
Lower IC50 value, i.e., stronger inhibition activation are shown in human liver microsome proteins strain LX-2, cultured rat hepatic stellate cells strain T-6
The effect of hepatic stellate cell proliferation.And the overwhelming majority has identical as positive control saikoside d or higher inhibition thin
The effect of born of the same parents' proliferation, shows the effect of stronger anti-hepatic stellate cell proliferation in vitro experiment, and it is fine to can be used as new anti-liver
The development and utilization of chemical drug object are tieed up, especially compound 1d and 1e is preferred.
Anti-hepatic fibrosis is tested in 3 rat body of embodiment
The present embodiment carries out anti-hepatic fibrosis experiment in rat body for compound of the present invention.
Experimental animal: male SD rat, SPF grades, 6-8 week old, weight 180+-20 is purchased from the Chinese military medicine academy of sciences
Experimental Animal Center, quality certification number: SCXK- (army) 2012-2004. normal adaptation rearing conditions: independent isolating rearging cage is raised
It supports, 18-21 DEG C of temperature, humidity > 40%, 150Pa pressure air blowing, circulation light is according to (12h illumination, 12h are dark), feed of freely intaking
(standard particle feed) changes a padding and feed every other day.
Experimental group: being only randomly divided into 18 groups for experimental rat 265, respectively normal group (10), model group (15),
Compound 1a group (15), compound 1b group (15), compound 1c group (15), compound 1d group (15), compound 1e
Group (15), compound 2a group (15), compound 2b group (15), compound 3a group (15), compound 3b group (15),
Compound 4a group (15), compound 4b group (15), compound 5a group (15), compound 5b group (15), compound 14
Group (15), compound 6a group (15) and saikoside 7b group (15).
(1) blank group, after adaptive feeding 1 week terminates, life is given once daily in continuous 3d in a manner of being injected intraperitoneally weekly
Salt water 0.1mL/100g is managed, gives physiological saline 0.1mL/100g daily with intraperitoneal injection, is administered 4 weeks；
(2) model group, after adaptive feeding 1 week terminates, continuous 3d is given once daily 1% in a manner of being injected intraperitoneally weekly
DMN, 0.1mL/100g give physiological saline 0.1mL/100g daily with intraperitoneal injection, are administered 4 weeks；
(3) compound 1a group, after adaptive feeding 1 week terminates, continuous 3d is given daily in a manner of being injected intraperitoneally weekly
1%DMN, 0.1mL/100g are given, gives compound 1,2mg/kg daily with intraperitoneal injection, is administered 4 weeks；
(4) compound 1b group, answering property are raised 1 week after terminating, and continuous 3d is given once daily in a manner of being injected intraperitoneally weekly
1%DMN, 0.1mL/100g give compound 2,2mg/kg daily with intraperitoneal injection, are administered 4 weeks；
(5) compound 1c group, answering property are raised 1 week after terminating, and continuous 3d is given once daily in a manner of being injected intraperitoneally weekly
1%DMN, 0.1mL/100g give compound 3,2mg/kg daily with intraperitoneal injection, are administered 4 weeks；
(6) compound 1d group, answering property are raised 1 week after terminating, and continuous 3d is given once daily in a manner of being injected intraperitoneally weekly
1%DMN, 0.1mL/100g give compound 4,2mg/kg daily with intraperitoneal injection, are administered 4 weeks；
(7) compound 1e group, answering property are raised 1 week after terminating, and continuous 3d is given once daily in a manner of being injected intraperitoneally weekly
1%DMN, 0.1mL/100g give compound 5,2mg/kg daily with intraperitoneal injection, are administered 4 weeks；
(8) compound 2a group, answering property are raised 1 week after terminating, and continuous 3d is given once daily in a manner of being injected intraperitoneally weekly
1%DMN, 0.1mL/100g give compound 6,2mg/kg daily with intraperitoneal injection, are administered 4 weeks；
(9) compound 2b group, answering property are raised 1 week after terminating, and continuous 3d is given once daily in a manner of being injected intraperitoneally weekly
1%DMN, 0.1mL/100g give compound 7,2mg/kg daily with intraperitoneal injection, are administered 4 weeks；
(10) compound 3a group, answering property are raised 1 week after terminating, and continuous 3d is given once daily in a manner of being injected intraperitoneally weekly
1%DMN, 0.1mL/100g give compound 8,2mg/kg daily with intraperitoneal injection, are administered 4 weeks；
(11) compound 3b group, answering property are raised 1 week after terminating, and continuous 3d is given once daily in a manner of being injected intraperitoneally weekly
1%DMN, 0.1mL/100g give compound 9,2mg/kg daily with intraperitoneal injection, are administered 4 weeks；
(12) compound 4a group, answering property are raised 1 week after terminating, and continuous 3d is given once daily in a manner of being injected intraperitoneally weekly
1%DMN, 0.1mL/100g give compound 10,2mg/kg daily with intraperitoneal injection, are administered 4 weeks；
(13) compound 4b group, answering property are raised 1 week after terminating, and continuous 3d is given once daily in a manner of being injected intraperitoneally weekly
1%DMN, 0.1mL/100g give compound 11,2mg/kg daily with intraperitoneal injection, are administered 4 weeks；
(14) compound 5a group, answering property are raised 1 week after terminating, and continuous 3d is given once daily in a manner of being injected intraperitoneally weekly
1%DMN, 0.1mL/100g give compound 12,2mg/kg daily with intraperitoneal injection, are administered 4 weeks；
(15) compound 5b group, answering property are raised 1 week after terminating, and continuous 3d is given once daily in a manner of being injected intraperitoneally weekly
1%DMN, 0.1mL/100g give compound 13,2mg/kg daily with intraperitoneal injection, are administered 4 weeks；
(16) compound 6a group, answering property are raised 1 week after terminating, and continuous 3d is given once daily in a manner of being injected intraperitoneally weekly
1%DMN, 0.1mL/100g give compound 14,2mg/kg daily with intraperitoneal injection, are administered 4 weeks；
(17) compound 7a group, answering property are raised 1 week after terminating, and continuous 3d is given once daily in a manner of being injected intraperitoneally weekly
1%DMN, 0.1mL/100g give compound 15,2mg/kg daily with intraperitoneal injection, are administered 4 weeks；
(18) saikoside d group, answering property are raised 1 week after terminating, and continuous 3d is given daily in a manner of being injected intraperitoneally weekly
1%DMN, 0.1mL/100g are given, gives saikoside d daily with intraperitoneal injection, 2mg/kg is administered 4 weeks；
Fasting 12h after rat feeding 4 weeks, 10% chloraldurate is with 0.35ml/100g intraperitoneal injection of anesthesia, heart extracting blood,
After the static 3-4h of the blood of taking-up, 3500r/min is centrifuged 10min, takes supernatant, it is spare that -80 DEG C of refrigerators are stored in after packing.Take the heart after blood
Dirty Saline perfusion quickly wins liver and spleen, and physiological saline cleans blood stains, and filter paper blots the weighing of post analysis balance, meter
Calculate liver index and spleen index.It wins rat liver lobus sinister to be placed in neutral fixer, standby pathology detect.Remaining liver freezes in -80
DEG C, it is used for subsequent Indexs measure.
15 compounds see the table below 3 to the influence result of the Serum Indexes of rat liver fibrosis treatment respectively.
Table 3
#P < 0.05, ##p < 0.01 .*P < 0.05 compared with normal group, p < 0.01 * * is compared with model group.
15 compounds see the table below 4 to the influence result of the liver index of rat liver fibrosis treatment respectively.
Table 4
#P < 0.05, ##p < 0.01 .*P < 0.05 compared with normal group, p < 0.01 * * is compared with model group.
Statistics indicate that, show that 15 compounds are lured in DMN in terms of rat blood serum index and liver index two by table 4
Rat Liver Fibrosis Model is led with preferable anti-hepatic fibrosis drug activity.And the compound 1-15 overwhelming majority has and sun
Property the equivalent pharmaceutical activity of control saikoside d.15 compounds show the work of stronger anti-hepatic fibrosis in vivo experiment
With can be used as new anti-hepatic fibrosis medicines development and utilization.
The anti-hepatic fibrosis activity of 3 molecular level of embodiment is tested
The present embodiment carries out the anti-hepatic fibrosis activity experiment of molecular level for 15 compounds of the present invention respectively.
MMP2 activity Elisa kit, MMP9 activity Elisa kit, 1 activity Elisa kit of TGF-β and
MMP2, MMP9,1 protein standard liquid of TGF-β are provided by Beijing Dong Ge Biotechnology Co., Ltd.
Five concentration gradients are respectively configured in 15 compounds, and each concentration gradient differs 10 times.Respectively by various concentration
15 compounds mix respectively from different protein standard liquid, and the activity of albumen in each sample is detected using Elisa kit.
The process for using of kit is as follows:
(1) prepare: taking out kit from refrigerator, room temperature rewarming balances 30min；
(2) match liquid: 20 times of concentrated cleaning solutions being diluted to former cleaning solution again with distilled water；
(3) sample to be tested is added: taking sufficient amount of enzyme mark coating plate, is fixed on frame, sample to be tested is respectively set
Hole and blank control wells record each hole site, and 10 μ L of sample to be tested, then plus 40 μ L (i.e. Sample Dilution 5 of Sample dilution is first added
Times)；Blank control wells are not added；
(4) incubate: insulating box incubates 30min；
(5) board-washing: discarding liquid, pat dry on blotting paper, and cleaning solution is filled it up in every hole, stands 1min, gets rid of cleaning solution, absorbs water
It pats dry on paper, so repeats board-washing 4 times；
(6) enzyme mark working solution: 50 μ L of enzyme mark working solution is added in every hole, and blank control wells are not added；
(7) incubate: 37 DEG C of insulating boxs incubate 30min；
(8) board-washing: discarding liquid, pat dry on blotting paper, and cleaning solution is filled it up in every hole, stands 1min, gets rid of cleaning solution, absorbs water
It pats dry on paper, so repeats board-washing 4 times；
(9) develop the color: 50 μ L of color developing agent A liquid is first added in every hole, adds 50 μ L of color developing agent B liquid, and plate vortex mixer mixes
30s, 37 DEG C are protected from light colour developing 15min；
(10) it terminates: taking out ELISA Plate, every hole adds 50 μ L of terminate liquid, terminates reaction；
(11) it measures: being returned to zero with blank well, after termination in 15min, with the light absorption value in each hole of 450nm wavelength measurement.
Experimental data is as shown in table 5.
Table 5
By table 5, statistics indicate that, the compound in the present invention is for albumen relevant to liver fibrosis: matrix metalloprotease
Enzyme -2 (MMP2), Matrix Metalloproteinase-9 (MMP9) and transforming growth factor-beta 1 (TGF-β 1) have lower IC50 value.I.e.
There is good combination and inhibits its bioactivity.
Although the present invention and its advantage has been described in detail it should be appreciated that without departing from by the attached claims
Defined by can carry out various changes, substitution and transformation in the case where the spirit and scope of the present invention.Moreover, the model of the application
Enclose the specific embodiment for being not limited only to process, equipment described in specification, means, method and steps.In the art is common
Technical staff is from the disclosure it will be readily understood that execution and corresponding reality described herein can be used according to the present invention
Apply the essentially identical function of example or process that obtain the result essentially identical with it, that existing and future is to be developed, equipment,
Means, method or step.Therefore, the attached claims are intended in the range of them include such process, equipment, hand
Section, method or step.
Claims (3)
1. a kind of Lignanoids compounds are preparing the application in anti-hepatic fibrosis medicines, the Lignanoids compounds are with formula
Structure shown in 1:
2. application according to claim 1, it is characterised in that: the Lignanoids compounds are extracted from natural HERBA BUPLEURI
It is isolated.
3. application according to claim 2, it is characterised in that: the preparation method of the Lignanoids compounds includes step
It is rapid:
1) it takes dry HERBA BUPLEURI pulverizing medicinal materials at coarse powder, measures 75% ethyl alcohol heating and refluxing extraction 2-4 times with 8-10 times, every time
1-2h, filtering, merging filtrate are concentrated under reduced pressure and remove ethyl alcohol, and stirring is diluted with water, and successively use petroleum ether, ethyl acetate, n-butanol
It is extracted with water；Extract liquor is collected, rotary evaporation obtains each extraction part；
2) ethyl acetate extraction part is dissolved in ethyl acetate makes its concentration reach 20-50mg/ml, crosses the silicon of 200-300 mesh
Rubber column gel column chromatography uses petroleum ether-ethyl acetate system with 4:1,1:1,1:8,1:20 according to volume ratio, and pure methanol system gradient is washed
It is de-；Eluent is collected, rotary evaporation obtains the elution fraction of 1:20；
3) elution fraction of 1:20 is dissolved in ethyl acetate makes its concentration reach 30-80mg/ml, crosses the silica gel of 200-300 mesh
Column chromatography uses petroleum ether-ethyl acetate system with 2:1 isocratic elution according to volume ratio；Each fraction collection 50ml, note first
A fraction is 1#, and number consecutively is merged by fraction polarity after rotary evaporation, obtain eluting fraction 94-109# to be separated and
110-128#；
4) eluting fraction 94-109# to be separated is dissolved in acetonitrile makes its concentration reach 30-80mg/ml, using reverse phase C18 column
Isocratic elution through 60% acetonitrile of row, the Detection wavelength of UV detector are 230-280nm, collect the chemical combination that 41-43min occurs
Object absorption peak；Fraction is named as compound 4b；
5) eluting fraction 110-128# to be separated is dissolved in acetonitrile makes its concentration reach 30-80mg/ml, using reverse phase C18
Isocratic elution of the column through 50% acetonitrile of row, the Detection wavelength of UV detector are 230-280nm, collect the change that 12-14min occurs
Close object absorption peak；Fraction is named as compound 1a, collects the compound absorption peak that 22-25min occurs；Fraction is named as compound
4a；
6) n-butanol portion by 3 times amount acetone soln heating and refluxing extraction 3 times, each 30min；Collect extracting solution, rotation
Evaporation, obtains the insoluble part of acetone of the acetone soluble fraction and extracting n-butyl alcohol of extracting n-butyl alcohol；
7) the acetone soluble fraction of extracting n-butyl alcohol is dissolved in acetone makes its concentration reach 20-50mg/ml, crosses 200-300 mesh
Silica gel column chromatography, use acetate-methanol system with 100:1,75:1,25:1, pure methanol system gradient according to volume ratio
Elution；Eluent is collected, rotary evaporation obtains the elution fraction of 75:1；
8) elution fraction of 75:1 is dissolved in acetone makes its concentration reach 30-80mg/ml, crosses the silicagel column color of 200-300 mesh
Spectrum, uses cyclohexane-acetone system with 4:1,3:2,1:2 gradient elution according to volume ratio；Each fraction collection 50ml, note first
A fraction is 1#, and number consecutively is merged after rotary evaporation by polarity, obtains eluting fraction 3-5# and 6-10# to be separated；
9) eluting fraction 3-5# to be separated is dissolved in acetonitrile makes its concentration reach 30-80mg/ml, is passed through using reverse phase C18 column
The isocratic elution of 55% acetonitrile of row, the Detection wavelength of UV detector are 230-280nm, collect the compound that 45-47min occurs
Absorption peak, fraction are named as compound 3a；The compound absorption peak that 48-49min occurs is collected, fraction is named as compound 5a；
The compound absorption peak that 61-63min occurs is collected, fraction is named as compound 2a；
9) eluting fraction 6-10# to be separated is dissolved in acetonitrile makes its concentration reach 30-80mg/ml, is passed through using reverse phase C18 column
The isocratic elution of 45% acetonitrile of row, the Detection wavelength of UV detector are 230-280nm, collect the compound that 30-32min occurs
Absorption peak, fraction are named as compound 2b；The compound absorption peak that 35-37min occurs is collected, fraction is named as compound 5b；
The compound absorption peak that 45-49min occurs is collected, fraction is named as compound 3b；
10) so that its concentration is reached 20-50mg/ml insoluble be partially soluble in acetone of the acetone of extracting n-butyl alcohol, cross 100-200
Purpose silica gel column chromatography uses acetate-methanol system with 100:1,4:1,1:1, pure methanol system gradient according to volume ratio
Elution；Eluent is collected, rotary evaporation obtains the elution fraction of 4:1；
11) elution fraction of 4:1 is dissolved in acetone makes its concentration reach 30-80mg/ml, crosses the silicagel column color of 200-300 mesh
Spectrum, uses acetate-methanol system with 3:4 isocratic elution according to volume ratio；Each fraction collection 50ml remembers first fraction
For 1#, number consecutively is merged after rotary evaporation by polarity, obtains eluting fraction 1-3#, 4-8# and 9-12# to be separated；
12) eluting fraction 1-3# to be separated is dissolved in acetonitrile makes its concentration reach 30-80mg/ml, is passed through using reverse phase C18 column
The isocratic elution of 35% acetonitrile of row, the Detection wavelength of UV detector are 230-280nm, collect the compound that 18-20min occurs
Absorption peak, fraction are named as compound 1e；The compound absorption peak that 26-29min occurs is collected, fraction is named as compound 1d；
13) eluting fraction 4-8# to be separated is dissolved in acetonitrile makes its concentration reach 30-80mg/ml, is passed through using reverse phase C18 column
The isocratic elution of 35% acetonitrile of row, the Detection wavelength of UV detector are 230-280nm, collect the compound that 31-33min occurs
Absorption peak, fraction are named as compound 6a；The compound absorption peak that 36-37min occurs is collected, fraction is named as compound 1b；
14) eluting fraction 4-8# to be separated is dissolved in acetonitrile makes its concentration reach 30-80mg/ml, is passed through using reverse phase C18 column
The isocratic elution of 30% acetonitrile of row, the Detection wavelength of UV detector are 230-280nm, collect the compound that 25-28min occurs
Absorption peak, fraction are named as compound 1c；The compound absorption peak that 48-50min occurs is collected, fraction is named as compound 7a.
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| CN201910902605.2A CN110652509A (en) | 2017-09-13 | 2017-09-13 | Application of diaryl butyrolactone lignan compound in preparing anti-hepatic fibrosis medicine |
| CN201910902604.8A CN110538177B (en) | 2017-09-13 | 2017-09-13 | Application of aryl naphthalene lignan compound in preparation of anti-hepatic fibrosis medicine |
| CN201710823479.2A CN107595838B (en) | 2017-09-13 | 2017-09-13 | A kind of Lignanoids compounds are preparing the application in anti-liver fiber drug |
| CN201910902661.6A CN110585193B (en) | 2017-09-13 | 2017-09-13 | Application of diaryl butyrolactone lignan compound in preparation of anti-hepatic fibrosis medicine |
| CN201910902603.3A CN110538176B (en) | 2017-09-13 | 2017-09-13 | Application of aryl naphthalene lignans compound in preparation of anti-hepatic fibrosis medicine |
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| CN201910902661.6A Division CN110585193B (en) | 2017-09-13 | 2017-09-13 | Application of diaryl butyrolactone lignan compound in preparation of anti-hepatic fibrosis medicine |
| CN201910902603.3A Division CN110538176B (en) | 2017-09-13 | 2017-09-13 | Application of aryl naphthalene lignans compound in preparation of anti-hepatic fibrosis medicine |
| CN201910902605.2A Division CN110652509A (en) | 2017-09-13 | 2017-09-13 | Application of diaryl butyrolactone lignan compound in preparing anti-hepatic fibrosis medicine |
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| CN201910902603.3A Active CN110538176B (en) | 2017-09-13 | 2017-09-13 | Application of aryl naphthalene lignans compound in preparation of anti-hepatic fibrosis medicine |
| CN201910902604.8A Active CN110538177B (en) | 2017-09-13 | 2017-09-13 | Application of aryl naphthalene lignan compound in preparation of anti-hepatic fibrosis medicine |
| CN201710823479.2A Active CN107595838B (en) | 2017-09-13 | 2017-09-13 | A kind of Lignanoids compounds are preparing the application in anti-liver fiber drug |
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| CN101254253A (en) * | 2008-04-11 | 2008-09-03 | 北京星昊医药股份有限公司 | Rhizoma saururi herba saururi lignans extract, preparation and uses thereof |
| JP2010116371A (en) * | 2008-11-14 | 2010-05-27 | Hamari Chemicals Ltd | Composition for prevention or amelioration of metabolic syndrome |
| CN103012118B (en) * | 2013-01-15 | 2014-08-20 | 云南民族大学 | Lignans compounds and preparation method and application thereof |
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| CN103467463B (en) * | 2013-09-18 | 2016-06-22 | 南开大学 | One lignans analog derivative and its production and use |
| CN104095859B (en) * | 2014-07-31 | 2016-12-28 | 顾玉奎 | The application in preparing anti-hepatic fibrosis medicines of O-(piperidyl) ethyl derivative of Cleistanone Cleistanone |
| CN104873491B (en) * | 2015-05-19 | 2017-09-12 | 中国人民解放军第二军医大学 | The application of deoxyschizandrin, schisandrin C in anti-hepatic fibrosis medicines are prepared |
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| CN110538176B (en) | 2022-08-02 |
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